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The tryptophan metabolite, kynurenine, is known to be produced at elevated levels within human cytomegalovirus (HCMV)-infected fibroblasts. Kynurenine is an endogenous aryl hydrocarbon receptor (AhR) ligand. Here we show that the AhR is activated following HCMV infection, and pharmacological inhibition of AhR or knockdown of AhR RNA reduced the accumulation of viral RNAs and infectious progeny. RNA-seq analysis of infected cells following AhR knockdown showed that the receptor alters the levels of numerous RNAs, including RNAs related to cell cycle progression. AhR knockdown alleviated the G1/S cell cycle block that is normally instituted in HCMV-infected fibroblasts, consistent with its known ability to regulate cell cycle progression and cell proliferation. In sum, AhR is activated by kynurenine and perhaps other ligands produced during HCMV infection, it profoundly alters the infected-cell transcriptome, and one outcome of its activity is a block to cell cycle progression, providing mechanistic insight to a long-known element of the virus-host cell interaction., Competing Interests: The authors declare no competing interest.

Pericentromeric human satellite II (HSATII) repeats are normally silent but can be actively transcribed in tumor cells, where increased HSATII copy number is associated with a poor prognosis in colon cancer, and in human cytomegalovirus (HCMV)-infected fibroblasts, where the RNA facilitates viral replication. Here, we report that HCMV infection or treatment of ARPE-19 diploid epithelial cells with DNA-damaging agents, etoposide or zeocin, induces HSATII RNA expression, and a kinase-independent function of ATM is required for the induction. Additionally, various breast cancer cell lines growing in adherent, two-dimensional cell culture express HSATII RNA at different levels, and levels are markedly increased when cells are infected with HCMV or treated with zeocin. High levels of HSATII RNA expression correlate with enhanced migration of breast cancer cells, and knockdown of HSATII RNA reduces cell migration and the rate of cell proliferation. Our investigation links high expression of HSATII RNA to the DNA damage response, centered on a noncanonical function of ATM, and demonstrates a role for the satellite RNA in tumor cell proliferation and movement., Competing Interests: Competing interest statement: Icahn School of Medicine at Mt. Sinai and Princeton University have submitted a provisional patent application based on the use of HSATII-specific LNAs for HSATII knockdown, and M.T.N. and T.S. are co-inventors.

Human cytomegalovirus (HCMV) manipulates many aspects of host cell biology to create an intracellular milieu optimally supportive of its replication and spread. Our study reveals that levels of several components of the purinergic signaling system, including the P2Y2 and P2X5 receptors, are elevated in HCMV-infected fibroblasts. Knockdown and drug treatment experiments demonstrated that P2Y2 enhances the yield of virus, whereas P2X5 reduces HCMV production. The HCMV IE1 protein induces P2Y2 expression; and P2Y2-mediated signaling is important for efficient HCMV gene expression, DNA synthesis, and the production of infectious HCMV progeny. P2Y2 cooperates with the viral UL37x1 protein to regulate cystolic Ca 2+ levels. P2Y2 also regulates PI3K/Akt signaling and infected cell motility. Thus, P2Y2 functions at multiple points within the viral replication cycle to support the efficient production of HCMV progeny, and it may facilitate in vivo viral spread through its role in cell migration., Competing Interests: Conflict of interest statement: T.S. is a founder of Forge Life Science (sirtuin modulators as antivirals), ImmVira (oncolytic herpesviruses), and PMV Pharmaceuticals (structural correctors for mutant p53 proteins), and has stock holdings in those companies. T.S. is on the board of directors of MeiraGTx (ophthalmology, gene therapy) and Vical (antifungal agents), and receives fees, stock, and stock options from these companies. None of these activities is related to data presented in the manuscript.

