Human Cytomegalovirus Gene Products Research Articles

Partners in Crime: Synergistic Antiapoptotic Effects of HCMV Gene Products in Infected Cells

The means by which human cytomegalovirus (HCMV) evades the immune system has been a hot research topic for the past three decades. While much of the research has focused on viral proteins that interfere with the host’s immune response, recent investigations suggest that proteins are not the only culprits. New findings by Hancock et al. indicate that HCMV-encoded microRNAs (miRNAs) work alongside viral proteins to produce synergistic antiapoptotic effects, shedding light on the mechanisms by which HCMV establishes latent infection in host cells.

Targeting the Host Mitochondria as a Novel Human Cytomegalovirus Antiviral Strategy.

Human cytomegalovirus (HCMV) exploits host mitochondrial function to promote viral replication. HCMV gene products have been described to directly interact and alter functional or structural aspects of host mitochondria. Current antivirals against HCMV, such as ganciclovir and letermovir, are designed against viral targets. Concerns with the current antivirals include toxicity and viral resistance. Targeting host mitochondrial function is a promising alternative or complimentary antiviral approach as (1) drugs targeting host mitochondrial function interact with host targets, minimizing viral resistance, and (2) host mitochondrial metabolism plays key roles in HCMV replication. This review describes how HCMV alters mitochondrial function and highlights pharmacological targets that can be exploited for novel antiviral development.

The Association of Human Cytomegalovirus Infection and Colorectal Cancer: A Clinical Analysis

Human cytomegalovirus (HCMV) commonly infects humans and establishes lifelong infection. It causes disease and increased mortality rates in patients with immunosuppression. HCMV gene products are found to be present in multiple human malignancies and target cellular functions involved in tumor development; additionally, a tumor-cytoreductive role of CMV has also been observed. The purpose of this study was to evaluate the correlation between CMV infection and the incidence of colorectal cancer (CRC).The data were provided by a national database that is compliant with Health Insurance Portability and Accountability Act (HIPAA). Using International Classification of Disease (ICD)-10 and ICD-9 diagnostic codes, the data were filtered to evaluate patients infected with HCMV versus patients never infected with HCMV. Patient data from 2010 to 2019 were assessed. Access to the database was granted by Holy Cross Health, Fort Lauderdale for the purpose of academic research. Standard statistical methods were used.Between January 2010 and December 2019, the query was analyzed and resulted in 14,235 patients after matching in the infected and control groups. The groups were matched by age range, sex, Charlson Comorbidity Index (CCI) score, and treatment. The incidence of CRC was 1.159% (165 patients) in the HCMV group and 2.845% (405 patients) in the control group. The difference after matching was statistically significant by a P-value < 2.2 × 10-16 with an odds ratio of 0.37 (95% confidence interval (CI) 0.32 - 0.42).The study shows a statistically significant correlation between CMV infection and a reduced incidence of CRC. Further evaluation is recommended to assess the potential of CMV in reducing CRC incidence.

ADAM9: A regulator between HCMV infection and function of smooth muscle cells.

Lots of epidemiological and clinical studies have shown that human cytomegalovirus (HCMV) is related to the pathogenesis of atherosclerosis. Released by inflammatory cells and vascular smooth muscle cell (VSMCs), metalloproteinases are observed in many pathological vessel conditions, including atherosclerosis and restenosis.This study was designed to investigate the effect of HCMV infection on the expression of metalloproteinases and their involvements in the HCMV-induced functional changes of VSMCs.Differential metalloproteinase after HCMV infection was assayed using reverse transcription-polymerase chain reaction (RT-PCR) microarray. The most significant increased a disintegrin and metalloprotease 9 (ADAM9) was chosen to investigate the mechanism of its specific increase after infection using the treatment of UV-irradiated replication-deficient HCMV, HCMV-infected cell lysate filters or Foscarnet. The function of proliferation, migration, production of inflammatoty factors and phenotypic transformation were determined by using cell counting kit-8, transwell, Enzyme-linked immunosorbent assay, RT-quantitative PCR(qPCR) and Western blot, respectively. Moreover, the effect of ADAM9 deficiency on HCMV replication was also determined using RT-qPCR and immunofluorescence.The expression levels of 6 genes were upregulated and 14 genes were downregulated at different time points after HCMV infection. Among these, the expression level of ADAM9 increased most significantly at each time point and the abnormal expression of ADAM9 might be induced by the early gene products of HCMV. Further studies found that ADAM9 promoted the proliferation, the migration, the production of inflammatory factors and the transit from the contractile phenotype (decreased ACTA2 expression) to the synthetic phenotype (increased osteopontin [OPN] expression). Knockdown theADAM9expression could rescue the decreased ACTA2 expression, but has no effect on OPN expression. ADAM-9 deficiency didn't affect the replication of HCMV.The findings of our study suggest that HCMV infection changed VSMC function through ADAM9 expression, which may contribute to the understanding of the underlying pathological mechanisms of HCMV-induced atherosclerosis.

