Oncomodulatory signals by regulatory proteins encoded by human cytomegalovirus: a novel role for viral infection in tumor progression

A high frequency of human cytomegalovirus (HCMV) genome and antigens in tumor samples of patients with different malignancies is now well documented, although the causative role for HCMV in the development of the neoplasias remains to be established. HCMV infection can modulate multiple cellular regulatory and signalling pathways in a manner similar to that of oncoproteins of small DNA tumor viruses such as human papilloma virus or adenoviruses. However, in contrast to these DNA tumor viruses, HCMV infection fails to transform susceptible normal human cells. There is now growing evidence that tumor cells with disrupted regulatory and signalling pathways enable HCMV to modulate their properties including stimulation of cell proliferation, survival, invasion, production of angiogenic factors, and immunogenic properties. In contrast to previously suggested "hit and run" transformation we suggest that persistence in tumor cells is essential for HCMV to fully express its oncomodulatory effects. These effects are observed particularly in persistent HCMV infection and are mediated mainly by activity of HCMV regulatory proteins. In persistently HCMV-infected tumor cell lines - a selection of novel, slowly growing virus variants with changes in coding sequences for virus regulatory proteins takes place. As a result, oncomodulatory effects of HCMV infection may lead to a shift to more malignant phenotype of tumor cells contributing to tumor progression.     

Ex vivo profiling of CD8+-T-cell responses to human cytomegalovirus reveals broad and multispecific reactivities in healthy virus carriers

Human cytomegalovirus (HCMV) can establish both nonproductive (latent) and productive (lytic) infections. Many of the proteins expressed during these phases of infection could be expected to be targets of the immune response; however, much of our understanding of the CD8(+)-T-cell response to HCMV is mainly based on the pp65 antigen. Very little is known about T-cell control over other antigens expressed during the different stages of virus infection; this imbalance in our understanding undermines the importance of these antigens in several aspects of HCMV disease pathogenesis. In the present study, an efficient and rapid strategy based on predictive bioinformatics and ex vivo functional T-cell assays was adopted to profile CD8(+)-T-cell responses to a large panel of HCMV antigens expressed during different phases of replication. These studies revealed that CD8(+)-T-cell responses to HCMV often contained multiple antigen-specific reactivities, which were not just constrained to the previously identified pp65 or IE-1 antigens. Unexpectedly, a number of viral proteins including structural, early/late antigens and HCMV-encoded immunomodulators (pp28, pp50, gH, gB, US2, US3, US6, and UL18) were also identified as potential targets for HCMV-specific CD8(+)-T-cell immunity. Based on this extensive analysis, numerous novel HCMV peptide epitopes and their HLA-restricting determinants recognized by these T cells have been defined. These observations contrast with previous findings that viral interference with the antigen-processing pathway during lytic infection would render immediate-early and early/late proteins less immunogenic. This work strongly suggests that successful HCMV-specific immune control in healthy virus carriers is dependent on a strong T-cell response towards a broad repertoire of antigens.     

An HCMV reading frame which has similarity with both the V and C regions of the TCR gamma chain.

Human cytomegalovirus (HCMV) is a member of the herpes virus family and is characterized by widespread infection, latency, persistence and reactivation and is a serious pathogen particularly of immunosuppressed patients. Previously HCMV was shown to encode a glycoprotein homologous to the MHC class I antigens. We report here that HCMV may encode another glycoprotein similar to another sub-group of the Ig superfamily. This putative glycoprotein gene shows similarity to both the constant and variable regions of the human T cell receptor gamma chain (TCR gamma). The level of homology is low but may be of interest as the predicted gene might have some role in virus infection or immune evasion.     

Monocyte-derived inhibitor of interleukin 1 induced by human cytomegalovirus.

It has previously been shown that human cytomegalovirus (HCMV) can exert immunosuppressive effects, and it has been suggested that these may be mediated by monocytes, although the mechanism is unclear. We showed that infection of human monocytes with the AD169 strain of HCMV abrogates their production of interleukin 1 (IL-1) activity. This was associated with the release from infected monocytes of an inhibitor of IL-1 activity which was also released after HCMV infection of the U937 macrophage-like cell line. The inhibitor of IL-1 activity is a protein with an apparent molecular weight of ca. 95,000. This action of HCMV strain AD169 was virus specific and required infectious virus but occurred without virus replication or detectable expression of viral proteins. This effect may account, at least in part, for the previously observed immunosuppressive properties of HCMV.     