Human CMV (HCMV) exhibits a broad cell tropism that depends on two virion glycoprotein complexes: a trimeric complex (gH/gL/gO) that facilitates viral infection primarily in fibroblasts and a pentameric complex (gH/gL/pUL128-pUL130-pUL131A) that mediates infection in epithelial and endothelial cells. We performed genome-wide CRISPR screens in which the PDGF receptor-α (PDGFRα) was identified as the most significant cellular gene product essential for infection by HCMV virions containing only trimeric complex (trimer-only virus). Trimer-only virus did not enter PDGFRα knockout fibroblasts. By using knockout fibroblasts, the extracellular domain of PDGFRα required for virus entry was mapped, and the intracellular tyrosine kinase domain was shown to be nonessential. In addition, direct cell-to-cell spread of virus from knockout cells transfected with trimer-only viral DNA was blocked, despite the production of infectious virus in the transfected cells. In contrast to trimer-only virus, wild-type HCMV virions containing both trimeric and pentameric complexes entered PDGFRα knockout cells, reinforcing the view that fibroblasts contain a second, independent receptor for the pentameric complex. Importantly, however, wild-type virus entered the knockout fibroblasts at reduced efficiency compared with parental fibroblasts, arguing that the cellular receptor for the virion pentameric complex is limiting or that virions are produced containing different relative amounts of the two glycoprotein complexes. Finally, ectopic expression of PDGFRα in ARPE-19 epithelial cells and THP-1 monocytic cells, which have little to no endogenous PDGFRα expression, markedly enhanced their susceptibility to trimer-only virions. In sum, our data clarify several key determinants of HCMV tropism., Competing Interests: The authors declare no conflict of interest.

Human cytomegalovirus (HCMV) is the prototypical human β-herpes virus. Here we perform a systems analysis of the HCMV host-cell transcriptome, using gene set enrichment analysis (GSEA) as an engine to globally map the host-pathogen interaction across two cell types. Our analysis identified several previously unknown signatures of infection, such as induction of potassium channels and amino acid transporters, derepression of genes marked with histone H3 lysine 27 trimethylation (H3K27me3), and inhibition of genes related to epithelial-to-mesenchymal transition (EMT). The repression of EMT genes was dependent on early viral gene expression and correlated with induction E-cadherin (CDH1) and mesenchymal-to-epithelial transition (MET) genes. Infection of transformed breast carcinoma and glioma stem cells similarly inhibited EMT and induced MET, arguing that HCMV induces an epithelium-like cellular environment during infection.

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.

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.

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.

Cell proteins can restrict the replication of viruses. Here, we identify the cellular BclAF1 protein as a human cytomegalovirus restriction factor and describe two independent mechanisms the virus uses to decrease its steady-state levels. Immediately following infection, the viral pp71 and UL35 proteins, which are delivered to cells within virions, direct the proteasomal degradation of BclAF1. Although BclAF1 reaccumulates through the middle stages of infection, it is subsequently down-regulated at late times by miR-UL112-1, a virus-encoded microRNA. In the absence of BclAF1 neutralization, viral gene expression and replication are inhibited. These data identify two temporally and mechanistically distinct functions used by human cytomegalovirus to down-regulate a cellular antiviral protein.

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.

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 has previously been shown to induce the accumulation of cyclooxygenase-2 RNA, protein, and enzyme activity. High doses of cyclooxygenase inhibitors substantially block viral replication in cultured fibroblasts. However, doses corresponding to the level of drug achieved in the plasma of patients have little effect on the replication of human cytomegalovirus in cultured cells. Here, we demonstrate that two nonsteroidal anti-inflammatory drugs, tolfenamic acid and indomethacin, markedly reduce direct cell-to-cell spread of human cytomegalovirus in cultured fibroblasts. The block is reversed by addition of prostaglandin E2, proving that it results from the action of the drugs on cyclooxygenase activity. Because direct cell-to-cell spread likely contributes importantly to pathogenesis of the virus, we suggest that nonsteroidal anti-inflammatory drugs might help to control human cytomegalovirus infections in conjunction with other anti-viral treatments.

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.

