Publications

2014
Casadevall, Arturo, and Thomas Shenk. “mBio addresses the pause in gain-of-function (GOF) experiments involving pathogens with pandemic potential (PPP).”. MBio 56 (2014). Web.
Casadevall, Arturo, et al.On the need for a national board to assess dual use research of concern.”. J Virol 88.12 (2014): , 88, 12, 6535-7. Web.
O'Connor, Christine M, et al.Quantitative proteomic discovery of dynamic epigenome changes that control human cytomegalovirus (HCMV) infection.”. Mol Cell Proteomics 13.9 (2014): , 13, 9, 2399-410. Web.Abstract
This work represents the first comprehensive quantitative analysis of global histone post-translational modifications (PTMs) from a virus infection, namely human cytomegalovirus (HCMV) infection. We used a nanoLC-MS/MS platform to identify and quantify the dynamic histone H3 and H4 PTMs expressed during HCMV replication in primary fibroblasts. Specifically, we examined the changes in histone PTMs over a 96 h time course to sample the immediate early (IE), early (E), and late (L) stages of viral infection. Several changes in histone H3 and H4 PTMs were observed, including a marked increase in H3K79me2 and H3K27me3K36me2, and a decrease in H4K16ac, highlighting likely epigenetic strategies of transcriptional activation and silencing during HCMV lytic infection. Heavy methyl-SILAC (hm-SILAC) was used to further confirm the histone methylation flux (especially for H3K79) during HCMV infection. We evaluated DOT1L (the H3K79 methyltransferase) mRNA levels in mock and HCMV-infected cells over a 96 h time course, and observed a significant increase in this methyltransferase as early as 24 hpi showing that viral infection up-regulates DOT1L expression, which drives H3K79me2. We then used shRNA to create a DOT1L knockdown cell population, and found that HCMV infection of the knockdown cells resulted in a 10-fold growth defect when compared with infected control cells not subjected to knockdown. This work documents multiple histone PTMs that occur in response to HCMV infection of fibroblasts, and provides a framework for evaluation of the role of epigenetic modifications in the virus-host interaction.
Dermody, Terence S, Rozanne M Sandri-Goldin, and Thomas Shenk. “Sequence changes associated with respiratory transmission of H7N1 influenza virus in mammals.”. J Virol 88.12 (2014): , 88, 12, 6533-4. Web.
Hwang, Jesse, et al.Estrogen-related receptor α is required for efficient human cytomegalovirus replication.”. Proc Natl Acad Sci U S A 111.52 (2014): , 111, 52, E5706-15. Web.Abstract
An shRNA-mediated screen of the 48 human nuclear receptor genes identified multiple candidates likely to influence the production of human cytomegalovirus in cultured human fibroblasts, including the estrogen-related receptor α (ERRα), an orphan nuclear receptor. The 50-kDa receptor and a 76-kDa variant were induced posttranscriptionally following infection. Genetic and pharmacological suppression of the receptor reduced viral RNA, protein, and DNA accumulation, as well as the yield of infectious progeny. In addition, RNAs encoding multiple metabolic enzymes, including enzymes sponsoring glycolysis (enolase 1, triosephosphate isomerase 1, and hexokinase 2), were reduced when the function of ERRα was inhibited in infected cells. Consistent with the effect on RNAs, a substantial number of metabolites, which are normally induced by infection, were either not increased or were increased to a reduced extent in the absence of normal ERRα activity. We conclude that ERRα is needed for the efficient production of cytomegalovirus progeny, and we propose that the nuclear receptor contributes importantly to the induction of a metabolic environment that supports optimal cytomegalovirus replication.
Mathias, Rommel A, et al.Sirtuin 4 is a lipoamidase regulating pyruvate dehydrogenase complex activity.”. Cell 159.7 (2014): , 159, 7, 1615-25. Web.Abstract
Sirtuins (SIRTs) are critical enzymes that govern genome regulation, metabolism, and aging. Despite conserved deacetylase domains, mitochondrial SIRT4 and SIRT5 have little to no deacetylase activity, and a robust catalytic activity for SIRT4 has been elusive. Here, we establish SIRT4 as a cellular lipoamidase that regulates the pyruvate dehydrogenase complex (PDH). Importantly, SIRT4 catalytic efficiency for lipoyl- and biotinyl-lysine modifications is superior to its deacetylation activity. PDH, which converts pyruvate to acetyl-CoA, has been known to be primarily regulated by phosphorylation of its E1 component. We determine that SIRT4 enzymatically hydrolyzes the lipoamide cofactors from the E2 component dihydrolipoyllysine acetyltransferase (DLAT), diminishing PDH activity. We demonstrate SIRT4-mediated regulation of DLAT lipoyl levels and PDH activity in cells and in vivo, in mouse liver. Furthermore, metabolic flux switching via glutamine stimulation induces SIRT4 lipoamidase activity to inhibit PDH, highlighting SIRT4 as a guardian of cellular metabolism.