Insight for Immunotherapy of HCMV Infection

Human cytomegalovirus (HCMV), a ubiquitous in humans, has a high prevalence rate. Young people are susceptible to HCMV infection in developing countries, while older individuals are more susceptible in developed countries. Most patients have no obvious symptoms from the primary infection. Studies have indicated that the virus has gradually adapted to the host immune system. Therefore, the control of HCMV infection requires strong immune modulation. With the recent advances in immunotherapy, its application to HCMV infections is receiving increasing attention. Here, we discuss the immune response to HCMV infection, the immune escape mechanism, and the different roles that HCMV plays in various types of immunotherapy, including vaccines, adoptive cell therapy, checkpoint blockade therapy, and targeted antibodies.     

THY-1 Cell Surface Antigen (CD90) Has an Important Role in the Initial Stage of Human Cytomegalovirus Infection

Human cytomegalovirus (HCMV) infects about 50% of the US population, is the leading infectious cause of birth defects, and is considered the most important infectious agent in transplant recipients. The virus infects many cell types in vivo and in vitro. While previous studies have identified several cellular proteins that may function at early steps of infection in a cell type dependent manner, the mechanism of virus entry is still poorly understood. Using a computational biology approach, correlating gene expression with virus infectivity in 54 cell lines, we identified THY-1 as a putative host determinant for HCMV infection in these cells. With a series of loss-of-function, gain-of-function and protein-protein interaction analyses, we found that THY-1 mediates HCMV infection at the entry step and is important for infection that occurs at a low m.o.i. THY-1 antibody that bound to the cell surface blocked HCMV during the initial 60 minutes of infection in a dose-dependent manner. Down-regulation of THY-1 with siRNA impaired infectivity occurred during the initial 60 minutes of inoculation. Both THY-1 antibody and siRNA inhibited HCMV-induced activation of the PI3-K/Akt pathway required for entry. Soluble THY-1 protein blocked HCMV infection during, but not after, virus internalization. Expression of exogenous THY-1 enhanced entry in cells expressing low levels of the protein. THY-1 interacted with HCMV gB and gH and may form a complex important for entry. However, since gB and gH have previously been shown to interact, it is uncertain if THY-1 directly binds to both of these proteins. Prior observations that THY-1 (a) interacts with αVβ3 integrin and recruits paxillin (implicated in HCMV entry), (b) regulates leukocyte extravasation (critical for HCMV viremia), and (c) is expressed on many cells targeted for HCMV infection including epithelial and endothelial cells, fibroblast, and CD34+/CD38- stem cells, all support a role for THY-1 as an HCMV entry mediator in a cell type dependent manner. THY-1 may function through a complex setting, that would include viral gB and gH, and other cellular factors, thus links virus entry with signaling in host cells that ultimately leads to virus infection.     

Incoming human cytomegalovirus pp65 (UL83) contained in apoptotic infected fibroblasts is cross-presented to CD8(+) T cells by dendritic cells

Human cytomegalovirus (HCMV) infection is well controlled mainly by cytotoxic CD8(+) T lymphocytes (CTL) directed against the matrix protein pp65 despite the numerous immune escape mechanisms developed by the virus. Dendritic cells (DCs) are key antigen-presenting cells for the generation of an immune response which have the capacity to acquire antigens via endocytosis of apoptotic cells and thus present peptides to major histocompatibility complex class I-restricted T cells. We examined whether this mechanism could contribute to the activation of anti-pp65 CTL. In this study, we show that infection by HCMV AD169 induced sensitization of MRC5 fibroblasts to tumor necrosis factor alpha-mediated apoptosis very early after virus inoculation and that pp65 contained in apoptotic cells came from the delivery of the matrix protein into the cell. We observed that immature DCs derived from peripheral monocytes were not permissive to HCMV AD169 infection but were able to internalize pp65-positive apoptotic infected MRC5 cells. We then demonstrated that following exposure to these apoptotic bodies, DCs could activate HLA-A2- or HLA-B35-restricted anti-pp65 CTL, suggesting that they acquired and processed properly fibroblast-derived pp65. Together, our data suggest that cross-presentation of incoming pp65 contained in apoptotic cells may provide a quick and efficient way to prime anti-HCMV CD8(+) T cells.     