Human cytomegalovirus infects multiple cell types, including fibroblasts and epithelial cells. It penetrates fibroblasts by fusion at the cell surface but is endocytosed into epithelial cells. In this report, we demonstrate by electron microscopy that the virus uses two different routes to enter retinal pigmented epithelial cells, depending on the cell type in which the infecting virus was produced. Virus produced in epithelial cells preferentially fuses with the plasma membrane, whereas fibroblast-derived virus mostly enters by receptor-mediated endocytosis. Treatment of epithelial cells with agents that block endosome acidification inhibited infection by virus produced in fibroblasts but had only a modest effect on infection by virus from epithelial cells. Epithelial cell-generated virions had higher intrinsic "fusion-from-without" activity than fibroblast-generated particles, and the two virus preparations triggered different cellular signaling responses, as evidenced by markedly different transcriptional profiles. We propose that the cell type in which a human cytomegalovirus particle is produced likely influences its subsequent spread and its contribution to pathogenesis.

The human CMV UL37x1-encoded protein, also known as the viral mitochondria-localized inhibitor of apoptosis, traffics to the endoplasmic reticulum and mitochondria of infected cells. It induces the fragmentation of mitochondria and blocks apoptosis. We demonstrate that UL37x1 protein mobilizes Ca(2+) from the endoplasmic reticulum into the cytosol. This release is accompanied by cell rounding, cell swelling, and reorganization of the actin cytoskeleton, and these morphological changes can be substantially blocked by a Ca(2+) chelating agent. The UL37x1-mediated release of Ca(2+) from the endoplasmic reticulum likely has multiple consequences, including induction of the unfolded protein response, modulation of mitochondrial function, induction of mitochondrial fission, and protection against apoptotic stimuli.

The human cytomegalovirus immediate-early 5-kb RNA previously has been shown to be a stable intron that is not required for efficient replication of the virus in cultured fibroblasts. Here we describe a murine cytomegalovirus 7.2-kb ortholog of the human cytomegalovirus 5-kb RNA. The 5' end of the 7.2-kb transcript maps to a consensus splice-donor site that is conserved among all cytomegaloviruses. We constructed mutant viruses lacking the entire 7.2-kb coding domain, the splice-donor site predicted to function in the generation of the intron or a hairpin predicted to stabilize the intron. All three mutant viruses failed to produce the 7.2-kb RNA, supporting our conclusion that it is a stable intron. Each of the mutants replicated with normal kinetics in cultured fibroblasts, but the mutants exhibited a clear defect within infected mice. Although the initial acute phase at 4 days after infection appeared to be normal, none of the mutant viruses progressed to the persistent phase, i.e., little virus was detected in the salivary gland at 14 days after infection. The intron functions as an in vivo virulence factor, facilitating progression from the acute to persistent phase of infection.

The DNA damage checkpoint pathway responds to DNA damage and induces a cell cycle arrest to allow time for DNA repair. Several viruses are known to activate or modulate this cellular response. Here we show that the ataxia-telangiectasia mutated checkpoint pathway, which responds to double-strand breaks in DNA, is activated in response to human cytomegalovirus DNA replication. However, this activation does not propagate through the pathway; it is blocked at the level of the effector kinase, checkpoint kinase 2 (Chk2). Late after infection, several checkpoint proteins, including ataxia-telangiectasia mutated and Chk2, are mislocalized to a cytoplasmic virus assembly zone, where they are colocalized with virion structural proteins. This colocalization was confirmed by immunoprecipitation of virion proteins with an antibody that recognizes Chk2. Virus replication was resistant to ionizing radiation, which causes double-strand breaks in DNA. We propose that human CMV DNA replication activates the checkpoint response to DNA double-strand breaks, and the virus responds by altering the localization of checkpoint proteins to the cytoplasm and thereby inhibiting the signaling pathway.

