Human Cytomegalovirus Proteins Research Articles

Decreased human leukocyte antigen A*02:01 frequency is associated with risk of glioma and existence of human cytomegalovirus: a case-control study in Northern China.

Human leukocyte antigens (HLAs) play an important role in host defense against viral infection and tumorigenesis. Human cytomegalovirus (HCMV) has been linked to glioma development. This study investigated the relationship between HLA distribution, presence of HCMV, and glioma development in a Han Chinese population. The study population included 150 glioma patients and 150 tumor-free brain injury control subjects (control-A) matched according to geography, ethnicity, age, and gender. HLA allele frequency was compared between the two groups using peripheral blood samples by PCR sequence-based typing. These data were also compared with HLA frequencies obtained from a Northern Chinese Han population database (control-B). HCMV DNA was detected in the peripheral blood of glioma patients and control group-A by nested PCR. The expression of HCMV proteins IE1-72 and pp65 in tumor tissues was evaluated by immunohistochemistry. The frequency of HLA-A*02:01 was decreased in glioma patients as compared to control group-A and -B (P<0.001 and P=0.001, respectively). The age/sex-adjusted odds ratio for HLA-A*02:01 positivity vs. negativity was 0.392 (95% confidence interval 0.225-0.683). HCMV was more frequently detected in the peripheral blood and tumor tissue of HLA-A*02:01-negative glioma patients. HLA-A*02:01 and HCMV were not associated with overall survival. There is a correlation between decreased HLA-A*0201 allele frequency and glioma susceptibility.

Low Expression of Estrogen Receptor-α and Progesterone Receptor in Human Breast Cancer Tissues Is Associated With High-Grade Human Cytomegalovirus Protein Expression

BackgroundThe underlying mechanisms for breast cancer (BC) are largely unknown. We investigated possible correlations between the expression levels of human cytomegalovirus (HCMV) proteins and established histopathological markers of BC, including expression of estrogen receptor (ER)-α, the progesterone receptor (PgR), and HER2. Materials and MethodsWe retrospectively examined paraffin-embedded biopsy specimens of BC (n = 62), ductal carcinoma in situ (n = 19), and adjacent normal breast tissue (n = 42) for HCMV immediate-early protein (IE), HCMV late antigen, HCMV DNA and RNA, and investigated possible correlations between them and expression of ER-α, PgR, and HER2. ResultsHCMV DNA and RNA were detected in all examined infiltrating BCs. High-grade positivity for HCMV-IE was detected in 77% of infiltrating BCs, 39% of ductal carcinomas in situ, and 7% of tumor-free breast tissue samples. HCMV expression correlated inversely with ER-α (P = .02) and PgR (P = .003) expression. HER2 expression was also reduced in HCMV-positive samples without reaching a level of statistical significance (P = .09). ConclusionThe negative correlation between high-grade expression HCMV-IE and hormone receptor expression suggests a role for HCMV in hormone receptor-negative BC tumors, possibly by forcing BC cells into a more aggressive phenotype.

RO0504985 is an inhibitor of CMGC kinase proteins and has anti-human cytomegalovirus activity

Public-private partnerships allow many previously unavailable compounds to be screened for antiviral activity. Here a screening method was used to identify an oxindole compound, RO0504985, from a Roche kinase inhibitor library that inhibited human cytomegalovirus (HCMV) protein production. RO0504985 was previously described as an inhibitor of cyclin-dependent kinase 2 (CDK2). However, using kinase selectivity assays it was found that RO0504985 was an inhibitor of several CMGC group kinase proteins, including CDK2. Using virus yield reduction assays it was observed that RO0504985 inhibited replication of different HCMV strains at low micromolar concentrations. Western blotting was used to investigate how RO0504985 inhibited HCMV replication. Treatment of HCMV infected cells with RO0504985 inhibited production of the immediate early viral IE2 proteins and the late viral protein pp28. Thus, RO0504985 inhibited HCMV replication by preventing production of specific HCMV proteins necessary for virus replication.

