Papazoglou I, Lee J-H, Cui Z, Li C, Fulgenzi G, Bahn YJ, Staniszewska-Goraczniak HM, Piñol RA, Hogue IB, Enquist LW, et al. A distinct hypothalamus-to-β cell circuit modulates insulin secretion. Cell Metab. 2022;34 (2) :285-298.e7.Abstract
The central nervous system has long been thought to regulate insulin secretion, an essential process in the maintenance of blood glucose levels. However, the anatomical and functional connections between the brain and insulin-producing pancreatic β cells remain undefined. Here, we describe a functional transneuronal circuit connecting the hypothalamus to β cells in mice. This circuit originates from a subpopulation of oxytocin neurons in the paraventricular hypothalamic nucleus (PVN), and it reaches the islets of the endocrine pancreas via the sympathetic autonomic branch to innervate β cells. Stimulation of PVN neurons rapidly suppresses insulin secretion and causes hyperglycemia. Conversely, silencing of these neurons elevates insulin levels by dysregulating neuronal signaling and secretory pathways in β cells and induces hypoglycemia. PVN neuronal activity is triggered by glucoprivation. Our findings reveal that a subset of PVN neurons form functional multisynaptic circuits with β cells in mice to regulate insulin secretion, and their function is necessary for the β cell response to hypoglycemia.
Esteves AD, Koyuncu OO, Enquist LW. A Pseudorabies Virus Serine/Threonine Kinase, US3, Promotes Retrograde Transport in Axons via Akt/mToRC1. J Virol. 2022 :JVI0175221.Abstract
Infection of peripheral axons by alpha herpesviruses (AHVs) is a critical stage in establishing a life-long infection in the host. Upon entering the cytoplasm of axons, AHV nucleocapsids and associated inner-tegument proteins must engage the cellular retrograde transport machinery to promote the long-distance movement of virion components to the nucleus. The current model outlining this process is incomplete and further investigation is required to discover all viral and cellular determinants involved as well as the temporality of the events. Using a modified tri-chamber system, we have discovered a novel role of the pseudorabies virus (PRV) serine/threonine kinase, US3, in promoting efficient retrograde transport of nucleocapsids. We discovered that transporting nucleocapsids move at similar velocities both in the presence and absence of a functional US3 kinase; however fewer nucleocapsids are moving when US3 is absent and move for shorter periods of time before stopping, suggesting US3 is required for efficient nucleocapsid engagement with the retrograde transport machinery. This led to fewer nucleocapsids reaching the cell bodies to produce a productive infection 12hr later. Furthermore, US3 was responsible for the induction of local translation in axons as early as 1hpi through the stimulation of a PI3K/Akt-mToRC1 pathway. These data describe a novel role for US3 in the induction of local translation in axons during AHV infection, a critical step in transport of nucleocapsids to the cell body. Neurons are highly polarized cells with axons that can reach centimeters in length. Communication between axons at the periphery and the distant cell body is a relatively slow process involving the active transport of chemical messengers. There's a need for axons to respond rapidly to extracellular stimuli. Translation of repressed mRNAs present within the axon occurs to enable rapid, localized responses independently of the cell body. AHVs have evolved a way to hijack local translation in the axons to promote their transport to the nucleus. We have determined the cellular mechanism and viral components involved in the induction of axonal translation. The US3 serine/threonine kinase of PRV activates Akt-mToRC1 signaling pathways early during infection to promote axonal translation. When US3 is not present, the number of moving nucleocapsids and their processivity are reduced, suggesting that US3 activity is required for efficient engagement of nucleocapsids with the retrograde transport machinery.
Burckhardt RM, Dennehy JJ, Poon LLM, Saif LJ, Enquist LW. Are COVID-19 Vaccine Boosters Needed? The Science behind Boosters. J Virol. 2021 :JVI0197321.Abstract
Waning vaccine-induced immunity coupled with the emergence of SARS-CoV-2 variants has led to increases in breakthrough infections, prompting consideration for vaccine booster doses. Boosters have been reported to be safe and increase SARS-CoV-2-specific neutralizing antibody levels, but how these doses impact the trajectory of the global pandemic and herd immunity is unknown. Information on immunology, epidemiology and equitable vaccine distribution should be considered when deciding the timing and eligibility for COVID-19 vaccine boosters.
Enquist LW, Dermody TS, DiMaio D. Introduction. Annu Rev Virol. 2021;8 (1) :i-ii.
Xydakis MS, Albers MW, Holbrook EH, Lyon DM, Shih RY, Frasnelli JA, Pagenstecher A, Kupke A, Enquist LW, Perlman S. Post-viral effects of COVID-19 in the olfactory system and their implications. Lancet Neurol. 2021.Abstract
BACKGROUND: The mechanisms by which any upper respiratory virus, including SARS-CoV-2, impairs chemosensory function are not known. COVID-19 is frequently associated with olfactory dysfunction after viral infection, which provides a research opportunity to evaluate the natural course of this neurological finding. Clinical trials and prospective and histological studies of new-onset post-viral olfactory dysfunction have been limited by small sample sizes and a paucity of advanced neuroimaging data and neuropathological samples. Although data from neuropathological specimens are now available, neuroimaging of the olfactory system during the acute phase of infection is still rare due to infection control concerns and critical illness and represents a substantial gap in knowledge. RECENT DEVELOPMENTS: The active replication of SARS-CoV-2 within the brain parenchyma (ie, in neurons and glia) has not been proven. Nevertheless, post-viral olfactory dysfunction can be viewed as a focal neurological deficit in patients with COVID-19. Evidence is also sparse for a direct causal relation between SARS-CoV-2 infection and abnormal brain findings at autopsy, and for trans-synaptic spread of the virus from the olfactory epithelium to the olfactory bulb. Taken together, clinical, radiological, histological, ultrastructural, and molecular data implicate inflammation, with or without infection, in either the olfactory epithelium, the olfactory bulb, or both. This inflammation leads to persistent olfactory deficits in a subset of people who have recovered from COVID-19. Neuroimaging has revealed localised inflammation in intracranial olfactory structures. To date, histopathological, ultrastructural, and molecular evidence does not suggest that SARS-CoV-2 is an obligate neuropathogen. WHERE NEXT?: The prevalence of CNS and olfactory bulb pathosis in patients with COVID-19 is not known. We postulate that, in people who have recovered from COVID-19, a chronic, recrudescent, or permanent olfactory deficit could be prognostic for an increased likelihood of neurological sequelae or neurodegenerative disorders in the long term. An inflammatory stimulus from the nasal olfactory epithelium to the olfactory bulbs and connected brain regions might accelerate pathological processes and symptomatic progression of neurodegenerative disease. Persistent olfactory impairment with or without perceptual distortions (ie, parosmias or phantosmias) after SARS-CoV-2 infection could, therefore, serve as a marker to identify people with an increased long-term risk of neurological disease.
Laval K, Enquist LW. The Potential Role of Herpes Simplex Virus Type 1 and Neuroinflammation in the Pathogenesis of Alzheimer's Disease. Front Neurol. 2021;12 :658695.Abstract
Alzheimer's disease (AD) is a neurodegenerative disease affecting ~50 million people worldwide. To date, there is no cure and current therapies have not been effective in delaying disease progression. Therefore, there is an urgent need for better understanding of the pathogenesis of AD and to rethink possible therapies. Herpes simplex virus type 1 (HSV1) has recently received growing attention for its potential role in sporadic AD. The virus is a ubiquitous human pathogen that infects mucosal epithelia and invades the peripheral nervous system (PNS) of its host to establish a reactivable, latent infection. Upon reactivation, HSV1 spreads back to the epithelium and initiates a new infection, causing epithelial lesions. Occasionally, the virus spreads from the PNS to the brain after reactivation. In this review, we discuss current work on the pathogenesis of AD and summarize research results that support a potential role for HSV1 in the infectious hypothesis of AD. We also highlight recent findings on the neuroinflammatory response, which has been proposed to be the main driving force of AD, starting early in the course of the disease. Relevant rodent models to study neuroinflammation in AD and novel therapeutic approaches are also discussed. Throughout this review, we focus on several aspects of HSV1 pathogenesis, including its primary role as an invader of the PNS, that should be considered in the etiology of AD. We also point out some of the contradictory data and remaining knowledge gaps that require further research to finally fully understand the cause of AD in humans.
Pearson VR, Bosse JB, Koyuncu OO, Scherer J, Toruno C, Robinson R, Abegglen LM, Schiffman JD, Enquist LW, Rall GF. Identification of African Elephant Polyomavirus in wild elephants and the creation of a vector expressing its viral tumor antigens to transform elephant primary cells. PLoS One. 2021;16 (2) :e0244334.Abstract
Wild elephant populations are declining rapidly due to rampant killing for ivory and body parts, range fragmentation, and human-elephant conflict. Wild and captive elephants are further impacted by viruses, including highly pathogenic elephant endotheliotropic herpesviruses. Moreover, while the rich genetic diversity of the ancient elephant lineage is disappearing, elephants, with their low incidence of cancer, have emerged as a surprising resource in human cancer research for understanding the intrinsic cellular response to DNA damage. However, studies on cellular resistance to transformation and herpesvirus reproduction have been severely limited, in part due to the lack of established elephant cell lines to enable in vitro experiments. This report describes creation of a recombinant plasmid, pAelPyV-1-Tag, derived from a wild isolate of African Elephant Polyomavirus (AelPyV-1), that can be used to create immortalized lines of elephant cells. This isolate was extracted from a trunk nodule biopsy isolated from a wild African elephant, Loxodonta africana, in Botswana. The AelPyV-1 genome contains open-reading frames encoding the canonical large (LTag) and small (STag) tumor antigens. We cloned the entire early region spanning the LTag and overlapping STag genes from this isolate into a high-copy vector to construct a recombinant plasmid, pAelPyV-1-Tag, which effectively transformed primary elephant endothelial cells. We expect that the potential of this reagent to transform elephant primary cells will, at a minimum, facilitate study of elephant-specific herpesviruses.
Enquist LW, DiMaio D, Dermody TS. Recurring Revolutions in Virology. Annu Rev Virol. 2021.
Alwine JC, Enquist LW, Dermody TS, Goodrum F. What Is the Price of Science?. mBio [Internet]. 2021;12 (2) :e00117-21. Publisher's VersionAbstract
The peer-reviewed scientific literature is the bedrock of science. However,
      scientific publishing is undergoing dramatic changes, which include the expansion of
      open access, an increased number of for-profit publication houses, and ready
      availability of preprint manuscripts that have not been peer reviewed. In this
      opinion article, we discuss the inequities and concerns that these changes have
Tanneti NS, Federspiel JD, Cristea IM, Enquist LW. The axonal sorting activity of pseudorabies virus Us9 protein depends on the state of neuronal maturation. PLoS Pathog. 2020;16 (12) :e1008861.Abstract
Alpha-herpesviruses establish a life-long infection in the nervous system of the affected host; while this infection is restricted to peripheral neurons in a healthy host, the reactivated virus can spread within the neuronal circuitry, such as to the brain, in compromised individuals and lead to adverse health outcomes. Pseudorabies virus (PRV), an alpha-herpesvirus, requires the viral protein Us9 to sort virus particles into axons and facilitate neuronal spread. Us9 sorts virus particles by mediating the interaction of virus particles with neuronal transport machinery. Here, we report that Us9-mediated regulation of axonal sorting also depends on the state of neuronal maturation. Specifically, the development of dendrites and axons is accompanied with proteomic changes that influence neuronal processes. Immature superior cervical ganglionic neurons (SCGs) have rudimentary neurites that lack markers of mature axons. Immature SCGs can be infected by PRV, but they show markedly reduced Us9-dependent regulation of sorting, and increased Us9-independent transport of particles into neurites. Mature SCGs have relatively higher abundances of proteins characteristic of vesicle-transport machinery. We also identify Us9-associated neuronal proteins that can contribute to axonal sorting and subsequent anterograde spread of virus particles in axons. We show that SMPD4/nsMase3, a sphingomyelinase abundant in lipid-rafts, associates with Us9 and is a negative regulator of PRV sorting into axons and neuronal spread, a potential antiviral function.
Van Cleemput J, Koyuncu OO, Laval K, Engel EA, Enquist LW. CRISPR/Cas9-constructed pseudorabies virus mutants reveal the importance of UL13 in alphaherpesvirus escape from genome silencing. J Virol. 2020.Abstract
Latent and recurrent productive infection of long-living cells, such as neurons, enables alphaherpesviruses to persist in their host populations. Still, the viral factors involved in these events remain largely obscure. Using a complementation assay in compartmented primary peripheral nervous system (PNS) neuronal cultures, we previously reported that productive replication of axonally-delivered genomes is facilitated by PRV tegument proteins. Here, we sought to unravel the role of tegument protein UL13 in this escape from silencing. We first constructed four new PRV mutants in the virulent Becker strain using CRISPR/Cas9-mediated gene replacement: (i) PRV Becker defective for UL13 expression (PRV ΔUL13), (ii) PRV where UL13 is fused to eGFP (PRV UL13-eGFP) and two control viruses (iii and iv) PRV where VP16 is fused with mTurquoise at either the N-terminus (PRV mTurq-VP16) or C-terminus (PRV VP16-mTurq). Live cell imaging of PRV capsids showed efficient retrograde transport after axonal infection with PRV UL13-eGFP, although we did not detect dual-color particles. Surprisingly, immunofluorescence staining of particles in mid-axons indicated that UL13 might be co-transported with PRV capsids in PNS axons. Superinfecting nerve cell bodies with UV-inactivated PRV ΔUL13 failed to efficiently promote escape from genome silencing when compared to UV-PRV wild type and UV-PRV UL13-eGFP superinfection. However, UL13 does not act directly in the escape from genome silencing, as AAV-mediated UL13 expression in neuronal cell bodies was not sufficient to provoke escape from genome silencing. Based on this, we suggest that UL13 may contribute to initiation of productive infection through phosphorylation of other tegument proteins. Alphaherpesviruses have mastered various strategies to persist in an immunocompetent host, including the induction of latency and reactivation in peripheral nervous system (PNS) ganglia. We recently discovered that the molecular mechanism underlying escape from latency by the alphaherpesvirus pseudorabies virus (PRV) relies on a structural viral tegument protein. This study aimed at unravelling the role of tegument protein UL13 in PRV escape from latency. First, we confirmed the use of CRISPR/Cas9-mediated gene replacement as a versatile tool to modify the PRV genome. Next, we used our new set of viral mutants and AAV vectors to conclude on the indirect role of UL13 in PRV escape from latency in primary neurons and on its spatial localization during retrograde capsid transport in axons. Based on these findings, we speculate that UL13 phosphorylates one or more tegument proteins, thereby priming these putative proteins to induce escape from genome silencing.
Enquist LW. (2020, Nov 30). Virology and cancer biology: DNA viruses associated with transformation and tumor formation. The discovery of tumor suppressor genes [Video file]. In The Biomedical & Life Sciences Collection, Henry Stewart Talks. Retrieved December 7, 2020, from 2020.
Enquist LW. (2020, Nov 30). Virology and cancer biology: the retroviruses and the discovery of oncogenes [Video file]. In The Biomedical & Life Sciences Collection, Henry Stewart Talks. Retrieved Dec 7, 2020, from 2020.
Koyuncu OO, Enquist LW, Engel EA. Invasion of the Nervous System. Curr Issues Mol Biol. 2020;41 :1-62.Abstract
In vertebrates, the nervous system (NS) is composed of a peripheral collection of neurons (the peripheral nervous system, PNS), a central set found in the brain and spinal cord (the central nervous system, CNS). The NS is protected by rather complicated multi-layer barriers that allow access to nutrients and facilitate contact with the peripheral tissues, but block entry of pathogens and toxins. Virus infections usually begin in peripheral tissues and if these barriers are weakened, they can spread into the PNS and more rarely into the CNS. Most viral infections of the NS are opportunistic or accidental pathogens that gain access via the bloodstream (e.g., HIV and various arboviruses). But a few have evolved to enter the NS efficiently by invading neurons directly and by exploiting neuronal cell biology (e.g., rhabdoviruses and alphaherpesviruses). Most NS infections are devastating and difficult to manage. Remarkably, the alphaherpesviruses establish life-long quiescent infections in the PNS, with rare but often serious CNS pathology. In this review, we will focus on how alphaherpesviruses gain access to and spread in the NS, with particular emphasis on bidirectional transport and spread within and between neurons and neural circuits, which is regulated by complex viral-host protein interactions. Finally, we will describe the wide use of alphaherpesviruses as tools to study nerve connectivity and function in animal models.
