Human Cytomegalovirus Persistence Research Articles

A Comparative Quantitative Proteomic Analysis of HCMV-Infected Cells Highlights pUL138 as a Multifunctional Protein.

Human cytomegalovirus (HCMV) is a widespread virus that can establish life-long latent infection in large populations. The establishment of latent infection prevents HCMV from being cleared by host cells, and HCMV reactivation from latency can cause severe disease and death in people with immature or compromised immune systems. To establish persistent and latent infection in healthy individuals, HCMV encodes a large array of proteins that can modulate different components and pathways of host cells. It has been reported that pUL138 encoded by the UL133-UL138 polycistronic locus promotes latent infection in primary CD34+ hematopoietic progenitor cells (HPCs) infected in vitro. In this study, recombinant HCMV HanUL138del was constructed by deleting the UL138 locus of Han, a clinical HCMV strain. Then, a comparative quantitative proteomic analysis of Han- and HanUL138del-infected MRC5 cells was performed to study the effect of pUL138 on host cells in the context of HCMV infection. Our results indicated that, during the early phase of HCMV infection, the innate immune response was differentially activated, while during the late phase of HCMV infection, multiple host proteins were differentially expressed between Han- and HanUL138del-infected cells, and these proteins are involved in the oxidation-reduction process, ER to Golgi vesicle-mediated transport, and extracellular matrix organization. Among these proteins, STEAP3, BORCS7, FAM172A, RELL1, and WDR48 were further demonstrated to affect HCMV infection. Our study provides a systematic view of the effect of pUL138 on the host cell proteome and highlights the proposition that multiple biological processes or host factors may be involved in the overall role of the UL133-UL138 polycistronic locus in HCMV persistence.

Persistent HCMV infection of a glioblastoma cell line contributes to the development of resistance to temozolomide

Glioblastoma multiforme (GBM) is the most aggressive form of primary human gliomas. While chemotherapy using the DNA alkylating agent temozolomide (TMZ) is a first line treatment for GBMs, the development of resistance to TMZ is a common limitation to successful treatment. Human Cytomegalovirus (HCMV) is a ubiquitous β-herpesvirus that establishes a lifelong infection latent infection in host haematopoetic cells, where lytic replication of the virus is silenced. HCMV can also establish a persistent infection in hosts, where low levels of virus are lytically produced. Furthermore, multiple studies have identified HCMV DNA and/or proteins in human GBM samples, and have shown that acute infection with HCMV confers a glioblastoma stem cell (GSC) phenotype, further supporting an oncomodulatory role for HCMV in GBM progression and severity. In this current study, we examined the long-term effects of HCMV persistence to cell viability, cell proliferation, and the development of TMZ resistance over time using a glioblastoma cell line known as LN-229. Persistent HCMV infections were established and maintained in this cell line for 30 weeks without the addition of new virus. Here, we report that HCMV persistence in this cell line resulted in increased cell viability, increased cell proliferation, and a marked resistance to the DNA alkylating agent, TMZ, over time, suggesting that low levels of lytically replicating HCMV could contribute to tumor progression in GBM.

Host Genetics of Cytomegalovirus Pathogenesis.