The ND10 Component Promyelocytic Leukemia Protein Acts as an E3 Ligase for SUMOylation of the Major Immediate Early Protein IE1 of Human Cytomegalovirus.

Previous studies identified the nuclear domain 10 (ND10) components promyelocytic leukemia protein (PML), hDaxx, and Sp100 as factors of an intrinsic immune response against human cytomegalovirus (HCMV). This antiviral function of ND10, however, is antagonized by viral effector proteins like IE1p72, which induces dispersal of ND10. Furthermore, we have shown that both major immediate early proteins of HCMV, IE1p72 and IE2p86, transiently colocalize with ND10 subnuclear structures and undergo modification by the covalent attachment of SUMO. Since recent reports indicate that PML acts as a SUMO E3 ligase, we asked whether the SUMOylation of IE1p72 and IE2p86 is regulated by PML. To address this, PML-depleted fibroblasts, as well as cells overexpressing individual PML isoforms, were infected with HCMV. Western blot experiments revealed a clear correlation between the degree of IE1p72 SUMO conjugation and the abundance of PML. On the other hand, the SUMOylation of IE2p86 was not affected by PML. By performing in vitro SUMOylation assays, we were able to provide direct evidence that IE1p72 is a substrate for PML-mediated SUMOylation. Interestingly, disruption of the RING finger domain of PML, which is proposed to confer SUMO E3 ligase activity, abolished PML-induced SUMOylation of IE1p72. In contrast, IE1p72 was still efficiently SUMO modified by a SUMOylation-defective PML mutant, indicating that intact ND10 bodies are not necessary for this effect. Thus, this is the first report that the E3 ligase PML is capable of stimulating the SUMOylation of a viral protein which is supposed to serve as a cellular mechanism to compromise specific functions of IE1p72.IMPORTANCE The major immediate early proteins of human cytomegalovirus, termed IE1p72 and IE2p86, have previously been shown to undergo posttranslational modification by covalent coupling to SUMO moieties at specific lysine residues. However, the enzymatic activities that are responsible for this modification have not been identified. Here, we demonstrate that the PML protein, which mediates an intrinsic immune response against HCMV, specifically serves as an E3 ligase for SUMO modification of IE1p72. Since SUMO modification of IE1p72 has previously been shown to interfere with STAT factor binding, thus compromising the interferon-antagonistic function of this viral effector protein, our finding highlights an additional mechanism through which PML is able to restrict viral infections.

Human cytomegalovirus-encoded viral cyclin-dependent kinase (v-CDK) UL97 phosphorylates and inactivates the retinoblastoma protein-related p107 and p130 proteins

The human cytomegalovirus (HCMV)-encoded viral cyclin-dependent kinase (v-CDK) UL97 phosphorylates the retinoblastoma (Rb) tumor suppressor. Here, we identify the other Rb family members p107 and p130 as novel targets of UL97. UL97 phosphorylates p107 and p130 thereby inhibiting their ability to repress the E2F-responsive E2F1 promoter. As with Rb, this phosphorylation, and the rescue of E2F-responsive transcription, is dependent on the L1 LXCXE motif in UL97 and its interacting clefts on p107 and p130. Interestingly, UL97 does not induce the disruption of all p107-E2F or p130-E2F complexes, as it does to Rb-E2F complexes. UL97 strongly interacts with p107 but not Rb or p130. Thus the inhibitory mechanisms of UL97 for Rb family protein-mediated repression of E2F-responsive transcription appear to differ for each of the Rb family proteins. The immediate early 1 (IE1) protein of HCMV also rescues p107- and p130-mediated repression of E2F-responsive gene expression, but it does not induce their phosphorylation and does not disrupt p107-E2F or p130-E2F complexes. The unique regulation of Rb family proteins by HCMV UL97 and IE1 attests to the importance of modulating Rb family protein function in HCMV-infected cells.

