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Human cytomegalovirus (HCMV) replication relies on a nucleocapsid coat of the 150 kDa, subfamily-specific tegument phosphoprotein (pp150) to regulate cytoplasmic virion maturation. While recent structural studies revealed pp150-capsid interactions, the role of specific amino-acids involved in these interactions have not been established experimentally. In this study, pp150 and the small capsid protein (SCP), one of pp150's binding partners found atop the major capsid protein (MCP), were subjected to mutational and structural analyses. Mutations to clusters of polar or hydrophobic residues along the pp150-SCP interface abolished viral replication, with no replication detected in mutant virus-infected cells. Notably, a single amino acid mutation (pp150 K255E) at the pp150-MCP interface significantly attenuated viral replication, unlike in pp150-deletion mutants where capsids degraded outside host nuclei. These functionally significant mutations targeting pp150-capsid interactions, particularly the pp150 K255E replication-attenuated mutant, can be explored to overcome the historical challenges of developing effective antivirals and vaccines against HCMV infection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Herpes stromal keratitis is the leading cause of infectious blindness in the western world. Infection by HSV1 is most common, but VZV and hCMV also infect the cornea. Multiple models of HSV1 corneal infection exist, but none for VZV and hCMV because of their host specificity. Here, we used commercially available 3D human corneal epithelial equivalents (HCEE) to study infection by these herpesviruses. HCEE was infected by HSV-1 and hCMV without requiring scarification and resulted in spreading infections. Spread of HSV-1 infection was rapid, while that of hCMV was slow. In contrast, infections with VZV required damage to the HCEE and did not spread. Acyclovir dramatically reduced replication of HSV-1 in this model. We conclude that highly quality-controlled, readily available HCEE is a useful model to study human-restricted herpesvirus infection of the human corneal epithelium and for screening of antiviral drugs for treating HSK in an 3D model system., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Human cytomegalovirus (HCMV) infects the chorioamnion, but whether these infections cause fetal membrane dysfunction remains poorly understood. We sought to assess whether guinea pig cytomegalovirus (GPCMV) infects amnion-derived cells in vitro, compare the inflammatory response of amnion cells to GPCMV and HCMV, and determine if GPCMV infects the amnion in vivo. We found that GPCMV replicates in primary guinea pig amnion derived cells and HPV16 E6/E7-transduced amniotic epithelial cells (AEC[E6/E7]s). HCMV and GPCMV infection of amnion cells increased the transcription of the chemokines CCL5/Ccl5, CXCL8/Cxcl8, and CXCL10/Cxcl10. Myd88-knockdown decreased Ccl5 and Cxc8 transcription in GPCMV-infected AEC[E6/E7]s. GPCMV was detected in the guinea pig amnion after primary maternal infection, revealing that guinea pigs are an appropriate model to study fetal membrane physiology after cytomegalovirus infection. As inflammation is known to cause fetal membrane weakening, the amnion's response to cytomegalovirus infection may cause preterm birth and other adverse pregnancy outcomes., (Copyright © 2020 Elsevier Inc. All rights reserved.)

We previously showed that CMV-induced CD4 + CD27 - CD28 - T cells have regulatory (Treg) function. Here we sought to identify the target/s and the mechanistic underpinning/s of this effect. CMV-induced CD4 + CD27 - CD28 - were sorted from CMV-stimulated PBMC and added to CMV-stimulated autologous PBMC cultures. Transwell experiments showed that the CMV-induced Treg mechanism of action required cell-to-cell contact. CMV-Treg significantly decreased proliferation of autologous CMV-stimulated CD8 + and, to a lesser extent, CD4 + T cells; reduced activation and increased apoptosis of CD4 + and CD8 + T cells; and increased apoptosis and expression of CTLA-4, T cell-inhibitory ligand, on dendritic cells. There was no effect on monocytes. Anti-PD-1, but not anti-CTLA-4, mAb-treatment increased proliferation of CD8 + T cells and decreased apoptosis of CD4 + and CD8 + T cells. Our data indicated that CD8 + T cells were the main target of CMV-specific Treg, which induced apoptosis of their targets using the PD-1 pathway., Competing Interests: Declaration of competing interest AW receives research grants from Merck & Co. Inc., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Among the Herpesviridae, human cytomegalovirus (HCMV) owns the largest genome and displays a huge coding potential. Here, we characterized the UL5 gene product (pUL5) of the clinical isolate TR strain. The protein was predicted as a 166-amino-acid membrane protein with a theoretical mass of 19 kDa. Recombinant virus expressing pUL5 with a tag allowed the identification of two pUL5 non-glycosylated species of approximately 19 and 9 kDa, expressed with early and late kinetic respectively. Experiments in infection confirmed that the lower molecular weight species was translated from an internal ATG in the UL5 open reading frame. Confocal microscopy analysis showed that pUL5 localized within the assembly compartment, but is not incorporated in the virion, as shown by Western blot on purified viral particles. Finally, pull-down experiments coupled with mass spectrometry analysis identified IQGAP1 as a pUL5 interactor, giving new hints on possible roles of pUL5 during HCMV infection., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