Koyuncu, Emre, et al.Sirtuins are evolutionarily conserved viral restriction factors.”. MBio 56 (2014). Web.Abstract
UNLABELLED: The seven human sirtuins are a family of ubiquitously expressed and evolutionarily conserved NAD(+)-dependent deacylases/mono-ADP ribosyltransferases that regulate numerous cellular and organismal functions, including metabolism, cell cycle, and longevity. Here, we report the discovery that all seven sirtuins have broad-range antiviral properties. We demonstrate that small interfering RNA (siRNA)-mediated knockdown of individual sirtuins and drug-mediated inhibition of sirtuin enzymatic activity increase the production of virus progeny in infected human cells. This impact on virus growth is observed for both DNA and RNA viruses. Importantly, sirtuin-activating drugs inhibit the replication of diverse viruses, as we demonstrate for human cytomegalovirus, a slowly replicating DNA virus, and influenza A (H1N1) virus, an RNA virus that multiplies rapidly. Furthermore, sirtuin defense functions are evolutionarily conserved, since CobB, the sirtuin homologue in Escherichia coli, protects against bacteriophages. Altogether, our findings establish sirtuins as broad-spectrum and evolutionarily conserved components of the immune defense system, providing a framework for elucidating a new set of host cell defense mechanisms and developing sirtuin modulators with antiviral activity. IMPORTANCE: We live in a sea of viruses, some of which are human pathogens. These pathogenic viruses exhibit numerous differences: DNA or RNA genomes, enveloped or naked virions, nuclear or cytoplasmic replication, diverse disease symptoms, etc. Most antiviral drugs target specific viral proteins. Consequently, they often work for only one virus, and their efficacy can be compromised by the rapid evolution of resistant variants. There is a need for the identification of host proteins with broad-spectrum antiviral functions, which provide effective targets for therapeutic treatments that limit the evolution of viral resistance. Here, we report that sirtuins present such an opportunity for the development of broad-spectrum antiviral treatments, since our findings highlight these enzymes as ancient defense factors that protect against a variety of viral pathogens.
2013
Grady, Sarah L, et al.Argininosuccinate synthetase 1 depletion produces a metabolic state conducive to herpes simplex virus 1 infection.”. Proc Natl Acad Sci U S A 110.51 (2013): , 110, 51, E5006-15. Web.Abstract
Herpes simplex virus 1 (HSV-1) infection triggers specific metabolic changes in its host cell. To explore the interactions between cellular metabolism and HSV-1 infection, we performed an siRNA screen of cellular metabolic genes, measuring their effect on viral replication. The screen identified multiple enzymes predicted to influence HSV-1 replication, including argininosuccinate synthetase 1 (AS1), which consumes aspartate as part of de novo arginine synthesis. Knockdown of AS1 robustly enhanced viral genome replication and the production of infectious virus. Using high-resolution liquid chromatography-mass spectrometry, we found that the metabolic phenotype induced by knockdown of AS1 in human fibroblasts mimicked multiple aspects of the metabolic program observed during HSV-1 infection, including an increase in multiple nucleotides and their precursors. Together with the observation that AS1 protein and mRNA levels decrease during wild-type infection, this work suggests that reduced AS1 activity is partially responsible for the metabolic program induced by infection.