Laval K, Maturana CJ, Enquist LW. Mouse Footpad Inoculation Model to Study Viral-Induced Neuroinflammatory Responses. J Vis Exp. 2020;(160).Abstract
This protocol describes a footpad inoculation model used to study the initiation and development of neuroinflammatory responses during alphaherpesvirus infection in mice. As alphaherpesviruses are main invaders of the peripheral nervous system (PNS), this model is suitable to characterize the kinetics of viral replication, its spread from the PNS to CNS, and associated neuroinflammatory responses. The footpad inoculation model allows virus particles to spread from a primary infection site in the footpad epidermis to sensory and sympathetic nerve fibers that innervate the epidermis, sweat glands, and dermis. The infection spreads via the sciatic nerve to the dorsal root ganglia (DRG) and ultimately through the spinal cord to the brain. Here, a mouse footpad is inoculated with pseudorabies virus (PRV), an alphaherpesvirus closely related to herpes simplex virus (HSV) and varicella-zoster virus (VZV). This model demonstrates that PRV infection induces severe inflammation, characterized by neutrophil infiltration in the footpad and DRG. High concentrations of inflammatory cytokines are subsequently detected in homogenized tissues by ELISA. In addition, a strong correlation is observed between PRV gene and protein expression (via qPCR and IF staining) in DRG and the production of pro-inflammatory cytokines. Therefore, the footpad inoculation model provides a better understanding of the processes underlying alphaherpesvirus-induced neuropathies and may lead to the development of innovative therapeutic strategies. In addition, the model can guide research on peripheral neuropathies, such as multiple sclerosis and associated viral-induced damage to the PNS. Ultimately, it can serve as a cost-effective in vivo tool for drug development.
Maturana CJ, Verpeut JL, Pisano TJ, Dhanerawala ZM, Esteves A, Enquist LW, Engel EA. Small Alphaherpesvirus Latency-Associated Promoters Drive Efficient and Long-Term Transgene Expression in the CNS. Mol Ther Methods Clin Dev. 2020;17 :843-857.Abstract
Recombinant adeno-associated viruses (rAAVs) are used as gene therapy vectors to treat central nervous system (CNS) diseases. Despite their safety and broad tropism, important issues need to be corrected such as the limited payload capacity and the lack of small gene promoters providing long-term, pan-neuronal transgene expression in the CNS. Commonly used gene promoters are relatively large and can be repressed a few months after CNS transduction, risking the long-term performance of single-dose gene therapy applications. We used a whole-CNS screening approach based on systemic delivery of AAV-PHP.eB, iDisco+ tissue-clearing and light-sheet microscopy to identify three small latency-associated promoters (LAPs) from the herpesvirus pseudorabies virus (PRV). These promoters are LAP1 (404 bp), LAP2 (498 bp), and LAP1_2 (880 bp). They drive chronic transcription of the virus-encoded latency-associated transcript (LAT) during productive and latent phases of PRV infection. We observed stable, pan-neuronal transgene transcription and translation from AAV-LAPs in the CNS for 6 months post AAV transduction. In several CNS areas, the number of cells expressing the transgene was higher for LAP2 than the large conventional EF1α promoter (1,264 bp). Our data suggest that the LAPs are suitable candidates for viral vector-based CNS gene therapies requiring chronic transgene expression after one-time viral-vector administration.
DiMaio D, Enquist LW, Dermody TS. Introduction: A New Coronavirus Emerges, This Time Causing a Pandemic. Annu Rev Virol. 2020.
Laval K, Enquist LW. The Neuropathic Itch Caused by Pseudorabies Virus. Pathogens. 2020;9 (4).Abstract
Pseudorabies virus (PRV) is an alphaherpesvirus related to varicella-zoster virus (VZV) and herpes simplex virus type 1 (HSV1). PRV is the causative agent of Aujeskzy's disease in swine. PRV infects mucosal epithelium and the peripheral nervous system (PNS) of its host where it can establish a quiescent, latent infection. While the natural host of PRV is the swine, a broad spectrum of mammals, including rodents, cats, dogs, and cattle can be infected. Since the nineteenth century, PRV infection is known to cause a severe acute neuropathy, the so called "mad itch" in non-natural hosts, but surprisingly not in swine. In the past, most scientific efforts have been directed to eradicating PRV from pig farms by the use of effective marker vaccines, but little attention has been given to the processes leading to the mad itch. The main objective of this review is to provide state-of-the-art information on the mechanisms governing PRV-induced neuropathic itch in non-natural hosts. We highlight similarities and key differences in the pathogenesis of PRV infections between non-natural hosts and pigs that might explain their distinctive clinical outcomes. Current knowledge on the neurobiology and possible explanations for the unstoppable itch experienced by PRV-infected animals is also reviewed. We summarize recent findings concerning PRV-induced neuroinflammatory responses in mice and address the relevance of this animal model to study other alphaherpesvirus-induced neuropathies, such as those observed for VZV infection.
Huang H, Koyuncu OO, Enquist LW. Pseudorabies Virus Infection Accelerates Degradation of the Kinesin-3 Motor KIF1A. J Virol. 2020;94 (9).Abstract
Alphaherpesviruses, including pseudorabies virus (PRV), are neuroinvasive pathogens that establish lifelong latency in peripheral ganglia following the initial infection at mucosal surfaces. The establishment of latent infection and subsequent reactivations, during which newly assembled virions are sorted into and transported anterogradely inside axons to the initial mucosal site of infection, rely on axonal bidirectional transport mediated by microtubule-based motors. Previous studies using cultured peripheral nervous system (PNS) neurons have demonstrated that KIF1A, a kinesin-3 motor, mediates the efficient axonal sorting and transport of newly assembled PRV virions. Here we report that KIF1A, unlike other axonal kinesins, is an intrinsically unstable protein prone to proteasomal degradation. Interestingly, PRV infection of neuronal cells leads not only to a nonspecific depletion of KIF1A mRNA but also to an accelerated proteasomal degradation of KIF1A proteins, leading to a near depletion of KIF1A protein late in infection. Using a series of PRV mutants deficient in axonal sorting and anterograde spread, we identified the PRV US9/gE/gI protein complex as a viral factor facilitating the proteasomal degradation of KIF1A proteins. Moreover, by using compartmented neuronal cultures that fluidically and physically separate axons from cell bodies, we found that the proteasomal degradation of KIF1A occurs in axons during infection. We propose that the PRV anterograde sorting complex, gE/gI/US9, recruits KIF1A to viral transport vesicles for axonal sorting and transport and eventually accelerates the proteasomal degradation of KIF1A in axons. Pseudorabies virus (PRV) is an alphaherpesvirus related to human pathogens herpes simplex viruses 1 and 2 and varicella-zoster virus. Alphaherpesviruses are neuroinvasive pathogens that establish lifelong latent infections in the host peripheral nervous system (PNS). Following reactivation from latency, infection spreads from the PNS back via axons to the peripheral mucosal tissues, a process mediated by kinesin motors. Here, we unveil and characterize the underlying mechanisms for a PRV-induced, accelerated degradation of KIF1A, a kinesin-3 motor promoting the sorting and transport of PRV virions in axons. We show that PRV infection disrupts the synthesis of KIF1A and simultaneously promotes the degradation of intrinsically unstable KIF1A proteins by proteasomes in axons. Our work implies that the timing of motor reduction after reactivation would be critical because progeny particles would have a limited time window for sorting into and transport in axons for further host-to-host spread.
Grose C, Enquist LW. The round trip model for severe herpes zoster caused by live attenuated varicella vaccine virus. J Med Virol. 2020.
Scherer J, Hogue IB, Yaffe ZA, Tanneti NS, Winer BY, Vershinin M, Enquist LW. A Kinesin-3 recruitment complex facilitates axonal sorting of enveloped alpha herpesvirus capsids. PLoS Pathog. 2020;16 (1) :e1007985.Abstract
Axonal sorting, the controlled passage of specific cargoes from the cell soma into the axon compartment, is critical for establishing and maintaining the polarity of mature neurons. To delineate axonal sorting events, we took advantage of two neuroinvasive alpha-herpesviruses. Human herpes simplex virus 1 (HSV-1) and pseudorabies virus of swine (PRV; suid herpesvirus 1) have evolved as robust cargo of axonal sorting and transport mechanisms. For efficient axonal sorting and subsequent egress from axons and presynaptic termini, progeny capsids depend on three viral membrane proteins (Us7 (gI), Us8 (gE), and Us9), which engage axon-directed kinesin motors. We present evidence that Us7-9 of the veterinary pathogen pseudorabies virus (PRV) form a tripartite complex to recruit Kif1a, a kinesin-3 motor. Based on multi-channel super-resolution and live TIRF microscopy, complex formation and motor recruitment occurs at the trans-Golgi network. Subsequently, progeny virus particles enter axons as enveloped capsids in a transport vesicle. Artificial recruitment of Kif1a using a drug-inducible heterodimerization system was sufficient to rescue axonal sorting and anterograde spread of PRV mutants devoid of Us7-9. Importantly, biophysical evidence suggests that Us9 is able to increase the velocity of Kif1a, a previously undescribed phenomenon. In addition to elucidating mechanisms governing axonal sorting, our results provide further insight into the composition of neuronal transport systems used by alpha-herpesviruses, which will be critical for both inhibiting the spread of infection and the safety of herpesvirus-based oncolytic therapies.
Laval K, Van Cleemput J, Vernejoul JB, Enquist LW. Alphaherpesvirus infection of mice primes PNS neurons to an inflammatory state regulated by TLR2 and type I IFN signaling. PLoS Pathog. 2019;15 (11) :e1008087.Abstract
Pseudorabies virus (PRV), an alphaherpesvirus closely related to Varicella-Zoster virus (VZV) and Herpes simplex type 1 (HSV1) infects mucosa epithelia and the peripheral nervous system (PNS) of its host. We previously demonstrated that PRV infection induces a specific and lethal inflammatory response, contributing to severe neuropathy in mice. So far, the mechanisms that initiate this neuroinflammation remain unknown. Using a mouse footpad inoculation model, we found that PRV infection rapidly and simultaneously induces high G-CSF and IL-6 levels in several mouse tissues, including the footpad, PNS and central nervous system (CNS) tissues. Interestingly, this global increase occurred before PRV had replicated in dorsal root ganglia (DRGs) neurons and also was independent of systemic inflammation. These high G-CSF and IL-6 levels were not caused by neutrophil infiltration in PRV infected tissues, as we did not detect any neutrophils. Efficient PRV replication and spread in the footpad was sufficient to activate DRGs to produce cytokines. Finally, by using knockout mice, we demonstrated that TLR2 and IFN type I play crucial roles in modulating the early neuroinflammatory response and clinical outcome of PRV infection in mice. Overall, these results give new insights into the initiation of virus-induced neuroinflammation during herpesvirus infections.
Enquist LW, Dermody TS, DiMaio D. Introduction. Annu Rev Virol. 2019;6 (1) :i-ii.
Berger S, Pho H, Fleury-Curado T, Bevans-Fonti S, Younas H, Shin M-K, Jun JC, Anokye-Danso F, Ahima RS, Enquist LW, et al. Intranasal Leptin Relieves Sleep-disordered Breathing in Mice with Diet-induced Obesity. Am J Respir Crit Care Med. 2019;199 (6) :773-783.Abstract
RATIONALE: Leptin treats upper airway obstruction and alveolar hypoventilation in leptin-deficient ob/ob mice. However, obese humans and mice with diet-induced obesity (DIO) are resistant to leptin because of poor permeability of the blood-brain barrier. We propose that intranasal leptin will bypass leptin resistance and treat sleep-disordered breathing in obesity. OBJECTIVES: To assess if intranasal leptin can treat obesity hypoventilation and upper airway obstruction during sleep in mice with DIO. METHODS: Male C57BL/6J mice were fed with a high-fat diet for 16 weeks. A single dose of leptin (0.4 mg/kg) or BSA (vehicle) were administered intranasally or intraperitoneally, followed by either sleep studies (n = 10) or energy expenditure measurements (n = 10). A subset of mice was treated with leptin daily for 14 days for metabolic outcomes (n = 20). In a separate experiment, retrograde viral tracers were used to examine connections between leptin receptors and respiratory motoneurons. MEASUREMENTS AND MAIN RESULTS: Acute intranasal, but not intraperitoneal, leptin decreased the number of oxygen desaturation events in REM sleep, and increased ventilation in non-REM and REM sleep, independently of metabolic effects. Chronic intranasal leptin decreased food intake and body weight, whereas intraperitoneal leptin had no effect. Intranasal leptin induced signal transducer and activator of transcription 3 phosphorylation in hypothalamic and medullary centers, whereas intraperitoneal leptin had no effect. Leptin receptor-positive cells were synaptically connected to respiratory motoneurons. CONCLUSIONS: In mice with DIO, intranasal leptin bypassed leptin resistance and significantly attenuated sleep-disordered breathing independently of body weight.
Enquist LW, Dermody TS, DiMaio D. Introduction. Annu Rev Virol. 2018;5 (1) :i.
DiMaio D, Dermody TS, Enquist LW. Reductio ad Intellectum. Annu Rev Virol. 2018;5 (1) :ii-iv.
Laval K, Vernejoul JB, Van Cleemput J, Koyuncu OO, Enquist LW. Virulent PRV infection induces a specific and lethal systemic inflammatory response in mice. J Virol. 2018.Abstract
Pseudorabies virus (PRV) is an alphaherpesvirus that infects the peripheral nervous system (PNS). The natural host of PRV is the swine, but it can infect most mammals, including cattle, rodents and dogs. In these non-natural hosts, PRV always causes a severe acute and lethal neuropathy called the "mad itch", which is uncommon in swine. So far, the pathophysiological and immunological processes leading to the development of the neuropathic itch, and death of the animal are unclear.Using a footpad inoculation model, we established that mice inoculated with PRV-Becker (virulent strain) develop a severe pruritus in the foot and become moribund at 82 hours post-inoculation (hpi). We found necrosis and inflammation with a massive neutrophil infiltration only in the footpad and DRGs by H&E staining. PRV load was detected in the foot, PNS and CNS tissues by quantitative-RT-PCR. Infected mice had elevated plasma levels of pro-inflammatory cytokines (IL6 and G-CSF) and chemokines (Gro-1 and MCP-1). Significant IL6 and G-CSF levels were detected in several tissues at 82hpi. High plasma levels of C-reactive protein confirmed the acute inflammatory response to PRV-Becker infection. Moreover, mice inoculated with PRV-Bartha (attenuated, live vaccine strain), did not develop pruritus at 82hpi. PRV-Bartha also replicated in the PNS, infection spread further in the brain than PRV-Becker. PRV-Bartha infection did not induce the specific and lethal systemic inflammatory response seen with PRV-Becker. Overall, we demonstrated the importance of inflammation in the clinical outcome of PRV infection in mice and provide new insights into the process of PRV-induced neuroinflammation.Pseudorabies virus (PRV) is an alphaherpesvirus related to human pathogens such as herpes simplex virus type 1 (HSV-1) and varicella-zoster virus (VZV). The natural host of PRV is the swine but it can infect most mammals. In susceptible animals other than pigs, PRV infection always causes a characteristic lethal pruritus known as the "mad itch". The role of the immune response in the clinical outcome of PRV infection is still poorly understood. Here, we show that a systemic host inflammatory response is responsible for the severe pruritus and acute death of mice infected with virulent PRV-Becker but not attenuated strain PRV-Bartha. In addition, we identified IL-6 and G-CSF as two main cytokines that play crucial roles in the regulation of this process. Our findings give new insights into neuroinflammatory diseases and strengthen further the similarities between VZV and PRV infections at the level of innate immunity.