The human cytomegalovirus (HCMV) UL34 gene encodes sequence-specific DNA-binding proteins (pUL34) which are required for viral replication. Interactions of pUL34 with DNA binding sites represses transcription of two viral immune evasion genes, US3 and US9. 12 additional predicted pUL34-binding sites are present in the HCMV genome (strain AD169) with three binding sites concentrated near the HCMV origin of lytic replication (oriLyt). We used ChIP-seq analysis of pUL34-DNA interactions to confirm that pUL34 binds to the oriLyt region during infection. Mutagenesis of the UL34-binding sites in an oriLyt-containing plasmid significantly reduced viral-mediated oriLyt-dependent DNA replication. Mutagenesis of these sites in the HCMV genome reduced the replication efficiencies of the resulting viruses. Protein-protein interaction analyses demonstrated that pUL34 interacts with the viral proteins IE2, UL44, and UL84, that are essential for viral DNA replication, suggesting that pUL34-DNA interactions in the oriLyt region are involved in the DNA replication cascade., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Glossina palipides salivary gland hypertrophy virus (GpSGHV) infects tsetse flies, which are vectors for African trypanosomosis. This virus represents a major challenge in insect mass rearing and has hampered the implementation of the sterile insect technique programs in the Member States of the International Atomic Energy Agency. GpSGHV virions consist of long rod-shaped particles over 9000Å in length, but little is known about their detailed structural organization. We show by cryo electron microscopy and cryo electron tomography that the GpSGHV virion has a unique, non-icosahedral helical structure. Its envelope exhibits regularly spaced spikes that protrude from the lipid bilayer and are arranged on a four-start helix. This study provides a detailed insight into the 3D architecture of GpSGHV, which will help to understand the viral life cycle and possibly allow the design of antiviral strategies in the context of tsetse fly infections., (Copyright © 2017 Elsevier Inc. All rights reserved.)

T98G cells have been shown to support long-term human cytomegalovirus (HCMV) genome maintenance without infectious virus release. However, it remains unclear whether these viral genomes could be reactivated. To address this question, a recombinant HCMV (rHCMV) containing a GFP gene was used to infect T98G cells, and the infected cells absent of infectious virus production were designated T98G-LrV. Upon dibutyryl cAMP plus IBMX (cAMP/IBMX) treatment, a serial of phenomena were observed, including GFP signal increase, viral genome replication, lytic genes expression and infectious viruses release, indicating the reactivation of HCMV in T98G-LrV cells from a latent status. Mechanistically, HCMV reactivation in the T98G-LrV cells induced by cAMP/IBMX was associated with the PKA-CREB signaling pathway. These results demonstrate that HCMV was latent in T98G-LrV cells and could be reactivated. The T98G-LrV cells represent an effective model for investigating the mechanisms of HCMV reactivation from latency in the context of neural cells., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Cytomegalovirus (CMV) infection can generate debilitating disease in immunocompromised individuals and neonates. It is also the most common infectious cause of congenital birth defects in infected fetuses. Available anti-CMV drugs are partially effective but are limited by some toxicity, potential viral resistance, and are not recommended for fetal exposure. Valproate, valpromide, and valnoctamide have been used for many years to treat epilepsy and mood disorders. We report for the first time that, in contrast to the virus-enhancing actions of valproate, structurally related valpromide and valnoctamide evoke a substantial and specific inhibition of mouse and human CMV in vitro. In vivo, both drugs safely attenuate mouse CMV, improving survival, body weight, and developmental maturation of infected newborns. The compounds appear to act by a novel mechanism that interferes with CMV attachment to the cell. Our work provides a novel potential direction for CMV therapeutics through repositioning of agents already approved for use in psychiatric disorders., Competing Interests: none., (Copyright © 2016 Elsevier Inc. All rights reserved.)

US28 transcripts have been detected in primary monocytes and in THP-1 monocytes infected with HCMV but US28 protein expression has not yet been demonstrated in these cell types. Moreover, the mechanism(s) by which US28 signals and contributes to viral pathogenesis in monocytes remains unclear. Here, we show that US28 protein is robustly expressed in HCMV infected THP-1 monocytes and that US28 can trigger Gαq dependent signaling in THP-1 cells infected with HCMV and in THP-1 cells stably expressing US28. US28 signaling in these cells is dependent on G-protein coupling, but independent of chemokine binding. Importantly, we demonstrate that this US28 signaling is functionally important as it stimulates the adhesion of monocytes to an endothelial monolayer. Our studies, which demonstrate that US28-driven Gαq signaling has profound effects on monocyte biology, suggest that US28 driven phenotypic changes in HCMV infected monocytes may play important roles in HCMV dissemination and/or pathogenesis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Human Cytomegalovirus (HCMV) infection is compromised in cells lacking p53, a transcription factor that mediates cellular stress responses. In this study we have investigated compromised functional virion production in cells with p53 knocked out (p53KOs). Infectious center assays found most p53KOs released functional virions. Analysis of electron micrographs revealed modestly decreased capsid production in infected p53KOs compared to wt. Substantially fewer p53KOs displayed HCMV-induced infoldings of the inner nuclear membrane (IINMs). In p53KOs, fewer capsids were found in IINMs and in the cytoplasm. The deficit in virus-induced membrane remodeling within the nucleus of p53KOs was mirrored in the cytoplasm, with a disproportionately smaller number of capsids re-enveloped. Reintroduction of p53 substantially recovered these deficits. Overall, the absence of p53 contributed to inhibition of the formation and function of IINMs and re-envelopment of the reduced number of capsids able to reach the cytoplasm., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Our electron microscopy study (Kuan et al., 2016) found HCMV nuclear capsid egress was significantly reduced in p53 knockout cells (p53KOs), correlating with inhibited formation of infoldings of the inner nuclear membrane (IINMs). Molecular examination of these phenomena has found p53KOs expressed UL97 and phosphorylated lamins, however the lamina failed to remodel. The nuclear egress complex (NEC) protein UL50 was expressed in almost all cells. UL50 re-localized to the inner nuclear membrane (INM) in ~90% of wt cells, but only ~35% of p53KOs. UL53 expression was significantly reduced in p53KOs, and cells lacking UL50 nuclear staining, expressed no UL53. Re-introduction of p53 into p53KOs largely recovered UL53 positivity and UL50 nuclear re-localization. Nuclear rim located UL50/53 puncta, which co-localized with the major capsid protein, were largely absent in p53KOs. We believe these puncta were IINMs. In the absence of p53, UL53 expression was inhibited, disrupting formation of the NEC/IINMs, and reducing functional virion secretion., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Human cytomegalovirus (HCMV) lacking TRS1 and IRS1 (HCMV[ΔI/ΔT]) cannot replicate in cell culture. Although both proteins can block the protein kinase R (PKR) pathway, they have multiple other activities and binding partners. It remains unknown which functions are essential for HCMV replication. To investigate this issue, we first identified a TRS1 mutant that is unable to bind to PKR. Like HCMV[ΔI/ΔT], a recombinant HCMV containing this mutant (HCMV[TRS1-Mut 1]) did not replicate in wild-type cells. However, HCMV[ΔI/ΔT] did replicate in cells in which PKR expression was reduced by RNA interference. Moreover, HCMV[ΔI/ΔT] and HCMV[TRS1-Mut 1] replicated to similar levels as virus containing wild-type TRS1 in cell lines in which PKR expression was knocked out by CRISPR/Cas9-mediated genome editing. These results demonstrate that the sole essential function of TRS1 is to antagonize PKR and that its other activities do not substantially enhance HCMV replication, at least in cultured human fibroblasts., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Human cytomegalovirus (HCMV) was recently shown to encode a large number of spliced mRNAs. While the nuclear export of unspliced viral transcripts has been extensively studied, the role of host mRNA export factors in HCMV mRNA trafficking remains poorly defined. We found that the eIF4AIII RNA helicase, a component of the exon junction complex, was necessary for efficient virus replication. Depletion of eIF4AIII limited viral DNA accumulation, export of viral mRNAs from the nucleus, and the production of progeny virus. However eIF4AIII was dispensable for the association of viral transcripts with ribosomes. We found that pateamine A, a natural compound that inhibits both eIF4AI/II and eIF4AIII, has potent antiviral activity and inhibits HCMV replication throughout the virus lytic cycle. Our results demonstrate that eIF4AIII is required for efficient HCMV replication, and suggest that eIF4A family helicases may be a new class of targets for the development of host-directed antiviral therapeutics., (Copyright © 2016. Published by Elsevier Inc.)

