Publications by Type: Thesis

Ahmad S. Investigating the Role of SMPD4 in Anterograde Sorting of Pseudorabies Virus in Neurons. Molecular Biology. 2020.Abstract

Pseudorabies virus (PRV) is an alpha herpesvirus of swine causing disease characterized by encephalomyelitis and inflammation of the respiratory tract. Efforts to studying PRV have led to discoveries uncovering the molecular mechanisms of herpesvirus pathogenesis and neuronal invasion. Consequently, PRV can be a powerful tool in exploring the neuroanatomical pathways of the brain, because infection spreads among synaptically connected neurons. PRV infection requires the viral Us9 protein to spread form pre-synaptically connected neurons to post-synaptically connected neurons. Us9 is a membrane protein found in lipid rafts and is required for sorting newly assembled virions into axons. Various studies suggest that sphingolipids, significant lipid raft constituents, and sphingomyelinases are necessary for viral intracellular transport. The host protein, SMPD4, is a neutral sphingomyelinase involved in ceramide production and biogenesis of exosomes. Previous work in this laboratory demonstrated that SMPD4 protein interacts with Us9. SMPD4 was the second most enriched host protein associated with GFP-Us9 in an immunoprecipitation and mass-spectrometry experiment. In this thesis, we studied SMPD4 through a series of immunofluorescence and western blot experiments. We used the neuronal cell line (Neuro2a) to study the function of SMPD4 and Us9 interaction. These cells can be differentiated into central nervous system (CNS) neurons. However, PRV infection in these cells was variable and the production of infection virus was low. Various environmental conditions were explored to optimize PRV infection of these cells. While natural mouse laminin coating induces the growth of two or more neurite-like projections per cell by 25%, it did not affect the percent of Neuro2a cells infected. The infectious cycle is not synchronous in Neuro2a cells, which may reflect the lack of synchrony of the cell cycle. In addition, infected cells appeared to round up and die, perhaps indicating an apoptotic response to infection. We detected SMPD4 using immunofluorescence microscopy. The protein appeared as individual puncta throughout the cell in uninfected and infected Neuro2a cells. Furthermore, we could observe colocalization between SMPD4 and viral capsids.

Salimbangon A. Investigating the Regulation of the Innate Immune Responses of PRV-Becker and PRV-Bartha Infections in Neurons In Vitro. Molecular Biology. 2020.Abstract

Pseudorabies virus (PRV) is a swine alphaherpesvirus that is closely related to human herpes simplex virus type 1 (HSV-1) and varicella-zoster virus (VZV). The virus also infects a large number of mammals, including sheep, dogs, and rodents. In these non-natural hosts, infection with a virulent wild-type PRV strain (PRV- Becker), causes a neuropathic itch also known as “mad itch” which is followed by an uncontrolled systemic inflammation, leading to sudden death. Using a mouse footpad inoculation model, it was demonstrated that PRV-Becker infection induces the increased production of two proinflammatory cytokines (IL-6 and G-CSF) in pe- ripheral nervous system (PNS) and central nervous system (CNS) tissues, very early after infection. This suggests that PRV infection activates the nervous system and initiates an early neuroinflammatory response that later escalates into a systemic in- flammation. However, the molecular mechanisms used by PRV to regulate the innate immune responses specifically upon infection of neuronal cells remain unclear. This study aimed to establish an in vitro system that could mimic PRV infection in vivo and help to dissect the mechanisms that initiate this neuroinflammatory response. Using murine neuroblastomas (neuro-2A cells), we showed that all confluent neuro- 2A cells are infected with PRV by 8 hours post inoculation (hpi). We found that the virulent PRV-Becker strain and the attenuated vaccine strain, PRV-Bartha, replicate to the same extent in confluent neuro-2A cells up to 10^5 plaque forming units (PFU) at 48 hpi. Compared to non-infected neuro-2A cells, PRV-Becker- and PRV-Bartha- infected cells show significant decreased IL-6 levels at 48 hpi. Moreover, PRV-Becker- and PRV-Bartha-infected neuro-2A cells do not produce significant amounts of G- CSF and type 1 interferon (IFN). Overall, we demonstrated that PRV infection of neuro-2A cells does not recapitulate the inflammatory cytokine production found after PRV infection of mice.