AIDS: a syndrome of immune dysregulation, dysfunction, and deficiency

Acquired immune deficiency syndrome (AIDS) is a disease caused by the human immunodeficiency virus (HIV) in which cellular immune functions are severely impaired. Acute infection and subsequent destruction of helper T cells, although occurring readily in cell cultures, do not appear to be the only mechanisms mediating helper T cell loss. Other mechanisms that may account for the loss of helper T cells include: T cell syncytia formation, decreased T cell production, and autoimmune-related destruction of helper T cells. Immune abnormalities seen early in the course of HIV infection include immune hyperactivation and autoimmune phenomena suggestive of immune dysregulation rather than immune deficiency. Many changes in immune function are, in fact, seen in HIV-seropositive patients who possess a normal number of helper T cells. Mechanisms (other than the loss of helper T cells) that may contribute to the immune abnormalities seen in these patients include noninfectious effects of HIV and HIV proteins, effects of HIV on non-T cells, autoimmune-related manifestations of HIV infection, and HIV-induced activation of normal immunosuppressive circuits.     

Cytomegalovirus (CMV)

Human cytomegalovirus (HCMV) is a ubiquitous beta human herpesvirus type 5. Compared to other human herpesviruses, HCMV is the largest, with a genome of approximately 235 kb containing approximately 250 ORFs with the potential to encode proteins. Usually, HCMV asymptomatically infects the host during childhood, and establishes life-long latency. The infection is life-threatening for infants and immunocompromised individuals, because of direct cytopathicity by viral replication, causing systemic organ injuries. Intrauterine infection occasionally causes microcephaly, sensorineural hearing loss and mental retardation. HCMV genome contains a number of accessory genes. Most of them are engaged in immune evasion or inhibition of cell death, possibly, resulting in a symbiosis between virus and host. CD34-positive myeloid progenitor cells are considered as a site of latency. However, the molecular mechanisms by which HCMV establishes and maintains latency and reactivates remain poorly understood. Recently in Japan, the decline of maternal HCMV seropositivity may increase the risk of intrauterine infection. It needs to immediately establish the protection against transplacental HCMV infection, such as a new type of neutralizing antibody or vaccine, which effectively interferes viral entry specific to endothelial and epithelial cells. Furthermore, HCMV infection might be considered as the most important factor for driving immune senescence in the elderly.     

Human Cytomegalovirus Latency-Associated Proteins Elicit Immune-Suppressive IL-10 Producing CD4+ T Cells

Human cytomegalovirus (HCMV) is a widely prevalent human herpesvirus, which, after primary infection, persists in the host for life. In healthy individuals, the virus is well controlled by the HCMV-specific T cell response. A key feature of this persistence, in the face of a normally robust host immune response, is the establishment of viral latency. In contrast to lytic infection, which is characterised by extensive viral gene expression and virus production, long-term latency in cells of the myeloid lineage is characterised by highly restricted expression of viral genes, including UL138 and LUNA. Here we report that both UL138 and LUNA-specific T cells were detectable directly ex vivo in healthy HCMV seropositive subjects and that this response is principally CD4⁺ T cell mediated. These UL138-specific CD4⁺ T cells are able to mediate MHC class II restricted cytotoxicity and, importantly, show IFNγ effector function in the context of both lytic and latent infection. Furthermore, in contrast to CD4⁺ T cells specific to antigens expressed solely during lytic infection, both the UL138 and LUNA-specific CD4⁺ T cell responses included CD4⁺ T cells that secreted the immunosuppressive cytokine cIL-10. We also show that cIL-10 expressing CD4⁺ T-cells are directed against latently expressed US28 and UL111A. Taken together, our data show that latency-associated gene products of HCMV generate CD4⁺ T cell responses in vivo, which are able to elicit effector function in response to both lytic and latently infected cells. Importantly and in contrast to CD4⁺ T cell populations, which recognise antigens solely expressed during lytic infection, include a subset of cells that secrete the immunosuppressive cytokine cIL-10. This suggests that HCMV skews the T cell responses to latency-associated antigens to one that is overall suppressive in order to sustain latent carriage in vivo.     