Detection of Human Cytomegalovirus Proteins in Paraffin-Embedded Breast Cancer Tissue Specimens-A Novel, Automated Immunohistochemical Staining Protocol.

Emerging evidence supports a significant association between human cytomegalovirus (HCMV) and human malignancies, suggesting HCMV as a human oncomodulatory virus. HCMV gene products are found in >90% of breast cancer tumors and seem to be correlated with more aggressive disease. The definitive diagnosis of HCMV relies on identification of virus inclusions and/or viral proteins by different techniques including immunohistochemical staining. In order to reduce biases and improve clinical value of HCMV diagnostics in oncological pathology, automation of the procedure is needed and this was the purpose of this study. Tumor specimens from 115 patients treated for primary breast cancer at Akershus University Hospital in Norway were available for the validation of the staining method in this retrospective study. We demonstrate that our method is highly sensitive and delivers excellent reproducibility for staining of HCMV late antigen (LA), which makes this method useful for future routine diagnostics and scientific applications.

Contributions of the Human Cytomegalovirus UL30-Associated Open Reading Frames to Infection.

Transposon-based insertional mutagenesis screens have assessed how disruption of numerous human cytomegalovirus (HCMV) open reading frames (ORFs) impacts in vitro viral replication. Insertional mutagenesis of the HCMV UL30 gene was previously found to substantially inhibit production of viral progeny. However, there are a number of putative UL30-associated ORFs, and it is unclear how they impact viral replication. Here, we report on the contributions of the eight UL30-associated ORFs to infection. We find that deletion of the canonically annotated UL30 ORF substantially reduces production of infectious virus at both high and low multiplicities of infection (MOI). This deletion likely has complex effects on viral replication, as we find that it reduces the expression of neighboring non-UL30-associated ORFs. Mutation of the initiating methionine of the canonical UL30 ORF indicated that it is dispensable for high- and low-MOI infection in the highly passaged AD169 strain, although it is important for low-MOI infection in the less-passaged TB40/E strain. Comutation of eight methionines in the UL30 region results in a low-MOI viral replication defect, as does mutation of the TATA box responsible for the most abundant UL30 transcript, which is found to be necessary for the accumulation of multiple UL30-associated protein isoforms during infection. In total, our data indicate the importance of the UL30-associated ORFs during low-MOI HCMV infection and further highlight the difficulty associated with the functional interrogation of broadly disruptive mutations: e.g., large deletions or transposon insertions.IMPORTANCE Viral genes and their products are the critical determinants of viral infection. Human cytomegalovirus (HCMV) encodes many gene products whose roles during viral infection have not been assessed. Elucidation of the contributions that various HCMV gene products make to infection provides insight into the infectious program, which could potentially be used to limit HCMV-associated morbidity, a major issue during congenital infection and in immunosuppressed populations. Here, we explored the role of HCMV's UL30-associated gene products and found that they are important for HCMV replication. Future work elucidating the mechanisms through which they contribute to viral infection could highlight novel avenues for therapeutic intervention.

Subversion of Immune Response by Human Cytomegalovirus.

Human cytomegalovirus (HCMV) is the most common cause of congenital infections and is an important pathogen in immunocompromised individuals. Despite a robust host immune system, HCMV able to replicate, evade host defenses, establish latency for life. A significant portion of HCMV genome dedicated to encode gene products for modulation of host immune response. Growing number of HCMV gene products are being recognized to play role in immune evasion. Information on viral immune evasion mechanisms by which HCMV persists in host will be useful in devising antiviral intervention strategies and development of new vaccines. This minireview provides a brief overview of immune evasion strategy adapted by HCMV by utilizing its gene products in modulation of host immune response.