Hsu Z-S. A comparative analysis of HSV-1 and PRV transcriptional activator VP16 in primary neurons. Molecular Biology. 2019.Abstract

The VP16 tegument protein of herpes simplex virus 1 (HSV-1) has been shown to have a role in reactivation of latent infection in the peripheral nervous system (PNS), but while it appears to activate viral gene transcription, it is unknown if this protein can also activate neuronal genes. Less research has been done on the VP16 homolog in the related pseudorabies virus (PRV) and any role it may play in activating neuronal genes. By using adeno-associated virus (AAV) vectors that encode either HSV VP16 or PRV VP16 (aka UL48), cultured superior cervical ganglia rat neurons (SCGs) can be transduced and made to express VP16 or UL48 independent of virus infection. Gene expression in SCGs transduced in this manner was compared using RNA-seq and RT-qPCR and it was found that the neuronal gene Jun was enriched in the presence of HSV VP16, Adcyap1 with PRV UL48, and Crem in the presence of both proteins. Subsequent analysis of subcellular localization in AAV vector-transduced and virus-infected SCGs showed that, while localization of Adcyap1 and Jun did not change with or without the presence of the VP16 proteins, Crem was nuclear only in the presence of PRV UL48. It appears that PRV UL48 may be increasing expression of Crem and Adcyap1 but only recruiting Crem to the nucleus for activation of viral gene expression. While the presence of HSV VP16 is connected to enrichment of Crem, that same nuclear localization is not observed, suggesting it may not play the same role in HSV-1 as in PRV.
Vernejoul J. Characterization of the Neuroinflammatory Response During Pseudorabies Virus Infection in Mice. Neuroscience. 2019.Abstract

When infected with the wild-type virus strain of pseudorabies virus (PRV-Becker), mice will exhibit symptoms including a “mad-itch” response, tremors, and weight loss, and eventually die. Interestingly, an attenuated version of PRV (PRV-Bartha) is significantly less virulent. PRV-Bartha elicits less severe symptoms in infected mice and these mice will, on average, live significantly longer. Past rtPCR analysis has shown that these viruses do not replicate beyond the PNS and CNS, yet peripheral organs express elevated levels of pro-inflammatory cytokines and chemokines at the time of PRV-Becker infected animal death. These results have suggested that it is the inflammatory response to PRV as opposed to viral replication in tissues that kills the mice. Currently, little is known about the exact causes of death and of pruritus in the mice when infected with PRV-Becker. It is also unknown why PRV-Bartha is so much less virulent than PRV-Becker. This report characterizes the virulence of the PRV-Becker strain in a murine model by comparing it to the attenuated, PRV-Bartha strain. rtPCR and ELISA analysis conducted in this study shows that PRV-Becker elevates G-CSF levels at early timepoints post infection and a systemic immune response consisting of both G-CSF and IL-6 across organs shortly follows this initial peak of G-CSF. Furthermore, we found that by effectively knocking out type-I IFN signaling in a mouse model, PRV-Bartha induces an immune response more similar to the PRVBecker strain. With IFN signaling absent, PRV-Bartha induces higher levels of G-CSF and viral load in the Dorsal Route Ganglion (DRG). These results promote a model of infection whereby, PRV-Becker is more virulent due to its ability to successfully evade the IFN response. Overall, this study furthers our understanding on the pathogenesis and virulence of PRV and provides potential molecular targets for future therapies.

Song B. A Comparative Analysis of Amyloid Precursor Protein Processing in Neuronal Cells Infected with HSV-1 and PRV. Neuroscience. 2019.Abstract

Recent epidemiological and experimental findings suggest that HSV-1, in particular, might contribute to the pathogenesis of Alzheimer’s disease (AD), although no causal relation has been demonstrated yet. This neurodegenerative disorder is often associated with intracellular and extracellular accumulation of amyloid beta (Aβ), a hallmark of amyloid plaque deposits, from the processing of amyloid precursor protein (APP). However, little if any research has been done on examining the effects of PRV infection on the processing of APP. The aim of this study was to identify and compare the effects of HSV-1 and PRV infection on APP processing in mouse neuroblastoma cells. We found that HSV-1 infection yielded an important C-terminal fragment cleavage product of APP and also induced elevated levels of Aβ intracellularly and extracellularly. Immunofluorescence imaging also showed that HSV induced aggregation of APP to the endomembrane system. These phenomena were not seen with PRV infection. We hypothesize that HSV-1 ICP34.5 may be critical for disruption of this processing pathway and that repeated HSV-1 reactivation could be a risk factor for AD.
Albrecht K. Pseudorabies Virus Replication and Spread in the Mouse Footpad Inoculation Model. Molecular Biology. 2018.Abstract