Koyuncu, Emre, et al.Saturated very long chain fatty acids are required for the production of infectious human cytomegalovirus progeny.”. PLoS Pathog 95 (2013): , 9, 5, e1003333. Web.Abstract
Human cytomegalovirus hijacks host cell metabolism, increasing the flux of carbon from glucose to malonyl-CoA, the committed precursor to fatty acid synthesis and elongation. Inhibition of acetyl-CoA carboxylase blocks the production of progeny virus. To probe further the role of fatty acid metabolism during infection, we performed an siRNA screen to identify host cell metabolic enzymes needed for the production of infectious cytomegalovirus progeny. The screen predicted that multiple long chain acyl-CoA synthetases and fatty acid elongases are needed during infection, and the levels of RNAs encoding several of these enzymes were upregulated by the virus. Roles for acyl-CoA synthetases and elongases during infection were confirmed by using small molecule antagonists. Consistent with a role for these enzymes, mass spectrometry-based fatty acid analysis with ¹³C-labeling revealed that malonyl-CoA is consumed by elongases to produce very long chain fatty acids, generating an approximately 8-fold increase in C26-C34 fatty acid tails in infected cells. The virion envelope was yet further enriched in C26-C34 saturated fatty acids, and elongase inhibitors caused the production of virions with lower levels of these fatty acids and markedly reduced infectivity. These results reveal a dependence of cytomegalovirus on very long chain fatty acid metabolism.
2012
Grady, Sarah L, et al.Herpes simplex virus 1 infection activates poly(ADP-ribose) polymerase and triggers the degradation of poly(ADP-ribose) glycohydrolase.”. J Virol 86.15 (2012): , 86, 15, 8259-68. Web.Abstract
Herpes simplex virus 1 infection triggers multiple changes in the metabolism of host cells, including a dramatic decrease in the levels of NAD(+). In addition to its role as a cofactor in reduction-oxidation reactions, NAD(+) is required for certain posttranslational modifications. Members of the poly(ADP-ribose) polymerase (PARP) family of enzymes are major consumers of NAD(+), which they utilize to form poly(ADP-ribose) (PAR) chains on protein substrates in response to DNA damage. PAR chains can subsequently be removed by the enzyme poly(ADP-ribose) glycohydrolase (PARG). We report here that the HSV-1 infection-induced drop in NAD(+) levels required viral DNA replication, was associated with an increase in protein poly(ADP-ribosyl)ation (PARylation), and was blocked by pharmacological inhibition of PARP-1/PARP-2 (PARP-1/2). Neither virus yield nor the cellular metabolic reprogramming observed during HSV-1 infection was altered by the rescue or further depletion of NAD(+) levels. Expression of the viral protein ICP0, which possesses E3 ubiquitin ligase activity, was both necessary and sufficient for the degradation of the 111-kDa PARG isoform. This work demonstrates that HSV-1 infection results in changes to NAD(+) metabolism by PARP-1/2 and PARG, and as PAR chain accumulation can induce caspase-independent apoptosis, we speculate that the decrease in PARG levels enhances the auto-PARylation-mediated inhibition of PARP, thereby avoiding premature death of the infected cell.
Terry, Laura J, et al.Human kinome profiling identifies a requirement for AMP-activated protein kinase during human cytomegalovirus infection.”. Proc Natl Acad Sci U S A 109.8 (2012): , 109, 8, 3071-6. Web.Abstract
Human cytomegalovirus (HCMV) modulates numerous cellular signaling pathways. Alterations in signaling are evident from the broad changes in cellular phosphorylation that occur during HCMV infection and from the altered activity of multiple kinases. Here we report a comprehensive RNAi screen, which predicts that 106 cellular kinases influence growth of the virus, most of which were not previously linked to HCMV replication. Multiple elements of the AMP-activated protein kinase (AMPK) pathway scored in the screen. As a regulator of carbon and nucleotide metabolism, AMPK is poised to activate many of the metabolic pathways induced by HCMV infection. An AMPK inhibitor, compound C, blocked a substantial portion of HCMV-induced metabolic changes, inhibited the accumulation of all HCMV proteins tested, and markedly reduced the production of infectious progeny. We propose that HCMV requires AMPK or related activity for viral replication and reprogramming of cellular metabolism.
Gudleski-O'Regan, Nicole, et al.Increased expression of LDL receptor-related protein 1 during human cytomegalovirus infection reduces virion cholesterol and infectivity.”. Cell Host Microbe 12.1 (2012): , 12, 1, 86-96. Web.Abstract
In response to virus infection, cells can alter protein expression to modify cellular functions and limit viral replication. To examine host protein expression during infection with human cytomegalovirus (HCMV), an enveloped DNA virus, we performed a semiquantitative, temporal analysis of the cell surface proteome in infected fibroblasts. We determined that resident low density lipoprotein related receptor 1 (LRP1), a plasma membrane receptor that regulates lipid metabolism, is elevated early after HCMV infection, resulting in decreased intracellular cholesterol. siRNA knockdown or antibody-mediated inhibition of LRP1 increased intracellular cholesterol and concomitantly increased the infectious virus yield. Virions produced under these conditions contained elevated cholesterol, resulting in increased infectivity. Depleting cholesterol from virions reduced their infectivity by blocking fusion of the virion envelope with the cell membrane. Thus, LRP1 restricts HCMV infectivity by controlling the availability of cholesterol for the virion envelope, and increased LRP1 expression is likely a defense response to infection.