The Role of RhoA, RhoB and RhoC GTPases in Cell Morphology, Proliferation and Migration in Human Cytomegalovirus (HCMV) Infected Glioblastoma Cells

Background/Aims: Rho GTPases are crucial regulators of the actin cytoskeleton, membrane trafficking and cell signaling and their importance in cell migration and invasion is well- established. The human cytomegalovirus (HCMV) is a widespread pathogen responsible for generally asymptomatic and persistent infections in healthy people. Recent evidence indicates that HCMV gene products are expressed in over 90% of malignant type glioblastomas (GBM). In addition, the HCMV Immediate Early-1 protein (IE1) is expressed in >90% of tumors analyzed. Methods: RhoA, RhoB and RhoC were individually depleted in U373MG glioblastoma cells as well as U373MG cells stably expressing the HCMV IE1 protein (named U373MG-IE1 cells) shRNA lentivirus vectors. Cell proliferation assays, migration as well as wound-healing assays were performed in uninfected and HCMV-infected cells. Results: The depletion of RhoA, RhoB and RhoC protein resulted in significant alterations in the morphology of the uninfected cells, which were further enhanced by the cytopathic effect caused by HCMV. Furthermore, in the absence or presence of HCMV, the knockdown of RhoB and RhoC proteins decreased the proliferation rate of the parental and the IE1-expressing glioblastoma cells, whereas the knockdown of RhoA protein in the HCMV infected cell lines restored their proliferation rate. In addition, wound healing assays in U373MG cells revealed that depletion of RhoA, RhoB and RhoC differentially reduced their migration rate, even in the presence or the absence of HCMV. Conclusion: Collectively, these data show for the first time a differential implication of Rho GTPases in morphology, proliferation rate and motility of human glioblastoma cells during HCMV infection, further supporting an oncomodulatory role of HCMV depending on the Rho isoforms' state.

Human cytomegalovirus antigens in malignant gliomas as targets for adoptive cellular therapy.

Malignant gliomas are the most common primary brain tumor in adults, with over 12,000 new cases diagnosed in the United States each year. Over the last decade, investigators have reliably identified human cytomegalovirus (HCMV) proteins, nucleic acids, and virions in most high-grade gliomas, including glioblastoma (GBM). This discovery is significant because HCMV gene products can be targeted by immune-based therapies. In this review, we describe the current level of understanding regarding the presence and role in pathogenesis of HCMV in GBM. We describe our success detecting and expanding HCMV-specific cytotoxic T lymphocytes to kill GBM cells and explain how these cells can be used as a platform for enhanced cellular therapies. We discuss alternative approaches that capitalize on HCMV infection to treat patients with HCMV-positive tumors. Adoptive cellular therapy for HCMV-positive GBM has been tried in a small number of patients with some benefit, but we reason why, to date, these approaches generally fail to generate long-term remission or cure. We conjecture how cellular therapy for GBM can be improved and describe the barriers that must be overcome to cure these patients.

Human cytomegalovirus viral load in tumor and peripheral blood samples of patients with malignant gliomas

Malignant gliomas are the most common primary brain tumors in adults. The disease has no known etiology, progresses rapidly, and is fatal despite current therapies. Human cytomegalovirus (HCMV) is a beta herpes virus that is trophic for glial cells and infects 50% to 90% of the adult human population. HCMV-mediated disease in immunosuppressed patients has highlighted the possible role of this virus in the development of other diseases, particularly inflammatory diseases such as vascular diseases, autoimmune diseases, and certain malignancies. Sensitive detection of viral DNA, mRNA, and antigens in tumor tissues, as well as seroepidemiologic evidence, suggest a link between HCMV and several human malignancies. HCMV gene products are proposed to dysregulate multiple cellular pathways involved in oncogenesis, such as cell cycle regulation, apoptosis, migration, and angiogenesis. These theories, currently being researched, suggest that HCMV acts as an oncomodulator in malignancies. We investigated the association between HCMV infection and reactivation, and malignant gliomas. An open, matched case-control, parallel group pilot study was performed in a tertiary referral center. The HCMV viral load in peripheral blood and tumor samples of 19 patients newly diagnosed with glioblastoma multiforme was compared with a matched control cohort comprising 19 patients newly diagnosed with non-malignant brain tumors. There was no significant correlation between peripheral blood and tumor tissue HCMV viral load in patients with glioblastoma multiforme compared to the control cohort. The findings of the present study did not support an oncomodulatory role for HCMV in malignant gliomas.