Human cytomegalovirus (HCMV) resides in a latent form in hematopoietic progenitors and undifferentiated cells within the myeloid lineage. Maturation and differentiation along the myeloid lineage triggers lytic replication. Here, we used peripheral blood monocytes and the monocytic cell line THP-1 to investigate the effects of 1,25-dihydroxyvitamin D3 on HCMV replication. Interestingly, 1,25-dihydroxyvitamin D3 induces lytic replication marked by upregulation of HCMV gene expression and production of infectious virus. Moreover, we demonstrate that the effects of 1,25-dihydroxyvitamin D3 correlate with maturation/differentiation of the monocytes and not by directly stimulating the MIEP. These results are somewhat surprising as 1,25-dihydroxyvitamin D3 typically boosts immunity to bacteria and viruses rather than driving the infectious life cycle as it does for HCMV. Defining the signaling pathways kindled by 1,25-dihydroxyvitamin D3 will lead to a better understanding of the underlying molecular mechanisms that determine the fate of HCMV once it infects cells in the myeloid lineage., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The UL84 gene of human cytomegalovirus (HCMV) is thought to be involved in the initiation of viral DNA replication, and is essential for replication of strains AD169 and Towne. Hence, discovery that strain TB40-BAC4 is viable in the absence of UL84 presented an enigma requiring an explanation. Data reported here show that strain TR also tolerated loss of UL84, whereas strains FIX, Merlin, Ph, and Toledo did not. UL84-independent growth required the viral replication origin. The genetic locus in TB40 that controls UL84 dependence was mapped to codon 388 of the UL122 gene, which encodes the immediate early 2 (IE2) 86kD protein. Introduction of this TB40-BAC4 variant (H388D) into FIX and Toledo clones converted these strains to UL84 independence. These results provide genetic evidence in virus-infected cells that supports the hypothesis that UL122 participates in the initiation of viral DNA replication by a mechanism involving transcription-mediated activation of the origin., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Urine and breast milk represent the main routes of human cytomegalovirus (HCMV) transmission but the contribution of renal and mammary epithelial cells to viral excretion remains unclear. We observed that kidney and mammary epithelial cells were permissive to HCMV infection and expressed immediate early, early and late antigens within 72 h of infection. During the first 24 h after infection, high titers of infectious virus were measured associated to the cells and in culture supernatants, independently of de novo synthesis of virus progeny. This phenomenon was not observed in HCMV-infected fibroblasts and suggested the sequestration and the release of HCMV by epithelial cells. This hypothesis was supported by confocal and electron microscopy analyses. The sequestration and progressive release of HCMV by kidney and mammary epithelial cells may play an important role in the excretion of the virus in urine and breast milk and may thereby contribute to HCMV transmission., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Translationally silenced mRNAs are recruited to two major classes of RNA granules in the cytoplasm, processing bodies (PBs) and stress granules (SGs). We show that PBs accumulated after human cytomegalovirus (HCMV) infection. PB assembly after HCMV infection was also detected in the presence of the protein synthesis inhibitor, cycloheximide, but required active RNA synthesis. UV-inactivated HCMV virions were sufficient to induce PB accumulation in HFF cells treated with cycloheximide. Viral IE1 RNA did not colocalize with PBs, and we could not detect an effect of PB accumulation on viral growth. These results may indicate that HCMV inhibits the colocalization of IE1 mRNA with PBs, preventing IE1 mRNA decay and translational inhibition., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The Major Immediate Early Promoter (MIEP) of human cytomegalovirus (HCMV) controls viral Immediate Early (IE) gene expression, which must be activated to initiate productive infection and repressed to establish latency. Regulation of the MIEP is critical for both viral spread and persistence. In addition to the Daxx-mediated intrinsic cellular defense that regulates the MIEP, the cell-type specific balance between cellular activators and repressors of the promoter may help dictate whether viral IE genes will be expressed or silenced. For example, in undifferentiated myeloid cells, transcriptional repressors of the MIEP may outnumber transcriptional activators, leading to promoter silencing and latency establishment. We created a recombinant viral genome in which a myeloid-active promoter replaced part of the MIEP. The viable virus generated failed to express the viral IE genes in an undifferentiated myeloid cell line. These observations have mechanistic implications regarding how viral IE gene expression is regulated during latency., (© 2013 Elsevier Inc. All rights reserved.)