Mechanism of Protein Kinase R Inhibition by Human Cytomegalovirus pTRS1.

Double-stranded RNAs (dsRNA) produced during human cytomegalovirus (HCMV) infection activate the antiviral kinase protein kinase R (PKR), which potently inhibits virus replication. The HCMV pTRS1 and pIRS1 proteins antagonize PKR to promote HCMV protein synthesis and replication; however, the mechanism by which pTRS1 inhibits PKR is unclear. PKR activation occurs in a three-step cascade. First, binding to dsRNA triggers PKR homodimerizaton. PKR dimers then autophosphorylate, leading to a conformational shift that exposes the binding site for the PKR substrate eIF2α. Consistent with previous in vitro studies, we found that pTRS1 bound and inhibited PKR. pTRS1 binding to PKR was not mediated by an RNA intermediate, and mutations in the pTRS1 RNA binding domain did not affect PKR binding or inhibition. Rather, mutations that disrupted the pTRS1 interaction with PKR ablated the ability of pTRS1 to antagonize PKR activation by dsRNA. pTRS1 did not block PKR dimerization and could bind and inhibit a constitutively dimerized PKR kinase domain. In addition, pTRS1 binding to PKR inhibited PKR kinase activity. Single amino acid point mutations in the conserved eIF2α binding domain of PKR disrupted pTRS1 binding and rendered PKR resistant to inhibition by pTRS1. Consistent with a critical role for the conserved eIF2α contact site in PKR binding, pTRS1 bound an additional eIF2α kinase, heme-regulated inhibitor (HRI), and inhibited eIF2α phosphorylation in response to an HRI agonist. Together our data suggest that pTRS1 inhibits PKR by binding to conserved amino acids in the PKR eIF2α binding site and blocking PKR kinase activity.IMPORTANCE The antiviral kinase PKR plays a critical role in controlling HCMV replication. This study furthered our understanding of how HCMV evades inhibition by PKR and identified new strategies for how PKR activity might be restored during infection to limit HCMV disease.

Abstract P6-06-02: Low expression levels of hormone receptors (ER-α and PGR) in human breast cancer samples is significantly associated with high-grade human cytomegalovirus-IEA

Abstract P6-06-02: Low expression levels of hormone receptors (ER-α and PGR) in human breast cancer samples is significantly associated with high-grade human cytomegalovirus-IEA

Human cytomegalovirus interleukin-10 enhances matrigel invasion of MDA-MB-231 breast cancer cells

BackgroundWhile some risk factors for breast cancer are well-known, the influence of other factors, particularly virus infection, remains unclear. Human cytomegalovirus (HCMV) is widespread in the general population, and both molecular and epidemiological evidence has indicated links between HCMV and breast cancer. The HCMV protein cmvIL-10 is a potent suppressor of immune function that has also been shown to promote proliferation and migration of breast cancer cells. In this study, the impact of cmvIL-10 on tumor cell invasion through a simulated basement membrane was investigated.ResultsMDA-MB-231 breast cancer cells exhibited invasion through a matrigel layer that was significantly enhanced in the presence of either purified cmvIL-10 or supernatants from HCMV-infected cells containing secreted cmvIL-10. Transcriptional profiling revealed that cmvIL-10 altered expression of several genes implicated in metastasis. Exposure to cmvIL-10 resulted in higher MMP-3 mRNA levels, greater protein expression, and increased enzymatic activity. Treatment with cmvIL-10 also increased expression of both urokinase plasminogen receptor (uPAR) and plasminogen activator inhibitor-1 (PAI-1), which can stimulate MMP-3 activity and have previously been identified as poor prognostic markers in breast cancer patients. Finally, MDA-MB-231 cells treated with cmvIL-10 showed significant downregulation of metastasis suppressor 1 (MTSS1), a scaffolding protein that regulates cytoskeletal rearrangements and is frequently lost in metastatic tumors.ConclusionsHCMV, and in particular the secreted viral cytokine, cmvIL-10, can induce cellular changes that facilitate cell migration and invasion. These findings indicate that HCMV may be associated with promoting the malignant spread of breast cancer cells and suggest that antiviral treatment may be a useful complement to chemotherapy in some patients.