Infection by alphaherpesviruses such as varicella-zoster virus (VZV) is a significant cause of neuropathic itch. VZV produces varicella or chickenpox upon primary infection, remains in a latent state in ganglia, and produces herpes zoster (HZ) or shingles if reactivated. Neuron damage from productive infection may lead to lasting pain or itch. In spite of various treatments and several vaccines, postherpetic itch continues to affect HZ patients. PHI is less studied than postherpetic neuralgia, and its mechanisms have not been defined. Pseudorabies virus (PRV), a swine alphaherpesvirus closely related to VZV, produces similar intense itching in non-natural hosts such as mice. Attenuated PRV-Bartha does not induce itch, so comparing PRV-Bartha and wildtype PRV-Becker will reveal the mechanisms of virus-induced neuropathic itch. To establish the mouse hind footpad inoculation model, we must first track how infection spreads and then identify inflammatory mediators responsible for pathology. This study focuses on the first objective; we aim to characterize viral spread and replication throughout the course of PRV-Becker infection. First, we detected PRV antigen in foot, bladder, kidney, and heart with immunohistochemistry at 72 hours post-inoculation (hpi). Using q-PCR to verify these results, we found PRV DNA in foot, dorsal root ganglia (DRG), spinal cord, and brain for both PRV-Becker (82 hpi) and PRV-Bartha (240 hpi). Finally, q-PCR at 24 and 48 hpi showed PRV-Becker replicates in the foot by 24 hpi. Infection spreads to the DRG and spinal cord between 48 and 82 hpi, correlating with development of itch around 66 hpi. Comparing these results with those from PRV-Bartha infection (ongoing experiments) and correlating with cytokine production will increase understanding of virus-induced neuropathic itch.

Vogeley G. Dissecting the Anterograde Spread Defect of HSV-1 Strain McIntyre in Neurons. Neuroscience. 2018.Abstract

Viruses are increasingly being used as tracers of neural connections in the nervous systems of animal models. The herpes simplex virus 1 (HSV-1) is a prime target for ongoing research, due to its broad host range, which includes rodents, higher primates and humans. Wild-type HSV-1 can spread in both directions (retrogradely and anterogradely) in a polysynaptic circuit. A strain of HSV-1 known as McIntyre exhibits a unique abrogation of anterograde spread in infected neurons and can only spread in a retrograde fashion. This thesis sought to determine the mechanisms behind this defect, with a specific focus on viral proteins gI and US9. The addition of wild-type gI and US9 proteins led to a significant but incomplete rescue of anterograde spread. Further work must be done to elucidate the effects of glycosylation, the mechanisms of motor recruitment, and the repair of other mutated genes on the spreading abilities of HSV-1 McIntyre. Given that HSV-1 is a chronic human pathogen with very high incidence, understanding its viral spread will be critical to improving antiviral treatments.