Protein Kinase R (PKR) inhibits translation initiation following double-stranded RNA (dsRNA) binding and thereby represses viral replication. Human cytomegalovirus (HCMV) encodes two noncanonical dsRNA binding proteins, IRS1 and TRS1, and the expression of at least one of these PKR antagonists is essential for HCMV replication. In this study, we investigated the role of dsRNA binding by TRS1 in PKR inhibition. We found that purified TRS1 binds specifically to dsRNA with an affinity lower than that of PKR. Point mutants in the TRS1 dsRNA binding domain that were deficient in rescuing the replication of vaccinia virus lacking its PKR antagonist E3L were unable to bind to dsRNA but retained the ability bind to PKR. Thus TRS1 binding to dsRNA and to PKR are separable. Overall, our results are most consistent with a model in which TRS1 binds simultaneously to both dsRNA and PKR to inhibit PKR activation., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Suitable host cell metabolic conditions are fundamental for the effective development of the human cytomegalovirus (HCMV) lytic cycle. Indeed, several studies have demonstrated the ability of this virus to interfere with cell cycle regulation, mainly by blocking proliferating cells in G1 or G1/S. In the present study, we demonstrate that HCMV deregulates the cell cycle of THP-1 macrophages (a cell line irreversibly arrested in G0) by pushing them into S and G2 phases. Moreover, we show that HCMV infection of THP-1 macrophages leads to Toll-like receptor 4 (TLR4) activation. Since various studies have indicated TLR4 to be involved in promoting cell proliferation, here we investigate the possible role of TLR4 in the observed HCMV-induced cell cycle perturbation. Our data strongly support TLR4 as a mediator of HCMV-triggered cell cycle activation in THP-1 macrophages favouring, in turn, the development of an efficient viral lytic cycle., (Copyright © 2013 Elsevier Inc. All rights reserved.)

At a low MOI (≤0.01), cytomegalovirus-associated programmed cell death terminates productive infection via a pathway triggered by the mitochondrial serine protease HtrA2/Omi. This infected cell death is associated with late phase replication events naturally suppressed by the viral mitochondrial inhibitor of apoptosis (vMIA). Here, higher MOI (ranging from 0.1-3.0) triggers cell death earlier during infection independent of viral DNA synthesis. Thus, MOI-dependent activating signals early, at high MOI, or late, at low MOI, during replication promote serine protease-dependent death that is suppressed by vMIA. Treatment with an antioxidant targeting reactive oxygen species (ROS) or the serine protease inhibitor N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) delays cell death, and the combination has an additive impact. These studies identify serine proteases and ROS as important factors triggering programmed cell death induced by vMIA-deficient virus, and show that this death pathway occurs earlier and reduces viral yields as the MOI is increased., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Human cytomegalovirus (HCMV) remains latent in cells of the myeloid lineage after primary infection. The THP-1 monocytic cell line is conditionally permissive for infection and has been used primarily to study the process of HCMV reactivation when the cells are induced to differentiate. In the present report, we characterized lytic infection in THP-1 derived macrophages using two strains of HCMV, Towne and BAC-derived TR. Our findings indicate that these cells express viral genes of all three kinetic classes and produce extracellular virus, but that there is a delay in these processes relative to productively infected fibroblasts. Importantly, our studies in THP-1 derived macrophages revealed strain-specific differences in pp65 trafficking and actin dynamics. Based on these observations, our studies indicate that differentiated THP-1 cells can serve as a valuable model for lytic infection., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Human cytomegalovirus transient lytic DNA replication relies on the cis-acting element oriLyt, six viral-encoded core proteins, the proposed DNA replication initiator protein UL84, IE2, IRS1 and the gene products from the UL112/113 loci. In an effort to elucidate cellular and viral-encoded factors that may play a role in oriLyt-dependent replication we used DNA-affinity purification and mass spectrometry to isolate and identify several previously unknown cellular and viral factors that interact with HCMV oriLyt DNA. These proteins include the multifunctional hnRNP-K, BUB3, HMGB1, PTB-1, UL83, UL112/113, and IRS1. Chromatin immunoprecipitation (ChIP) assays confirmed an interaction of several of these factors with oriLyt. Co-immunoprecipitation experiments detected an interaction between UL84 and hnRNP-K in infected and transfected cells. Knockdown of hnRNP K expression by siRNA inhibited the amplification of oriLyt in the transient assay. Together, these data suggest a possible regulatory role in DNA replication for several previously unidentified viral and cellular factors., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The human cytomegalovirus DNA polymerase subunit UL44 is a phosphoprotein, but its sites and roles of phosphorylation have not been investigated. We compared sites of phosphorylation of UL44 in vitro by the viral protein kinase UL97 and cyclin-dependent kinase 1 with those in infected cells. Transient treatment of infected cells with a UL97 inhibitor greatly reduced labeling of two minor UL44 phosphopeptides. Viruses containing alanine substitutions of most UL44 residues that are phosphorylated in infected cells exhibited at most modest effects on viral DNA synthesis and yield. However, substitution of highly phosphorylated sites adjacent to the nuclear localization signal abolished viral replication. The results taken together are consistent with UL44 being phosphorylated directly by UL97 during infection, and a crucial role for phosphorylation-mediated nuclear localization of UL44 for viral replication, but lend little support to the widely held hypothesis that UL97-mediated phosphorylation of UL44 is crucial for viral DNA synthesis., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Cynomolgus macaques have been widely used as an animal model in preclinical biomedical research and are becoming more popular among HIV/SIV vaccine researchers. Here we report the isolation and characterization of a cytomegalovirus from cynomolgus macaques (CyCMV). CyCMV was isolated from a healthy captive-bred 4-year-old cynomolgus macaque of Filipino origin. The virus was identified by its characteristic growth properties in cell culture, ultrastructural morphology and sequence of viral DNA polymerase and glycoprotein B (gB). CyCMV gB shows 77% identity and 88% homology to rhesus cytomegalovirus (RhCMV) gB and 58% identity and 76% homology to human cytomegalovirus gB at the amino acid level. Phylogenetic analysis using known CMV gB protein sequences show that CyCMV is more closely related to RhCMV than to other primate CMVs. CyCMV down-regulates MHC class I expression on infected cells and we show that the colony-bred cynomolgus macaques have detectable CyCMV-specific humoral and cell-mediated immune responses., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The UL84 gene of human cytomegalovirus is implicated in the initiation of viral DNA replication during lytic infection. UL84 is essential for replication of a cloned viral origin of lytic replication (oriLyt) in vitro and mutants of strains AD169 or Towne with deletions or insertions in UL84 fail to grow in cells permissive for wild type virus. Here we show that UL84 is dispensable for replication of a strain TB40/E clone derived from a bacterial artificial chromosome. The genomes of the fibroblast-adapted strains AD169 and Towne are altered substantially from the consensus for strains that have not been propagated extensively in cell culture. The parental TB40/E genome conforms to the consensus genomic organization. Accordingly, natural HCMV strains may possess replication capability that extends beyond the known oriLyt-dependent replication system of laboratory strains., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The class E genome of human cytomegalovirus (HCMV) contains long and short segments that invert due to recombination between flanking inverted repeats, causing the genome to isomerize into four distinct isomers. To determine if isomerization is important for HCMV replication, one copy of each repeat was deleted. The resulting virus replicated in cultured human fibroblasts with only a slight growth impairment. Restriction and Southern analyses confirmed that its genome is locked in the prototypic arrangement and unable to isomerize. We conclude that efficient replication of HCMV in fibroblasts does not require (i) the ability to undergo genome isomerization, (ii) genes that lie partially within the deleted repeats, or (iii) diploidy of genes that lie wholly within repeats. The simple genomic structure of this virus should facilitate studies of genome circularization, latency or persistence, and concatemer packaging as such studies are hindered by the complexities imposed by isomerization.