Rare Human Codons and HCMV Translational Regulation

Restriction of protein synthesis characterizes human cytomegalovirus (HCMV) latency in the human host. In analyzing the molecular factors that hinder HCMV expression, the present study shows that HCMV genes frequently use 6 rare codons, i.e., GCG (Ala), CCG (Pro), CGT (Arg), CGC (Arg), TCG (Ser), and ACG (Thr). In some instances, the rare host codons are clustered along viral nucleotide sequences and represent the majority in sequences encoding short alanine and proline repeats. Given the positive correlation between codon usage, tRNA content, and protein production, the results support the hypothesis that HCMV usage of rare human codons might hinder HCMV protein synthesis, in this way leading to HCMV latency.

The E3 Ubiquitin Ligase TMEM129 Is a Tri-Spanning Transmembrane Protein.

Misfolded proteins from the endoplasmic reticulum (ER) are transported back into the cytosol for degradation via the ubiquitin-proteasome system. The human cytomegalovirus protein US11 hijacks this ER-associated protein degradation (ERAD) pathway to downregulate human leukocyte antigen (HLA) class I molecules in virus-infected cells, thereby evading elimination by cytotoxic T-lymphocytes. Recently, we identified the E3 ubiquitin ligase transmembrane protein 129 (TMEM129) as a key player in this process, where interference with TMEM129 activity in human cells completely abrogates US11-mediated class I degradation. Here, we set out to further characterize TMEM129. We show that TMEM129 is a non-glycosylated protein containing a non-cleaved signal anchor sequence. By glycosylation scanning mutagenesis, we show that TMEM129 is a tri-spanning ER-membrane protein that adopts an Nexo–Ccyto orientation. This insertion in the ER membrane positions the C-terminal really interesting new gene (RING) domain of TMEM129 in the cytosol, making it available to catalyze ubiquitination reactions that are required for cytosolic degradation of secretory proteins.

Persistent Cytomegalovirus Infection in Amniotic Membranes of the Human Placenta

Human cytomegalovirus (HCMV) is the leading viral cause of birth defects, including microcephaly, neurological deficits, hearing impairment, and vision loss. We previously reported that epithelial cells in amniotic membranes of placentas from newborns with intrauterine growth restriction and underlying congenital HCMV infection contain viral proteins in cytoplasmic vesicles. Herein, we immunostained amniotic membranes from 51 placentas from symptomatic and asymptomatic congenital infection with HCMV DNA in amniotic fluid and/or newborn saliva, intrauterine growth restriction, preterm deliveries, and controls. We consistently observed HCMV proteins in amniotic epithelial cells (AmEpCs) from infected placentas, sometimes with aberrant morphology. Primary AmEpCs isolated from mid-gestation placentas infected with pathogenic VR1814 proliferated and released infectious progeny for weeks, producing higher virus titers than late-gestation cells that varied by donor. In contrast to intact virion assembly compartments in differentiated retinal pigment epithelial cells, infected AmEpCs made dispersed multivesicular bodies. Primary AmEpCs and explants of amniochorionic membranes from mid-gestation placentas formed foci of infection, and interferon-β production was prolonged. Infected AmEpCs up-regulated anti-apoptotic proteins survivin and Bcl-xL by mechanisms dependent and independent of the activated STAT3. Amniotic membranes naturally expressed both survivin and Bcl-xL, indicating that fetal membranes could foster persistent viral infection. Our results suggest strengthening innate immune responses and reducing viral functions could suppress HCMV infection in the fetal compartment.

Human Cytomegalovirus DNA Quantification and Gene Expression in Gliomas of Different Grades.