O'Connor, Christine M, and Thomas Shenk. “Human cytomegalovirus pUL78 G protein-coupled receptor homologue is required for timely cell entry in epithelial cells but not fibroblasts.”. J Virol 86.21 (2012): , 86, 21, 11425-33. Web.Abstract
Human cytomegalovirus (HCMV) encodes four putative G protein-coupled receptors, including pUL78, whose rodent orthologues are known to be important for replication and spread in their hosts. To investigate the mechanism by which pUL78 contributes to viral replication and pathogenesis, we generated a derivative of the TB40/E clinical isolate of HCMV that is unable to express the receptor. Consistent with previous findings using laboratory strains of the virus, the mutant replicated normally in fibroblasts. Although laboratory strains are restricted to growth in fibroblasts, clinical isolates grow in many cell types, including epithelial and endothelial cells, in which the pUL78-deficient TB40/E derivative exhibited a growth defect. Infection with the mutant virus resulted in a significant decrease in viral RNA and protein expression. Although there was no difference in binding of the virus to the cell, we detected a delay in the entry and subsequent delivery of virion DNA and protein to the nuclei of epithelial cells following infection with the UL78 mutant virus. Taken together, our results demonstrate that pUL78 supports infection at a point after binding but before entry in epithelial cells, a cell type important for in vivo viral replication and spread.
2011
Vastag, Livia, et al.Divergent effects of human cytomegalovirus and herpes simplex virus-1 on cellular metabolism.”. PLoS Pathog 77 (2011): , 7, 7, e1002124. Web.Abstract
Viruses rely on the metabolic network of the host cell to provide energy and macromolecular precursors to fuel viral replication. Here we used mass spectrometry to examine the impact of two related herpesviruses, human cytomegalovirus (HCMV) and herpes simplex virus type-1 (HSV-1), on the metabolism of fibroblast and epithelial host cells. Each virus triggered strong metabolic changes that were conserved across different host cell types. The metabolic effects of the two viruses were, however, largely distinct. HCMV but not HSV-1 increased glycolytic flux. HCMV profoundly increased TCA compound levels and flow of two carbon units required for TCA cycle turning and fatty acid synthesis. HSV-1 increased anapleurotic influx to the TCA cycle through pyruvate carboxylase, feeding pyrimidine biosynthesis. Thus, these two related herpesviruses drive diverse host cells to execute distinct, virus-specific metabolic programs. Current drugs target nucleotide metabolism for treatment of both viruses. Although our results confirm that this is a robust target for HSV-1, therapeutic interventions at other points in metabolism might prove more effective for treatment of HCMV.
O'Connor, Christine M, and Thomas Shenk. “Human cytomegalovirus pUS27 G protein-coupled receptor homologue is required for efficient spread by the extracellular route but not for direct cell-to-cell spread.”. J Virol 85.8 (2011): , 85, 8, 3700-7. Web.Abstract
Human cytomegalovirus (HCMV) encodes multiple G protein-coupled receptor (GPCR) homologues, including pUS27, pUS28, pUL33, and pUL78. To explore the function of pUS27, we constructed pUS27-deficient derivates of two clinical isolates of HCMV. BFX-GFPstopUS27 is a FIX variant with a single base pair change in the US27 open reading frame, generating a stop codon that ablates accumulation of the GPCR homologue, and TB40/E-mCherrydlUS27 lacks the entire US27 coding region. BFX-GFPstopUS27 generated 10-fold less extracellular progeny in fibroblasts, and TB40/E-mCherrydlUS27 exhibited a similar defect in endothelial cells. The pUS27-deficient FIX derivative produced normal quantities of viral DNA and viral proteins tested, and a late virion protein was appropriately localized to the cytoplasmic assembly zone. After infection at a low multiplicity with wild-type FIX virus, neutralizing antibody reduced the accumulation of intracellular viral DNA and intracellular virions, as would be expected if the virus is limited to direct cell-to-cell spread by neutralization of extracellular virus. In contrast, the antibody had little effect on the spread of the BFX-GFPstopUS27 virus. Further, after infection at a low multiplicity, the pUS27-deficient TB40/E virus exhibited a growth defect in endothelial cells, where the clinical isolate normally generates extracellular virus, but the TB40/E derivative exhibited little defect in epithelial cells, where the wild-type virus does not produce extracellular virus. Thus, mutants lacking pUS27 rely primarily on direct cell-to-cell spread, and we conclude that the viral GCPR homologue acts at a late stage of the HCMV replication cycle to support spread of virus by the extracellular route.