Functional annotation of human cytomegalovirus gene products: an update.

Human cytomegalovirus is an opportunistic double-stranded DNA virus with one of the largest viral genomes known. The 235 kB genome is divided in a unique long (UL) and a unique short (US) region which are flanked by terminal and internal repeats. The expression of HCMV genes is highly complex and involves the production of protein coding transcripts, polyadenylated long non-coding RNAs, polyadenylated anti-sense transcripts and a variety of non-polyadenylated RNAs such as microRNAs. Although the function of many of these transcripts is unknown, they are suggested to play a direct or regulatory role in the delicately orchestrated processes that ensure HCMV replication and life-long persistence. This review focuses on annotating the complete viral genome based on three sources of information. First, previous reviews were used as a template for the functional keywords to ensure continuity; second, the Uniprot database was used to further enrich the functional database; and finally, the literature was manually curated for novel functions of HCMV gene products. Novel discoveries were discussed in light of the viral life cycle. This functional annotation highlights still poorly understood regions of the genome but more importantly it can give insight in functional clusters and/or may be helpful in the analysis of future transcriptomics and proteomics studies.

Expression of insulin-like growth factor binding protein-2 (IGFBP-2) gene in negative and positive human cytomegalovirus glioblastoma multiforme tissues

Glioblastoma multiforme (GBM), a WHO grade IV malignant glioma, is the most common and lethal primary brain tumor in adults and has but few treatments. The median survival of glioblastoma patients is 12months. The (possible) relationship between human cytomegalovirus (HCMV) infection and cancer has been investigated for decades. Detection of viral DNA, mRNA and/or antigens in tumor tissues suggests that HCMV infection has a role to play in the etiology of several human malignancies. HCMV gene products can promote the various signaling pathways critical to tumor growth, including platelet derived growth factor receptor, phosphatidyl inositol 3-kinases (PI3K/AKT), signal transducer and activator of transcription 3 and glycogen synthase kinase 3 beta that are involved in apoptosis, angiogenesis, invasion and immune evasion. Insulin-like growth factor binding protein 2 (IGFBP2) is a biomarker of the PI3K/AKT pathway so we decided to evaluate the expression of this gene in 3 groups: HCMV-negative GBM tissues, HCMV-positive GBM tissues and non-tumor tissues. The presence of HCMV was assessed according to our previous article. HCMV was present in%75 of glioblastoma tissues. Then RNA was extracted, cDNA was synthesized, and real-time PCR was performed. Then, the rate of increased expression was calculated using the Livac or 2(-ΔΔCt). ΔCt of samples in the three groups were compared using analysis of variance (ANOVA). The expression of IGFBP2 gene relative to GAPDH gene in HCMV-negative glioblastoma tissues and HCMV-positive glioblastoma tissues, respectively, was increased 5.486 and 15.032 times compared to non-neoplastic brain tissues. ANOVA tests showed that the difference of mean ΔCt for IGFBP2 gene between healthy subjects and patients with HCMV-positive and HCMV-negative glioblastoma tumors statistically significant.

Cytomegalovirus and brain tumor

First described in 2002, the presence and role of human cytomegalovirus (HCMV) infection in glioblastoma (GBM) has remained a controversial topic. New research indicates HCMV gene products likely promote GBM pathogenesis and that therapies aimed at HCMV might influence disease progression. Recently, investigators have begun to analyze HCMV genome and proteins present in GBM cells in vivo. Furthermore, the research has demonstrated that several HCMV gene products that have oncomodulatory properties are expressed in GBM and may be impacting tumor pathogenesis in vivo. These HCMV gene products modulate GBM proliferation, apoptosis, angiogenesis, invasion and immune evasion. A recent mouse model provides mechanistic information as to how CMV may promote gliomagenesis in the setting of tumor suppressor dysfunction and STAT3 signaling. In addition, clinical outcomes of GBM patients are associated with the degree of HCMV infection. Novel therapies aimed at direct antiviral and immunotherapy approaches to HCMV suggest that these modalities may impact the future treatment of this disease. A more precise understanding of the role of HCMV infection in gliomagenesis and GBM pathogenesis could reveal novel therapeutic and preventive strategies.