Hogue IB, Card PJ, Rinaman L, Goraczniak HS, Enquist LW. Characterization of the neuroinvasive profile of a pseudorabies virus recombinant expressing the mTurquoise2 reporter in single and multiple injection experiments. J Neurosci Methods. 2018;308 :228-239.Abstract
BACKGROUND: Viral transneuronal tracing has become a well established technology used to define the synaptic architecture of polysynaptic neural networks. NEW METHOD: In this report we define the neuroinvasive profile and reporter expression of a new recombinant of the Bartha strain of pseudorabies virus (PRV). The new recombinant, PRV-290, expresses the mTurquoise2 fluorophor and is designed to complement other isogenic recombinants of Bartha that express different reporters of infection. Results & Comparison with Existing Methods: PRV-290 was injected either alone or in combination with isogenic recombinants of PRV that express enhanced green fluorescent protein (EGFP; PRV-152) or monomeric red fluorescent protein (mRFP; PRV-614). Circuits previously defined using PRV-152 and PRV-614 were used for the analysis. The data demonstrate that PRV-290 is a retrograde transneuronal tracer with temporal kinetics similar to those of its isogenic recombinants. Stable expression of the diffusible mTurquoise2 reporter filled infected neurons, with the extent and intensity of labeling increasing with advancing post inoculation survival. In multiple injection experiments, PRV-290 established productive infections in neurons also replicating PRV-152 and/or PRV-614. This novel demonstration of three recombinants infecting individual neurons represents an important advance in the technology. CONCLUSION: Collectively, these data demonstrate that PRV-290 is a valuable addition to the viral tracer toolbox for transneuronal tracing of neural circuitry.
MacGibeny MA, Koyuncu OO, Wirblich C, Schnell MJ, Enquist LW. Retrograde axonal transport of rabies virus is unaffected by interferon treatment but blocked by emetine locally in axons. PLOS Pathogens [Internet]. 2018;14 (7) :1-28. Publisher's VersionAbstract
Author summary Rabies virus (RABV) and alpha herpesviruses (αHV) (e.g. herpes simplex virus) evolved to enter the nervous system efficiently each time they infect a host. In most mammals, RABV reaches the brain, causing a fatal encephalitis. Whereas, αHV remain in the peripheral nervous system in a quiescent but reactivatable state. Despite distinct clinical outcomes, both RABV and αHV must invade axons and repurpose the axon transport machinery to travel long distances toward the neuronal cell bodies where virus replication occurs. How virus particles hijack the transport machinery and how axons respond to and regulate infection are questions of significant interest. We investigated how axonal RABV transport is regulated by exposing axons to interferons or protein synthesis inhibitors, both of which restrict transport of αHV particles. Unlike αHV infection, exposure of isolated axons to interferons has no effect on RABV neuroinvasion. However, RABV transport is blocked by axonal exposure to the translation elongation inhibitor, emetine, via a mechanism that does not depend on protein synthesis inhibition. The effect of emetine is not due to a global inhibition of axon transport because emetine does not limit axonal transport of cellular vesicles. Therefore, emetine may be a novel inhibitory modulator of RABV axonal transport.
Koyuncu OO, MacGibeny MA, Enquist LW. Latent versus productive infection: the alpha herpesvirus switch. Future Virol. 2018;13 (6) :431-443.Abstract
Alpha herpesviruses are common pathogens of mammals. They establish a productive infection in many cell types, but a life-long latent infection occurs in PNS neurons. A vast majority of the human population has latent HSV-1 infections. Currently, there is no cure to clear latent infections. Even though HSV-1 is among the best studied viral pathogens, regulation of latency and reactivation is not well understood due to several challenges including a lack of animal models that precisely recapitulate latency/reactivation episodes; a difficulty in modeling latency; and a limited understanding of neuronal biology. In this review, we discuss insights gained from latency models with a focus on the neuronal and viral factors that determine the mode of infection.
Hogue IB, Jean J, Esteves AD, Tanneti NS, Scherer J, Enquist LW. Functional Carboxy-Terminal Fluorescent Protein Fusion to Pseudorabies Virus Small Capsid Protein VP26. J Virol. 2018;92 (1).Abstract
Fluorescent protein fusions to herpesvirus capsids have proven to be a valuable method to study virus particle transport in living cells. Fluorescent protein fusions to the amino terminus of small capsid protein VP26 are the most widely used method to visualize pseudorabies virus (PRV) and herpes simplex virus (HSV) particles in living cells. However, these fusion proteins do not incorporate to full occupancy and have modest effects on virus replication and pathogenesis. Recent cryoelectron microscopy studies have revealed that herpesvirus small capsid proteins bind to capsids via their amino terminus, whereas the carboxy terminus is unstructured and therefore may better tolerate fluorescent protein fusions. Here, we describe a new recombinant PRV expressing a carboxy-terminal VP26-mCherry fusion. Compared to previously characterized viruses expressing amino-terminal fusions, this virus expresses more VP26 fusion protein in infected cells and incorporates more VP26 fusion protein into virus particles, and individual virus particles exhibit brighter red fluorescence. We performed single-particle tracking of fluorescent virus particles in primary neurons to measure anterograde and retrograde axonal transport, demonstrating the usefulness of this novel VP26-mCherry fusion for the study of viral intracellular transport.IMPORTANCE Alphaherpesviruses are among the very few viruses that are adapted to invade the mammalian nervous system. Intracellular transport of virus particles in neurons is important, as this process underlies both mild peripheral nervous system infection and severe spread to the central nervous system. VP26, the small capsid protein of HSV and PRV, was one of the first herpesvirus proteins to be fused to a fluorescent protein. Since then, these capsid-tagged virus mutants have become a powerful tool to visualize and track individual virus particles. Improved capsid tags will facilitate fluorescence microscopy studies of virus particle intracellular transport, as a brighter particle will improve localization accuracy of individual particles and allow for shorter exposure times, reducing phototoxicity and improving the time resolution of particle tracking in live cells.
Koyuncu OO, MacGibeny MA, Hogue IB, Enquist LW. Compartmented neuronal cultures reveal two distinct mechanisms for alpha herpesvirus escape from genome silencing. PLoS Pathog. 2017;13 (10) :e1006608.Abstract
Alpha herpesvirus genomes encode the capacity to establish quiescent infections (i.e. latency) in the peripheral nervous system for the life of their hosts. Multiple times during latency, viral genomes can reactivate to start a productive infection, enabling spread of progeny virions to other hosts. Replication of alpha herpesviruses is well studied in cultured cells and many aspects of productive replication have been identified. However, many questions remain concerning how a productive or a quiescent infection is established. While infections in vivo often result in latency, infections of dissociated neuronal cultures in vitro result in a productive infection unless lytic viral replication is suppressed by DNA polymerase inhibitors or interferon. Using primary peripheral nervous system neurons cultured in modified Campenot tri-chambers, we previously reported that reactivateable, quiescent infections by pseudorabies virus (PRV) can be established in the absence of any inhibitor. Such infections were established in cell bodies only when physically isolated axons were infected at a very low multiplicity of infection (MOI). In this report, we developed a complementation assay in compartmented neuronal cultures to investigate host and viral factors in cell bodies that prevent establishment of quiescent infection and promote productive replication of axonally delivered genomes (i.e. escape from silencing). Stimulating protein kinase A (PKA) signaling pathways in isolated cell bodies, or superinfecting cell bodies with either UV-inactivated PRV or viral light particles (LP) promoted escape from genome silencing and prevented establishment of quiescent infection but with different molecular mechanisms. Activation of PKA in cell bodies triggers a slow escape from silencing in a cJun N-terminal kinase (JNK) dependent manner. However, escape from silencing is induced rapidly by infection with UVPRV or LP in a PKA- and JNK-independent manner. We suggest that viral tegument proteins delivered to cell bodies engage multiple signaling pathways that block silencing of viral genomes delivered by low MOI axonal infection.
Enquist LW, Scherer J. Alphaherpesviruses: parasites of theperipheral nervous system. Future Virology [Internet]. 2017;12 (10) :555-559. Publisher's Version
Enquist LW, Dermody TS, DiMaio D. Introduction. Annu Rev Virol. 2017;4 (1) :i-ii.
Pomeranz LE, Ekstrand MI, Latcha KN, Smith GA, Enquist LW, Friedman JM. Gene Expression Profiling with Cre-Conditional Pseudorabies Virus Reveals a Subset of Midbrain Neurons That Participate in Reward Circuitry. J Neurosci. 2017;37 (15) :4128-4144.Abstract
The mesolimbic dopamine pathway receives inputs from numerous regions of the brain as part of a neural system that detects rewarding stimuli and coordinates a behavioral response. The capacity to simultaneously map and molecularly define the components of this complex multisynaptic circuit would thus advance our understanding of the determinants of motivated behavior. To accomplish this, we have constructed pseudorabies virus (PRV) strains in which viral propagation and fluorophore expression are activated only after exposure to Cre recombinase. Once activated in Cre-expressing neurons, the virus serially labels chains of presynaptic neurons. Dual injection of GFP and mCherry tracing viruses simultaneously illuminates nigrostriatal and mesolimbic circuitry and shows no overlap, demonstrating that PRV transmission is confined to synaptically connected neurons. To molecularly profile mesolimbic dopamine neurons and their presynaptic inputs, we injected Cre-conditional GFP virus into the NAc of (anti-GFP) nanobody-L10 transgenic mice and immunoprecipitated translating ribosomes from neurons infected after retrograde tracing. Analysis of purified RNA revealed an enrichment of transcripts expressed in neurons of the dorsal raphe nuclei and lateral hypothalamus that project to the mesolimbic dopamine circuit. These studies identify important inputs to the mesolimbic dopamine pathway and further show that PRV circuit-directed translating ribosome affinity purification can be broadly applied to identify molecularly defined neurons comprising complex, multisynaptic circuits.SIGNIFICANCE STATEMENT The mesolimbic dopamine circuit integrates signals from key brain regions to detect and respond to rewarding stimuli. To further define this complex multisynaptic circuit, we constructed a panel of Cre recombinase-activated pseudorabies viruses (PRVs) that enabled retrograde tracing of neural inputs that terminate on Cre-expressing neurons. Using these viruses and Retro-TRAP (translating ribosome affinity purification), a previously reported molecular profiling method, we developed a novel technique that provides anatomic as well as molecular information about the neural components of polysynaptic circuits. We refer to this new method as PRV-Circuit-TRAP (PRV circuit-directed TRAP). Using it, we have identified major projections to the mesolimbic dopamine circuit from the lateral hypothalamus and dorsal raphe nucleus and defined a discrete subset of transcripts expressed in these projecting neurons, which will allow further characterization of this important pathway. Moreover, the method we report is general and can be applied to the study of other neural circuits.
Enquist LW, Leib DA. Intrinsic and Innate Defenses of Neurons: Détente with the Herpesviruses. J Virol. 2017;91 (1).Abstract
Neuroinvasive herpesviruses have evolved to efficiently infect and establish latency in neurons. The nervous system has limited capability to regenerate, so immune responses therein are carefully regulated to be nondestructive, with dependence on atypical intrinsic and innate defenses. In this article we review studies of some of these noncanonical defense pathways and how herpesvirus gene products counter them, highlighting the contributions that primary neuronal in vitro models have made to our understanding of this field.
Enquist LW, Dermody TS, DiMaio D. Introduction. Annu Rev Virol. 2016;3 (1) :v.
Johnson BN, Lancaster KZ, Hogue IB, Meng F, Kong YL, Enquist LW, McAlpine MC. 3D printed nervous system on a chip. Lab Chip. 2016;16 (8) :1393-400.Abstract
Bioinspired organ-level in vitro platforms are emerging as effective technologies for fundamental research, drug discovery, and personalized healthcare. In particular, models for nervous system research are especially important, due to the complexity of neurological phenomena and challenges associated with developing targeted treatment of neurological disorders. Here we introduce an additive manufacturing-based approach in the form of a bioinspired, customizable 3D printed nervous system on a chip (3DNSC) for the study of viral infection in the nervous system. Micro-extrusion 3D printing strategies enabled the assembly of biomimetic scaffold components (microchannels and compartmented chambers) for the alignment of axonal networks and spatial organization of cellular components. Physiologically relevant studies of nervous system infection using the multiscale biomimetic device demonstrated the functionality of the in vitro platform. We found that Schwann cells participate in axon-to-cell viral spread but appear refractory to infection, exhibiting a multiplicity of infection (MOI) of 1.4 genomes per cell. These results suggest that 3D printing is a valuable approach for the prototyping of a customized model nervous system on a chip technology.
Rosario W, Singh I, Wautlet A, Patterson C, Flak J, Becker TC, Ali A, Tamarina N, Philipson LH, Enquist LW, et al. The Brain-to-Pancreatic Islet Neuronal Map Reveals Differential Glucose Regulation From Distinct Hypothalamic Regions. Diabetes. 2016;65 (9) :2711-23.Abstract
The brain influences glucose homeostasis, partly by supplemental control over insulin and glucagon secretion. Without this central regulation, diabetes and its complications can ensue. Yet, the neuronal network linking to pancreatic islets has never been fully mapped. Here, we refine this map using pseudorabies virus (PRV) retrograde tracing, indicating that the pancreatic islets are innervated by efferent circuits that emanate from the hypothalamus. We found that the hypothalamic arcuate nucleus (ARC), ventromedial nucleus (VMN), and lateral hypothalamic area (LHA) significantly overlap PRV and the physiological glucose-sensing enzyme glucokinase. Then, experimentally lowering glucose sensing, specifically in the ARC, resulted in glucose intolerance due to deficient insulin secretion and no significant effect in the VMN, but in the LHA it resulted in a lowering of the glucose threshold that improved glucose tolerance and/or improved insulin sensitivity, with an exaggerated counter-regulatory response for glucagon secretion. No significant effect on insulin sensitivity or metabolic homeostasis was noted. Thus, these data reveal novel direct neuronal effects on pancreatic islets and also render a functional validation of the brain-to-islet neuronal map. They also demonstrate that distinct regions of the hypothalamus differentially control insulin and glucagon secretion, potentially in partnership to help maintain glucose homeostasis and guard against hypoglycemia.
Bosse JB, Enquist LW. The diffusive way out: Herpesviruses remodel the host nucleus, enabling capsids to access the inner nuclear membrane. Nucleus. 2016;7 (1) :13-9.Abstract
Herpesviruses are large DNA viruses that utilize the host nucleus for genome replication as well as capsid assembly. After maturation, these 125 nm large capsid assemblies must cross the nucleoplasm to engage the nuclear envelope and bud into the cytoplasm. Here we summarize our recent findings how this motility is facilitated. We suggest that herpesvirus induced nuclear remodeling allows capsids to move by diffusion in the nucleus and not by motor-dependent transport.
Scherer J, Yaffe ZA, Vershinin M, Enquist LW. Dual-color Herpesvirus Capsids Discriminate Inoculum from Progeny and Reveal Axonal Transport Dynamics. J Virol. 2016.Abstract
Alpha herpesviruses, such as herpes simplex virus and pseudorabies virus (PRV), are neuroinvasive dsDNA viruses that establish life-long latency in peripheral nervous system (PNS) neurons of their native hosts. Following reactivation, the infection can spread back to the initial mucosal site of infection or, in rare cases, to the central nervous system with usually serious outcomes. During entry and egress, viral capsids depend on microtubule-based molecular motors for efficient and fast transport. In axons of PNS neurons, cytoplasmic dynein provides force for retrograde movements towards the soma, and kinesins move cargo in the opposite, anterograde direction. The dynamic properties of virus particles in cells can be imaged by fluorescent protein fusions to the small capsid protein VP26, which are incorporated into capsids. However, single-color fluorescent protein tags fail to distinguish virus inoculum from progeny. Therefore, we established a dual-color system by growing a recombinant PRV expressing a red fluorescent VP26 fusion (PRV180) on a stable cell line expressing a green VP26 fusion (PK15-mNG-VP26). The resulting dual-color virus preparation (PRV180G) contains capsids tagged with both red and green fluorescent proteins, and 97% of particles contain detectable levels of mNG-VP26. After replication in neuronal cells, all PRV180G progeny exclusively contain mRFP-VP26 tagged capsids. We used PRV180G for an analysis of axonal capsid transport dynamics in PNS neurons. Fast dual-color total internal reflection fluorescence (TIRF) microscopy, single particle tracking and motility analyses reveal robust, bidirectional capsid motility mediated by cytoplasmic dynein and kinesin during entry, whereas egressing progeny particles are exclusively transported by kinesins. IMPORTANCE: Alpha herpesviruses are neuroinvasive viruses that infect the peripheral nervous system (PNS) of infected hosts as an integral part of their life cycle. Establishment of a quiescent or latent infection in PNS neurons is a hallmark of most alpha herpesviruses. Spread of infection to the central nervous system is surprisingly rare in natural hosts, but can be fatal. Pseudorabies virus (PRV) is a broad host range, swine alpha herpesvirus that enters neuronal cells and utilizes intracellular transport processes to establish infection and to spread between cells. By using a virus preparation with fluorescent viral capsids that change color depending on the stage of the infectious cycle, we find that, during entry, axons of PNS neurons support robust, bidirectional capsid motility, similar to cellular cargo, towards the cell body. In contrast, progeny particles appear to be transported unidirectionally by kinesin motors towards distal egress sites.