Human cytomegalovirus replicates in many different cell types, including epithelial cells, endothelial cells, and fibroblasts. However, laboratory strains of the virus, many of which were developed as attenuated vaccine candidates by serial passage in fibroblasts, have lost the ability to infect epithelial and endothelial cells. Their growth is restricted primarily to fibroblasts, due to mutations in the UL131-UL128 locus. We now demonstrate that two products of this locus, pUL130 and pUL128, form a complex with gH and gL, but not gO. The AD169 laboratory strain, which lacks a functional UL131 protein, produces virions containing only the gH-gL-gO complex. An epithelial and endothelial cell tropic AD169 variant in which the UL131 ORF has been repaired, termed BADrUL131, produces virions that carry both gH-gL-gO and gH-gL-pUL128-pUL130 complexes. Antibodies against pUL130 and pUL128 block infection of epithelial and endothelial cells by BADrUL131 and the fusion-inducing factor X clinical human cytomegalovirus isolate but do not affect the efficiency with which fibroblasts are infected.

The human cytomegalovirus UL99-coded pp28 is a myristoylated phosphoprotein located in the virion tegument domain, which resides between the capsid and envelope. A previous study has demonstrated that BADsubUL99, a pp28-deficient mutant virus, fails to assemble enveloped virus particles. Capsids, coated with tegument proteins, accumulate in the cytoplasm of mutant virus-infected cells. This phenotype indicates that pp28 is required for the acquisition of an envelope; it presumably acts by directing tegument-associated capsids to bud through an intracellular membrane derived from the cell's secretory apparatus that has been modified to contain viral transmembrane glycoproteins. Here we demonstrate that BADsubUL99 can spread from cell to cell, even though highly sensitive assays fail to detect infectious virus progeny in cultures of infected fibroblasts. We propose that, in the absence of pp28, tegument-coated capsids might nevertheless bud through cellular membranes, including the plasma membrane. If this suggestion is correct, the enveloped particle could potentially infect an adjacent cell to mediate the cell-to-cell spread that is observed. This mode of spread might also occur after infection with wild-type virus, and it could facilitate immune evasion, assuming that the resulting particles do not have a normal complement of virus-coded envelope glycoproteins.

The human cytomegalovirus 72-kDa immediate-early (IE)1 and 86-kDa IE2 proteins are expressed at the start of infection, and they are believed to exert much of their function through promiscuous transcriptional activation of viral and cellular gene expression. Here, we show that the impaired growth of an IE1-deficient mutant virus in human fibroblasts is efficiently rescued by histone deacetylase (HDAC) inhibitors of three distinct chemical classes. In the absence of IE1 expression, the viral major IE and UL44 early promoters exhibited decreased de novo acetylation of histone H4 during the early phase of infection, and the hypoacetylation correlated with reduced transcription and accumulation of the respective gene products. Consistent with these findings, IE1 interacts specifically with HDAC3 within infected cells. We also demonstrate an interaction between IE2 and HDAC3. We propose that the ability to modify chromatin is fundamental to transcriptional activation by IE1 and, likely, IE2 as well.

The human cytomegalovirus pUL21.5 protein is a small, secreted glycoprotein whose mRNA is packaged into virions. We demonstrate that pUL21.5 protein is a soluble CC chemokine receptor that functions as a decoy to modulate the host immune response to infection. In contrast to other viral chemokine-binding proteins, which interact promiscuously with multiple chemokines, pUL21.5 selectively binds RANTES (regulated upon activation, normal T cell expressed and secreted) with high affinity. By binding RANTES, pUL21.5 blocks RANTES interaction with its cellular receptors. We propose that human cytomegalovirus directs the synthesis of a secreted, virus-coded protein that modulates the host antiviral response even before the newly infecting viral genome becomes transcriptionally active.

