Many scientists attempt to publish their work in a journal with the highest possible journal impact factor (IF). Despite widespread condemnation of the use of journal IFs to assess the significance of published work, these numbers continue to be widely misused in publication, hiring, funding, and promotion decisions (1, 2).

Many scientists attempt to publish their work in a journal with the highest possible journal impact factor (IF). Despite widespread condemnation of the use of journal IFs to assess the significance of published work, these numbers continue to be widely misused in publication, hiring, funding, and promotion decisions (1, 2).

Replication of human cytomegalovirus (HCMV) is regulated in part by cellular kinases and the single viral Ser/Thr kinase, pUL97. The virus-coded kinase augments the replication of HCMV by enabling nuclear egress and altering cell cycle progression. These roles are accomplished through direct phosphorylation of nuclear lamins and the retinoblastoma protein, respectively. In an effort to identify additional pUL97 substrates, we analyzed the phosphoproteome of SILAC-labeled human fibroblasts during infection with either wild-type HCMV or a pUL97 kinase-dead mutant virus. Phosphopeptides were enriched over a titanium dioxide matrix and analyzed by high-resolution MS. We identified 157 unambiguous phosphosites from 106 cellular and 17 viral proteins whose phosphorylation required UL97. Analysis of peptides containing these sites allowed the identification of several candidate pUL97 phosphorylation motifs, including a completely novel phosphorylation motif, LxSP. Substrates harboring the LxSP motif were enriched in nucleocytoplasmic transport functions, including a number of components of the nuclear pore complex. These results extend the known functions of pUL97 and suggest that modulation of nuclear pore function may be important during HCMV replication.

Human cytomegalovirus (HCMV) infection rewires host-cell metabolism, upregulating flux from glucose into acetyl-CoA to feed fatty acid metabolism, with saturated very-long-chain fatty acids (VLFCAs) required for production of infectious virion progeny. The human genome encodes seven elongase enzymes (ELOVL) that extend long-chain fatty acids into VLCFA. Here, we identify ELOVL7 as pivotal for HCMV infection. HCMV induces ELOVL7 by more than 150-fold. This induction is dependent on mTOR and SREBP-1. ELOVL7 knockdown or mTOR inhibition impairs HCMV-induced fatty acid elongation, HCMV particle release, and infectivity per particle. ELOVL7 overexpression enhances HCMV replication. During HCMV infection, mTOR activity is maintained by the viral protein pUL38. Expression of pUL38 is sufficient to induce ELOVL7, and pUL38-deficient virus is partially defective in ELOVL7 induction and fatty acid elongation. Thus, through its ability to modulate mTOR and SREBP-1, HCMV induces ELOVL7 to synthesize the saturated VLCFA required for efficient virus replication.

Viruses rely on the host translation machinery for the synthesis of viral proteins. Human cells have evolved sensors that recognize viral RNAs and inhibit mRNA translation in order to limit virus replication. Understanding how viruses manipulate the host translation machinery to gain access to ribosomes and disable the antiviral response is therefore a critical aspect of the host/pathogen interface. In this study, we used a proteomics approach to identify human cytomegalovirus (HCMV) proteins that might contribute to viral mRNA translation. The HCMV TRS1 protein (pTRS1) associated with the 7-methylguanosine mRNA cap, increased the total level of protein synthesis, and colocalized with mRNAs undergoing translation initiation during infection. pTRS1 stimulated translation of a nonviral reporter gene and increased the translation of a reporter containing an HCMV 5' untranslated region (5'UTR) to a greater extent. The preferential effect of pTRS1 on translation of an mRNA containing a viral 5'UTR required the pTRS1 RNA and double-stranded RNA-dependent protein kinase (PKR)-binding domains, and was likely the result of PKR inhibition. However, pTRS1 also stimulated the total level of protein synthesis and translation directed by an HCMV 5'UTR in cells lacking PKR. Thus our results demonstrate that pTRS1 stimulates translation through both PKR-dependent and PKR-independent mechanisms.

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.

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.

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.

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.