Hogue IB, Scherer J, Enquist LW. Exocytosis of Alphaherpesvirus Virions, Light Particles, and Glycoproteins Uses Constitutive Secretory Mechanisms. MBio. 2016;7 (3).Abstract
UNLABELLED: Many molecular and cell biological details of the alphaherpesvirus assembly and egress pathway remain unclear. Recently we developed a live-cell fluorescence microscopy assay of pseudorabies virus (PRV) exocytosis, based on total internal reflection fluorescence (TIRF) microscopy and a virus-encoded pH-sensitive fluorescent probe. Here, we use this assay to distinguish three classes of viral exocytosis in a nonpolarized cell type: (i) trafficking of viral glycoproteins to the plasma membrane, (ii) exocytosis of viral light particles, and (iii) exocytosis of virions. We find that viral glycoproteins traffic to the cell surface in association with constitutive secretory Rab GTPases and exhibit free diffusion into the plasma membrane after exocytosis. Similarly, both virions and light particles use these same constitutive secretory mechanisms for egress from infected cells. Furthermore, we show that viral light particles are distinct from cellular exosomes. Together, these observations shed light on viral glycoprotein trafficking steps that precede virus particle assembly and reinforce the idea that virions and light particles share a biogenesis and trafficking pathway. IMPORTANCE: The alphaherpesviruses, including the important human pathogens herpes simplex virus 1 (HSV-1), HSV-2, and varicella-zoster virus (VZV), are among the few viruses that have evolved to exploit the mammalian nervous system. These viruses typically cause mild recurrent herpetic or zosteriform lesions but can also cause debilitating herpes encephalitis, more frequently in very young, old, immunocompromised, or nonnatural hosts. Importantly, many of the molecular and cellular mechanisms of viral assembly and egress remain unclear. This study addresses the trafficking of viral glycoproteins to the plasma membrane, exocytosis of light particles, and exocytosis of virions. Trafficking of glycoproteins affects immune evasion and pathogenesis and may precede virus particle assembly. The release of light particles may also contribute to immune evasion and pathogenesis. Finally, exocytosis of virions is important to understand, as this final step in the virus replication cycle produces infectious extracellular particles capable of spreading to the next round of host cells.
Christensen MH, Jensen SB, Miettinen JJ, Luecke S, Prabakaran T, Reinert LS, Mettenleiter T, Chen ZJ, Knipe DM, Sandri-Goldin RM, et al. HSV-1 ICP27 targets the TBK1-activated STING signalsome to inhibit virus-induced type I IFN expression. EMBO J. 2016;35 (13) :1385-99.Abstract
Herpes simplex virus (HSV) 1 stimulates type I IFN expression through the cGAS-STING-TBK1 signaling axis. Macrophages have recently been proposed to be an essential source of IFN during viral infection. However, it is not known how HSV-1 inhibits IFN expression in this cell type. Here, we show that HSV-1 inhibits type I IFN induction through the cGAS-STING-TBK1 pathway in human macrophages, in a manner dependent on the conserved herpesvirus protein ICP27. This viral protein was expressed de novo in macrophages with early nuclear localization followed by later translocation to the cytoplasm where ICP27 prevented activation of IRF3. ICP27 interacted with TBK1 and STING in a manner that was dependent on TBK1 activity and the RGG motif in ICP27. Thus, HSV-1 inhibits expression of type I IFN in human macrophages through ICP27-dependent targeting of the TBK1-activated STING signalsome.
Harris GM, Madigan NN, Lancaster KZ, Enquist LW, Windebank AJ, Schwartz J, Schwarzbauer JE. Nerve Guidance by a Decellularized Fibroblast Extracellular Matrix. Matrix Biol. 2016.Abstract
Spinal cord and peripheral nerve injuries require the regeneration of nerve fibers across the lesion site for successful recovery. Providing guidance cues and soluble factors to promote neurite outgrowth and cell survival can enhance repair. The extracellular matrix (ECM) plays a key role in tissue repair by controlling cell adhesion, motility, and growth. In this study, we explored the ability of a mesenchymal ECM to support neurite outgrowth from neurons in the superior cervical ganglia (SCG). Length and morphology of neurites extended on a decellularized fibroblast ECM were compared to those on substrates coated with laminin, a major ECM protein in neural tissue, or fibronectin, the main component of a mesenchymal ECM. Average radial neurite extension was equivalent on laminin and on the decellularized ECM, but contrasted with the shorter, curved neurites observed on the fibronectin substrate. Differences between neurites on fibronectin and on other substrates were confirmed by fast Fourier transform analyses. To control the direction of neurite outgrowth, we developed an ECM with linearly aligned fibril organization by orienting the fibroblasts that deposit the matrix on a polymeric surface micropatterned with a striped chemical interface. Neurites projected from SCGs appeared to reorient in the direction of the pattern. These results highlight the ability of a mesenchymal ECM to enhance neurite extension and to control the directional outgrowth of neurites. This micropatterned decellularized ECM architecture has potential as a regenerative microenvironment for nerve repair.
Song R, Koyuncu OO, Greco TM, Diner BA, Cristea IM, Enquist LW. Two Modes of the Axonal Interferon Response Limit Alphaherpesvirus Neuroinvasion. MBio. 2016;7 (1) :e02145-15.Abstract
UNLABELLED: Infection by alphaherpesviruses, including herpes simplex virus (HSV) and pseudorabies virus (PRV), typically begins at epithelial surfaces and continues into the peripheral nervous system (PNS). Inflammatory responses are induced at the infected peripheral site prior to invasion of the PNS. When the peripheral tissue is first infected, only the innervating axons are exposed to this inflammatory milieu, which includes the interferons (IFNs). The fundamental question is how do PNS cell bodies respond to these distant, potentially damaging events experienced by axons. Using compartmented cultures that physically separate neuron axons from cell bodies, we found that pretreating isolated axons with beta interferon (IFN-β) or gamma interferon (IFN-γ) significantly diminished the number of herpes simplex virus 1 (HSV-1) and PRV particles moving in axons toward the cell bodies in a receptor-dependent manner. Exposing axons to IFN-β induced STAT1 phosphorylation (p-STAT1) only in axons, while exposure of axons to IFN-γ induced p-STAT1 accumulation in distant cell body nuclei. Blocking transcription in cell bodies eliminated antiviral effects induced by IFN-γ, but not those induced by IFN-β. Proteomic analysis of IFN-β- or IFN-γ-treated axons identified several differentially regulated proteins. Therefore, unlike treatment with IFN-γ, IFN-β induces a noncanonical, local antiviral response in axons. The activation of a local IFN response in axons represents a new paradigm for cytokine control of neuroinvasion. IMPORTANCE: Neurons are highly polarized cells with long axonal processes that connect to distant targets. PNS axons that innervate peripheral tissues are exposed to various situations that follow infection, inflammation, and damage of the tissue. After viral infection in the periphery, axons represent potential front-line barriers to PNS infection and damage. Indeed, most viral infections do not spread to the PNS, yet the mechanisms responsible are not well studied. We devised an experimental system to study how axons respond to inflammatory cytokines that would be produced by infected tissues. We found that axons respond differentially to type I and type II interferons. The response to type I interferon (IFN-β) is a rapid axon-only response. The response to type II interferon (IFN-γ) involves long-distance signaling to the PNS cell body. These responses to two interferons erect an efficient and rapid barrier to PNS infection.
Wojaczynski GJ, Engel EA, Steren KE, Enquist LW, Patrick Card J. The neuroinvasive profiles of H129 (herpes simplex virus type 1) recombinants with putative anterograde-only transneuronal spread properties. Brain Struct Funct. 2015;220 (3) :1395-420.Abstract
The use of viruses as transneuronal tracers has become an increasingly powerful technique for defining the synaptic organization of neural networks. Although a number of recombinant alpha herpesviruses are known to spread selectively in the retrograde direction through neural circuits only one strain, the H129 strain of herpes simplex virus type 1, is reported to selectively spread in the anterograde direction. However, it is unclear from the literature whether there is an absolute block or an attenuation of retrograde spread of H129. Here, we demonstrate efficient anterograde spread, and temporally delayed retrograde spread, of H129 and three novel recombinants. In vitro studies revealed no differences in anterograde and retrograde spread of parental H129 and its recombinants through superior cervical ganglion neurons. In vivo injections of rat striatum revealed a clear bias of anterograde spread, although evidence of deficient retrograde transport was also present. Evidence of temporally delayed retrograde transneuronal spread of H129 in the retina was observed following injection of the lateral geniculate nucleus. The data also demonstrated that three novel recombinants efficiently express unique fluorescent reporters and have the capacity to infect the same neurons in dual infection paradigms. From these experiments we conclude that H129 and its recombinants not only efficiently infect neurons through anterograde transneuronal passage, but also are capable of temporally delayed retrograde transneuronal spread. In addition, the capacity to produce dual infection of projection targets following anterograde transneuronal passage provides an important addition to viral transneuronal tracing technology.
Taylor MP, Enquist LW. Axonal spread of neuroinvasive viral infections. Trends Microbiol. 2015;23 (5) :283-8.Abstract
Neuroinvasive viral infections invade the nervous system, often eliciting serious disease and death. Members of four viral families are both neuroinvasive and capable of transmitting progeny virions or virion components within the long neuronal extensions known as axons. Axons provide physical structures that enable viral infection to spread within the host while avoiding extracellular immune responses. Technological advances in the analysis of in vivo neural circuits, neuronal culturing, and live imaging of fluorescent fusion proteins have enabled an unprecedented view into the steps of virion assembly, transport, and egress involved in axonal spread. In this review we summarize the literature supporting anterograde (axon to cell) spread of viral infection, describe the various strategies of virion transport, and discuss the effects of spread on populations of neuroinvasive viruses.
Carpenter JE, Clayton AC, Halling KC, Bonthius DJ, Buckingham EM, Jackson W, Dotzler SM, Card PJ, Enquist LW, Grose C. Defensive Perimeter in the Central Nervous System: Predominance of Astrocytes and Astrogliosis during Recovery from Varicella-Zoster Virus Encephalitis. J Virol. 2015;90 (1) :379-91.Abstract
UNLABELLED: Varicella-zoster virus (VZV) is a highly neurotropic virus that can cause infections in both the peripheral nervous system and the central nervous system. Several studies of VZV reactivation in the peripheral nervous system (herpes zoster) have been published, while exceedingly few investigations have been carried out in a human brain. Notably, there is no animal model for VZV infection of the central nervous system. In this report, we characterized the cellular environment in the temporal lobe of a human subject who recovered from focal VZV encephalitis. The approach included not only VZV DNA/RNA analyses but also a delineation of infected cell types (neurons, microglia, oligodendrocytes, and astrocytes). The average VZV genome copy number per cell was 5. Several VZV regulatory and structural gene transcripts and products were detected. When colocalization studies were performed to determine which cell types harbored the viral proteins, the majority of infected cells were astrocytes, including aggregates of astrocytes. Evidence of syncytium formation within the aggregates included the continuity of cytoplasm positive for the VZV glycoprotein H (gH) fusion-complex protein within a cellular profile with as many as 80 distinct nuclei. As with other causes of brain injury, these results suggested that astrocytes likely formed a defensive perimeter around foci of VZV infection (astrogliosis). Because of the rarity of brain samples from living humans with VZV encephalitis, we compared our VZV results with those found in a rat encephalitis model following infection with the closely related pseudorabies virus and observed similar perimeters of gliosis. IMPORTANCE: Investigations of VZV-infected human brain from living immunocompetent human subjects are exceedingly rare. Therefore, much of our knowledge of VZV neuropathogenesis is gained from studies of VZV-infected brains obtained at autopsy from immunocompromised patients. These are not optimal samples with which to investigate a response by a human host to VZV infection. In this report, we examined both flash-frozen and paraffin-embedded formalin-fixed brain tissue of an otherwise healthy young male with focal VZV encephalitis, most likely acquired from VZV reactivation in the trigeminal ganglion. Of note, the cellular response to VZV infection mimicked the response to other causes of trauma to the brain, namely, an ingress of astrocytes and astrogliosis around an infectious focus. Many of the astrocytes themselves were infected; astrocytes aggregated in clusters. We postulate that astrogliosis represents a successful defense mechanism by an immunocompetent human host to eliminate VZV reactivation within neurons.
Hogue IB, Bosse JB, Engel EA, Scherer J, Hu J-R, del Rio T, Enquist LW. Fluorescent Protein Approaches in Alpha Herpesvirus Research. Viruses. 2015;7 (11) :5933-61.Abstract
In the nearly two decades since the popularization of green fluorescent protein (GFP), fluorescent protein-based methodologies have revolutionized molecular and cell biology, allowing us to literally see biological processes as never before. Naturally, this revolution has extended to virology in general, and to the study of alpha herpesviruses in particular. In this review, we provide a compendium of reported fluorescent protein fusions to herpes simplex virus 1 (HSV-1) and pseudorabies virus (PRV) structural proteins, discuss the underappreciated challenges of fluorescent protein-based approaches in the context of a replicating virus, and describe general strategies and best practices for creating new fluorescent fusions. We compare fluorescent protein methods to alternative approaches, and review two instructive examples of the caveats associated with fluorescent protein fusions, including describing several improved fluorescent capsid fusions in PRV. Finally, we present our future perspectives on the types of powerful experiments these tools now offer.
Engel EA, Song R, Koyuncu OO, Enquist LW. Investigating the biology of alpha herpesviruses with MS-based proteomics. Proteomics. 2015;15 (12) :1943-56.Abstract
Viruses are intracellular parasites that can only replicate and spread in cells of susceptible hosts. Alpha herpesviruses (α-HVs) contain double-stranded DNA genomes of at least 120 kb, encoding for 70 or more genes. The viral genome is contained in an icosahedral capsid that is surrounded by a proteinaceous tegument layer and a lipid envelope. Infection starts in epithelial cells and spreads to the peripheral nervous system. In the natural host, α-HVs establish a chronic latent infection that can be reactivated and rarely spread to the CNS. In the nonnatural host, viral infection will in most cases spread to the CNS with often fatal outcome. The host response plays a crucial role in the outcome of viral infection. α-HVs do not encode all the genes required for viral replication and spread. They need a variety of host gene products including RNA polymerase, ribosomes, dynein, and kinesin. As a result, the infected cell is dramatically different from the uninfected cell revealing a complex and dynamic interplay of viral and host components required to complete the virus life cycle. In this review, we describe the pivotal contribution of MS-based proteomics studies over the past 15 years to understand the complicated life cycle and pathogenesis of four α-HV species from the alphaherpesvirinae subfamily: Herpes simplex virus-1, varicella zoster virus, pseudorabies virus and bovine herpes virus-1. We describe the viral proteome dynamics during host infection and the host proteomic response to counteract such pathogens.
Koyuncu OO, Song R, Greco TM, Cristea IM, Enquist LW. The number of alphaherpesvirus particles infecting axons and the axonal protein repertoire determines the outcome of neuronal infection. MBio. 2015;6 (2).Abstract
UNLABELLED: Infection by alphaherpesviruses invariably results in invasion of the peripheral nervous system (PNS) and establishment of either a latent or productive infection. Infection begins with long-distance retrograde transport of viral capsids and tegument proteins in axons toward the neuronal nuclei. Initial steps of axonal entry, retrograde transport, and replication in neuronal nuclei are poorly understood. To better understand how the mode of infection in the PNS is determined, we utilized a compartmented neuron culturing system where distal axons of PNS neurons are physically separated from cell bodies. We infected isolated axons with fluorescent-protein-tagged pseudorabies virus (PRV) particles and monitored viral entry and transport in axons and replication in cell bodies during low and high multiplicities of infection (MOIs of 0.01 to 100). We found a threshold for efficient retrograde transport in axons between MOIs of 1 and 10 and a threshold for productive infection in the neuronal cell bodies between MOIs of 1 and 0.1. Below an MOI of 0.1, the viral genomes that moved to neuronal nuclei were silenced. These genomes can be reactivated after superinfection by a nonreplicating virus, but not by a replicating virus. We further showed that viral particles at high-MOI infections compete for axonal proteins and that this competition determines the number of viral particles reaching the nuclei. Using mass spectrometry, we identified axonal proteins that are differentially regulated by PRV infection. Our results demonstrate the impact of the multiplicity of infection and the axonal milieu on the establishment of neuronal infection initiated from axons. IMPORTANCE: Alphaherpesvirus genomes may remain silent in peripheral nervous system (PNS) neurons for the lives of their hosts. These genomes occasionally reactivate to produce infectious virus that can reinfect peripheral tissues and spread to other hosts. Here, we use a neuronal culture system to investigate the outcome of axonal infection using different numbers of viral particles and coinfection assays. We found that the dynamics of viral entry, transport, and replication change dramatically depending on the number of virus particles that infect axons. We demonstrate that viral genomes are silenced when the infecting particle number is low and that these genomes can be reactivated by superinfection with UV-inactivated virus, but not with replicating virus. We further show that viral invasion rapidly changes the profiles of axonal proteins and that some of these axonal proteins are rate limiting for efficient infection. Our study provides new insights into the establishment of silent versus productive alphaherpesvirus infections in the PNS.