Human cytomegalovirus attenuates interleukin-1beta and tumor necrosis factor alpha proinflammatory signaling by inhibition of NF-kappaB activation

Viral infection is associated with a vigorous inflammatory response characterized by cellular infiltration and release of the proinflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-alpha). In the present study, we identified a novel function of human cytomegalovirus (HCMV) that results in inhibition of IL-1 and TNF-alpha signaling pathways. The effect on these pathways was limited to cells infected with the virus, occurred at late times of infection, and was independent of cell type or virus strain. IL-1 and TNF-alpha signaling pathways converge at a point upstream of NF-kappaB activation and involve phosphorylation and degradation of the NF-kappaB inhibitory molecule IkappaBalpha. The HCMV inhibition of IL-1 and TNF-alpha pathways corresponded to a suppression of NF-kappaB activation. Analysis of IkappaBalpha phosphorylation and degradation suggested that HCMV induced two independent blocks in NF-kappaB activation, which occurred upstream from the point of convergence of the IL-1 and TNF-alpha pathways. We believe that the ability of HCMV to inhibit these two major proinflammatory pathways reveals a critical aspect of HCMV biology, with possible importance for immune evasion, as well as establishment of infection in cell types persistently infected by this virus.     

Human cytomegalovirus differentially controls B cell and T cell responses through effects on plasmacytoid dendritic cells

Plasmacytoid dendritic cells (PDCs), the main producers of type I IFN in response to viral infection, are essential in antiviral immunity. In this study, we assessed the effect of human CMV (HCMV) infection on PDC function and on downstream B and T cell responses in vitro. HCMV infection of human PDCs was nonpermissive, as immediate-early but not late viral Ags were detected. HCMV led to partial maturation of PDCs and up-regulated MHC class II and CD83 molecules but not the costimulatory molecules CD80 and CD86. Regardless of viral replication, PDCs secreted cytokines after contact with HCMV, including IFN-alpha secretion that was blocked by inhibitory CpG, suggesting an engagement of the TLR7 and/or TLR9 pathways. In the presence of B cell receptor stimulation, soluble factors produced by HCMV-matured PDCs triggered B cell activation and proliferation. Through PDC stimulation, HCMV prompted B cell activation, but only induced Ab production in the presence of T cells or T cell secreted IL-2. Conversely, HCMV hampered the allostimulatory ability of PDCs, leading to decreased proliferation of CD4(+) and CD8(+) T cells. These findings reveal a novel mechanism by which HCMV differentially controls humoral and cell-mediate immune responses through effects on PDCs.     

Interactions between human immunodeficiency virus type 1 and human cytomegalovirus in human term syncytiotrophoblast cells coinfected with both viruses.

Human cytomegalovirus (HCMV) and human immunodeficiency virus type 1 (HIV-1) may interact in the pathogenesis of AIDS. The placental syncytiotrophoblast layer serves as the first line of defense of the fetus against viruses. We analyzed the patterns of replication of HIV-1 and HCMV in singly an dually infected human term syncytiotrophoblast cells cultured in vitro. Syncytiotrophoblast cells exhibited restricted permissiveness for HIV-1, while HCMV replication was restricted at the level of immediate-early and early gene products in the singly infected cells. We found that the syncytiotrophoblasts as an overlapping cell population could be coinfected with HIV-1 and HCMV. HIV-1 replication was markedly upregulated by previous or simultaneous infection of the cells with HCMV, whereas prior HIV-1 infection of the cells converted HCMV infection from a nonpermissive to a permissive one. No simultaneous enhancement of HCMV and HIV-1 expression was observed in the dually infected cell cultures. Major immediate-early proteins of HCMV were necessary for enhancement of HIV-1 replication, and interleukin-6 production induced by HCMV and further increased by replicating HIV-1 synergized with these proteins to produce this effect. Permissive replication cycle of HCMV was induced by the HIV-1 tat gene product. We were unable to detect HIV-1 (HCMV) or HCMV (HIV-1) pseudotypes in supernatant fluids from dually infected cell cultures. Our results suggest that interactions between HIV-1 and HCMV in coinfected syncytiotrophoblast cells may contribute to the transplacental transmission of both viruses.     