The human cytomegalovirus immediate-early protein pUL37x1 induces the release of Ca(2+) stores from the endoplasmic reticulum into the cytosol. This release causes reorganization of the cellular actin cytoskeleton with concomitant cell rounding. Here we demonstrate that pUL37x1 activates Ca(2+)-dependent protein kinase Cα (PKCα). Both PKCα and Rho-associated protein kinases are required for actin reorganization and cell rounding; however, only PKCα is required for the efficient production of virus progeny, arguing that HCMV depends on the kinase for a second function. PKCα activation is also needed for the production of large (1-5 μm) cytoplasmic vesicles late after infection. The production of these vesicles is blocked by inhibition of fatty acid or phosphatidylinositol-3-phosphate biosynthesis, and the failure to produce vesicles is correlated with substantially reduced production of enveloped virus capsids. These results connect earlier work identifying a requirement for lipid synthesis with specific morphological changes, and support the argument that the PKCα-induced large vesicles are either required for the efficient production of mature virus particles or serve as a marker for the process.

Viruses face a multitude of challenges when they infect a host cell. Cells have evolved innate defenses to protect against pathogens, and an infecting virus may induce a stress response that antagonizes viral replication. Further, the metabolic, oxidative, and cell cycle state may not be conducive to the viral infection. But viruses are fabulous manipulators, inducing host cells to use their own characteristic mechanisms and pathways to provide what the virus needs. This article centers on the manipulation of host cell metabolism by human cytomegalovirus (HCMV). We review the features of the metabolic program instituted by the virus, discuss the mechanisms underlying these dramatic metabolic changes, and consider how the altered program creates a synthetic milieu that favors efficient HCMV replication and spread.

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.

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.

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.

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.

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.

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.

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.

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.

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.

The human cytomegalovirus (HCMV) virion protein pUL83 (also termed pp65) inhibits the expression of interferon-inducible cellular genes. In this work we demonstrate that pUL83 is also important for efficient induction of transcription from the viral major immediate-early promoter. Infection with a mutant virus containing a premature translation termination codon in the UL83 open reading frame (ORF) (UL83Stop) resulted in decreased transcription from the major immediate-early promoter in a time- and multiplicity-dependent manner. Expression of pUL83 alone is capable of transactivating the promoter in a reporter assay, and pUL83 associates with the promoter in infected cells. To investigate the mechanism by which the protein regulates the major immediate-early promoter, we utilized a mutant virus expressing an epitope-tagged pUL83 from its own promoter to identify protein binding partners for pUL83 during infection. We identified and confirmed the interaction of pUL83 with cellular IFI16 family members throughout the course of HCMV infection. pUL83 recruits IFI16 to the major immediate-early promoter, and IFI16 binding at the promoter is dependent upon the presence of pUL83. Consistent with the results obtained with the UL83Stop virus, infection of IFI16 knockdown cells with wild-type virus resulted in decreased levels of immediate-early transcripts compared to those of control cells. These data identify a previously unknown role for pUL83 in the initiation of the human cytomegalovirus gene expression cascade.

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.

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.

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.

Histone deacetylation plays a pivotal role in regulating human cytomegalovirus gene expression. In this report, we have identified candidate HDAC1-interacting proteins in the context of infection by using a method for rapid immunoisolation of an epitope-tagged protein coupled with mass spectrometry. Putative interactors included multiple human cytomegalovirus-coded proteins. In particular, the interaction of pUL38 and pUL29/28 with HDAC1 was confirmed by reciprocal immunoprecipitations. HDAC1 is present in numerous protein complexes, including the HDAC1-containing nucleosome remodeling and deacetylase protein complex, NuRD. pUL38 and pUL29/28 associated with the MTA2 component of NuRD, and shRNA-mediated knockdown of the RBBP4 and CHD4 constituents of NuRD inhibited HCMV immediate-early RNA and viral DNA accumulation; together this argues that multiple components of the NuRD complex are needed for efficient HCMV replication. Consistent with a positive acting role for the NuRD elements during viral replication, the growth of pUL29/28- or pUL38-deficient viruses could not be rescued by treating infected cells with the deacetylase inhibitor, trichostatin A. Transient expression of pUL29/28 enhanced activity of the HCMV major immediate-early promoter in a reporter assay, regardless of pUL38 expression. Importantly, induction of the major immediate-early reporter activity by pUL29/28 required functional NuRD components, consistent with the inhibition of immediate-early RNA accumulation within infected cells after knockdown of RBBP4 and CHD4. We propose that pUL29/28 modifies the NuRD complex to stimulate the accumulation of immediate-early RNAs.