Bosse JB, Tanneti NS, Hogue IB, Enquist LW. Open LED Illuminator: A Simple and Inexpensive LED Illuminator for Fast Multicolor Particle Tracking in Neurons. PLoS One. 2015;10 (11) :e0143547.Abstract
Dual-color live cell fluorescence microscopy of fast intracellular trafficking processes, such as axonal transport, requires rapid switching of illumination channels. Typical broad-spectrum sources necessitate the use of mechanical filter switching, which introduces delays between acquisition of different fluorescence channels, impeding the interpretation and quantification of highly dynamic processes. Light Emitting Diodes (LEDs), however, allow modulation of excitation light in microseconds. Here we provide a step-by-step protocol to enable any scientist to build a research-grade LED illuminator for live cell microscopy, even without prior experience with electronics or optics. We quantify and compare components, discuss our design considerations, and demonstrate the performance of our LED illuminator by imaging axonal transport of herpes virus particles with high temporal resolution.
Bosse JB, Hogue IB, Feric M, Thiberge SY, Sodeik B, Brangwynne CP, Enquist LW. Remodeling nuclear architecture allows efficient transport of herpesvirus capsids by diffusion. Proc Natl Acad Sci U S A. 2015;112 (42) :E5725-33.Abstract
The nuclear chromatin structure confines the movement of large macromolecular complexes to interchromatin corrals. Herpesvirus capsids of approximately 125 nm assemble in the nucleoplasm and must reach the nuclear membranes for egress. Previous studies concluded that nuclear herpesvirus capsid motility is active, directed, and based on nuclear filamentous actin, suggesting that large nuclear complexes need metabolic energy to escape nuclear entrapment. However, this hypothesis has recently been challenged. Commonly used microscopy techniques do not allow the imaging of rapid nuclear particle motility with sufficient spatiotemporal resolution. Here, we use a rotating, oblique light sheet, which we dubbed a ring-sheet, to image and track viral capsids with high temporal and spatial resolution. We do not find any evidence for directed transport. Instead, infection with different herpesviruses induced an enlargement of interchromatin domains and allowed particles to diffuse unrestricted over longer distances, thereby facilitating nuclear egress for a larger fraction of capsids.
Hagen C, Dent KC, Zeev-Ben-Mordehai T, Grange M, Bosse JB, Whittle C, Klupp BG, Siebert AC, Vasishtan D, Bäuerlein FJB, et al. Structural Basis of Vesicle Formation at the Inner Nuclear Membrane. Cell. 2015;163 (7) :1692-701.Abstract
Vesicular nucleo-cytoplasmic transport is becoming recognized as a general cellular mechanism for translocation of large cargoes across the nuclear envelope. Cargo is recruited, enveloped at the inner nuclear membrane (INM), and delivered by membrane fusion at the outer nuclear membrane. To understand the structural underpinning for this trafficking, we investigated nuclear egress of progeny herpesvirus capsids where capsid envelopment is mediated by two viral proteins, forming the nuclear egress complex (NEC). Using a multi-modal imaging approach, we visualized the NEC in situ forming coated vesicles of defined size. Cellular electron cryo-tomography revealed a protein layer showing two distinct hexagonal lattices at its membrane-proximal and membrane-distant faces, respectively. NEC coat architecture was determined by combining this information with integrative modeling using small-angle X-ray scattering data. The molecular arrangement of the NEC establishes the basic mechanism for budding and scission of tailored vesicles at the INM.
Johnson BN, Lancaster KZ, Zhen G, He J, Gupta MK, Kong YL, Engel EA, Krick KD, Ju A, Meng F, et al. 3D Printed Anatomical Nerve Regeneration Pathways. Adv Funct Mater. 2015;25 (39) :6205-6217.Abstract
An imaging-coupled 3D printing methodology for the design, optimization, and fabrication of a customized nerve repair technology for complex injuries is presented. The custom scaffolds are deterministically fabricated via a microextrusion printing principle which enables the simultaneous incorporation of anatomical geometries, biomimetic physical cues, and spatially controlled biochemical gradients in a one-pot 3D manufacturing approach.
Wu BW, Engel EA, Enquist LW. Characterization of a replication-incompetent pseudorabies virus mutant lacking the sole immediate early gene IE180. MBio. 2014;5 (6) :e01850.Abstract
UNLABELLED: The alphaherpesvirus pseudorabies virus (PRV) encodes a single immediate early gene called IE180. The IE180 protein is a potent transcriptional activator of viral genes involved in DNA replication and RNA transcription. A PRV mutant with both copies of IE180 deleted was constructed 20 years ago (S. Yamada and M. Shimizu, Virology 199:366-375, 1994, doi:10.1006/viro.1994.1134), but propagation of the mutant depended on complementing cell lines that expressed the toxic IE180 protein constitutively. Recently, Oyibo et al. constructed a novel set of PRV IE180 mutants and a stable cell line with inducible IE180 expression (H. Oyibo, P. Znamenskiy, H. V. Oviedo, L. W. Enquist, A. Zador, Front. Neuroanat. 8:86, 2014, doi:10.3389/fnana.2014.00086), which we characterized further here. These mutants failed to replicate new viral genomes, synthesize immediate early, early, or late viral proteins, and assemble infectious virions. The PRV IE180-null mutant did not form plaques in epithelial cell monolayers and could not spread from primary infected neurons to second-order neurons in culture. PRV IE180-null mutants lacked the property of superinfection exclusion. When PRV IE180-null mutants infected cells first, subsequent superinfecting viruses were not blocked in cell entry and formed replication compartments in epithelial cells, fibroblasts, and neurons. Cells infected with PRV IE180-null mutants survived as long as uninfected cells in culture while expressing a fluorescent reporter gene. Transcomplementation with IE180 in epithelial cells restored all mutant phenotypes to wild type. The conditional expression of PRV IE180 protein enables the propagation of replication-incompetent PRV IE180-null mutants and will facilitate construction of long-term single-cell-infecting PRV mutants for precise neural circuit tracing and high-capacity gene delivery vectors. IMPORTANCE: Pseudorabies virus (PRV) is widely used for neural tracing in animal models. The virus replicates and spreads between synaptically connected neurons. Current tracing strains of PRV are cytotoxic and kill infected cells. Infected cells exclude superinfection with a second virus, limiting multiple virus infections in circuit tracing. By removing the only immediate early gene of PRV (called IE180), the mutant virus will not replicate or spread in epithelial cells, fibroblasts, or neurons. The wild-type phenotype can be restored by transcomplementation of infected cells with IE180. The PRV IE180-null mutant can express fluorescent reporters for weeks in cells with no toxicity; infected cells survive as long as uninfected cells. Infection with the mutant virus allows superinfection of the same cell with a second virus that can enter and replicate. The PRV IE180-null mutant will permit conditional long-term tracing in animals and is a high-capacity vector for gene delivery.
Oyibo HK, Znamenskiy P, Oviedo HV, Enquist LW, Zador AM. Long-term Cre-mediated retrograde tagging of neurons using a novel recombinant pseudorabies virus. Front Neuroanat. 2014;8 :86.Abstract
Brain regions contain diverse populations of neurons that project to different long-range targets. The study of these subpopulations in circuit function and behavior requires a toolkit to characterize and manipulate their activity in vivo. We have developed a novel set of reagents based on Pseudorabies Virus (PRV) for efficient and long-term genetic tagging of neurons based on their projection targets. By deleting IE180, the master transcriptional regulator in the PRV genome, we have produced a mutant virus capable of infection and transgene expression in neurons but unable to replicate in or spread from those neurons. IE180-null mutants showed no cytotoxicity, and infected neurons exhibited normal physiological function more than 45 days after infection, indicating the utility of these engineered viruses for chronic experiments. To enable rapid and convenient construction of novel IE180-null recombinants, we engineered a bacterial artificial chromosome (BAC) shuttle-vector system for moving new constructs into the PRV IE180-null genome. Using this system we generated an IE180-null recombinant virus expressing the site-specific recombinase Cre. This Cre-expressing virus (PRV-hSyn-Cre) efficiently and robustly infects neurons in vivo and activates transgene expression from Cre-dependent vectors in local and retrograde projecting populations of neurons in the mouse. We also generated an assortment of recombinant viruses expressing fluorescent proteins (mCherry, EGFP, ECFP). These viruses exhibit long-term labeling of neurons in vitro but transient labeling in vivo. Together these novel IE180-null PRV reagents expand the toolkit for targeted gene expression in the brain, facilitating functional dissection of neuronal circuits in vivo.
Bosse JB, Virding S, Thiberge SY, Scherer J, Wodrich H, Ruzsics Z, Koszinowski UH, Enquist LW. Nuclear herpesvirus capsid motility is not dependent on F-actin. MBio. 2014;5 (5) :e01909-14.Abstract
A considerable part of the herpesvirus life cycle takes place in the host nucleus. While much progress has been made to understand the molecular processes required for virus replication in the nucleus, much less is known about the temporal and spatial dynamics of these events. Previous studies have suggested that nuclear capsid motility is directed and dependent on actin filaments (F-actin), possibly using a myosin-based, ATP-dependent mechanism. However, the conclusions from these studies were indirect. They either relied on the effects of F-actin depolymerizing drugs to deduce an F-actin dependency or they visualized nuclear F-actin but failed to show a direct link to capsid motility. Moreover, no direct link between nuclear capsid motility and a molecular motor has been established. In this report, we reinvestigate the involvement of F-actin in nuclear herpesvirus capsid transport. We show for representative members of all three herpesvirus subfamilies that nuclear capsid motility is not dependent on nuclear F-actin and that herpesvirus infection does not induce nuclear F-actin in primary fibroblasts. Moreover, in these cells, three F-actin-inhibiting drugs failed to effect capsid motility. Only latrunculin A treatment stalled nuclear capsids but did so by an unexpected effect: the drug induced actin rods in the nucleus. Immobile capsids accumulated around actin rods, and immunoprecipitation experiments suggested that capsid motility stopped because latrunculin-induced actin rods nonspecifically bind nuclear capsids. Interestingly, capsid motility was unaffected in cells that do not induce actin rods. Based on these data, we conclude that herpesvirus nuclear capsid motility is not dependent on F-actin. Importance: Herpesviruses are large DNA viruses whose replication is dependent on the host nucleus. However, we do not understand how key nuclear processes, including capsid assembly, genome replication, capsid packaging, and nuclear egress, are dynamically connected in space and time. Fluorescence live-cell microscopy revealed that nuclear capsids are highly mobile early in infection. Two studies suggested that this motility might be due to active myosin-based transport of capsids on nuclear F-actin. However, direct evidence for such motor-based transport is lacking. We revisited this phenomenon and found no evidence that nuclear capsid motility depended on F-actin. Our results reopen the question of how nuclear herpesvirus capsids move in the host nucleus.
Hogue IB, Bosse JB, Hu J-R, Thiberge SY, Enquist LW. Cellular mechanisms of alpha herpesvirus egress: live cell fluorescence microscopy of pseudorabies virus exocytosis. PLoS Pathog. 2014;10 (12) :e1004535.Abstract
Egress of newly assembled herpesvirus particles from infected cells is a highly dynamic process involving the host secretory pathway working in concert with viral components. To elucidate the location, dynamics, and molecular mechanisms of alpha herpesvirus egress, we developed a live-cell fluorescence microscopy method to visualize the final transport and exocytosis of pseudorabies virus (PRV) particles in non-polarized epithelial cells. This method is based on total internal reflection fluorescence (TIRF) microscopy to selectively image fluorescent virus particles near the plasma membrane, and takes advantage of a virus-encoded pH-sensitive probe to visualize the precise moment and location of particle exocytosis. We performed single-particle tracking and mean squared displacement analysis to characterize particle motion, and imaged a panel of cellular proteins to identify those spatially and dynamically associated with viral exocytosis. Based on our data, individual virus particles travel to the plasma membrane inside small, acidified secretory vesicles. Rab GTPases, Rab6a, Rab8a, and Rab11a, key regulators of the plasma membrane-directed secretory pathway, are present on the virus secretory vesicle. These vesicles undergo fast, directional transport directly to the site of exocytosis, which is most frequently near patches of LL5β, part of a complex that anchors microtubules to the plasma membrane. Vesicles are tightly docked at the site of exocytosis for several seconds, and membrane fusion occurs, displacing the virion a small distance across the plasma membrane. After exocytosis, particles remain tightly confined on the outer cell surface. Based on recent reports in the cell biological and alpha herpesvirus literature, combined with our spatial and dynamic data on viral egress, we propose an integrated model that links together the intracellular transport pathways and exocytosis mechanisms that mediate alpha herpesvirus egress.
Koyuncu OO, Perlman DH, Enquist LW. Efficient retrograde transport of pseudorabies virus within neurons requires local protein synthesis in axons. Cell Host Microbe. 2013;13 (1) :54-66.Abstract
After replicating in epithelial cells, alphaherpesviruses such as pseudorabies virus (PRV) invade axons of peripheral nervous system neurons and undergo retrograde transport toward the distant cell bodies. Although several viral proteins engage molecular motors to facilitate transport, the initial steps and neuronal responses to infection are poorly understood. Using compartmented neuron cultures to physically separate axon infection from cell bodies, we found that PRV infection induces local protein synthesis in axons, including proteins involved in cytoskeletal remodeling, intracellular trafficking, signaling, and metabolism. This rapid translation of axonal mRNAs is required for efficient PRV retrograde transport and infection of cell bodies. Furthermore, induction of axonal damage, which also induces local protein synthesis, prior to infection reduces virion trafficking, suggesting that host damage signals and virus particles compete for retrograde transport. Thus, similar to axonal damage, virus infection induces local protein translation in axons, and viruses likely exploit this response for invasion.
Kramer T, Enquist LW. Directional spread of alphaherpesviruses in the nervous system. Viruses. 2013;5 (2) :678-707.Abstract
Alphaherpesviruses are pathogens that invade the nervous systems of their mammalian hosts. Directional spread of infection in the nervous system is a key component of the viral lifecycle and is critical for the onset of alphaherpesvirus-related diseases. Many alphaherpesvirus infections originate at peripheral sites, such as epithelial tissues, and then enter neurons of the peripheral nervous system (PNS), where lifelong latency is established. Following reactivation from latency and assembly of new viral particles, the infection typically spreads back out towards the periphery. These spread events result in the characteristic lesions (cold sores) commonly associated with herpes simplex virus (HSV) and herpes zoster (shingles) associated with varicella zoster virus (VZV). Occasionally, the infection spreads transsynaptically from the PNS into higher order neurons of the central nervous system (CNS). Spread of infection into the CNS, while rarer in natural hosts, often results in severe consequences, including death. In this review, we discuss the viral and cellular mechanisms that govern directional spread of infection in the nervous system. We focus on the molecular events that mediate long distance directional transport of viral particles in neurons during entry and egress.
Sun XR, Badura A, Pacheco DA, Lynch LA, Schneider ER, Taylor MP, Hogue IB, Enquist LW, Murthy M, Wang SS-H. Fast GCaMPs for improved tracking of neuronal activity. Nat Commun. 2013;4 :2170.Abstract
The use of genetically encodable calcium indicator proteins to monitor neuronal activity is hampered by slow response times and a narrow Ca(2+)-sensitive range. Here we identify three performance-limiting features of GCaMP3, a popular genetically encodable calcium indicator protein. First, we find that affinity is regulated by the calmodulin domain's Ca(2+)-chelating residues. Second, we find that off-responses to Ca(2+) are rate-limited by dissociation of the RS20 domain from calmodulin's hydrophobic pocket. Third, we find that on-responses are limited by fast binding to the N-lobe at high Ca(2+) and by slow binding to the C-lobe at lower Ca(2+). We develop Fast-GCaMPs, which have up to 20-fold accelerated off-responses and show that they have a 200-fold range of K(D), allowing coexpression of multiple variants to span an expanded range of Ca(2+) concentrations. Finally, we show that Fast-GCaMPs track natural song in Drosophila auditory neurons and generate rapid responses in mammalian neurons, supporting the utility of our approach.