Human cytomegalovirus (HCMV) encodes four glycoproteins, termed gpUS2, gpUS3, gpUS6 and gpUS11 that interfere with MHC class I biosynthesis and antigen presentation. Despite gpUS2-11 expression, however, HCMV infection is efficiently controlled by cytolytic CD8 T lymphocytes (CTL). To address the role of gpUS2 and gpUS11 in antigen presentation during viral infection, HCMV mutants were generated that expressed either gpUS2 or gpUS11 alone without coexpression of the three other proteins. Fibroblasts infected with these viruses showed reduced HLA-A2 and HLA-B7 surface expression. Surprisingly, however, CTL directed against the tegument protein pp65 and the regulatory IE1 protein still recognized and lysed mutant virus infected fibroblasts. Yet, suppression of IE1 derived peptide presentation by gpUS2 or gpUS11 was far more pronounced. The results show that gpUS2 and gpUS11 alone only incompletely protect HCMV infected fibroblasts from CTL recognition and underline the importance of studying infected cells to elucidate HCMV immune evasion.

Expression of the major immediate-early (MIE) genes of human cytomegalovirus (HCMV) in the human thyroid papillary carcinoma cell line TPC-1 is repressed at the transcriptional level. However, treatment of these cells with hexamethylene bisacetamide (HMBA), a chemical inducer of differentiation, for 12 to 24 h before infection enabled the cells to support IE1 and IE2 gene expression and consequently HCMV replication. In HMBA-treated cells the transcription factor NF-kappaB was induced and the MIE promoter (MIEP) was activated. The presence of a NF-kappaB inhibitory peptide SN-50 or expression of a dominant negative IkappaBalpha protein during the HMBA pretreatment period efficiently prevented the HMBA-induced MIEP activation and MIE protein synthesis. Moreover, introduction of mutations into the NF-kappaB binding sites in the MIEP in a plasmid expressing the IE1 protein diminished its ability to express the protein in HMBA-treated cells. Therefore, the NF-kappaB activity previously induced in HMBA-treated cells and the NF-kappaB sites in the MIEP were shown to be essential for HCMV to respond to HMBA action and to express the MIE genes. Investigation of the mechanisms by which HMBA activates NF-kappaB revealed that degradation of IkappaBalpha and translocation of the phosphorylated NF-kappaB p65 subunit to the nucleus, both of which are known to be critical steps in NF-kappaB activation, are stimulated in the HMBA-treated cells. These results indicate that treatment of nonpermissive TPC-1 cells with HMBA induces MIE gene permissiveness by up-regulating NF-kappaB activity.

Initiation of human cytomegalovirus (HCMV) productive infection is dependent on the major immediate early (MIE) genes ie1 and ie2. Several putative binding sites for CCAAT displacement protein (CDP or CUX1) were identified within the MIE promoter/regulatory region. Binding assays demonstrated binding of CUX1 to MIE-region oligonucleotides containing the CUX1 core binding sequence ATCGAT and mutagenesis of this sequence abrogated CUX1 binding. Furthermore, CUX1 repressed expression of a luciferase reporter construct controlled by the MIE promoter, and mutation of CUX1 binding sites within the promoter diminished this repressive function of CUX1. In the context of virus infection of HEK293 cells transfected with the CUX1 expression vector, CUX1 showed evidence of association with the HCMV MIE regulatory region and inhibited the capacity of the virus to express ie1 and ie2 transcripts, suggesting that this cellular factor regulates MIE gene expression following virus entry. These data identify a role for CUX1 in repressing HCMV gene expression essential for initiation of the replicative cycle.

The human cytomegalovirus tegument protein pp71 localizes to the nucleus immediately upon infection, and functions to initiate viral gene expression. Analysis of a series of random insertion mutations revealed that sequences within the mid region (MR) of pp71 are important for localization to the nucleus. Fusion of MR sequences with eGFP revealed that amino acids 94 to 300 were sufficient to target proteins to the nucleus. Random substitution mutagenesis within this domain resulted in two double substitution mutants, pp71P203T/T223M and pp71T228M/L275Q, with a predominantly cytoplasmic localization. Disruption of nuclear targeting resulted in relocalization of the fusion proteins to a distinct perinuclear region. Using tandem mass spectrometry, we determined that threonine 223 can be phosphorylated. Mutation of this residue to a phosphomimetic amino acid resulted in abrogation of nuclear targeting. These results strongly suggest that the intracellular trafficking of pp71 is regulated by phosphorylation.