The human cytomegalovirus virion is composed of a DNA genome packaged in an icosahedral capsid, surrounded by a tegument of protein and RNA, all enclosed within a glycoprotein-studded envelope. Achieving this intricate virion architecture requires a coordinated process of assembly and egress. We show here that pUL71, a component of the virion tegument with a previously uncharacterized function, is required for the virus-induced reorganization of host cell membranes, which is necessary for efficient viral assembly and egress. A mutant that did not express pUL71 was able to efficiently accumulate viral genomes and proteins that were tested but was defective for the production and release of infectious virions. The protein localized to vesicular structures at the periphery of the viral assembly compartment, and during infection with a pUL71-deficient virus, these structures were grossly enlarged and aberrantly contained a cellular marker of late endosomes/lysosomes. Mutant virus preparations exhibited less infectivity per unit genome than wild-type virus preparations, due to aggregation of virus particles and their association with membrane fragments. Finally, mutant virus particles accumulated within the cytoplasm of infected cells and were localized to the periphery of large structures with properties of lysosomes, whose formation was kinetically favored in mutant-virus-infected cells. Together, these observations point to a role for pUL71 in the establishment and/or maintenance of a functional viral assembly compartment that is required for normal virion trafficking and egress from infected cells.

We have identified a spliced transcript that contains sequences from the HCMV UL29 and UL28 open reading frames. It contains amino-terminal UL29 sequences followed by UL28 sequences, and it includes a poly(A) signal derived from the 3'-untranslated region following the UL26 open reading frame. UL29/28 RNA is expressed with early kinetics, and a virus containing a FLAG epitope inserted at the amino terminus of UL29 expressed a tagged approximately 79-kDa protein, pUL29/28, that was detected at 6 h postinfection. The virus also expressed a less-abundant tagged 41-kDa protein, which corresponds in size to a protein that could be produced by translation of an unspliced UL29/28 transcript. Consistent with this prediction, both unspliced and spliced UL29/28 transcript was present in RNA isolated from polysomes. FLAG-tagged protein from the UL29/28 locus accumulated within nuclear viral replication centers during the early phase of infection. Late after infection it was present in the cytoplasm as well, and the protein was present and resistant to proteinase treatment in partially purified preparations of viral particles. Disruption of the UL29/28 locus by mutation resulted in a 10-fold decrease in the levels of DNA replication along with a similar reduction in virus yield. Quantitative reverse transcription-PCR analysis revealed an approximately 2-fold decrease in immediate-early gene expression at 4 to 10 h postinfection compared to the wild-type virus, and transient expression of pUL29/28 activated the major immediate-early promoter. Our results argue that the UL29/28 locus contributes to activation of immediate-early gene expression.

Human cytomegalovirus DNA is packaged in virions without histones but associates with histones upon reaching the nucleus of an infected cell. Since transcription is modulated by the interplay of histone modifications, we used chromatin immunoprecipitation to detect acetylation and methylation of histone H3 at viral promoters at different times during the viral replication cycle. Histone H3 at immediate-early promoters is acetylated at the start of infection, while it is initially methylated at early and late promoters. Acetylation at immediate-early promoters is dynamic, with a high level of activating modifications at 3 and 6 h postinfection (hpi), followed by a marked reduction at 12 hpi. All viral promoters, as well as nonpromoter regions, are modified with activating acetylations at 24 to 72 hpi. The transient reduction in histone H3 acetylation at the major immediate-early promoter depends on the cis-repressive sequence to which the UL122-coded IE2 protein binds. A mutant virus lacking this element exhibited decreased IE2 binding at the major immediate-early promoter and failed to show reduced acetylation of histone H3 residing at this promoter at 12 hpi. Our results demonstrate that cytomegalovirus chromatin undergoes dynamic, promoter-specific histone modifications early in the infectious cycle, after which the entire chromosome becomes highly acetylated.

Rhesus cytomegalovirus (RhCMV) is an emerging model for human cytomegalovirus (HCMV) pathogenesis that facilitates experimental CMV infection of a natural primate host closely related to humans. We have generated a library of RhCMV mutants with lesions in genes whose HCMV orthologues have been characterized as nonessential for replication in human fibroblasts, and we characterized their replication in rhesus fibroblasts and epithelial cells. The RhCMV mutants grew well in fibroblasts, as predicted by earlier studies with HCMV. However, mutations in four genes caused replication defects in rhesus retinal pigment epithelial cells: Rh01 (an HCMV TRL1 orthologue), Rh159 (HCMV UL148), Rh160 (HCMV UL132), and Rh203 (HCMV US22). Growth of the Rh01-deficient mutant was examined in detail. After entry into epithelial cells, the mutant expressed representative viral proteins, accumulated viral DNA, and generated infectious virus, but it failed to spread efficiently. We conclude that Rh01 is a cell tropism determinant that has the potential to dramatically affect virus spread and pathogenesis.