Gliomas are the most common type of primary brain tumors. The most aggressive type, Glioblastoma multiforme (GBM), is one of the deadliest human diseases, with an average survival at diagnosis of about 1 year. Previous evidence suggests a link between human cytomegalovirus (HCMV) and gliomas. HCMV has been shown to be present in these tumors and several viral proteins can have oncogenic properties in glioma cells. Here we have investigated the presence of HCMV DNA, RNA and proteins in fifty-two gliomas of different grades of malignancy. The UL83 viral region, the early beta 2.7 RNA and viral protein were detected in 73%, 36% and 57% by qPCR, ISH and IHC, respectively. Positivity of the viral targets and viral load was independent of tumor type or grade suggesting no correlation between viral presence and tumor progression. Our results demonstrate high prevalence of the virus in gliomas from Brazilian patients, contributing to a better understanding of the association between HCMV infection and gliomas worldwide and supporting further investigations of the virus oncomodulatory properties.

Repair of a Mutation Disrupting the Guinea Pig Cytomegalovirus Pentameric Complex Acquired during Fibroblast Passage Restores Pathogenesis in Immune-Suppressed Guinea Pigs and in the Context of Congenital Infection

Guinea pig cytomegalovirus (GPCMV) provides a valuable model for congenital cytomegalovirus transmission. Salivary gland (SG)-passaged stocks of GPCMV are pathogenic, while tissue culture (TC) passage in fibroblasts results in attenuation. Nonpathogenic TC-derived virus N13R10 (cloned as a bacterial artificial chromosome [BAC]) has a 4-bp deletion that disrupts GP129, which encodes a subunit of the GPCMV pentameric complex (PC) believed to govern viral entry into select cell types, and GP130, an overlapping open reading frame (ORF) of unknown function. To determine if this deletion contributes to attenuation of N13R10, markerless gene transfer in Escherichia coli was used to construct virus r129, a variant of N13R10 in which the 4-bp deletion is repaired. Virions from r129 were found to contain GP129 as well as two other PC subunit proteins, GP131 and GP133, whereas these three PC subunits were absent from N13R10 virions. Replication of r129 in fibroblasts appeared unaltered compared to that of N13R10. However, following experimental challenge of immunocompromised guinea pigs, r129 induced significant weight loss, longer duration of viremia, and dramatically higher (up to 1.5 × 10(6)-fold) viral loads in blood and end organs compared to N13R10. In pregnant guinea pigs, challenge with doses of r129 virus of ≥5 × 10(6) PFU resulted in levels of maternal viremia, congenital transmission, pup viral loads, intrauterine growth restriction, and pup mortality comparable to that induced by pathogenic SG virus, although higher doses of r129 were required. These results suggest that the GP129-GP130 mutation is a significant contributor to attenuation of N13R10, likely by abrogating expression of a functional PC. Tissue culture adaptation of cytomegaloviruses rapidly selects for mutations, deletions, and rearrangements in the genome, particularly for viruses passaged in fibroblast cells. Some of these mutations are focused in the region of the genome encoding components of the pentameric complex (PC), in particular homologs of human cytomegalovirus (HCMV) proteins UL128, UL130, and UL131A. These mutations can attenuate the course of infection when the virus is reintroduced into animals for vaccine and pathogenesis studies. This study demonstrates that a deletion that arose during the process of tissue culture passage can be repaired, with subsequent restoration of pathogenicity, using BAC-based mutagenesis. Restoration of pathogenicity by repair of a frameshift mutation in GPCMV gene GP129 using this approach provides a valuable genetic platform for future studies using the guinea pig model of congenital CMV infection.