The human cytomegalovirus UL111A region is active during both productive and latent phases of infection. During productive infection, the virus expresses ORF79, a protein with oncogenic properties, and cmvIL-10, a functional homolog of human IL-10. During latent infection of myeloid progenitor cells, an alternately spliced variant of cmvIL-10, termed latency-associated (LA) cmvIL-10 has previously been identified. To determine whether LAcmvIL-10 transcription occurs during productive infection, we performed 5' and 3' RACE to map UL111A-region transcripts in productively infected human foreskin fibroblasts (HFFs). This analysis revealed the presence of a singly spliced UL111A-region transcript predicted to encode LAcmvIL-10. This transcript was expressed in HFFs with early (beta) kinetics, a temporal class that differs from that of ORF79 (alpha kinetics) and cmvIL-10 (gamma kinetics). These data identify and map a transcript encoding a latency-associated homolog of IL-10 which is expressed by the virus during the productive phase of infection.

The cellular Daxx protein represses human cytomegalovirus (HCMV) gene expression from the major immediate early promoter. HCMV prevents Daxx-mediated silencing during lytic infection by delivering the viral pp71 tegument protein to the nucleus, where pp71 binds to and induces the proteasomal degradation of Daxx. In this study, we show that a functional ubiquitin pathway is not required for the proteasomal degradation of the endogenous Daxx protein by tegument-delivered pp71 in HCMV-infected cells, demonstrating that the pp71-mediated degradation of Daxx occurs through a proteasome-dependent, ubiquitin-independent pathway.

Human cytomegalovirus (HCMV) reactivation from latency causes disease in individuals who are immunocompromised or immunosuppressed. Activation of the major immediate-early (MIE) promoter is thought to be an initial step for reactivation. We determined whether expression of the MIE gene products in trans was sufficient to circumvent an HCMV latent-like state in an undifferentiated transformed human promonocytic (THP)-1 cell model system. Expression of the functional MIE proteins was achieved with a replication-defective adenovirus vector, Ad-IE1/2, which contains the MIE gene locus. Expression of the MIE proteins by Ad-IE1/2 prior to HCMV infection induced viral early gene expression accompanied by an increase in active chromatin signals. Expression of the anti-apoptotic protein encoded by UL37x1 increased viral early gene expression. However, viral DNA replication and production of infectious virus was not detected. As expected, cellular differentiation with phorbol 12-myristate 13-acetate and hydrocortisone induced virus production. Cellular differentiation is required for efficient viral reactivation.

The human cytomegalovirus (HCMV) US12 gene family is a group of 10 predicted seven-transmembrane domain proteins that have some features in common with G-protein-coupled receptors. Little is known of their patterns of expression, localization, or functional interactions. Here, we studied the intracellular localization of three US12 family members, US14, US17, and US18, with respect to various intracellular markers and the cytoplasmic virion assembly compartment (AC). The three proteins have distinct patterns of expression, which include associations with the AC. US14 is often distributed in a uniform granular manner throughout the cytoplasm, concentrating in the AC in some cells. US17 is expressed in a segmented manner, with its N-terminal domain localizing to the periphery of what we show here to be the AC and the C-terminal domain localizing to nuclei and the cytoplasm [Das, S., Skomorovska-Prokvolit, Y., Wang, F. Z., Pellett, P.E., 2006. Infection-dependent nuclear localization of US17, a member of the US12 family of human cytomegalovirus-encoded seven-transmembrane proteins. J. Virol. 80, 1191-1203]. Here, we show that the C-terminal domain is present at the center of the AC, in close association with markers of early endosomes; the N-terminal staining corresponds to an area stained by markers for the Golgi and trans-Golgi. US18 is distributed throughout the cytoplasm, concentrating in the AC at later stages of infection; it is localized more to the periphery of the AC than are US14 and US17C, in association with markers of the trans-Golgi. Although not detected in virions, their structures and localization in various zones within the AC suggest possible roles for these proteins in the process of virion maturation and egress.

Mesenchymal stem cells (MSCs) in bone marrow (BM) regulate the differentiation and proliferation of adjacent hematopoietic precursor cells and contribute to the regeneration of mesenchymal tissues, including bone, cartilage, fat and connective tissue. BM is an important site for the pathogenesis of human cytomegalovirus (HCMV) where the virus establishes latency in hematopoietic progenitors and can transmit after reactivation to neighboring cells. Here we demonstrate that BM-MSCs are permissive to productive HCMV infection, and that HCMV alters the function of MSCs: (i) by changing the repertoire of cell surface molecules in BM-MSCs, HCMV modifies the pattern of interaction between BM-MSCs and hematopoietic cells; (ii) HCMV infection of BM-MSCs undergoing adipogenic or osteogenic differentiation impaired the process of differentiation. Our results suggest that by altering BM-MSC biology, HCMV may contribute to the development of various diseases.

Studies of human cytomegalovirus (HCMV) UL97 kinase deletion mutant (DeltaUL97) indicated a multi-step role for this kinase in early and late phases of the viral life cycle, namely, in DNA replication, capsid maturation and nuclear egress. Here, we addressed its possible involvement in cytoplasmic steps of HCMV assembly. Using the DeltaUL97 and the UL97 kinase inhibitor NGIC-I, we demonstrate that the absence of UL97 kinase activity results in a modified subcellular distribution of the viral structural protein assembly sites, from compact structures impacting upon the nucleus to diffuse perinuclear structures punctuated by large vacuoles. Infection by either wild type or DeltaUL97 viruses induced a profound reorganization of wheat germ agglutinin (WGA)-positive Golgi-related structures. Importantly, the viral-induced Golgi remodeling along with the reorganization of the nuclear architecture was substantially altered in the absence of UL97 kinase activity. These findings suggest that UL97 kinase activity might contribute to organization of the viral cytoplasmic assembly sites.