Human cytomegalovirus (HCMV) is a ubiquitous herpes virus (human herpes virus 5) with the highest morbidity and mortality rates compared to other herpes viruses. Risk groups include very young, elderly, transplant recipient, and immunocompromised individuals. HCMV may cause retinitis, encephalitis, hepatitis, esophagitis, colitis, pneumonia, neonatal infection sequelae, inflammatory, and age-related diseases. With an arsenal of genes in its large genome dedicated to host immune evasion, HCMV can block intrinsic cellular defenses and interfere with cellular immune responses. HCMV also encodes chemokines, chemokine receptors, and cytokines. Therefore, genes involved in human viral defense mechanisms and those encoding proteins targeted by the CMV proteins are candidates for host control of CMV infection and reactivation. Although still few in number, host genetic studies are producing valuable insights into biological processes involved in HCMV pathogenesis and HCMV-related diseases. For example, genetic variants in the immunoglobulin GM light chain can influence the antibody responsiveness to CMV glycoprotein B and modify risk of HCMV-related diseases. Moreover, CMV infection following organ transplantation has been associated with variants in genes encoding toll-like receptors (TLRs), programmed death-1 (PD-1), and interleukin-12p40 (IL-12B). A KIR haplotype (2DS4+) is proposed to be protective for CMV activation among hematopoietic stem cell transplant patients. Polymorphisms in the interferon lambda 3/4 (IFNL3/4) region are shown to influence susceptibility to CMV replication among solid organ transplant patients. Interestingly, the IFNL3/4 region is also associated with AIDS-related CMV retinitis susceptibility in HIV-infected patients. Likewise, interleukin-10 receptor 1 (IL-10R1) variants are shown to influence CMV retinitis development in patients with AIDS. Results from genome-wide association studies suggest a possible role for microtubule network and retinol metabolism in anti-CMV antibody response. Nevertheless, further genetic epidemiological studies with large cohorts, functional studies on the numerous HCMV genes, and immune response to chronic and latent states of infection that contribute to HCMV persistence are clearly necessary to elucidate the genetic mechanisms of CMV infection, reactivation, and pathogenesis.

Defining the Transcriptional Landscape during Cytomegalovirus Latency with Single-Cell RNA Sequencing.

ABSTRACTPrimary infection with human cytomegalovirus (HCMV) results in a lifelong infection due to its ability to establish latent infection, with one characterized viral reservoir being hematopoietic cells. Although reactivation from latency causes serious disease in immunocompromised individuals, our molecular understanding of latency is limited. Here, we delineate viral gene expression during natural HCMV persistent infection by analyzing the massive transcriptome RNA sequencing (RNA-seq) atlas generated by the Genotype-Tissue Expression (GTEx) project. This systematic analysis reveals that HCMV persistence in vivo is prevalent in diverse tissues. Notably, we find only viral transcripts that resemble gene expression during various stages of lytic infection with no evidence of any highly restricted latency-associated viral gene expression program. To further define the transcriptional landscape during HCMV latent infection, we also used single-cell RNA-seq and a tractable experimental latency model. In contrast to some current views on latency, we also find no evidence for any highly restricted latency-associated viral gene expression program. Instead, we reveal that latency-associated gene expression largely mirrors a late lytic viral program, albeit at much lower levels of expression. Overall, our work has the potential to revolutionize our understanding of HCMV persistence and suggests that latency is governed mainly by quantitative changes, with a limited number of qualitative changes, in viral gene expression.

Human Cytomegalovirus Requires Epidermal Growth Factor Receptor Signaling To Enter and Initiate the Early Steps in the Establishment of Latency in CD34+ Human Progenitor Cells.

The establishment of human cytomegalovirus (HCMV) latency and persistence relies on the successful infection of hematopoietic cells, which serve as sites of viral persistence and contribute to viral spread. Here, using blocking antibodies and pharmacological inhibitors, we document that HCMV activation of the epidermal growth factor receptor (EGFR) and downstream phosphatidylinositol 3-kinase (PI3K) mediates viral entry into CD34+ human progenitor cells (HPCs), resulting in distinct cellular trafficking and nuclear translocation of the virus compared to that in other immune cells, such as we have documented in monocytes. We argue that the EGFR allows HCMV to regulate the cellular functions of these replication-restricted cells via its signaling activity following viral binding. In addition to regulating HCMV entry/trafficking, EGFR signaling may also shape the early steps required for the successful establishment of viral latency in CD34+ cells, as pharmacological inhibition of EGFR increases the transcription of lytic IE1/IE2 mRNA while curbing the expression of latency-associated UL138 mRNA. EGFR signaling following infection of CD34+ HPCs may also contribute to changes in hematopoietic potential, as treatment with the EGFR kinase (EGFRK) inhibitor AG1478 alters the expression of the cellular hematopoietic cytokine interleukin 12 (IL-12) in HCMV-infected cells but not in mock-infected cells. These findings, along with our previous work with monocytes, suggest that EGFR likely serves as an important determinant of HCMV tropism for select subsets of hematopoietic cells. Moreover, our new data suggest that EGFR is a key receptor for efficient viral entry and that the ensuing signaling regulates important early events required for successful infection of CD34+ HPCs by HCMV.IMPORTANCE HCMV establishes lifelong persistence within the majority of the human population without causing overt pathogenesis in healthy individuals. Despite this, reactivation of HCMV from its latent reservoir in the bone marrow causes significant morbidity and mortality in immunologically compromised individuals, such as bone marrow and solid organ transplant patients. Lifelong persistent infection has also been linked with the development of various cardiovascular diseases in otherwise healthy individuals. Current HCMV therapeutics target lytic replication, but not the latent viral reservoir; thus, an understanding of the molecular basis for viral latency and persistence is paramount to controlling or eliminating HCMV infection. Here, we show that the viral signalosome activated by HCMV binding to its entry receptor, EGFR, in CD34+ HPCs initiates early events necessary for successful latent infection of this cell type. EGFR and associated signaling players may therefore represent promising targets for mitigating HCMV persistence.