Human cytomegalovirus has a complex double-stranded DNA genome of approximately 240,000 bp that contains approximately 150 ORFs likely to encode proteins, most of whose functions are not well understood. We have used an infectious bacterial artificial chromosome to introduce 413 defined insertion and substitution mutations into the human cytomegalovirus AD169 genome by random and site-directed transposon mutagenesis. Mutations were produced in all unique ORFs with a high probability of encoding proteins for which mutants have not been previously documented and in many previously characterized ORFs. The growth of selected mutants was assayed in cultured human fibroblasts, and we now recognize 41 essential, 88 nonessential, and 27 augmenting ORFs. Most essential and augmenting genes are located in the central region, and nonessential genes generally cluster near the ends of the viral genome.

The initial interaction of human cytomegalovirus with fibroblasts triggers, and then partially blocks, an innate immune response pathway that leads to the induction of IFN-responsive genes and proinflammatory chemokines. Infection of fibroblasts with human cytomegalovirus inhibited their ability to respond to exogenous IFN. Consistent with the observation that the block did not depend on de novo viral protein synthesis, ectopic expression of the viral UL83-coded pp65, an abundant virion protein, inhibited IFN signaling. Furthermore, DNA array analysis showed that infection with a pp65-deficient mutant virus caused a much stronger induction of many IFN-response and proinflammatory chemokine RNAs than infection with wild-type virus. The nuclear DNA-binding activities of transcription factors NF-kappaB and IRF1 were induced to a much greater extent after infection with the pp65-deficient mutant than with wild-type virus. IFN-stimulated gene factor 3 DNA-binding was modestly enhanced, whereas IRF3 activity was not affected by mutation of pp65. Together, these results imply that pp65, which is delivered to newly infected cells in the virion, antagonizes a pathway that affects NF-kappaB and IRF1 and prevents the accumulation of mRNAs encoded by numerous cellular antiviral genes.

Most of the substrates degraded by the proteasome are marked with polyubiquitin chains. However, there are a limited number of examples of nonubiquitinated proteins that are degraded by the proteasome. Here, we describe the degradation of the retinoblastoma family of tumor suppressor proteins by the proteasome in the absence of polyubiquitination. The retinoblastoma protein (p105), p107, and p130 are each targeted for degradation by the pp71 protein, which is encoded by the UL82 gene of human cytomegalovirus. It functions to direct their degradation in the absence of other viral proteins. While the pp71-mediated degradation of the retinoblastoma family of proteins requires proteasome function, it occurs without the attachment of ubiquitin to the substrates and in the absence of a functioning ubiquitin-conjugation system.

Human cytomegalovirus (HCMV) resides latently in hematopoietic cells of the bone marrow. Although viral genomes can be found in CD14+ monocytes and CD34+ progenitor cells, the primary reservoir for latent cytomegalovirus is unknown. We analyzed human hematopoietic subpopulations infected in vitro with a recombinant virus that expresses a green fluorescent protein marker gene. Although many hematopoietic cell subsets were infected in vitro, CD14+ monocytes and various CD34+ subpopulations were infected with the greatest efficiency. We have developed an in vitro system in which to study HCMV infection and latency in CD34+ cells cultured with irradiated stromal cells. Marker gene expression was substantially reduced by 4 days postinfection, and infectious virus was not made during the culture period. However, viral DNA sequences were maintained in infected CD34+ cells for >20 days in culture, and, importantly, virus replication could be reactivated by coculture with human fibroblasts. Using an HCMV gene array, we examined HCMV gene expression in CD34+ cells. The pattern of viral gene expression was distinct from that observed during productive or nonproductive infections. Some of these expressed viral genes may function in latency and are targets for further analysis. Altered gene expression in hematopoietic progenitors may be indicative of the nature and outcome of HCMV infection.