Kratchmarov R, Kramer T, Greco TM, Taylor MP, Ch'ng TH, Cristea IM, Enquist LW. Glycoproteins gE and gI are required for efficient KIF1A-dependent anterograde axonal transport of alphaherpesvirus particles in neurons. J Virol. 2013;87 (17) :9431-40.Abstract
Alphaherpesviruses, including pseudorabies virus (PRV), spread directionally within the nervous systems of their mammalian hosts. Three viral membrane proteins are required for efficient anterograde-directed spread of infection in neurons, including Us9 and a heterodimer composed of the glycoproteins gE and gI. We previously demonstrated that the kinesin-3 motor KIF1A mediates anterograde-directed transport of viral particles in axons of cultured peripheral nervous system (PNS) neurons. The PRV Us9 protein copurifies with KIF1A, recruiting the motor to transport vesicles, but at least one unidentified additional viral protein is necessary for this interaction. Here we show that gE/gI are required for efficient anterograde transport of viral particles in axons by mediating the interaction between Us9 and KIF1A. In the absence of gE/gI, viral particles containing green fluorescent protein (GFP)-tagged Us9 are assembled in the cell body but are not sorted efficiently into axons. Importantly, we found that gE/gI are necessary for efficient copurification of KIF1A with Us9, especially at early times after infection. We also constructed a PRV recombinant that expresses a functional gE-GFP fusion protein and used affinity purification coupled with mass spectrometry to identify gE-interacting proteins. Several viral and host proteins were found to associate with gE-GFP. Importantly, both gI and Us9, but not KIF1A, copurified with gE-GFP. We propose that gE/gI are required for efficient KIF1A-mediated anterograde transport of viral particles because they indirectly facilitate or stabilize the interaction between Us9 and KIF1A.
Granstedt AE, Brunton BW, Enquist LW. Imaging the transport dynamics of single alphaherpesvirus particles in intact peripheral nervous system explants from infected mice. MBio. 2013;4 (3) :e00358-13.Abstract
ABSTRACT Alphaherpesvirus particles travel long distances in the axons of neurons using host microtubule molecular motors. The transport dynamics of individual virions in neurons have been assessed in cultured neurons, but imaging studies of single particles in tissue from infected mice have not been reported. We developed a protocol to image explanted, infected peripheral nervous system (PNS) ganglia and associated innervated tissue from mice infected with pseudorabies virus (PRV). This ex vivo preparation allowed us to visualize and track individual virions over time as they moved from the salivary gland into submandibular ganglion neurons of the PNS. We imaged and tracked hundreds of virions from multiple mice at different time points. We quantitated the transport velocity, particle stalling, duty cycle, and directionality at various times after infection. Using a PRV recombinant that expressed monomeric red fluorescent protein (mRFP)-VP26 (red capsid) and green fluorescent protein (GFP)-Us9 (green membrane protein), we corroborated that anterograde transport in axons occurs after capsids are enveloped. We addressed the question of whether replication occurs initially in the salivary gland at the site of inoculation or subsequently in the neurons of peripheral innervating ganglia. Our data indicate that significant amplification of infection occurs in the peripheral ganglia after transport from the site of infection and that these newly made particles are transported back to the salivary gland. It is likely that this reseeding of the infected gland contributes to massive invasion of the innervating PNS ganglia. We suggest that this "round-trip" infection process contributes to the characteristic peripheral neuropathy of PRV infection. IMPORTANCE Much of our understanding of molecular mechanisms of alphaherpesvirus infection and spread in neurons comes from studying cultured primary neurons. These techniques enabled significant advances in our understanding of the viral and neuronal components needed for efficient replication and directional spread between cells. However, in vitro systems cannot recapitulate the environment of innervated tissue in vivo with associated defensive properties, such as innate immunity. Therefore, in this report, we describe a system to image the progression of infection by single virus particles in tissue harvested from infected animals. We explanted intact innervated tissue from infected mice and imaged fluorescent virus particles in infected axons of the specific ganglionic neurons. Our measurements of virion transport dynamics are consistent with published in vitro results. Importantly, this system enabled us to address a fundamental biological question about the amplification of a herpesvirus infection in a peripheral nervous system circuit.
Granstedt AE, Bosse JB, Thiberge SY, Enquist LW. In vivo imaging of alphaherpesvirus infection reveals synchronized activity dependent on axonal sorting of viral proteins. Proc Natl Acad Sci U S A. 2013;110 (37) :E3516-25.Abstract
A clinical hallmark of human alphaherpesvirus infections is peripheral pain or itching. Pseudorabies virus (PRV), a broad host range alphaherpesvirus, causes violent pruritus in many different animals, but the mechanism is unknown. Previous in vitro studies have shown that infected, cultured peripheral nervous system (PNS) neurons exhibited aberrant electrical activity after PRV infection due to the action of viral membrane fusion proteins, yet it is unclear if such activity occurs in infected PNS ganglia in living animals and if it correlates with disease symptoms. Using two-photon microscopy, we imaged autonomic ganglia in living mice infected with PRV strains expressing GCaMP3, a genetically encoded calcium indicator, and used the changes in calcium flux to monitor the activity of many neurons simultaneously with single-cell resolution. Infection with virulent PRV caused these PNS neurons to fire synchronously and cyclically in highly correlated patterns among infected neurons. This activity persisted even when we severed the presynaptic axons, showing that infection-induced firing is independent of input from presynaptic brainstem neurons. This activity was not observed after infections with an attenuated PRV recombinant used for circuit tracing or with PRV mutants lacking either viral glycoprotein B, required for membrane fusion, or viral membrane protein Us9, required for sorting virions and viral glycoproteins into axons. We propose that the viral fusion proteins produced by virulent PRV infection induce electrical coupling in unmyelinated axons in vivo. This action would then give rise to the synchronous and cyclical activity in the ganglia and contribute to the characteristic peripheral neuropathy.
Kratchmarov R, Taylor MP, Enquist LW. Role of Us9 phosphorylation in axonal sorting and anterograde transport of pseudorabies virus. PLoS One. 2013;8 (3) :e58776.Abstract
Alphaherpes viruses, such as pseudorabies virus (PRV), undergo anterograde transport in neuronal axons to facilitate anterograde spread within hosts. Axonal sorting and anterograde transport of virions is dependent on the viral membrane protein Us9, which interacts with the host motor protein Kif1A to direct transport. Us9-Kif1A interactions are necessary but not sufficient for these processes, indicating that additional cofactors or post-translational modifications are needed. In this study, we characterized two conserved serine phosphorylation sites (S51 and S53) in the PRV Us9 protein that are necessary for anterograde spread in vivo. We assessed the subcellular localization of phospho-Us9 subspecies during infection of neurons and found that the phospho-form is detectable on the majority, but not all, of axonal vesicles containing Us9 protein. In biochemical assays, phospho-Us9 was enriched in lipid raft membrane microdomains, though Us9 phosphorylation did not require prior lipid raft association. During infections of chambered neuronal cultures, we observed only a modest reduction in anterograde spread capacity for diserine mutant Us9, and no defect for monoserine mutants. Conversely, mutation of the kinase recognition sequence residues adjacent to the phosphorylation sites completely abrogated anterograde spread. In live-cell imaging analyses, anterograde transport of virions was reduced during infection with a recombinant PRV strain expressing GFP-tagged diserine mutant Us9. Phosphorylation was not required for Us9-Kif1A interaction, suggesting that Us9-Kif1A binding is a distinct step from the activation and/or stabilization of the transport complex. Taken together, our findings indicate that, while not essential, Us9 phosphorylation enhances Us9-Kif1A-based transport of virions in axons to modulate the overall efficiency of long-distance anterograde spread of infection.
Koyuncu OO, Hogue IB, Enquist LW. Virus infections in the nervous system. Cell Host Microbe. 2013;13 (4) :379-93.Abstract
Virus infections usually begin in peripheral tissues and can invade the mammalian nervous system (NS), spreading into the peripheral (PNS) and more rarely the central (CNS) nervous systems. The CNS is protected from most virus infections by effective immune responses and multilayer barriers. However, some viruses enter the NS with high efficiency via the bloodstream or by directly infecting nerves that innervate peripheral tissues, resulting in debilitating direct and immune-mediated pathology. Most viruses in the NS are opportunistic or accidental pathogens, but a few, most notably the alpha herpesviruses and rabies virus, have evolved to enter the NS efficiently and exploit neuronal cell biology. Remarkably, the alpha herpesviruses can establish quiescent infections in the PNS, with rare but often fatal CNS pathology. Here we review how viruses gain access to and spread in the well-protected CNS, with particular emphasis on alpha herpesviruses, which establish and maintain persistent NS infections.
Taylor MP, Kobiler O, Enquist LW. Alphaherpesvirus axon-to-cell spread involves limited virion transmission. Proc Natl Acad Sci U S A. 2012;109 (42) :17046-51.Abstract
The spread of viral infection within a host can be restricted by bottlenecks that limit the size and diversity of the viral population. An essential process for alphaherpesvirus infection is spread from axons of peripheral nervous system neurons to cells in peripheral epithelia (anterograde-directed spread, ADS). ADS is necessary for the formation of vesicular lesions characteristic of reactivated herpesvirus infections; however, the number of virions transmitted is unknown. We have developed two methods to quantitate ADS events using a compartmentalized neuronal culture system. The first method uses HSV-1 and pseudorabies virus recombinants that express one of three different fluorescent proteins. The fluorescence profiles of cells infected with the virus mixtures are used to quantify the number of expressed viral genomes. Strikingly, although epithelial or neuronal cells express 3-10 viral genomes after infection by free virions, epithelial cells infected by HSV-1 or pseudorabies virus following ADS express fewer than two viral genomes. The second method uses live-cell fluorescence microscopy to track individual capsids involved in ADS. We observed that most ADS events involve a single capsid infecting a target epithelial cell. Together, these complementary analyses reveal that ADS events are restricted to small numbers of viral particles, most often a single virion, resulting in a single viral genome initiating infection.
Kramer T, Enquist LW. Alphaherpesvirus infection disrupts mitochondrial transport in neurons. Cell Host Microbe. 2012;11 (5) :504-14.Abstract
Mitochondria are dynamic organelles that are essential for cellular metabolism but can be functionally disrupted during pathogen infection. In neurons, mitochondria are transported on microtubules via the molecular motors kinesin-1 and dynein and recruited to energy-requiring regions such as synapses. Previous studies showed that proteins from pseudorabies virus (PRV), an alphaherpesvirus, localize to mitochondria and affect mitochondrial function. We show that PRV and herpes simplex virus type 1 (HSV-1) infection of rodent superior cervical ganglion (SCG) neurons disrupts mitochondrial motility and morphology. During PRV infection, glycoprotein B (gB)-dependent fusion events result in electrical coupling of neurons and increased action potential firing rates. Consequently, intracellular [Ca(2+)] increases and alters mitochondrial dynamics through a mechanism involving the Ca(2+)-sensitive cellular protein Miro and reduced recruitment of kinesin-1 to mitochondria. This disruption in mitochondrial dynamics is required for efficient growth and spread of PRV, indicating that altered mitochondrial transport enhances alphaherpesvirus pathogenesis and infection.
Kramer T, Greco TM, Taylor MP, Ambrosini AE, Cristea IM, Enquist LW. Kinesin-3 mediates axonal sorting and directional transport of alphaherpesvirus particles in neurons. Cell Host Microbe. 2012;12 (6) :806-14.Abstract
During infection of the nervous system, alphaherpesviruses-including pseudorabies virus (PRV)-use retrograde axonal transport to travel toward the neuronal cell body and anterograde transport to traffic back to the cell periphery upon reactivation from latency. The PRV protein Us9 plays an essential but unknown role in anterograde viral spread. To determine Us9 function, we identified viral and host proteins that interact with Us9 and explored the role of KIF1A, a microtubule-dependent kinesin-3 motor involved in axonal sorting and transport. Viral particles are cotransported with KIF1A in axons of primary rat superior cervical ganglion neurons, and overexpression or disruption of KIF1A function, respectively, increases and reduces anterograde capsid transport. Us9 and KIF1A interact early during infection with the aid of additional viral protein(s) but exhibit diminished binding at later stages, when capsids typically stall in axons. Thus, alphaherpesviruses repurpose the axonal transport and sorting pathway to spread within their hosts.
Kramer T, Greco TM, Enquist LW, Cristea IM. Proteomic characterization of pseudorabies virus extracellular virions. J Virol. 2011;85 (13) :6427-41.Abstract
Pseudorabies virus (PRV), a member of the Alphaherpesvirinae, has a complex multilayered extracellular virion that is structurally conserved among other herpesviruses. PRV virions contain a double-stranded DNA genome within a proteinaceous capsid surrounded by the tegument, a layer of viral and cellular proteins. The envelope layer, which encloses the capsid and tegument, contains viral transmembrane proteins anchored in a phospholipid bilayer. The viral and host proteins contained within virions execute important functions during viral spread and pathogenesis, but a detailed understanding of the composition of PRV virions has been lacking. In this report, we present the first comprehensive proteomic characterization of purified PRV virions by mass spectrometry using two complementary approaches. To exclude proteins present in the extracellular medium that may nonspecifically associate with virions, we also analyzed virions treated with proteinase K and samples prepared from mock-infected cells. Overall, we identified 47 viral proteins associated with PRV virions, 40 of which were previously localized to the capsid, tegument, and envelope layers using traditional biochemical approaches. Additionally, we identified seven viral proteins that were previously undetected in virions, including pUL8, pUL20, pUL32, pUL40 (RR2), pUL42, pUL50 (dUTPase), and Rsp40/ICP22. Furthermore, although we did not enrich for posttranslational modifications, we detected phosphorylation of four virion proteins: pUL26, pUL36, pUL46, and pUL48. Finally, we identified 48 host proteins associated with PRV virions, many of which have known functions in important cellular pathways such as intracellular signaling, mRNA translation and processing, cytoskeletal dynamics, and membrane organization. This analysis extends previous work aimed at determining the composition of herpesvirus virions and provides novel insights critical for understanding the mechanisms underlying PRV entry, assembly, egress, spread, and pathogenesis.
Taylor MP, Koyuncu OO, Enquist LW. Subversion of the actin cytoskeleton during viral infection. Nat Rev Microbiol. 2011;9 (6) :427-39.Abstract
Viral infection converts the normal functions of a cell to optimize viral replication and virion production. One striking observation of this conversion is the reconfiguration and reorganization of cellular actin, affecting every stage of the viral life cycle, from entry through assembly to egress. The extent and degree of cytoskeletal reorganization varies among different viral infections, suggesting the evolution of myriad viral strategies. In this Review, we describe how the interaction of viral proteins with the cell modulates the structure and function of the actin cytoskeleton to initiate, sustain and spread infections. The molecular biology of such interactions continues to engage virologists in their quest to understand viral replication and informs cell biologists about the role of the cytoskeleton in the uninfected cell.
Zhao Y, Huang W, Zhu L, Hu J. Difluoromethyl 2-pyridyl sulfone: a new gem-difluoroolefination reagent for aldehydes and ketones. Org Lett. 2010;12 (7) :1444-7.Abstract
Difluoromethyl 2-pyridyl sulfone, a previously unknown compound, was found to act as a novel and efficient gem-difluoroolefination reagent for both aldehydes and ketones. It was found that the fluorinated sulfinate intermediate in the reaction is relatively stable, which can be observed by (19)F NMR and trapped with CH(3)I.