The p53–microRNA-34a axis regulates cellular entry receptors for tumor-associated human herpes viruses

A growing number of reports indicate the frequent presence of DNA sequences and gene products of human cytomegalovirus in various tumors as compared to adjacent normal tissues, the brain tumors being studied most intensely. The mechanisms underlying the tropism of human cytomegalovirus to the tumor cells or to the cells of tumor origin, as well as the role of the host’s genetic background in virus-associated oncogenesis are not well understood. It is also not clear why cytomegalovirus can be detected in many but not in all tumor specimens. Our in silico prediction results indicate that microRNA-34a may be involved in replication of some human DNA viruses by targeting and downregulating the genes encoding a diverse group of proteins, such as platelet-derived growth factor receptor-alpha, complement component receptor 2, herpes simplex virus entry mediators A, B, and C, and CD46. Notably, while their functions vary, these surface molecules have one feature in common: they serve as cellular entry receptors for human DNA viruses (cytomegalovirus, Epstein–Barr virus, human herpes virus 6, herpes simplex viruses 1 and 2, and adenoviruses) that are either proven or suspected to be linked with malignancies. MicroRNA-34a is strictly dependent on its transcriptional activator tumor suppressor protein p53, and both p53 and microRNA-34a are frequently mutated or downregulated in various cancers. We hypothesize that p53–microRNA-34a axis may alter susceptibility of cells to infection with some viruses that are detected in tumors and either proven or suspected to be associated with tumor initiation and progression.

Human CD1d molecules are resistant to human cytomegalovirus US2- and US11-mediated degradation

Natural killer T (NKT) cells may play a crucial role in controlling viral infection by bridging the innate and adaptive immune systems. These cells are activated by lipids presented by CD1d molecules, which are structurally homologous to major histocompatibility complex class I (MHC-I) molecules. Although human cytomegalovirus (HCMV) can avoid T cell recognition by down-regulating MHC-I-mediated antigen presentation, it remains unknown whether it can also interfere with CD1d-mediated lipid presentation. Here, we show that CD1d is resistant to rapid degradation induced by the HCMV gene products US2 and US11, which cause dislocation of MHC-I molecules from the endoplasmic reticulum (ER) to the cytosol for destruction by proteasomes. The resistance of CD1d to US11 is mainly due to the short cytosolic tail of CD1d; a hybrid CD1d protein, whose cytosolic tail was replaced with that of HLA-A2.1, was efficiently degraded by US11. Finally, we found that HCMV infection did not significantly influence the cell surface expression of CD1d. Thus, these results suggest that antigen presentation by CD1d is largely unaffected by the multiple immune-modulating functions of HCMV.

The Human Cytomegalovirus Gene Products Essential for Late Viral Gene Expression Assemble into Prereplication Complexes before Viral DNA Replication

The regulation of human cytomegalovirus (HCMV) late gene expression by viral proteins is poorly understood, and these viral proteins could be targets for novel antivirals. HCMV open reading frames (ORFs) UL79, -87, and -95 encode proteins with homology to late gene transcription factors of murine gammaherpesvirus 68 ORFs 18, 24, and 34, respectively. To determine whether these HCMV proteins are also essential for late gene transcription of a betaherpesvirus, we mutated HCMV ORFs UL79, -87, and -95. Cells were infected with the recombinant viruses at high and low multiplicities of infection (MOIs). While viral DNA was detected with the recombinant viruses, infectious virus was not detected unless the wild-type viral proteins were expressed in trans. At a high MOI, mutation of ORF UL79, -87, or -95 had no effect on the level of major immediate-early (MIE) gene expression or viral DNA replication, but late viral gene expression from the UL44, -75, and -99 ORFs was not detected. At a low MOI, preexpression of UL79 or -87, but not UL95, in human fibroblast cells negatively affected the level of MIE viral gene expression and viral DNA replication. The products of ORFs UL79, -87, and -95 were expressed as early viral proteins and recruited to prereplication complexes (pre-RCs), along with UL44, before the initiation of viral DNA replication. All three HCMV ORFs are indispensable for late viral gene expression and viral growth. The roles of UL79, -87, and -95 in pre-RCs for late viral gene expression are discussed.