Payen J. Investigating the role of UL47 gene in Pseudorabies Virus in vitro infection. Molecular Biology. 2017.Abstract
Pseudorabies virus (PRV) is an alpha herpesvirus native to pigs that infects the peripheral nervous system and establishes a latent infection, allowing for future reactivation. Infection by PRV in non-native species such as dog and cattle leads to the rabies-like symptoms of Aujezsky’s disease including: severe phantom itch, genital infection and death. Unlike with bovine herpesvirus (BoHV-1), where the UL47 gene product has been shown to down-regulate host cells’ anti-viral defenses, the mechanism by which PRV modulates host defense is not yet fully understood. In this study, we characterize the role of the PRV UL47 gene product, VP13/14, in epithelial cell infection, and we propose VP13/14 may play a role in reducing the IFN-β response in host cells. At late time points of in vitro PRV infection, phosphorylation of STAT1 is reduced. STAT1 is a transcription factor that triggers host defenses in response to IFN-β. While UL47 plays a role in infection and appears to effect phosphorylation of STAT1, its mechanism remains to be explained. A UL47 null virus and a C-terminal UL47-GFP fusion virus were reconstituted from bacterial artificial chromosomes and characterized. While showing otherwise normal protein expression of several major viral proteins, both viruses exhibited a small plaque phenotype. Furthermore, the UL47 null virus demonstrated a log deficiency in titer at 24 hours post infection.
Jean J. Construction and Characterization of a novel PRV recombinant expressing a C Terminal VP26-mCherry fusion protein. Molecular Biology. 2016.Abstract
Fluorescent protein-based methodologies have revolutionized the field of virology, allowing us to visualize the location and activity of pseudorabies virus (PRV) particles in vitro (G. A. Smith & Enquist, 2002). Several limitations, including brightness and stability have limited such tools for microscopy analysis (Hogue et al., 2015). While N terminal fusions of fluorescent proteins to capsid proteins like VP26 (pUL35) have been well utilized, little is known about the effectiveness of C Terminal fusions. Included in this study are the first recombinant PRV strains expressing C terminal VP26-mCherry. The properties of this reporter virus, PRV 1028, were compared to previously constructed N terminal recombinants containing either mRFP or mCherry. PRV 1028 had similar single-step replication kinetics compared to wild-type PRV Becker and traditional FP– VP26 strains. Despite slightly reduced plaque diameter, we find that our newly synthesized PRV 1028 incorporates more fluorescent VP26 fusion proteins in progeny virus particles. As a result, PRV 1028 capsids have a higher fluorescent intensity in comparison to their N terminal counterparts. Based on these findings, our novel PRV recombinant will allow for optimized fluorescent-based methodologies.
Yaffe Z. Towards An Understanding Of PRV Transport And Spread Using Dual-colored Virions. Molecular Biology. 2015.Abstract
Pseudorabies virus (PRV) is an alphaherpesvirus that is closely related to human herpes simplex virus. Although PRV infects many mammals, no human cases of PRV infection have been reported. PRV is therefore an ideal candidate for laboratory research of alphaherpesvirus infections. The mechanisms involved in the PRV infectious cycle have not been completely resolved. We performed two sets of in vitro experiments to further elucidate the dynamics of PRV axonal transport and spread in epithelial cells. We modified a single-color system for tracking of neuronal PRV infections to distinguish entering from egressing virus particles. We allowed the capsid-tagged strain PRV 180 to replicate on PK15 cells expressing mNeonGreen-VP26, yielding PRV 180G. As PRV 180G has a non-genetically encoded, secondary fluorescent tag, it can be distinguished from its red progeny. We see up to 92% of exocytosed particles incorporate mNG-VP26 at 14 hpi. However, integration of mNG-VP26 into PRV affects axonal transport efficiency. Dual-colored PRV shows promise as a tool for detailed analysis of PRV transport and for investigation of the fate of virion components during replication. In accordance with prior research, we find PRV 151 (diffusible GFP) plaques spread through PK15 monolayers 36% faster than the ~4.5 hour replication cycle would permit. To gain insight into routes of enhanced spread, we studied PRV transport through PK15 cell monolayers. We detected PRV 151 in the medium below non-permeable PK15 cells layers as early as one hour post-infection. These results suggest that productive PRV transmission through epithelial cells can occur through a non-replicative and active mechanism, which greatly impacts our understanding of PRV infection in vivo.
Huang T. Aedes aegypti and Dengue Transmission. Molecular Biology. 2015.Abstract

The geographical distribution of the four dengue viruses and their primary vector, Aedes aegypti (A. aegypti), has recently undergone a rapid expansion, establishing endemicity in most of the tropics and subtropics. This places a third of the world’s population at risk of dengue infection, making dengue the most important mosquito-borne viral disease today. Since the 1970’s, the incidence of dengue infections has increased drastically and urban dengue hemorrhagic fever (DHF) has emerged as an urgent global health problem. DHF epidemic activity is currently of particular concern in Southeast Asia, where it is one of the leading causes of childhood mortality. This thesis seeks to understand the mechanisms underlying the re-emergence and emergence of dengue and DHF epidemic activity, respectively, and how they direct available and future preventative measures. The historical and epidemiological record suggests that World War II facilitated the worldwide spread of A. aegypti and the four dengue viruses, establishing hyperendemicity in Southeast Asia. This, along with the particular customs and behaviors of the region, presumably stimulated the emergence of DHF epidemic activity in the 1950’s. Until a safe and effective tetravalent vaccine becomes available, the prevention of dengue rests solely on vector control programs. A more thorough understanding of the transmission cycles between A. aegypti dengue viruses and human hots is needed for the development of more successful vector control programs.