Valo HK, Laaksonen PH, Peltonen LJ, Linder MB, Hirvonen JT, Laaksonen TJ. Multifunctional hydrophobin: toward functional coatings for drug nanoparticles. ACS Nano. 2010;4 (3) :1750-8.Abstract
Efficient delivery of nanosized drug formulations to the desired body sites is not always reached despite the rapid development of pharmaceutical nanotechnologies. In spite of the undoubted effect of the size for increased bioavailability and controlled drug delivery, submicrometer formulations also require a deeper level of design. The surface properties of the particles determine the stability of the particles, interactions with the body, and targeting potentials of drugs. Thus, the efficacy of the drug can be increased utilizing the surface layer of the nanoparticles. Influencing the surface characters of the drug is the main focus of the present work, which introduces a method for preparing nanoparticles with functional sites from low-solubility drugs using hydrophobin (HFB) proteins. Particles were prepared by precipitating a lipophilic drug (beclomethasone dipropionate) in water in the presence of the HFB proteins. Particle size below 200 nm could easily be reached with increasing HFB concentration. The particles were shown to be stable for at least 5 h in suspension, and they could be stored for longer periods of time after freeze-drying. Labeling studies using green fluorescent protein (GFP) genetically fused to a HFB clearly demonstrated that the surface of the nanoparticles was covered with the hydrophobins and that the surface could be further modified by utilizing fusion proteins. This provides a template for a variety of different functional surface-bound groups that could be tailored by modifying the hydrophilic side of the HFB via protein bioengineering. In this study, the combination of proteins and traditional pharmaceutical technology was used to synthesize functionalized protein-coated nanoparticles for drug delivery purposes.
Granstedt AE, Kuhn B, Wang SS-H, Enquist LW. Calcium imaging of neuronal circuits in vivo using a circuit-tracing pseudorabies virus. Cold Spring Harb Protoc. 2010;2010 (4) :pdb.prot5410.Abstract
Pseudorabies virus (PRV) is a neuroinvasive virus of the herpes family that has a broad host range but does not infect higher-order primates. PRV characteristically travels along chains of synaptically connected neurons and has been used extensively for elucidating neural circuits in the peripheral and central nervous system in vivo. The recombinant virus PRV369 is an attenuated retrograde tracer that encodes G-CaMP2, a fluorescent calcium sensor protein that is stable at physiological pH and mammalian temperature. This protocol describes the use of PRV369 to express G-CaMP2 in a neuronal circuit and to monitor its activity in a living animal, specifically in the submandibular ganglia (SMG), the peripheral parasympathetic ganglia that innervate the salivary glands. The procedure describes the delivery of PRV369 to the glands and shows how SMG neurons can then be imaged post-inoculation to explore connectivity and activity.
Szpara ML, Kobiler O, Enquist LW. A common neuronal response to alphaherpesvirus infection. J Neuroimmune Pharmacol. 2010;5 (3) :418-27.Abstract
Alphaherpesviruses are a subfamily of the Herpesviridae that can invade the nervous system and establish either lytic or latent infections. The establishment of latent infection can occur only in neurons, indicating a unique virus-host interaction in these cells. Here, we compare results from seven microarray studies that focused on the host response of either neural tissue or isolated neurons to alphaherpesvirus infection. These studies utilized either herpes simplex virus type 1 or pseudorabies virus as the infectious agent. From these data, we have found common host responses spanning a variety of infection models in different species, with different herpesvirus strains, and during all phases of infection including lytic, latent, and reactivation. The repeated observation of transcriptional effects on these genes and gene families indicates their likely importance in host defenses or the viral infectious process. We discuss the possible role of these different genes and genes families in alphaherpesvirus infection.
Kobiler O, Lipman Y, Therkelsen K, Daubechies I, Enquist LW. Herpesviruses carrying a Brainbow cassette reveal replication and expression of limited numbers of incoming genomes. Nat Commun. 2010;1 :146.Abstract
Whether all the infectious herpesvirus particles entering a cell are able to replicate and/or express their genomes is not known. Here, we developed a general method to determine the number of viral genomes expressed in an infected cell. We constructed and analysed fluorophore expression from a recombinant pseudorabies virus (PRV263) carrying a Brainbow cassette (Cre-conditional expression of different fluorophores). Using three isogenic strains derived from PRV263, each expressing a single fluorophore, we analysed the colour composition of cells infected with these three viruses at different multiplicities. We estimate that fewer than seven incoming genomes are expressed per cell. In addition, those templates that are expressed are the genomes selected for replication and packaging into virions. This finite limit on the number of viral genomes that can be expressed is an intrinsic property of the infected cell and may be influenced by viral and cellular factors.
Curanović D, Ch'ng TH, Szpara M, Enquist L. Compartmented neuron cultures for directional infection by alpha herpesviruses. Curr Protoc Cell Biol. 2009;Chapter 26 :Unit 26.4.Abstract
Compartmented neuronal cultures allow experimenters to establish separate fluid environments for neuronal axons and the soma from which they emanate. Physical isolation of cell bodies and axons is achieved by culturing neurons in tri-chambered Teflon rings. Dissociated ganglia are plated in one end compartment of the trichamber, and axonal growth is guided underneath watertight silicone grease barriers into a separate compartment. Since the axons and cell bodies are located in different compartments, they can be infected and assayed separately. We describe the assembly and use of compartmented neuronal cultures for in vitro study of directional infection of neurons by alpha herpesviruses. Selective application of viral inoculum to only one compartment ensures that the remainder of the neuron is not contaminated by input inoculum. This allows for quantification of viral spread, and unambiguous interpretation of immunofluorescence and electron microscopy images.
Lyman MG, Enquist LW. Herpesvirus interactions with the host cytoskeleton. J Virol. 2009;83 (5) :2058-66.
Curanovic D, Enquist LW. Virion-incorporated glycoprotein B mediates transneuronal spread of pseudorabies virus. J Virol. 2009;83 (16) :7796-804.Abstract
Transneuronal spread of pseudorabies virus (PRV) is a multistep process that requires several virally encoded proteins. Previous studies have shown that PRV glycoprotein B (gB), a component of the viral fusion machinery, is required for the transmission of infection to postsynaptic, second-order neurons. We sought to identify the gB-mediated step in viral transmission. We determined that gB is not required for the sorting of virions into axons of infected neurons, anterograde transport, or the release of virions from the axon. trans or cis expression of gB on the cell surface was not sufficient for transneuronal spread of the virus; instead, efficient incorporation of gB into virions was required. Additionally, neuron-to-cell spread of PRV most likely does not proceed through syncytial connections. We conclude that, upon gB-independent release of virions at the site of neuron-cell contacts, the virion-incorporated gB/gH/gL fusion complex mediates entry into the axonally contacted cell by fusion of the closely apposed membranes.
Granstedt AE, Szpara ML, Kuhn B, Wang SS-H, Enquist LW. Fluorescence-based monitoring of in vivo neural activity using a circuit-tracing pseudorabies virus. PLoS One. 2009;4 (9) :e6923.Abstract
The study of coordinated activity in neuronal circuits has been challenging without a method to simultaneously report activity and connectivity. Here we present the first use of pseudorabies virus (PRV), which spreads through synaptically connected neurons, to express a fluorescent calcium indicator protein and monitor neuronal activity in a living animal. Fluorescence signals were proportional to action potential number and could reliably detect single action potentials in vitro. With two-photon imaging in vivo, we observed both spontaneous and stimulated activity in neurons of infected murine peripheral autonomic submandibular ganglia (SMG). We optically recorded the SMG response in the salivary circuit to direct electrical stimulation of the presynaptic axons and to physiologically relevant sensory stimulation of the oral cavity. During a time window of 48 hours after inoculation, few spontaneous transients occurred. By 72 hours, we identified more frequent and prolonged spontaneous calcium transients, suggestive of neuronal or tissue responses to infection that influence calcium signaling. Our work establishes in vivo investigation of physiological neuronal circuit activity and subsequent effects of infection with single cell resolution.
McCarthy KM, Tank DW, Enquist LW. Pseudorabies virus infection alters neuronal activity and connectivity in vitro. PLoS Pathog. 2009;5 (10) :e1000640.Abstract
Alpha-herpesviruses, including human herpes simplex virus 1 & 2, varicella zoster virus and the swine pseudorabies virus (PRV), infect the peripheral nervous system of their hosts. Symptoms of infection often include itching, numbness, or pain indicative of altered neurological function. To determine if there is an in vitro electrophysiological correlate to these characteristic in vivo symptoms, we infected cultured rat sympathetic neurons with well-characterized strains of PRV known to produce virulent or attenuated symptoms in animals. Whole-cell patch clamp recordings were made at various times after infection. By 8 hours of infection with virulent PRV, action potential (AP) firing rates increased substantially and were accompanied by hyperpolarized resting membrane potentials and spikelet-like events. Coincident with the increase in AP firing rate, adjacent neurons exhibited coupled firing events, first with AP-spikelets and later with near identical resting membrane potentials and AP firing. Small fusion pores between adjacent cell bodies formed early after infection as demonstrated by transfer of the low molecular weight dye, Lucifer Yellow. Later, larger pores formed as demonstrated by transfer of high molecular weight Texas red-dextran conjugates between infected cells. Further evidence for viral-induced fusion pores was obtained by infecting neurons with a viral mutant defective for glycoprotein B, a component of the viral membrane fusion complex. These infected neurons were essentially identical to mock infected neurons: no increased AP firing, no spikelet-like events, and no electrical or dye transfer. Infection with PRV Bartha, an attenuated circuit-tracing strain delayed, but did not eliminate the increased neuronal activity and coupling events. We suggest that formation of fusion pores between infected neurons results in electrical coupling and elevated firing rates, and that these processes may contribute to the altered neural function seen in PRV-infected animals.
Lyman MG, Curanovic D, Enquist LW. Targeting of pseudorabies virus structural proteins to axons requires association of the viral Us9 protein with lipid rafts. PLoS Pathog. 2008;4 (5) :e1000065.Abstract
The pseudorabies virus (PRV) Us9 protein plays a central role in targeting viral capsids and glycoproteins to axons of dissociated sympathetic neurons. As a result, Us9 null mutants are defective in anterograde transmission of infection in vivo. However, it is unclear how Us9 promotes axonal sorting of so many viral proteins. It is known that the glycoproteins gB, gC, gD and gE are associated with lipid raft microdomains on the surface of infected swine kidney cells and monocytes, and are directed into the axon in a Us9-dependent manner. In this report, we determined that Us9 is associated with lipid rafts, and that this association is critical to Us9-mediated sorting of viral structural proteins. We used infected non-polarized and polarized PC12 cells, a rat pheochromocytoma cell line that acquires many of the characteristics of sympathetic neurons in the presence of nerve growth factor (NGF). In these cells, Us9 is highly enriched in detergent-resistant membranes (DRMs). Moreover, reducing the affinity of Us9 for lipid rafts inhibited anterograde transmission of infection from sympathetic neurons to epithelial cells in vitro. We conclude that association of Us9 with lipid rafts is key for efficient targeting of structural proteins to axons and, as a consequence, for directional spread of PRV from pre-synaptic to post-synaptic neurons and cells of the mammalian nervous system.
Feierbach B, Piccinotti S, Bisher M, Denk W, Enquist LW. Alpha-herpesvirus infection induces the formation of nuclear actin filaments. PLoS Pathog. 2006;2 (8) :e85.Abstract
Herpesviruses are large double-stranded DNA viruses that replicate in the nuclei of infected cells. Spatial control of viral replication and assembly in the host nucleus is achieved by the establishment of nuclear compartments that serve to concentrate viral and host factors. How these compartments are established and maintained remains poorly understood. Pseudorabies virus (PRV) is an alpha-herpesvirus often used to study herpesvirus invasion and spread in the nervous system. Here, we report that PRV and herpes simplex virus type 1 infection of neurons results in formation of actin filaments in the nucleus. Filamentous actin is not found in the nucleus of uninfected cells. Nuclear actin filaments appear physically associated with the viral capsids, as shown by serial block-face scanning electron micropscopy and confocal microscopy. Using a green fluorescent protein-tagged viral capsid protein (VP26), we show that nuclear actin filaments form prior to capsid assembly and are required for the efficient formation of viral capsid assembly sites. We find that actin polymerization dynamics (e.g., treadmilling) are not necessary for the formation of these sites. Green fluorescent protein-VP26 foci co-localize with the actin motor myosin V, suggesting that viral capsids travel along nuclear actin filaments using myosin-based directed transport. Viral transcription, but not viral DNA replication, is required for actin filament formation. The finding that infection, by either PRV or herpes simplex virus type 1, results in formation of nuclear actin filaments in neurons, and that PRV infection of an epithelial cell line results in a similar phenotype is evidence that F-actin plays a conserved role in herpesvirus assembly. Our results suggest a mechanism by which assembly domains are organized within infected cells and provide insight into how the viral infectious cycle and host actin cytoskeleton are integrated to promote the infection process.
Paulus C, Sollars PJ, Pickard GE, Enquist LW. Transcriptome signature of virulent and attenuated pseudorabies virus-infected rodent brain. J Virol. 2006;80 (4) :1773-86.Abstract
Mammalian alphaherpesviruses normally establish latent infections in ganglia of the peripheral nervous system in their natural hosts. Occasionally, however, these viruses spread to the central nervous system (CNS), where they cause damaging, often fatal, infections. Attenuated alphaherpesvirus derivatives have been used extensively as neuronal circuit tracers in a variety of animal models. Their circuit-specific spread provides a unique paradigm to study the local and global CNS response to infection. Thus, we systematically analyzed the host gene expression profile after acute pseudorabies virus (PRV) infection of the CNS using Affymetrix GeneChip technology. Rats were injected intraocularly with one of three selected virulent and attenuated PRV strains. Relative levels of cellular transcripts were quantified from hypothalamic and cerebellar tissues at various times postinfection. The number of cellular genes responding to infection correlated with the extent of virus dissemination and relative virulence of the PRV strains. A total of 245 out of 8,799 probe sets, corresponding to 182 unique cellular genes, displayed increased expression ranging from 2- to more than 100-fold higher than in uninfected tissue. Over 60% thereof were categorized as immune, proinflammatory, and other cellular defense genes. Additionally, a large fraction of infection-induced transcripts represented cellular stress responses, including glucocorticoid- and redox-related pathways. This is the first comprehensive in vivo analysis of the global transcriptional response of the mammalian CNS to acute alphaherpesvirus infection. The differentially regulated genes reported here are likely to include potential diagnostic and therapeutic targets for viral encephalitides and other neurodegenerative or neuroinflammatory diseases.
del Rio T, Ch'ng TH, Flood EA, Gross SP, Enquist LW. Heterogeneity of a fluorescent tegument component in single pseudorabies virus virions and enveloped axonal assemblies. J Virol. 2005;79 (7) :3903-19.Abstract
The molecular mechanisms responsible for long-distance, directional spread of alphaherpesvirus infections via axons of infected neurons are poorly understood. We describe the use of red and green fluorescent protein (GFP) fusions to capsid and tegument components, respectively, to visualize purified, single extracellular virions and axonal assemblies after pseudorabies virus (PRV) infection of cultured neurons. We observed heterogeneity in GFP fluorescence when GFP was fused to the tegument component VP22 in both single extracellular virions and discrete puncta in infected axons. This heterogeneity was observed in the presence or absence of a capsid structure detected by a fusion of monomeric red fluorescent protein to VP26. The similarity of the heterogeneous distribution of these fluorescent protein fusions in both purified virions and in axons suggested that tegument-capsid assembly and axonal targeting of viral components are linked. One possibility was that the assembly of extracellular and axonal particles containing the dually fluorescent fusion proteins occurred by the same process in the cell body. We tested this hypothesis by treating infected cultured neurons with brefeldin A, a potent inhibitor of herpesvirus maturation and secretion. Brefeldin A treatment disrupted the neuronal secretory pathway, affected fluorescent capsid and tegument transport in the cell body, and blocked subsequent entry into axons of capsid and tegument proteins. Electron microscopy demonstrated that in the absence of brefeldin A treatment, enveloped capsids entered axons, but in the presence of the inhibitor, unenveloped capsids accumulated in the cell body. These results support an assembly process in which PRV capsids acquire a membrane in the cell body prior to axonal entry and subsequent transport.
del Rio T, DeCoste CJ, Enquist LW. Actin is a component of the compensation mechanism in pseudorabies virus virions lacking the major tegument protein VP22. J Virol. 2005;79 (13) :8614-9.Abstract
Despite being a major component of the pseudorabies virus tegument, VP22 is not required for PRV replication, virulence, or neuroinvasion (T. del Rio, H. C. Werner, and L. W. Enquist, J. Virol. 76:774-782, 2002). In the absence of VP22, tegument assembly compensates in a limited fashion with increased incorporation of cellular actin. Infection of epithelial cell lines expressing fluorescent actin fusion proteins resulted in the incorporation of filamentous and nonfilamentous actin into individual virions that were predominately light, noninfectious particles. We conclude that cellular actin is incorporated in the tegument of wild-type virions and is part of a compensation mechanism for VP22-null virions.