Adenovirus-vectored T cell vaccine for hepacivirus shows reduced effectiveness against a CD8 T cell escape variant in rats

There is an urgent need for a vaccine to prevent chronic infection by hepatitis C virus (HCV) and its many genetic variants. The first human vaccine trial, using recombinant viral vectors that stimulate pan-genotypic T cell responses against HCV non-structural proteins, failed to demonstrate efficacy despite significant preclinical promise. Understanding the factors that govern HCV T cell vaccine success is necessary for design of improved immunization strategies. Using a rat model of chronic rodent hepacivirus (RHV) infection, we assessed the impact of antigenic variation and immune escape upon success of a conceptually analogous RHV T cell vaccine. Naïve Lewis rats were vaccinated with a recombinant human adenovirus expressing RHV non-structural proteins (NS)3-5B and later challenged with a viral variant containing immune escape mutations within major histocompatibility complex (MHC) class I-restricted epitopes (escape virus). Whereas 7 of 11 (64%) rats cleared infection caused by wild-type RHV, only 3 of 12 (25%) were protected against heterologous challenge with escape virus. Uncontrolled replication of escape virus was associated with durable CD8 T cell responses targeting escaped epitopes alone. In contrast, clearance of escape virus correlated with CD4 T cell helper immunity and maintenance of CD8 T cell responses against intact viral epitopes. Interestingly, clearance of wild-type RHV infection after vaccination conferred enhanced protection against secondary challenge with escape virus. These results demonstrate that the efficacy of an RHV T cell vaccine is reduced when challenge virus contains escape mutations within MHC class I-restricted epitopes and that failure to sustain CD8 T cell responses against intact epitopes likely underlies immune failure in this setting. Further investigation of the immune responses that yield protection against diverse RHV challenges in this model may facilitate design of broadly effective HCV vaccines.     

Factors affecting human cytomegalovirus gene expression in human monocyte cell lines

To understand the mechanisms for establishing and reactivating monocytes and macrophages from latency by human cytomegalovirus (HCMV), human monocyte cell lines were infected and HCMV gene expression was investigated. Indirect immunofluorescence assay (IFA) with monoclonal antibody to HCMV major immediate early (MIE) IE1 or IE2 proteins revealed that HCMV MIE genes were expressed at low levels in relatively more differentiated THP-1 cells with TPA treatment after virus infection (posttreatment). Less differentiated cells such as U937 or HL60 did not support MIE gene expression even after TPA treatment. If THP-1 cells were pretreated before virus infection with TPA and became differentiated at the time of HCMV infection, MIE gene expression increased by 5-6 fold. Therefore, the relative degree of monocyte cell differentiation appears to be an important factor for regulating HCMV gene expression. Further IFA studies using monoclonal antibodies specific for IE1 or IE2 proteins indicate that the sequence and general pattern of IE1 and IE2 gene expression in THP-1 cells treated with TPA were similar to those in permissive human fibroblast cells with some delay in time. Formation of the replication compartment detected with monoclonal antibody to HCMV polymerase accessory protein UL44 in THP-1 cells suggests a fully productive replication process of HCMV in these cells. Monocytes are known to be induced to differentiate by hydrocortisone (HC), tumor necrosis factor (TNF)-alpha or interferon (IFN)-gamma. HC, which is known to stimulate HCMV replication in permissive human fibroblast (HF) cells, enhanced HCMV gene expression by 2-3 fold in TPA-pre or posttreated THP-1 cells, but TNF-alpha or IFN-gamma had little effect. Nitric oxide (NO) is released by immune cells in the defense against foreign stimuli and was shown to inhibit HCMV gene expression in HF cells. Increasing NO by nitroprusside significantly reduced HCMV gene expression in THP-1 cells. Therefore, it appears that the expression of HCMV immediate early genes in THP-1 cells treated with TPA closely resembles those in permissive HF cells.     