Lee J. Quantifying and Analyzing the Puzzle of Genetic Mosaic Segments in Mycobacteriophages. Computer Science. 2015.Abstract

Bacteriophage genomes are known to have a "mosaic" structure, which means that a given genome may contain regions highly similar to some phages interspersed with regions highly similar to other phages mixed with unique regions. This paper describes an algorithm to identify individual mosaic segments and quantifies the extent of phage mosaicism in phages that infect a common host, the bacterium M. smegmatis. The results verify previous work referencing the high amounts of phage mosaicism – on average, about half of any given genome of the over 800 in PhagesDB is made up of short mosaic segments that are shared with at least one other phage. Because novel phages are still being discovered frequently, the amount of mosaicism may be even higher. This paper also examines trends in the distribution of mosaic segments based on cluster, date found, and geography, and explores methods to build tree-based phylogenies based on the number of horizontal transfer events among phages.

Jeon DT. A Collusion in Coinfection: Evaluating the Synergistic Mechanisms Between Human Immunodeficiency Virus and Mycobacterium tuberculosis. Molecular Biology. 2014.Abstract
The convergence of the Human Immunodeficiency Virus-1 (HIV) and Mycobacterium tuberculosis (Mtb) pandemics affects 1.1 million people worldwide. The synergistic interplay between these two pathogens may explain the enhanced risk of TB in HIV infection and the increased progression of HIV pathogenesis in the co-presence of Mtb infection. However, the molecular pathogenesis of coinfection that increasingly impairs immune function has yet to be fully elucidated. Most studies have focused on HIV-mediated depletion of CD4+ T cells as the major mechanism that drives bidirectional effects.In contrast, this thesis evaluates possible mechanisms of how HIV and Mtb may cooperate to modulate the innate immune system, specifically alveolar macrophages. Macrophages are one of the first cells infected by Mtb or HIV, and infected macrophages contribute significantly to the pathogenesis of each infection. The hypotheses assessed here thus focus on how coinfected macrophages contribute to the exacerbated symptoms and mortality rate in HIV/Mtb coinfected individuals. The risk for tuberculosis (TB) develops early on in HIV infection at relatively normal CD4+ T cell counts but increases as immunosuppression worsens. By assessing the available literature on coinfection, I argue that the pathogens impair macrophage functions, including interactions with CD4+ T cells, which ultimately contributes to the enhanced morbidity and mortality. Dysregulation of macrophage functions contribute to the synergistic effects of coinfection and merits further study. Future research should focus on using models like lung organoids, animal models, and new imaging technologies to clarify the role of innate immunity in coinfection. Developing molecular epidemiological tools will also be critical to understanding transmission of Mtb in HIV+ individuals. These techniques will help elucidate host-pathogen dynamics and innate immunity in response to a broad range of infectious pathogens. Furthermore, as the risk of TB increases early in HIV infection, prevention and early treatment are critical. A comparative case study of coinfected populations in South Africa and the U.S. is used to contrast policies for expanding treatment and prevention coverage in areas of high or low TB/HIV burden respectively.
Al-Jabban J. Investigation of the Role of Dynein Intermediate Chain (IC) Isoforms in Pseudorabies Virus Post-­Entry Retrograde Transport. Molecular Biology. 2014.Abstract
Alpha herpesviruses are a family of neurotropic DNA viruses that replicate quickly in epithelial cells, and establish a latent phase in sensory ganglia neurons. Herpes simplex virus 1 (HSV‐1) and pseudorabies virus (PRV) are typical members of this family. A trademark of alpha herpesviruses is the bidirectional (anterograde and retrograde) transport capability of virions within and in between neurons. The machinery and mechanisms underlying retrograde transport of viral particles after entry remain nebulous, particularly in neurons. We know that viral nucleocapsids remain attached to the inner tegument proteins such as UL36, and interaction of this protein with motor protein dynein is required for retrograde transport of viral capsids. Moreover, PRV infection induces new protein synthesis in axons, which results in the local translation of several cytoskeletal, trafficking, and signaling proteins including a dynein regulator. Here I aim to examine the role of the dynein intermediate chain (IC) isoforms IC‐1B, neuronal, and IC‐2C, ubiquitous, in retrograde transport of PRV particles and mitochondria. I