Liu, Sean TH, et al.Synaptic vesicle-like lipidome of human cytomegalovirus virions reveals a role for SNARE machinery in virion egress.”. Proc Natl Acad Sci U S A 108.31 (2011): , 108, 31, 12869-74. Web.Abstract
Human cytomegalovirus induces and requires fatty acid synthesis. This suggests an essential role for lipidome remodeling in viral replication. We used mass spectrometry to quantify glycerophospholipids in mock-infected and virus-infected fibroblasts, as well as in virions. Although the lipid composition of mock-infected and virus-infected fibroblasts was similar, virions were markedly different. The virion envelope contained twofold more phosphatidylethanolamines and threefold less phosphatidylserines than the host cell. This indicates that the virus buds from a membrane with a different lipid composition from the host cell as a whole. Compared with published datasets, the virion envelope showed the greatest similarity to the synaptic vesicle lipidome. Synaptosome-associated protein of 25 kDa (SNAP-25) is a component of the complex that mediates exocytosis of synaptic vesicles in neurons; and its homolog, SNAP-23, functions in exocytosis in many other cell types. Infection induced the relocation of SNAP-23 to the cytoplasmic viral assembly zone, and knockdown of SNAP-23 inhibited the production of virus. We propose that cytomegalovirus capsids acquire their envelope by budding into vesicles with a lipid composition similar to that of synaptic vesicles, which subsequently fuse with the plasma membrane to release virions from the cell.
2010
Aoyagi, Mariko, Miguel Gaspar, and Thomas E Shenk. “Human cytomegalovirus UL69 protein facilitates translation by associating with the mRNA cap-binding complex and excluding 4EBP1.”. Proc Natl Acad Sci U S A 107.6 (2010): , 107, 6, 2640-5. Web.Abstract
4EBP1 is phosphorylated by the mTORC1 kinase. When mTORC1 activity is inhibited, hypophosphorylated 4EBP1 binds and sequesters eIF4E, a component of the mRNA cap-binding complex, and blocks translation. As a consequence, mTORC1 activity is needed to maintain active translation. The human cytomegalovirus pUL38 protein preserves mTORC1 activity, keeping most of the E4BP1 in the infected cell in a hyperphosphorylated, inactive state. Here we report that a second viral protein, pUL69, also antagonizes the activity of 4EBP1, but by a separate mechanism. pUL69 interacts directly with eIF4A1, an element of the cap-binding complex, and the poly(A)-binding protein, which binds to the complex. When pUL69 accumulates during infection with wild-type virus, 4EBP1 is excluded from the complex. However, 4EBP1 is present in the cap-binding complex after infection with a pUL69-deficient virus, coincident with reduced accumulation of several late virus-coded proteins. We propose that pUL69 supports translation in human cytomegalovirus-infected cells by excluding hypophosphorylated 4EBP1 from the cap-binding complex.