Ch'ng TH, Enquist LW. Efficient axonal localization of alphaherpesvirus structural proteins in cultured sympathetic neurons requires viral glycoprotein E. J Virol. 2005;79 (14) :8835-46.Abstract
Pseudorabies virus (PRV) glycoprotein E (gE) is a type I viral membrane protein that facilitates the anterograde spread of viral infection from the peripheral nervous system to the brain. In animal models, a gE-null mutant infection spreads inefficiently from presynaptic neurons to postsynaptic neurons (anterograde spread of infection). However, the retrograde spread of infection from post- to presynaptic neurons remains unaffected. Here we show that gE is required for wild-type localization of viral structural proteins in axons of infected neurons. During a gE-null PRV infection, a subset of viral glycoproteins, capsids, and tegument proteins enter and localize to the axon inefficiently. This defect is most obvious in the distal axon and growth cones. However, axonal entry and localization of other viral membrane proteins and endogenous cellular proteins remains unaffected. Neurons infected with gE-null mutants produce wild-type levels of viral structural proteins and infectious virions in the cell body. Our results indicate that reduced axonal targeting of viral structural proteins is a compelling explanation for the lack of anterograde spread in neural circuits following infection by a gE-null mutant.
Ch'ng TH, Enquist LW. Neuron-to-cell spread of pseudorabies virus in a compartmented neuronal culture system. J Virol. 2005;79 (17) :10875-89.Abstract
Alphaherpesviruses are parasites of the peripheral nervous system in their natural hosts. After the initial infection of peripheral tissues such as mucosal cells, these neurotropic viruses will invade the peripheral nervous system that innervates the site of infection via long-distance axonal transport of the viral genome. In natural hosts, a latent and a nonproductive infection is usually established in the neuronal cell bodies. Upon reactivation, the newly replicated genome will be assembled into capsids and transported back to the site of entry, where a localized infection of the epithelial or mucosal cells will produce infectious virions that can infect naïve hosts. In this paper, we describe an in vitro method for studying neuron-to-cell spread of alphaherpesviruses using a compartmented culture system. Using pseudorabies virus as a model, we infected neuron cell bodies grown in Teflon chambers and observed spread of infection to nonneuronal cells plated in a different compartment. The cells are in contact with the neurons via axons that penetrate the Teflon barrier. We demonstrate that wild-type neuron-to-cell spread requires intact axons and the presence of gE, gI, and Us9 proteins, but does not require gD. We also provide ultrastructural evidence showing that capsids enclosed within vesicles can be found along the entire length of the axon during viral egress.
Wang F, Tang W, McGraw HM, Bennett J, Enquist LW, Friedman HM. Herpes simplex virus type 1 glycoprotein e is required for axonal localization of capsid, tegument, and membrane glycoproteins. J Virol. 2005;79 (21) :13362-72.Abstract
Herpes simplex virus type 1 (HSV-1) glycoprotein E (gE) promotes cell-to-cell spread at basolateral surfaces of epithelial cells, but its activity in neurons is less clear. We used the mouse retina infection model and neuronal cell cultures to define the spread phenotype of gE mutant viruses. Wild-type (WT) and gE-null (NS-gEnull) viruses both infected retina ganglion cell neurons; however, NS-gEnull viral antigens failed to reach the optic nerve, which indicates a defect in axonal localization. We evaluated two Fc receptor-negative gE mutant viruses containing four amino acid inserts in the gE ectodomain. One mutant virus failed to spread from the retina into the optic nerve, while the other spread normally. Therefore, the gE ectodomain is involved in axonal localization, and the Fc receptor and neuronal spread are mediated by overlapping but distinct gE domains. In the retina infection model, virus can travel to the brain via the optic nerve from presynaptic to postsynaptic neurons (anterograde direction) or via nerves that innervate the iris and ciliary body from postsynaptic to presynaptic neurons (retrograde direction). WT virus infected the brain by anterograde and retrograde routes, whereas NS-gEnull virus failed to travel by either pathway. The site of the defect in retrograde spread remains to be determined; however, infection of rat superior cervical ganglia neurons in vitro indicates that gE is required to target virion components to the axon initial segment. The requirement for gE in axonal targeting and retrograde spread highlights intriguing similarities and differences between HSV-1 and pseudorabies virus gE.
Ch'ng TH, Flood AE, Enquist LW. Culturing primary and transformed neuronal cells for studying pseudorabies virus infection. Methods Mol Biol. 2005;292 :299-316.Abstract
This chapter discusses the culture of primary sympathetic neurons (superior cervical ganglia) from rat embryos and PC12 cells differentiated into neurons for use in viral infection experiments. Methods are described for the use of a neurotropic herpesvirus, pseudorabies virus (PRV), to analyze the assembly, egress, and transport of viral antigens in neurons.
Ray N, Enquist LW. Transcriptional response of a common permissive cell type to infection by two diverse alphaherpesviruses. J Virol. 2004;78 (7) :3489-501.Abstract
Pseudorabies virus (PRV) and herpes simplex virus type 1 (HSV-1) are distantly related alphaherpesviruses whose natural hosts are pigs and humans, respectively. Adult infections of natural hosts are mild and rarely lethal. However, both viruses are also able to infect other hosts, often with lethal effects. In this report, we use the paradigm of infection of a common permissive cell type and microarray analysis to determine if these two diverse alphaherpesviruses engage similar or different cellular pathways to obtain a common outcome: productive infection. We compared cellular gene expression in growth-arrested, primary rat embryonic fibroblasts that were mock infected or infected with either purified PRV-Becker or HSV-1(F). Infections by either virus affect the transcription of more than 1,500 cellular genes by threefold or more. Few differences are detected early, and the majority of changes occur during the late stages of infection. Remarkably, the transcripts of about 500 genes are regulated in common, while the rest are regulated in a virus-specific manner. Genes whose expression is affected by infection fall into a diverse group of functional classes and cellular pathways. Furthermore, a comparison of the cellular response to HSV-1 infection of primary human and rat fibroblasts revealed unexpected diversity in the transcript profiles.
Smith GA, Pomeranz L, Gross SP, Enquist LW. Local modulation of plus-end transport targets herpesvirus entry and egress in sensory axons. Proc Natl Acad Sci U S A. 2004;101 (45) :16034-9.Abstract
The core structures of many viruses move within cells by association with host cytoskeletal motor proteins; however, the mechanisms by which intracellular viral particles are transported toward sites of replication or the cell periphery at distinct stages of infection remain to be understood. The regulation of herpesvirus directional transport in sensory neurons was examined by tracking individual viral capsids within axons at multiple frames per s. After entry into axons, capsids underwent bidirectional and saltatory movement to the cell body independently of endosomes. A comparison of entry transport to a previous analysis of capsid axonal transport during egress revealed that capsid targeting in and out of cells occurs by modulation of plus-end, but not minus-end, motion. Entry transport was unperturbed by the presence of egressing virus from a prior infection, indicating that transport direction is not modulated globally by viral gene expression, but rather directly by a component of the viral particle.
Brittle EE, Reynolds AE, Enquist LW. Two modes of pseudorabies virus neuroinvasion and lethality in mice. J Virol. 2004;78 (23) :12951-63.Abstract
We describe two distinct modes of neuroinvasion and lethality after murine flank inoculation with virulent and attenuated strains of pseudorabies virus (PRV). Mice infected with virulent (e.g., PRV-Becker, PRV-Kaplan, or PRV-NIA3) strains self-mutilate their flank skin in response to virally induced pruritus, die rapidly with no identifiable symptoms of central nervous system (CNS) infection such as behavioral abnormalities, and have little infectious virus or viral antigen in the brain. In distinct contrast, animals infected with an attenuated PRV vaccine strain (PRV-Bartha) survive approximately three times longer than wild-type PRV-infected animals, exhibit severe CNS abnormalities, and have an abundance of infectious virus in the brain at the time of death. Interestingly, these animals have no skin lesions and do not appear pruritic at any time during infection. The severe pruritus and relatively earlier time until death induced by wild-type PRV infection may reflect the peripheral nervous system (PNS) and immune responses to infection rather than a fatal, virally induced CNS pathology. Based on previously characterized afferent (sensory) and efferent (motor) neuronal pathways that innervate the skin, we deduced that wild-type virulent strains transit through the PNS via both afferent and efferent routes, whereas PRV-Bartha travels by only efferent routes in the PNS en route to the brain.
Ray N, Bisher ME, Enquist LW. Cyclooxygenase-1 and -2 are required for production of infectious pseudorabies virus. J Virol. 2004;78 (23) :12964-74.Abstract
We have recently shown that cyclooxygenase-2 (COX-2) transcription is markedly induced after herpes simplex virus type 1 and pseudorabies virus (PRV) infections of rat embryonic fibroblast (REF) cells (N. Ray and L. W. Enquist, J. Virol. 78:3489-3501, 2004). For this study, we investigated the role of cyclooxygenase induction in the replication and growth of PRV. We demonstrate here a concordant increase in COX-2 mRNA and protein levels after the infection of REF cells. Inhibitors blocking the activity of cyclooxygenases caused a dramatic reduction in PRV growth. Viral growth could be restored if prostaglandin E(2), the final product of COX-2 activity, was added simultaneously with the COX inhibitors. Immediate-early protein IE180, major capsid protein VP5, and glycoprotein expression were slightly reduced in the presence of COX-2 inhibitors, but expression of the early protein EP0 was not affected by COX inhibition. Viral DNA replication was marginally reduced in the presence of a COX-1/2 inhibitor, but there was no defect in viral DNA cleavage. Electron microscopy analysis revealed an increased number of unusual empty capsid structures in the nuclei of cells infected with PRV in the presence of a COX-1/2 inhibitor. These capsid structures shared some characteristics with procapsids but had a novel appearance by negative staining. Our data establish a role for COX-1 and COX-2 in facilitating the efficient growth and replication of PRV in primary cells.
Silva MC, Yu Q-C, Enquist L, Shenk T. Human cytomegalovirus UL99-encoded pp28 is required for the cytoplasmic envelopment of tegument-associated capsids. J Virol. 2003;77 (19) :10594-605.Abstract
The human cytomegalovirus UL99-encoded pp28 is a myristylated phosphoprotein that is a constituent of the virion. The pp28 protein is positioned within the tegument of the virus particle, a protein structure that resides between the capsid and envelope. In the infected cell, pp28 is found in a cytoplasmic compartment derived from the Golgi apparatus, where the virus buds into vesicles to acquire its final membrane. We have constructed two mutants of human cytomegalovirus that fail to produce the pp28 protein, a substitution mutant (BADsubUL99) and a point mutant (BADpmUL99), and we have propagated them by complementation in pp28-expressing fibroblasts. Both mutant viruses are profoundly defective for growth in normal fibroblasts; no infectious virus could be detected after infection. Whereas normal levels of viral DNA and late proteins were observed in mutant virus-infected cells, large numbers of tegument-associated capsids accumulated in the cytoplasm that failed to acquire an envelope. We conclude that pp28 is required for the final envelopment of the human cytomegalovirus virion in the cytoplasm.
Enquist LW, Card PJ. Recent advances in the use of neurotropic viruses for circuit analysis. Curr Opin Neurobiol. 2003;13 (5) :603-6.Abstract
In the past decade, the literature describing how viruses can be used to define the synaptic architecture of the brain has increased dramatically. Early studies focused on determining the specificity of viral transport through synaptic connections. Analysis of the assembly and intracellular transport of viruses as well as of the role of the brain response to infection were central to this literature. With the growing acceptance that the transport of viruses is circuit-related, attention has shifted to application of the method to define the functional architecture of neural systems. The development of attenuated recombinant viruses that maintain neuroinvasiveness has been instrumental to the generation of increasingly powerful experimental approaches for the functional dissection of neural circuits. These approaches include the use of recombinant viruses that express unique reporters to address issues of axon collateralization in complex circuits, the use of green-fluorescent-protein-expressing recombinants to characterize the electrophysiological properties of projection-specific neurons in live slices of brain, and the exploitation of the Cre recombinase system for conditional replication of virus in phenotypically defined populations of neurons.
Yu D, Smith GA, Enquist LW, Shenk T. Construction of a self-excisable bacterial artificial chromosome containing the human cytomegalovirus genome and mutagenesis of the diploid TRL/IRL13 gene. J Virol. 2002;76 (5) :2316-28.Abstract
The full-length genome of human cytomegalovirus strain AD169 was cloned as an infectious bacterial artificial chromosome (BAC) plasmid, pAD/Cre. The BAC vector, flanked by LoxP sites, was inserted immediately after the Us28 open reading frame without deletion of any viral sequences. The BAC vector contained the Cre recombinase-encoding gene disrupted by an intron under control of the simian virus 40 early promoter. When pAD/Cre was transfected into primary human foreskin fibroblast cells, Cre was expressed and mediated site-specific recombination between the two LoxP sites, excising the BAC DNA backbone. This gave rise to progeny virus that was wild type with the exception of an inserted 34-bp LoxP site. We performed site-directed mutagenesis on pAD/Cre to generate a series of viruses in which the TRL/IRL13 diploid genes were disrupted and subsequently repaired. The mutants reach the same titer as the wild-type virus, indicating that the TRL/IRL13 open reading frames are not required for virus growth in cell culture. The sequence of the TRL13 open reading frame in the low-passage Toledo strain of human cytomegalovirus is quite different from the corresponding region in the AD169 strain. One of multiple changes is a frameshift mutation. As a consequence, strain Toledo encodes a putative TRL13 protein whose C-terminal domain is larger (extending through the TRL14 coding region) and encodes in a reading frame different from that of strain AD169. We speculate that the strain AD169 coding region has drifted during passage in the laboratory. We propose that TRL13 has been truncated in strain AD169 and that the partially overlapping TRL14 open reading frame is not functional. This view is consistent with the presence of both TRL13 and -14 on all mRNAs that we have mapped from this region, an organization that would include the much longer strain Toledo TRL13 open reading frame on the mRNAs.
Tomishima MJ, Enquist LW. In vivo egress of an alphaherpesvirus from axons. J Virol. 2002;76 (16) :8310-7.Abstract
Many alphaherpesviruses establish a latent infection in the peripheral nervous systems of their hosts. This life cycle requires the virus to move long distances in axons toward the neuron's cell body during infection and away from the cell body during reactivation. While the events underlying entry of the virion into neurons during infection are understood in principle, no such consensus exists regarding viral egress from neurons after reactivation. In this study, we challenged two different models of viral egress from neurons by using pseudorabies virus (PRV) infection of the rat retina: does PRV egress solely from axon terminals, or can the virus egress from axon shafts as well as axon terminals? We took advantage of PRV gD mutants that are not infectious as extracellular particles but are capable of spreading by cell-cell contact. We observed that both wild-type virus and a PRV gD null mutant are capable of spreading from axons to closely apposed nonneuronal cells within the rat optic nerve after intravitreal infection. However, infection does not spread from these infected nonneuronal cells. We suggest that viral egress can occur sporadically along the length of infected axons and is not confined solely to axon terminals. Moreover, it is likely that extracellular particles are not involved in nonneuronal cell infections. Taking these together with previous data, we suggest a model of viral egress from neurons that unifies previous apparently contradictory data.
Summanen P. Recent taxonomic changes for anaerobic gram-positive and selected gram-negative organisms. Clin Infect Dis. 1993;16 Suppl 4 :S168-74.Abstract
The strains formerly classified as Streptococcus anginosus or "Streptococcus milleri" have now been recognized as three distinct species, Streptococcus anginosus, Streptococcus constellatus, and Streptococcus intermedius. Streptococcus morbillorum has been transferred into the genus Gemella. Four new species within the genus Peptostreptococcus were recently named. A recent addition to the genus Clostridium is Clostridium argentinense, which includes Clostridium botulinum type G. Two new species of Actinomyces have been introduced: Actinomyces georgiae and Actinomyces gerencseriae. Arachnia propionica was shown to be related to propionibacteria and has been renamed Propionibacterium propionicum. Eubacterium yurii was named to contain "test-tube brush" bacteria found in subgingival plaque. Lactobacillus uli and Lactobacillus rimae are obligately anaerobic lactobacilli found in periodontal pockets. Bilophila is a new genus of gram-negative bacilli. Wolinella recta and Wolinella curva are now included in the genus Campylobacter. The taxonomic position of Mobiluncus, currently assigned to the family Bacteroidaceae, remains uncertain.