will demonstrate the functionality of our IC­‐GFP isoforms using a variety of in vitro studies (Chapter 2). Then, I will examine the interaction and co­‐transport of PRV capsids with the IC­‐GFP isoforms using in vitro analysis and live cell imaging in primary neurons and RAT‐2 cells. I will also study the effects of axon damage in PRV co­‐transport with the IC‐ GFP isoforms (Chapter 3). Lastly, I will investigate the role of the IC‐GFP isoforms in co­‐transport of mitochondria in SCG neurons and the effects of axonal damage on mitochondria transport and motility (Chapter 4). Through these experiments, I illustrate the pivotal role of different IC isoforms in retrograde transport of PRV virions and organelles in neurons.
Charles D. Sex Differences in APOE ԑ4-Associated Risk for Primary Progressive Aphasia. Molecular Biology. 2014.Abstract
Primary progressive aphasia (PPA), a language neurodegenerative disorder caused by atrophy of the frontal and temporal lobes, has three variants: The nonfluent/agrammatic variant (naPPA) is marked by slowed speech and difficulty with complex grammar; the semantic variant (svPPA), by impaired naming and single-word comprehension; and the logopenic variant (lvPPA), by difficulty with sentence repetition. Alzheimer’s disease pathology (AD-P), found in approximately one-third of PPA cases, is left- lateralized in PPA compared to a more symmetric distribution in Alzheimer’s disease (AD). Because the APOE ε4 allele is a major risk factor for AD, I hypothesized that ε4 carriers were at increased risk for lvPPA, which is often associated with AD-P. Odds ratios analyses on genetic data from the National Alzheimer’s Coordinating Cen- ter and the University of Pennsylvania Frontotemporal Degeneration Center showed that the ε4 allele increased risk for svPPA and lvPPA, but not naPPA. Additionally, this research extends previous work indicating an increased risk of PPA for women with the ε2/ε4 genotype. Analyses stratified by sex revealed that female, but not male, ε4 carriers had a statistically significant increase in risk for all PPA variants. These findings suggest a sex difference in risk that may be related to estrogen-APOE interactions in women.
Wu BW. Characterization of the Replication Incompetent IE180 Null Pseudorabies Virus. Molecular Biology. 2014.Abstract
Pseudorabies virus (PRV) is an economically significant porcine alphaherpesvirus that traverses the nervous system via synaptically connected neurons, causing ataxia, pruritus, and seizures before host death. The master transcriptional regulator gene IE180 was removed from the PRV genome, thus producing a replication-incompetent strain. Epithelial cells and neurons infected with IE180 null PRV survived significantly longer than those infected with wild-type PRV. Survival times exceeded a month post-infection and were dependent on the multiplicity of infection. In neurons, viral entry at the axons dramatically increased survival time relative to viral entry at the cell body. IE180 null PRV could only spread in epithelial cells when trans- complemented with the IE180 gene. IE180 null PRV was transported from the axon terminal to the cell body and could not spread to neighboring neurons. The NF-κB immune response pathway was activated only in cells that were heavily infected by IE180 null PRV. Epithelial cells, neurons, and fibroblasts infected with IE180 null PRV permitted entry of a secondary virus and therefore do not exhibit super-infection exclusion. IE180 trans-complementation restored super-infection exclusion. Fluorescently-labeled IE180 null PRV strains can serve as efficient long range and long-term neuronal tracers controlled by IE180 trans-complementation.
Hu J-R. Pseudorabies Virus Entry and Egress Visualized by Fluorescence Microscopy and a pH-Sensitive Reporter. Molecular Biology. 2013.Abstract
Conflicting reports have emerged as to whether entry of alpha-herpesvirus particles occurs by receptor-mediated membrane fusion or pH-dependent endocytosis, and whether the method of entry differs by cell type. At the other end of the virus replication cycle, it is unclear whether egress of alpha-herpesvirus particles occurs at terminal synapses, varicosities, or inter-varicosity sites of neuronal axons. We aimed to explore the pathway of virus entry and the sites of virus egress through live cell imaging of virus particles tagged with a pH-sensitive fluorescent protein, pHluorin. Here, we characterized PRV483, a novel recombinant of PRV Becker encoding both a fusion between capsid protein VP26 and monomeric red fluorescent protein, as well as a fusion between envelope glycoprotein M and pHluorin. These studies mark the first time, to our knowledge, that a pH-sensitive fluorescent protein has been used to investigate virus particle egress. We imaged rat superior cervical ganglion neurons infected