The M78 protein of murine cytomegalovirus exhibits sequence features of a G protein-coupled receptor. It is synthesized with early kinetics, it becomes partially colocalized with Golgi markers, and it is incorporated into viral particles. We have constructed a viral substitution mutant, SMsubM78, which lacks most of the M78 ORF. The mutant produces a reduced yield in cultured 10.1 fibroblast and IC21 macrophage cell lines. The defect is multiplicity dependent and greater in the macrophage cell line. Consistent with its growth defect in cultured cells, the mutant exhibits reduced pathogenicity in mice, generating less infectious progeny than wild-type virus in all organs assayed. SMsubM78 fails to efficiently activate accumulation of the viral m123 immediate-early mRNA in infected macrophages. M78 facilitates the accumulation of the immediate-early mRNA in cycloheximide-treated cells, arguing that it acts in the absence of de novo protein synthesis. We conclude that the M78 G protein-coupled receptor homologue is delivered to cells as a constituent of the virion, and it acts to facilitate the accumulation of immediate-early mRNA.

The human cytomegalovirus UL82 gene encodes a protein (pp71) that is localized in the tegument domain of the virus particle. The UL82 gene product is delivered to the nucleus at the time of infection, and it is believed to function in gene activation. We have constructed a human cytomegalovirus mutant, ADsubUL82, that lacks a substantial portion of the UL82 coding region. It was propagated on human diploid fibroblasts expressing the UL82 gene product, and it was possible to produce a mutant virus lacking the UL82 protein by passaging virus stocks for one cycle of growth on normal, noncomplementing fibroblasts. The UL82-deficient mutant displays a multiplicity-dependent growth defect in normal human fibroblasts. The growth of ADsubUL82 is severely restricted at low input multiplicities (0.01-0.1 plaque-forming units per cell), producing a yield that is reduced by a factor of about 10(5) in comparison to wild-type virus. At higher input multiplicities (10 plaque-forming units per cell), ADsubUL82 grew nearly as well as the wild-type virus. By using a human cytomegalovirus gene array, we demonstrated that UL82 functions to facilitate virus mRNA accumulation very early during the human cytomegalovirus replication cycle. The growth phenotype associated with the UL82 mutant seems to result from its inability to efficiently activate human cytomegalovirus immediate early genes.

Human cytomegalovirus blocks cell-cycle progression in the G(1) compartment upon infection of primary human fibroblasts. The virus-coded UL69 protein can institute a G(1) block when expressed in cells in the absence of virus infection. We have constructed a cytomegalovirus mutant, TNsubUL69, that lacks the UL69 coding region. This virus grows slowly in fibroblasts, but produces a wild-type yield after an extended delay. It grows with normal kinetics in cells coinfected with a recombinant retrovirus, retroUL69, which expresses UL69 protein, demonstrating that its growth defect results from the mutation in the UL69 gene. UL69 protein is packaged within virus particles, and it was possible for us to produce two types of virus stocks. TNsubUL69(+pUL69) lacks the UL69 gene but contains UL69 protein in virus particles. It is produced by growth in fibroblasts that are coinfected with retroUL69. TNsubUL69(-pUL69) lacks the UL69 gene and protein. It is produced by growth in fibroblasts that do not contain UL69 protein. The mutant virions lacking both the UL69 gene and protein fail to induce a cell-cycle block with normal efficiency, whereas the mutant particles lacking the gene but containing the protein can institute the block. These results are consistent with the view that the UL69 protein contributes to the cytomegalovirus-induced cell-cycle block, and they suggest that UL69 protein delivered to cells within virions can induce the block without the synthesis of additional UL69 protein encoded by the infecting viral genome.

Recently, several proteins have been identified that are related in their sequence to the p53 tumor-suppressor protein. One of these proteins, which is termed p73, exhibits sequence homology to the p53 transcriptional activation, DNA binding, and oligomerization domains. The adenovirus E1B 55-kDa protein, the adenovirus E4orf6 protein, and SV40 T antigen each can bind to p53 and inhibit p53 function. Here we demonstrate that the adenovirus E4orf6 protein, but not the E1B 55-kDa protein or T antigen, interacts with p73. The E4orf6 protein inhibits p73-mediated transcriptional activation and cell killing in a manner similar to its effect on p53. Thus, only a subset of viral oncoproteins that antagonize p53 function also interacts with the related p73 protein.