Evolving evidence implicates cytomegalovirus as a promoter of malignant glioma pathogenesis

Human cytomegalovirus (HCMV) was first reported to be strongly associated with human malignant gliomas in 2002. HCMV is a herpesvirus that causes congenital brain infection and multi-organ disease in immumocompromised individuals. Malignant gliomas are the most common and aggressive adult brain tumors and glioblastoma multiforme (GBM), the highest grade glioma, is associated with a life expectancy of less than two years. HCMV gene products encode for multiple proteins that can promote the various signaling pathways critical to tumor growth, including those involved in mitogenesis, mutagenesis, apoptosis, inflammation, angiogenesis, invasion and immuno-evasion. Several groups have now demonstrated that human malignant gliomas are universally infected with HCMV and express gene products that can promote key signaling pathways in glioma pathogenesis. In this review I discuss specific HCMV gene products that we and others have recently found to be expressed in GBM in vivo, including the HCMV IE1, US28, gB and IL-10 proteins. The roles these HCMV gene products play in dysregulating key pathways in glioma biology, including the PDGFR, AKT, STAT3, and monocyte/microglia function are discussed. Finally, I review emerging human clinical trials for GBM based on anti-HCMV strategies.

The TRC8 E3 ligase ubiquitinates MHC class I molecules before dislocation from the ER

The US2 and US11 gene products of human cytomegalovirus promote viral evasion by hijacking the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway. US2 and US11 initiate dislocation of newly translocated major histocompatibility complex class I (MHC I) from the ER to the cytosol for proteasome-mediated degradation, thereby decreasing cell surface MHC I. Despite being instrumental in elucidating the mammalian ERAD pathway, the responsible E3 ligase or ligases remain unknown. Using a functional small interfering RNA library screen, we now identify TRC8 (translocation in renal carcinoma, chromosome 8 gene), an ER-resident E3 ligase previously implicated as a hereditary kidney cancer gene, as required for US2-mediated MHC I ubiquitination. Depletion of TRC8 prevents MHC I ubiquitination and dislocation by US2 and restores cell surface MHC I. TRC8 forms an integral part of a novel multiprotein ER complex that contains MHC I, US2, and signal peptide peptidase. Our data show that the TRC8 E3 ligase is required for MHC I dislocation from the ER and identify a new complex associated with mammalian ERAD.

Identification of TRIM23 as a cofactor involved in the regulation of NF-kappaB by human cytomegalovirus.

Human cytomegalovirus (HCMV) regulates NF-kappaB during infection by a variety of mechanisms. For example, the HCMV gene product, UL144, is known to activate NF-kappaB in a tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6)-dependent manner, causing the upregulation of the chemokine CCL22 (MDC). Viral UL144 is expressed from the UL/b' region of the HCMV genome at early times postinfection and is a TNFR1-like homologue. Despite this homology to the TNFR1 receptor superfamily, UL144 does not bind to members of the TNF ligand superfamily. We show here that the upregulation of NF-kappaB by UL144 is dependent upon cellular tripartite motif 23 (TRIM23) protein. We propose a mechanism by which UL144 activates NF-kappaB through a direct interaction with the cellular protein TRIM23 in a complex containing TRAF6. In contrast, TRIM23 is not involved in conventional double-stranded RNA signaling via NF-kappaB. Therefore, we present a novel role for TRIM23 that is specific to UL144-mediated activation of NF-kappaB during the course of virus infection.

Relation between glioma and human cytomegalovirus

Human cytomegalovirus (HCMV), a betaherpesvirus, is ubiquitous in human population.Multiple HCMV gene products are expressed in malignant gliomas.Neural stem cells and glial cells are suscep-tible to HCMV.HCMV can induce malignant transformation and dysregulate key cellular pathways involved in host immune response, mutagenesis, cell cycle, apoptosis, and angio-genesis, and may be oncogenie in human.So, HCMV may play an active role in glioma genesis and development. Key words: Glioma; Pathogenesis; Cytomegalovirus