with PRV483, starting around 10 minutes post infection to observe virus entry, and 10 hours post infection to observe virus egress. In entry experiments, doubly-labeled puncta were observed, albeit rarely, to undergo directional movement, consistent with microtubule-based transport, after attachment to the axon, suggesting that endocytosis may be a mode of entry used by some portion of virus particles. In egress experiments, red puncta were observed to accumulate at varicosities and become dual-labeled, suggesting that virus egress occurs at varicosities. The findings from this exploratory study question the existing consensus on herpesvirus entry pathway in neurons and provide clues to herpesvirus sites of egress from neurons.
Kratchmarov R. Molecular mechanisms of Us9-mediated anterograde transport of alphaherpes viruses. Molecular Biology. 2013.Abstract
Alphaherpes viruses infect the nervous system of their hosts and undergo long-distance transport in neuronal axons during different steps of the life cycle. Active viral replication at the neuronal soma produces progeny virions, which must undergo anterograde transport down the axon to facilitate anterograde spread within the host. Throughout this work, the molecular mechanisms underlying anterograde transport are explored using pseudorabies virus infection as a model system. Several functional domains in the viral protein Us9, which is essential for anterograde transport, were characterized through a convergence of methodologies including live-cell imaging of fluorescent viruses as well as in vitro spread and biochemical assays. In Chapter 1, GFP-Us9 fusion proteins are employed to characterize the role of a dityrosine motif in the protein in anterograde transport of virion structural components. The dityrosine motif was required for anterograde neuron-to-cell spread in vitro as well as for axonal targeting of virion structural components. GFP-Us9 fusion proteins are then employed further in Chapter 2 to characterize the Us9 diserine motif. Interestingly, unlike the dityrosine motif, mutagenesis of the diserine motif resulted in only a modest defect in spread and was found to modulate the efficiency of anterograde transport. In Chapter 3, the requirement of Us9 for transport of viral membrane proteins is assessed for particles that do not constitute mature virions. Interestingly, we established that the viral glycoprotein M is capable of undergoing anterograde transport independently of Us9. A summation of our current understanding of the anterograde transport mechanism, known as the Married Model, is then presented in Chapter 4 through a critical analysis of experimental techniques in published works. Finally, Appendix A contains a preliminary investigation of glycoprotein E functionality with respect to Us9-Kif1A interactions and anterograde transport of virions. Together, these results expand our understanding of Us9 functionality and biochemical properties.
Brooks JNK. A Set of Herpes Simplex Virus type 1 Recombinants for Use in Neural Circuit Tracing. Molecular Biology. 2013.Abstract
Two powerful tools for advanced visualization of neurons and neural circuits have recently been developed. The first uses neuroinvasive α Herpesviruses as self-amplifying markers that spread only between snaptically connected neurons. These viruses can be used to selectively label a population of neurons based on genetic and/or connectivity criteria. Further, several known viral strains are defective for either anterograde or retrograde transport form the soma, offering neuroscientists the ability to answer direction-specific questions. The Herpes Simplex Virus type 1 (HSV-1) strain H129 is the only α Herpesvirus which has been observed to move almost exclusively in the anterograde-only direction while HSV-1 MacIntyre moves only in the retrograde direction from the site of infection. The second tool is the Brainbow 1.0L cassette, which expresses the red fluorescent protein tdTomato unless it undergoes Cre-meditated recombination to express either Cyan or Yellow fluorescent protein. The recombination event selectively labels Cre-expressing neurons, and generates a diversity of color that distinguishes individual cells within the target population. Recently neurovirologists have modified α herpesviruses to express Brainbow. These Brainbow viruses address the weaknesses in each of the above tools: 1. As informational circuits are not genetically defined, transgenic Brainbow animals could not be used to explore neural circuits; 2. Viral infection with only one labeling protein cannot distinguish between individual neurons in a circuit, particularly at the synaptic level. Here, I have constructed Brainbow- and Cre-encoding HSV-1 unidirectional recombinants.
Smith AC. Involvement of Kinesin-1 in Pseudorabies Virus Transport. Molecular Biology. 2009.