Human cytomegalovirus persistence

Viral persistence is the rule following infection with all herpesviruses. The β-herpesvirus, human cytomegalovirus (HCMV), persists through chronic and latent states of infection. Both of these states of infection contribute to HCMV persistence and to the high HCMV seroprevalence worldwide. The chronic infection is poorly defined molecularly, but clinically manifests as low-level virus shedding over extended periods of time and often in the absence of symptoms. Latency requires long-term maintenance of viral genomes in a reversibly quiescent state in the immunocompetent host. In this review, we focus on recent advances in the biology of HCMV persistence, particularly with respect to the latent mode of persistence. Latently infected individuals harbour HCMV genomes in haematopoietic cells and maintain large subsets of HCMV-specific T-cells. In the last few years, impressive advances have been made in understanding virus-host interactions important to HCMV infection, many of which will profoundly impact HCMV persistence. We discuss these advances and their known or potential impact on viral latency. As herpesviruses are met with similar challenges in achieving latency and often employ conserved strategies to persist, we discuss current and future directions of HCMV persistence in the context of the greater body of knowledge regarding α- and γ-herpesviruses persistence.

Usefulness of Interleukin-10 Detection in Lung Transplant Patients With Human Cytomegalovirus Infection With Respect to Virus Persistence

Human cytomegalovirus (HCMV) infection in lung transplant patients induces an inflammatory response, including local production of cytokines involved in viral clearance. The aim of this study was to evaluate the potential value of monitoring interleukin (IL)-10 with respect to HCMV persistence in blood and/or bronchoalveolar lavage (BAL). A quantitative polymerase chain reaction assay was used for HCMV-DNA detection in plasma and BAL. IL-10 was measured with an enzyme-linked immunosorbent assay in blood and with BAL in 101 lung transplant patients. IL-10 levels were correlated with clinical outcome. A total of 23 patients of 35 (66%) with detectable HCMV in plasma and/or BAL exhibited increased levels of IL-10 in plasma and/or BAL. Complete clearance of HCMV was observed after 168 (median 130) days in the IL-10-positive group (n=23) in comparison with 87 (median 58) days in the IL-10-negative group (n=12; P<0.024). In the seven HCMV-positive patients with positive IL-10 levels in BAL only, HCMV persisted in BAL for a median of 579 days without signs of systemic infection (positive plasma levels) or clinical symptoms. We show that in lung transplant patients with elevated levels of IL-10 in plasma and/or BAL, HCMV clearance is prolonged because of the influence of anti-inflammatory cytokines.