Moorman, Nathaniel J, and Thomas Shenk. “Rapamycin-resistant mTORC1 kinase activity is required for herpesvirus replication.”. J Virol 84.10 (2010): , 84, 10, 5260-9. Web.Abstract
Human cytomegalovirus (HCMV) infection has been shown to activate the mTORC1 signaling pathway. However, the phosphorylation of mTORC1 targets is differentially sensitive to the mTORC1 inhibitor rapamycin, and the drug inhibits HCMV replication to a modest extent. Using Torin1, a newly developed inhibitor that targets the catalytic site of mTOR kinase, we show that HCMV replication requires both rapamycin-sensitive and rapamycin-resistant mTOR activity. The treatment of infected cells with Torin1 inhibits the phosphorylation of rapamycin-sensitive and rapamycin-resistant mTOR targets and markedly blocks the production of virus progeny. The blockade of mTOR signaling with Torin1, but not rapamycin, disrupts the assembly of the eIF4F complex and increases the association of the translational repressor 4EBP1 to the 7-methylguanosine cap-binding complex. Torin1 does not affect HCMV entry and only modestly reduces the accumulation of the immediate-early and early viral proteins that were tested despite the disruption of the eIF4F complex. In contrast, Torin1 significantly decreases the accumulation of viral DNA and the pUL99 viral late protein. Similar mTOR signaling events were observed during murine cytomegalovirus (MCMV) infection, and we utilized murine fibroblasts containing several different mutations to dissect the mechanism by which Torin1 inhibits MCMV replication. This approach demonstrated that mTORC2 and the Akt1 and Akt2 kinases are not required for the Torin1-mediated inhibition of cytomegalovirus replication. The inhibition of MCMV replication by Torin1 was rescued in cells lacking 4EBP1, demonstrating that the inactivation of 4EBP1 by mTORC1 is critical for cytomegalovirus replication. Finally, we show that Torin1 inhibits the replication of representative members of the alpha-, beta-, and gammaherpesvirus families, demonstrating the potential of mTOR kinase inhibitors as broad-spectrum antiviral agents.
Moorman, Nathaniel J, et al.A targeted spatial-temporal proteomics approach implicates multiple cellular trafficking pathways in human cytomegalovirus virion maturation.”. Mol Cell Proteomics 95 (2010): , 9, 5, 851-60. Web.Abstract
The assembly of infectious virus particles is a complex event. For human cytomegalovirus (HCMV) this process requires the coordinated expression and localization of at least 60 viral proteins that comprise the infectious virion. To gain insight into the mechanisms controlling this process, we identified protein binding partners for two viral proteins, pUL99 (also termed pp28) and pUL32 (pp150), which are essential for HCMV virion assembly. We utilized HCMV strains expressing pUL99 or pUL32 carboxyl-terminal green fluorescent protein fusion proteins from their native location in the HCMV genome. Based on the presence of ubiquitin in the pUL99 immunoisolation, we discovered that this viral protein colocalizes with components of the cellular endosomal sorting complex required for transport (ESCRT) pathway during the initial stages of virion assembly. We identified the nucleocapsid and a large number of tegument proteins as pUL32 binding partners, suggesting that events controlling trafficking of this viral protein in the cytoplasm regulate nucleocapsid/tegument maturation. The finding that pUL32, but not pUL99, associates with clathrin led to the discovery that the two viral proteins traffic via distinct pathways during the early stages of virion assembly. Additional investigation revealed that the majority of the major viral glycoprotein gB initially resides in a third compartment. Analysis of the trafficking of these three viral proteins throughout a time course of virion assembly allowed us to visualize their merger into a single large cytoplasmic structure during the late stages of viral assembly. We propose a model of HCMV virion maturation in which multiple components of the virion traffic independently of one another before merging.
Hargett, Danna, and Thomas E Shenk. “Experimental human cytomegalovirus latency in CD14+ monocytes.”. Proc Natl Acad Sci U S A 107.46 (2010): , 107, 46, 20039-44. Web.Abstract
CD14(+) monocytes are a reservoir for latent human cytomegalovirus, and virus replication is reactivated during their differentiation to macrophages or dendritic cells. It has not been clear whether the virus can establish latency upon direct infection of monocytes or whether it must first become quiescent in a progenitor cell that subsequently differentiates to generate a monocyte. We report that infection of primary human monocytes with a clinical strain of human cytomegalovirus exhibits the hallmarks of latency. We established conditions for culturing monocytes that prevent differentiation for at least 25 d, as evidenced by cell surface marker expression. Infection of these monocytes with the FIX clinical strain resulted in transient accumulation of many viral lytic RNAs and sustained expression of four previously described latency-associated transcripts. The amount of viral DNA remained constant after infection, and cell surface and total HLA-DR proteins were substantially reduced on a continuing basis after infection. When treated with cytokine mixtures that stimulate differentiation to a macrophage or dendritic cell phenotype, infected monocytes reactivated virus replication and produced infectious progeny. Treatment of infected monocytes with IL-6 alone also was sufficient for reactivation, and the particles produced after exposure to this cytokine were about fivefold more infectious than virions produced by other treatments. We propose that in vivo microenvironments influence not only the efficiency of reactivation but also the infectivity of the virions produced from latently infected monocytes.

Pages