Rhesus cytomegalovirus infection of rhesus macaques has emerged as a model for human cytomegalovirus pathogenesis. The UL128-UL131 locus of the human virus is a primary determinant for viral entry into epithelial cells, an important cell type during cytomegalovirus infection. Rhesus cytomegalovirus strain 68-1 spreads slowly when grown in cultured rhesus epithelial cells, and it does not code for ORFs corresponding to UL128 and the second exon of UL130. We repaired the UL128-UL131 locus of strain 68-1, using rhesus cytomegalovirus strain 180.92 as template, to generate BRh68-1.1. We also repaired a mutation in the UL36 ORF in BRh68-1.1 to make BRh68-1.2. Both repaired derivatives replicate much more efficiently than parental 68-1 virus in rhesus epithelial cells, suggesting that strain 68-1 may be attenuated. Intriguingly, BRh68-1.1 and BRh68-1.2 replicate efficiently in cultured human epithelial cells and endothelial cells. The extended human cell host range of the repaired viruses raises the possibility that rhesus cytomegalovirus-like viruses will be found in humans.

Human cytomegalovirus proteins alter host cells to favor virus replication. These viral proteins include pUL38, which prevents apoptosis. To characterize the mode of action of pUL38, we modified the viral genome to encode an epitope-tagged pUL38 and used rapid immunoaffinity purification to isolate pUL38-interacting host proteins, which were then identified by mass spectrometry. One of the cellular proteins identified was TSC2, a constituent of the tuberous sclerosis tumor suppressor protein complex (TSC1/2). TSC1/2 integrates stress signals and regulates the mammalian target of rapamycin complex 1 (mTORC1), a protein complex that responds to stress by limiting protein synthesis and cell growth. We showed that pUL38 interacts with TSC1 and TSC2 in cells infected with wild-type cytomegalovirus. Furthermore, TSC1/2 failed to regulate mTORC1 in cells expressing pUL38, and these cells exhibited the enlarged size characteristic of cytomegalovirus infection. Thus, pUL38 supports virus replication at least in part by blocking cellular responses to stress.

Viruses rely on the metabolic network of their cellular hosts to provide energy and building blocks for viral replication. We developed a flux measurement approach based on liquid chromatography-tandem mass spectrometry to quantify changes in metabolic activity induced by human cytomegalovirus (HCMV). This approach reliably elucidated fluxes in cultured mammalian cells by monitoring metabolome labeling kinetics after feeding cells (13)C-labeled forms of glucose and glutamine. Infection with HCMV markedly upregulated flux through much of the central carbon metabolism, including glycolysis. Particularly notable increases occurred in flux through the tricarboxylic acid cycle and its efflux to the fatty acid biosynthesis pathway. Pharmacological inhibition of fatty acid biosynthesis suppressed the replication of both HCMV and influenza A, another enveloped virus. These results show that fatty acid synthesis is essential for the replication of two divergent enveloped viruses and that systems-level metabolic flux profiling can identify metabolic targets for antiviral therapy.

A quantitative algorithm was developed and applied to predict target genes of microRNAs encoded by herpesviruses. Although there is almost no conservation among microRNAs of different herpesvirus subfamilies, a common pattern of regulation emerged. The algorithm predicts that herpes simplex virus 1, human cytomegalovirus, Epstein-Barr virus, and Kaposi's sarcoma-associated herpesvirus all employ microRNAs to suppress expression of their own genes, including their immediate-early genes. In the case of human cytomegalovirus, a virus-coded microRNA, miR-112-1, was predicted to target the viral immediate-early protein 1 mRNA. To test this prediction, mutant viruses were generated that were unable to express the microRNA, or encoded an immediate-early 1 mRNA lacking its target site. Analysis of RNA and protein within infected cells demonstrated that miR-UL112-1 inhibits expression of the major immediate-early protein. We propose that herpesviruses use microRNA-mediated suppression of immediate-early genes as part of their strategy to enter and maintain latency.