Roles of Phosphatidylinositol 3-Kinase and NF-κB in Human Cytomegalovirus-Mediated Monocyte Diapedesis and Adhesion: Strategy for Viral Persistence

Infected peripheral blood monocytes are proposed to play a key role in the hematogenous dissemination of human cytomegalovirus (HCMV) to tissues, a critical step in the establishment of HCMV persistence and the development of HCMV-associated diseases. We recently provided evidence for a unique strategy involved in viral dissemination: HCMV infection of primary human monocytes promotes their transendothelial migration and differentiation into proinflammatory macrophages permissive for the replication of the original input virus. To decipher the mechanism of hematogenous spread, we focused on the viral dysregulation of early cellular processes involved in transendothelial migration. Here, we present evidence that both phosphatidylinositol 3-kinase [PI(3)K] and NF-kappaB activities were crucial for the HCMV induction of monocyte motility and firm adhesion to endothelial cells. We found that the beta(1) integrins, the beta(2) integrins, intracellular adhesion molecule 1 (ICAM-1), and ICAM-3 were upregulated following HCMV infection and that they played a key role in the firm adhesion of infected monocytes to the endothelium. The viral regulation of adhesion molecule expression is complex, with PI(3)K and NF-kappaB affecting the expression of each adhesion molecule at different stages of the expression cascade. Our data demonstrate key roles for PI(3)K and NF-kappaB signaling in the HCMV-induced cellular changes in monocytes and identify the biological rationale for the activation of these pathways in infected monocytes, which together suggest a mechanism for how HCMV promotes viral spread to and persistence within host organs.

Characterization and immunological analysis of the rhesus cytomegalovirus homologue (Rh112) of the human cytomegalovirus UL83 lower matrix phosphoprotein (pp65)

Rhesus cytomegalovirus (RhCMV) contains two open reading frames (Rh111 and Rh112) that encode proteins homologous to the phosphoprotein 65 (pp65) of the human cytomegalovirus (HCMV) UL83 gene. As HCMV pp65 elicits protective immune responses in infected humans and represents an important vaccination target, one RhCMV homologue of HCMV pp65, pp65-2 (Rh112), was characterized and analysed for its ability to induce host immune responses. Similar to its HCMV counterpart, RhCMV pp65-2 was expressed as a late gene, localized to the nucleus within pp65-2-expressing cells and was present within infectious virions. Longitudinal and cross-sectional studies of pp65-2 immunity in naturally infected rhesus macaques showed that humoral responses to pp65-2 were elicited early during infection, but were not always sustained over time. In contrast, pp65-2-specific T-cell responses, examined by gamma interferon ELISPOT, were broadly detectable in all of the animals studied during primary infection and persisted in the vast majority of RhCMV-seropositive monkeys. Moreover, there was considerable inter-animal variability in the pattern of the immune responses to pp65-2. Together, these results demonstrated that RhCMV pp65-2 exhibited biological and immunological homology to HCMV pp65. Thus, the rhesus macaque model of HCMV persistence and pathogenesis should be relevant for addressing pp65-based vaccine modalities.

Nonhuman Primate Models of Intrauterine Cytomegalovirus Infection

Congenital human cytomegalovirus (HCMV) infection has long been recognized as a threat to the developing fetus, even though studies have shown that only a subset of congenital infections results in clinical signs of disease. Among the estimated 8000 children who develop sequelae from congenital CMV infection each year in the United States alone, most suffer permanent developmental defects within the central nervous system. Because there is currently no approved vaccine for HCMV, and anti-HCMV drugs are not administered to gravid women with congenital infection because of potential toxicity to the fetus, there is a clear clinical need for effective strategies that minimize infection in the mother, transplacental transmission of the virus, and/or fetal disease. Animal models provide a method to understand the mechanisms of HCMV persistence and pathogenesis, and allow for testing of novel strategies that limit prenatal infection and disease. The rhesus macaque model is especially well suited for these tasks because monkeys and humans share strong developmental, immunological, anatomical, and biochemical similarities due to their close phylogenetic relationship. This nonhuman primate model provides an invaluable system to accelerate the clinical development of promising new therapies for the treatment of human disease. This review addresses salient findings with the macaque model as they relate to HCMV infection and potential avenues of discovery, including studies of intrauterine CMV infection. The complexity of the natural history of HCMV is discussed, along with the ethical and logistical issues associated with studies during pregnancy, the recent contributions of animal research in this field of study, and future prospects for increasing our understanding of immunity against HCMV disease.

The human eye (retina): a site of persistent HCMV infection?