Human cytomegalovirus infection in tumor specimens of Iranian patients with glioma

Human cytomegalovirus (HCMV) causes persistent infection in humans and severe diseases in fetus andimmunocompromised individuals. Although HCMV is not currently implicated in human cancer, emerging evidence suggests that HCMV infection might be specifically associated with some human malignancies including glioma. Glioma is one of the most common brain tumors affecting children and adults. In this study, we used Real-Time (RT) PCR and immunohistochemistry techniques for detection of HCMV infection in glioma brain tumor biopsies. Paraffin embedded tumor tissues were obtained from patients who had been diagnosed with glioma. After designing of specific primers for the HCMV US28 region, a RT-PCR method was developed for HCMV DNA detection. Immunohistochemistry was performed on the same samples by using monoclonal antibodies specific for immediate earlyprotein (IE)-72 and IE 86 protein of HCMV. The results of RT-PCR on 4 of 18 patients (22/2 %) were positive. Two of the patients with HCMV positive RT-PCR results, passed away. Seven patients (38.8%) were positive with the IHC assay. It was also shown that in patients with higher grade of glioma, higher level of positive cells was observed using IE72 and IE 86 antibodies. Considering the results and controversies associated with reports from other regions of the world, a more comprehensive study using this and other diagnostic methods are suggested in Iranian patients with glioma.

Intracellular Distribution of Capsid-Associated pUL77 of Human Cytomegalovirus and Interactions with Packaging Proteins and pUL93.

DNA packaging into procapsids is a common multistep process during viral maturation in herpesviruses. In human cytomegalovirus (HCMV), the proteins involved in this process are terminase subunits pUL56 and pUL89, which are responsible for site-specific cleavage and insertion of the DNA into the procapsid via portal protein pUL104. However, additional viral proteins are required for the DNA packaging process. We have shown previously that the plasmid that encodes capsid-associated pUL77 encodes another potential player during capsid maturation. Pulse-chase experiments revealed that pUL77 is stably expressed during HCMV infection. Time course analysis demonstrated that pUL77 is expressed in the early late part of the infectious cycle. The sequence of pUL77 was analyzed to find nuclear localization sequences (NLSs), revealing monopartite NLSm at the N terminus and bipartite NLSb in the middle of pUL77. The potential NLSs were inserted into plasmid pHM829, which encodes a chimeric protein with β-galactosidase and green fluorescent protein. In contrast to pUL56, neither NLSm nor NLSb was sufficient for nuclear import. Furthermore, we investigated by coimmunoprecipitation whether packaging proteins, as well as pUL93, the homologue protein of herpes simplex virus 1 pUL17, are interaction partners of pUL77. The interactions between pUL77 and packaging proteins, as well as pUL93, were verified. We showed that the capsid-associated pUL77 is another potential player during capsid maturation of HCMV. Protein UL77 (pUL77) is a conserved core protein of HCMV. This study demonstrates for the first time that pUL77 has early-late expression kinetics during the infectious cycle and an intrinsic potential for nuclear translocation. According to its proposed functions in stabilization of the capsid and anchoring of the encapsidated DNA during packaging, interaction with further DNA packaging proteins is required. We identified physical interactions with terminase subunits pUL56 and pUL89 and another postulated packaging protein, pUL93, in infected, as well as transfected, cells.

The Essential Human Cytomegalovirus Proteins pUL77 and pUL93 Are Structural Components Necessary for Viral Genome Encapsidation.