The human cytomegalovirus (HCMV) induces a replicative senescence program after arresting host cell cycle progression so as to create a favorable environment for its replication. Here, we report that HCMV infection stimulates the expression of p16(INK4a), a direct effector of the senescence phenotype. The increase in p16(INK4a) gene expression was due to an increase in gene transcription, since the expression of a reporter gene driven by the p16(INK4a)-encoding CDKN2A gene promoter was strongly induced by HCMV infection. The results of deletion and mutational analysis of the CDKN2A promoter further suggest the involvement of Ets transcription factors in HCMV-mediated stimulation of p16(INK4a) gene expression. The significance of p16(INK4a) upregulation during the HCMV replicative cycle is underscored by the finding that virus replication was severely impaired in fibroblasts homozygous for an intragenic deletion in CDKN2A locus and devoid of functional p16(INK4a). Moreover, a retrovirus-mediated p16(INK4a) small interfering RNA (p16-siRNA) effectively reduced viral replication, thus providing direct evidence that p16(INK4a) upregulation plays a positive role for HCMV replication.

Regulation of DNA repair mechanisms during the viral replication cycle may have consequences for the virus with regards to genomic variability, adaptation, and replication of viral DNA. We have studied the activities and expression patterns of key enzymes involved in the first two steps of base excision repair (BER) of DNA in primary fibroblasts infected by human cytomegalovirus (HCMV). Infected cells were very proficient for removal of uracil and 5' hydrolysis of AP sites (AP endonuclease activity) as compared to the mock-infected cells, suggesting a direct role in generating free ends at uracil lesions in DNA for initiation of viral replication. Furthermore, the capacity to initiate repair of alkylated and oxidized base lesions were reduced in HCMV-infected cells, indicating increased mutation frequencies that could promote genetic variability. We hypothesize that modulation of BER activities may play an important role in HCMV pathogenesis to ensure efficient replication and genomic variation of viral DNA.

The p53 protein is stabilized during infection of primary human fibroblasts with human cytomegalovirus (HCMV). However, the p53 in HCMV-infected cells is unable to activate its downstream targets. HCMV accomplishes this inactivation, at least in part, by sequestering p53 into viral replication centers within the cell's nucleus soon after they are established. In order to better understand the interplay between HCMV and p53 and the mechanism of sequestration, we constructed a panel of mutant p53-GFP fusion constructs for use in transfection/infection experiments. These mutants affected several post-translational modification sites and several sites within the central sequence-specific DNA-binding domain of the protein. Two categories of p53 sequestration were observed when the mutant constructs were transfected into primary fibroblasts and then infected at either high or low multiplicity. The first category, including all of the post-translational modification mutants, showed sequestration comparable to a wild-type (wt) control, while the second category, mutants affecting the DNA-binding core, were not specifically sequestered above control GFP levels. This suggested that the DNA-binding ability of the protein was required for sequestration. When the HCMV genome was analyzed for p53 consensus binding sites, 21 matches were found, which localized either to the promoters or the coding regions of viral proteins involved in DNA replication and processing as well as structural proteins. An analysis of in vivo binding to these identified sites via chromatin immunoprecipitation assays revealed differential binding to several of the sites over the course of infection.

Human herpesvirus 6 (HHV-6) is the only human herpesvirus encoding U94/rep, homologue to the parvovirus non-structural gene rep68/78. Results to date suggest that HHV-6 U94/rep might regulate viral gene expression and have a role in viral latency. To determine the effect of U94/REP upon viral replication, the protein was produced. The purified U94/REP retained the characteristic immunological features. It was internalized and localized in the nucleus of human cells, showing marked inhibitory activity on the replication of HHV-6 (both variants A and B). The effect of U94/REP was dose-dependent and sensitive to treatment with single-stranded but not double-stranded DNA. U94/REP inhibited the replication of other betaherpesviruses, HHV-7 and human cytomegalovirus, but had no effect on herpes simplex virus. These results confirm the action of U94/rep latency gene in the regulation of HHV-6 replication with implications for co-reactivations and latency of human betaherpesviruses.

Infection of fibroblasts by human cytomegalovirus (HCMV) rapidly activates the NF-kappaB signaling pathway, which we documented promotes efficient transactivation of the major immediate-early promoter (DeMeritt, I.B., Milford, L.E., Yurochko, A.D. (2004). Activation of the NF-kappaB pathway in human cytomegalovirus-infected cells is necessary for efficient transactivation of the major immediate-early promoter. J. Virol. 78, 4498-4507). Because a second, sustained increase in NF-kappaB activity following the initial phase of NF-kappaB activation was also observed, we investigated the role that this prolonged NF-kappaB activation played in viral replication and late gene expression. We first investigated HCMV replication in cells in which NF-kappaB activation was blocked by pretreatment with NF-kappaB inhibitors: HCMV replication was significantly decreased in these cultures. A decrease in replication was also observed when NF-kappaB was inhibited up to 48 h post-infection, suggesting a previously unidentified role for NF-kappaB in the regulation of the later class of viral genes.

The Human Cytomegalovirus (HCMV) UL72 gene is considered to be the equivalent of the dUTPase gene of the Alpha- and Gamma-herpesviruses. To characterize its function, the expression profiles of UL72 at both the RNA and the protein level were determined. The gene is expressed with a late kinetics and the corresponding UL72 46-kDa protein accumulates late during infection in the cytoplasm of infected cells. The pUL72 was expressed in E. coli and the purified recombinant protein did not display a detectable dUTPase activity. The viral yields of reconstituted HCMV RVDeltaUL72 viruses carrying a deletion within the UL72 ORF demonstrated a moderate growth defect following low MOI infections, whereas their DNA synthesis profiles were not significantly different from those of the parental HCMV RVAD169. These results demonstrate that the UL72 gene product is not a dUTPase and is not essential for replication in human fibroblasts.