Human cytomegalovirus (HCMV) retinitis frequently occurs in severely naturally and iatrogenically immunocompromised patients. It has been shown that the immune-privileged retina is a major site of HCMV infection in AIDS patients. It is conceivable either that during the immunosuppression HCMV infection reactivates in various other organs viremically affecting the retina or that HCMV persisting in the retina may locally reactivate and result in HCMV retinitis. As there is still controversy about the sites of HCMV latency and persistence we investigated 75 eyes of HIV-seronegative patients undergoing enucleation due to a variety of malignant and non-viral benign ophthalmic disorders for the retinal presence of HCMV antigen and DNA. None of the analyzed patients had symptoms of HCMV retinitis. Immunohistologic staining as well as Taq Man DNA PCR analysis showed all samples to be free of HCMV. Our data suggest that the human eye is rather unlikely to be a site of productive or latent HCMV persistence.

Thrombin induces Sp1-mediated antiviral effects in cytomegalovirus-infected human retinal pigment epithelial cells

Human cytomegalovirus (HCMV) retinitis causing retinal detachment and destruction of the blood-retina barrier is closely related to retinal hemorrhage/coagulation. However, the effects of procoagulants on HCMV (re)activation in retinal cells have not been investigated yet. Therefore, we studied whether thrombin modulates the expression of HCMV immediate early (IE) and late (L) genes in cultured human retinal pigment epithelial cells (RPE). Thrombin specifically stimulated the protease-activated receptor-1 (PAR-1) on RPE and, surprisingly, inhibited basal and 12,0-tetradecanoylphorbol 13-acetate-stimulated HCMV IE gene expression in infected RPE. On the other hand, HCMV strongly induced Sp1 DNA binding activity, which was prevented by thrombin/PAR1-mediated Sp1 hyperphosphorylation. Our data suggest that thrombin/PAR-1 may inhibit Sp1-dependent HCMV replication, which might be an important regulatory mechanism for HCMV persistence and replication in RPE.

Mechanisms of human cytomegalovirus persistence and latency.

Human cytomegalovirus (HCMV) is a ubiquitous beta-herpesvirus that causes severe disease primarily in immunosuppressed individuals. A major characteristic of HCMV with obvious clinical importance is the ability of the virus to establish lifelong infection within the host following the initial acute infection. One strategy used by HCMV to maintain itself within the host is the establishment of cellular sites of persistent infection and viral latency. Recent studies have identified endothelial cells and monocyte-derived macrophages (MDM) as sites of HCMV persistence and latency. These studies show that endothelial cell origin and MDM differentiation pathway are critical factors that influence the characteristics of HCMV replication in these cell types. The specific HCMV genes involved in endothelial cell and MDM tropism are unknown. However, studies in the closely related murine cytomegalovirus (MCMV) model have provided considerable insight into viral genes that enable replication in these cell types. This review will focus on mechanisms of HCMV replication in endothelial cells and MDM, and on the viral genes involved in regulation of viral replication in these important cell types.

Mechanisms of human cytomegalovirus persistence and latency

Mechanisms of human cytomegalovirus persistence and latency

A novel mechanism for persistence of human cytomegalovirus in macrophages

Human cytomegalovirus (HCMV) infection of monocyte-derived macrophages (MDM) results in delayed and nonlytic productive viral growth. During late stages of replication, infectious virus remains cell associated in cytoplasmic vacuoles. In order to understand HCMV survival and persistence in MDM, we examined mechanisms involved in the formation and trafficking of HCMV-containing vacuoles in these cells. Utilizing double-label immunofluorescence with antibodies to viral and cellular proteins, HCMV-containing vacuoles were associated with the Golgi apparatus marker mannosidase II but not with markers to early endosomes (transferrin receptor and rab5) or late endosomes and early lysosomes (LAMP-1 and -2). In addition, as late-stage viral infection progressed in MDM, the cells displayed increasing abnormalities in the Golgi apparatus. Analysis of structural features of infected cells revealed the disruption of the microtubule network. These observations suggest a novel mechanism by which HCMV is vacuolized in MDM, avoiding degradation and release from the cell.