Several essential viral proteins are proposed to participate in genome encapsidation of human cytomegalovirus (HCMV), among them pUL77 and pUL93, which remain largely uncharacterized. To gain insight into their properties, we generated an HCMV mutant expressing a pUL77-monomeric enhanced green fluorescent protein (mGFP) fusion protein and a pUL93-specific antibody. Immunoblotting demonstrated that both proteins are incorporated into capsids and virions. Conversely to data suggesting internal translation initiation sites within the UL93 open reading frame (ORF), we provide evidence that pUL93 synthesis commences at the first start codon. In infected cells, pUL77-mGFP was found in nuclear replication compartments and dot-like structures, colocalizing with capsid proteins. Immunogold labeling of nuclear capsids revealed that pUL77 is present on A, B, and C capsids. Pulldown of pUL77-mGFP revealed copurification of pUL93, indicating interaction between these proteins, which still occurred when capsid formation was prevented. Correct subnuclear distribution of pUL77-mGFP required pUL93 as well as the major capsid protein (and thus probably the presence of capsids), but not the tegument protein pp150 or the encapsidation protein pUL52, demonstrating that pUL77 nuclear targeting occurs independently of the formation of DNA-filled capsids. When pUL77 or pUL93 was missing, generation of unit-length genomes was not observed, and only empty B capsids were produced. Taken together, these results show that pUL77 and pUL93 are capsid constituents needed for HCMV genome encapsidation. Therefore, the task of pUL77 seems to differ from that of its alphaherpesvirus orthologue pUL25, which exerts its function subsequent to genome cleavage-packaging. The essential HCMV proteins pUL77 and pUL93 were suggested to be involved in viral genome cleavage-packaging but are poorly characterized both biochemically and functionally. By producing a monoclonal antibody against pUL93 and generating an HCMV mutant in which pUL77 is fused to a fluorescent protein, we show that pUL77 and pUL93 are capsid constituents, with pUL77 being similarly abundant on all capsid types. Each protein is required for genome encapsidation, as the absence of either pUL77 or pUL93 results in a genome packaging defect with the formation of empty capsids only. This distinguishes pUL77 from its alphaherpesvirus orthologue pUL25, which is enriched on DNA-filled capsids and exerts its function after the viral DNA is packaged. Our data for the first time describe an HCMV mutant with a fluorescent capsid and provide insight into the roles of pUL77 and pUL93, thus contributing to a better understanding of the HCMV encapsidation network.

Analysis of the role of autophagy inhibition by two complementary human cytomegalovirus BECN1/Beclin 1-binding proteins

ABSTRACTAutophagy is activated early after human cytomegalovirus (HCMV) infection but, later on, the virus blocks autophagy. Here we characterized 2 HCMV proteins, TRS1 and IRS1, which inhibit autophagy during infection. Expression of either TRS1 or IRS1 was able to block autophagy in different cell lines, independently of the EIF2S1 kinase, EIF2AK2/PKR. Instead, TRS1 and IRS1 interacted with the autophagy protein BECN1/Beclin 1. We mapped the BECN1-binding domain (BBD) of IRS1 and TRS1 and found it to be essential for autophagy inhibition. Mutant viruses that express only IRS1 or TRS1 partially controlled autophagy, whereas a double mutant virus expressing neither protein stimulated autophagy. A mutant virus that did not express IRS1 and expressed a truncated form of TRS1 in which the BBD was deleted, failed to control autophagy. However, this mutant virus had similar replication kinetics as wild-type virus, suggesting that autophagy inhibition is not critical for viral replication. In fact, using pharmacological modulators of autophagy and inhibition of autophagy by shRNA knockdown, we discovered that stimulating autophagy enhanced viral replication. Conversely, inhibiting autophagy decreased HCMV infection. Thus, our results demonstrate a new proviral role of autophagy for a DNA virus.

Human Cytomegalovirus pTRS1 and pIRS1 Antagonize Protein Kinase R To Facilitate Virus Replication.