Degradation of RIG-I following cytomegalovirus infection is independent of apoptosis

Many viruses have evolved strategies to either evade or hijack host cell immune programs, as a means of promoting their own reproduction. For example, the human cytomegalovirus (HCMV) immediate-early protein vMIA/UL37ex1 inhibits host cell apoptosis, and its expression during infection aids virus replication. Here it is shown that stable expression of vMIA/UL37ex1 reduces cleavage of the innate immune response-proteins MAVS and RIG-I by caspases during apoptosis. Unexpectedly, it is demonstrated that RIG-I, but not MAVS, is degraded during HCMV infection. This process occurs in a non-apoptotic manner, and provides new evidence that HCMV may have evolved a unique strategy to evade RIG-I-mediated immune responses.     

Role of interferon alpha (IFN-alpha) and interferon gamma (IFN-gamma) in the control of the infection of monocyte-like cells with human cytomegalovirus (HCMV)

The role of cytokines in the control of HCMV infection has been studied in THP-1 cells, a macrophage-like cell model and in MRC-5 cells. HCMV replication was studied by immune detection of viral immediate-early antigens (IEA) and virus yield was evaluated in MRC-5 cells by immunoperoxidase staining. Pretreatment of MRC-5 and phorbol 12-myristate 13-acetate (PMA)-treated THP-1 cells with IFN-alpha or IFN-gamma for 24 hr prior to the infection reduced the number of infected cells and virus yield. A synergistic anti-CMV activity in synthesis of early proteins was obtained with these cytokines in combination with TNF-alpha in differentiated THP-1 cells only. Treatment of HCMV-infected differentiated THP-1 cells or MRC-5 cells with IFN-alpha or IFN-gamma alone had no inhibitory effect on virus replication, however the virus yield was reduced with ganciclovir. A synergistic anti-CMV activity in virus yield was obtained only when infected differentiated THP-1 cells were treated with ganciclovir in combination with IFN-gamma. The current study shows that IFN-alpha and IFN-gamma can play a role in the reduction of HCMV replication in macrophage-like cells and in the efficiency of therapies with ganciclovir in this cell type and that the anti-CMV effect of cytokines may be different in fibroblasts and in macrophage-like cells.     

Disruption of Type I Interferon Induction by HIV Infection of T Cells

Our main objective of this study was to determine how Human Immunodeficiency Virus (HIV) avoids induction of the antiviral Type I Interferon (IFN) system. To limit viral infection, the innate immune system produces important antiviral cytokines such as the IFN. IFN set up a critical roadblock to virus infection by limiting further replication of a virus. Usually, IFN production is induced by the recognition of viral nucleic acids by innate immune receptors and subsequent downstream signaling. However, the importance of IFN in the defense against viruses has lead most pathogenic viruses to evolve strategies to inhibit host IFN induction or responses allowing for increased pathogenicity and persistence of the virus. While the adaptive immune responses to HIV infection have been extensively studied, less is known about the balance between induction and inhibition of innate immune defenses, including the antiviral IFN response, by HIV infection. Here we show that HIV infection of T cells does not induce significant IFN production even IFN I Interferon production. To explain this paradox, we screened HIV proteins and found that two HIV encoded proteins, Vpu and Nef, strongly antagonize IFN induction, with expression of these proteins leading to loss of expression of the innate immune viral RNA sensing adaptor protein, IPS-1 (IFN-β promoter stimulator-1). We hypothesize that with lower levels of IPS-1 present, infected cells are defective in mounting antiviral responses allowing HIV to replicate without the normal antiviral actions of the host IFN response. Using cell lines as well as primary human derived cells, we show that HIV targeting of IPS-1 is key to limiting IFN induction. These findings describe how HIV infection modulates IFN induction providing insight into the mechanisms by which HIV establishes infection and persistence in a host.