The virally encoded chemokine receptors US28 from human cytomegalovirus and ORF74 from human herpesvirus 8 are both constitutively active. We show that both receptors constitutively activate the transcription factors nuclear factor of activated T cells (NFAT) and cAMP response element binding protein (CREB) and that both pathways are modulated by their respective endogenous receptor ligands. By addition of specific pathway modulators against the G protein subunit Galphai, phospholipase C, protein kinase C, calcineurin, p38 MAP kinase, and MEK1, we find that the constitutive and ligand-dependent inductions are mediated by multiple yet similar pathways in both receptors. The NFAT and CREB transcription factors and their upstream activators are known inducers of host and virally encoded genes. We propose that the activity of these virally encoded chemokine receptors coordinates host and potentially viral gene expression similarly. As ORF74 is a known inducer of neoplasia, these findings may have important implications for cytomegalovirus-associated pathogenicity.

The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAP II) ordinarily exists in electrophoretically distinct hypophosphorylated and hyperphosphorylated forms. Human cytomegalovirus infection induced forms of this subunit whose electrophoretic mobilities were intermediate without decreases in abundance of the original forms. Phosphatase treatment nearly eliminated the intermediate migrating forms. In vitro, the viral protein kinase, UL97, phosphorylated this subunit, a recombinant protein containing the CTD, and peptides containing the CTD consensus sequence, YSPTSPS. Phosphorylation occurred predominantly on serine 5 and was substantially reduced when either serine 2 or 5 was already phosphorylated. The abundance of the intermediate and hypophosphorylated forms was reduced at most twofold during infections in which UL97 was genetically or pharmacologically inhibited. These results identify a new pattern of RNA polymerase II modification induced by virus infection and a viral enzyme that phosphorylates the CTD in vitro, but only possibly in vivo.

Analysis of placentas infected with human cytomegalovirus (CMV) suggested that viral transmission could involve differentiating/invasive cytotrophoblasts in villi that attach the placenta to the uterine wall. To parse the cellular components in this process, we developed a coculture system of polarized uterine microvascular endothelial cell (UtMVEC) infection with an endothelial cell-tropic pathogenic strain of CMV. Then we evaluated the potential role of neutrophils and endothelial cells in the spread of infection to differentiating cytotrophoblasts. As shown by immunocytochemistry and analysis of viral replication, CMV preferentially infected endothelial cells via apical membranes and disrupted cell junction proteins, thereby altering paracellular permeability and cell polarity. Neutralizing antibodies to CMV glycoprotein B, an envelope component that facilitates virion penetration, blocked plaque formation in polarized UtMVEC. Neutrophils transmitted CMV infection to UtMVEC, which in turn infected cytotrophoblasts. However, neutrophils did not directly infect cytotrophoblasts. These findings implicate endothelial cells from the uterine microvasculature as a potential source for CMV infection of endovascular cytotrophoblasts of the anchoring villi. Possibly the cytokine/chemokine milieu in the pregnant uterus could attract immune cells that infect endothelial cells in hybrid fetal-maternal vessels. In turn, these cells could infect endovascular cytotrophoblasts, one possible initiation point of a cascade that results in retrograde placental CMV infection.

Human cytomegalovirus (HCMV) infection of fibroblasts induces the proinflammatory mediator macrophage migration inhibitory factor (MIF). Our in vitro experiments show that active HCMV infection alone is required to induce an early and sustained induction of MIF mRNA and protein production. Unlike in other infection models, in which MIF has been described to be released from preformed stores, our data conclusively show that HCMV infection triggers de novo synthesis and subsequent secretion of MIF. The kinetics of MIF protein production during HCMV infection points to an efficacious immune modulation, in which lymphocytes and monocytes are initially recruited by the early release of chemokines to sites of infection and locally activated by increasing concentrations of MIF.

A cell fusion assay using fusion-from-without (FFWO) recombinant adenoviruses (RAds) and specific antibody showed a role in fusion modulation for glycoprotein gO, the recently identified third component of the gH/gL gCIII complex of human cytomegalovirus (HCMV). As in HCMV, RAd gO expressed multiple glycosylated species with a mature product of 125 kDa. Coexpression with gH/gL RAds showed gCIII reconstitution in the absence of other HCMV products and stabilisation by intermolecular disulfide bonds. Properties of HCMV clinical isolate, Pt, also implicated gO in cell spread. Compared to laboratory strain AD169, Pt was resistant to gH antibody plaque inhibition, but mature gH was identical. However, the gO sequences were highly divergent (20%), with further variation in laboratory strain Towne gO (34%). Thus, gO forms gCIII with gH/gL, performs in cell fusion, and is a newly identified HCMV hypervariable locus which may influence gCIII's function in mediating infection.

Human cytomegalovirus (HCMV) causes a broad spectrum of clinical manifestations in immunocompromised patients, including infection of the gastrointestinal tract. To investigate the role of epithelial cells in the gastrointestinal HCMV disease, we used the intestinal epithelial cell line Caco-2, which is permissive for HCMV replication. In differentiated Caco-2 cells, we showed previously that HCMV infection proceeds preferentially from the basolateral membrane, suggesting that receptors for HCMV may be contained predominantly in the basolateral membrane (A. Esclatine et al., 2000, J. Virol. 74, 513-517). Therefore, we examined expression and localization in Caco-2 cells of heparan sulfate (HS) proteoglycan and annexin II, previously implicated in initial events of HCMV infection. We observed that annexin II is expressed in Caco-2 cells, but is not essential for entry of HCMV. We showed that, during the differentiation process, HS, initially present on the entire surface of the membrane of undifferentiated cells, ultimately became sequestered at the basolateral cell surface of fully differentiated cells. We established by biochemical assays that membrane-associated HS proteoglycan mediates both viral attachment to, and subsequent infection of, Caco-2 cells, regardless of the cell differentiation state. Thus, the redistribution of HS is implicated in the basolateral entry of HCMV into differentiated Caco-2 cells., (Copyright 2001 Academic Press.)