Human cytomegalovirus (HCMV) counteracts host defenses that otherwise act to limit viral protein synthesis. One such defense is the antiviral kinase protein kinase R (PKR), which inactivates the eukaryotic initiation factor 2 (eIF2) translation initiation factor upon binding to viral double-stranded RNAs. Previously, the viral TRS1 and IRS1 proteins were found to antagonize the antiviral kinase PKR outside the context of HCMV infection, and the expression of either pTRS1 or pIRS1 was shown to be necessary for HCMV replication. In this study, we found that expression of either pTRS1 or pIRS1 is necessary to prevent PKR activation during HCMV infection and that antagonism of PKR is critical for efficient viral replication. Consistent with a previous study, we observed decreased overall levels of protein synthesis, reduced viral protein expression, and diminished virus replication in the absence of both pTRS1 and pIRS1. In addition, both PKR and eIF2α were phosphorylated during infection when pTRS1 and pIRS1 were absent. We also found that expression of pTRS1 was both necessary and sufficient to prevent stress granule formation in response to eIF2α phosphorylation. Depletion of PKR prevented eIF2α phosphorylation, rescued HCMV replication and protein synthesis, and reversed the accumulation of stress granules in infected cells. Infection with an HCMV mutant lacking the pTRS1 PKR binding domain resulted in PKR activation, suggesting that pTRS1 inhibits PKR through a direct interaction. Together our results show that antagonism of PKR by HCMV pTRS1 and pIRS1 is critical for viral protein expression and efficient HCMV replication. To successfully replicate, viruses must counteract host defenses that limit viral protein synthesis. We have identified inhibition of the antiviral kinase PKR by the viral proteins TRS1 and IRS1 and shown that this is a critical step in HCMV replication. Our results suggest that inhibiting pTRS1 and pIRS1 function or restoring PKR activity during infection may be a successful strategy to limit HCMV disease.

Positive Expression of Human Cytomegalovirus Phosphoprotein 65 in Atherosclerosis.

Previous studies showed that human cytomegalovirus (HCMV) is associated with atherosclerosis. However, local vascular atherosclerosis related HCMV infection and protein expression remain unclear. This study aimed to assess the relationship between HCMV infection and atherosclerosis. Formalin-fixed, paraffin-embedded peripheral artery specimens were obtained from 15 patients with atherosclerosis undergoing vascular surgery from 2008 to 2010 at Zhongnan Hospital, Wuhan University. Pathological analyses were carried out after hematoxylin and eosin (H&E) and Masson trichrome staining. In situ hybridization and immunohistochemistry with two different monoclonal antibodies were employed to detect HCMV nucleic acids and proteins, respectively. H&E and Masson trichrome staining showed homogeneous extracellular matrix in femoral artery, while smooth muscle fibers were interlaced with collagen fibers; in carotid artery, inflammatory cell infiltration, foam cell vascular change, cholesterol crystals, and layered collagen fibers were observed. In situ hybridization showed no expression of HCMV nucleic acids in all 15 cases. Immunohistochemical staining for protein immediate-early protein (IE1 72) was negative in all cases, while phosphoprotein 65 (pp65) expression was detected in 14 cases. A high rate of positive pp65 signals was found in patients with atherosclerosis, suggesting that local HCMV infection may be associated with the pathogenesis of atherosclerosis. Further studies on this relationship are warranted.

A Diverse Repertoire of CD4 T Cells Targets the Immediate-Early 1 Protein of Human Cytomegalovirus.

T-cell responses to the immediate-early 1 (IE-1) protein of human cytomegalovirus (HCMV) are associated with protection from viral disease. Thus, IE-1 is a promising target for immunotherapy. CD8 T-cell responses to IE-1 are generally strong. In contrast, CD4 T-cell responses to IE-1 were described to be comparatively infrequent or undetectable in HCMV carriers, and information on their target epitopes and their function has been limited. To analyze the repertoire of IE-1-specific CD4 T cells, we expanded them from healthy donors with autologous IE-1-expressing mini-Epstein–Barr virus-transformed B-cell lines and established IE-1-specific CD4 T-cell clones. Clones from seven out of seven HCMV-positive donors recognized endogenously processed IE-1 epitopes restricted through HLA-DR, DQ, or DP. Three to seven IE-1 epitopes were recognized per donor. Cumulatively, about 27 different HLA/peptide class II complexes were recognized by 117 IE-1-specific clones. Our results suggest that a highly diversified repertoire of IE-1-specific CD4 T cells targeting multiple epitopes is usually present in healthy HCMV carriers. Therefore, multiepitope approaches to immunomonitoring and immunotherapy will make optimal use of this potentially important class of HCMV-specific effector cells.