KSHV Viral Protein Kinase Interacts with USP9X to Modulate the Viral Lifecycle.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi sarcoma (KS), the plasmablastic form of multicentric Castleman's disease, and primary effusion lymphoma. In sub-Saharan Africa, KS is the most common HIV-related malignancy and one of the most common childhood cancers. Immunosuppressed patients, including HIV-infected patients, are more prone to KSHV-associated disease. KSHV encodes a viral protein kinase (vPK) that is expressed from ORF36. KSHV vPK contributes to the optimal production of infectious viral progeny and upregulation of protein synthesis. To elucidate the interactions of vPK with cellular proteins in KSHV-infected cells, we used a bottom-up proteomics approach and identified host protein ubiquitin-specific peptidase 9X-linked (USP9X) as a potential interactor of vPK. Subsequently, we validated this interaction using a co-immunoprecipitation assay. We report that both the ubiquitin-like and the catalytic domains of USP9X are important for association with vPK. To uncover the biological relevance of the USP9X/vPK interaction, we investigated whether the knockdown of USP9X would modulate viral reactivation. Our data suggest that depletion of USP9X inhibits both viral reactivation and the production of infectious virions. Understanding how USP9X influences the reactivation of KSHV will provide insights into how cellular deubiquitinases regulate viral kinase activity and how viruses co-opt cellular deubiquitinases to propagate infection. Hence, characterizing the roles of USP9X and vPK during KSHV infection constitutes a first step toward identifying a potentially critical interaction that could be targeted by future therapeutics. IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi sarcoma (KS), the plasmablastic form of multicentric Castleman's disease, and primary effusion lymphoma. In sub-Saharan Africa, KS is the most common HIV-related malignancy. KSHV encodes a viral protein kinase (vPK) that aids viral replication. To elucidate the interactions of vPK with cellular proteins in KSHV-infected cells, we used an affinity purification approach and identified host protein ubiquitin-specific peptidase 9X-linked (USP9X) as a potential interactor of vPK. Depletion of USP9X inhibits both viral reactivation and the production of infectious virions. Overall, our data suggest a proviral role for USP9X.

The Immune-Specific E3 Ubiquitin Ligase MARCH1 Is Upregulated during Human Cytomegalovirus Infection to Regulate Iron Levels.

Human cytomegalovirus (HCMV) modulates numerous cellular pathways to facilitate infection. Iron is essential to many cellular processes and is often incorporated into proteins and enzymes involved in oxidative phosphorylation and DNA synthesis and repair, among others. Despite its prominent role in the cell, little is known about the regulation of iron metabolism during HCMV infection. Herein, we observe modulation of the transferrin receptor (TfR) during infection and a corresponding change in the cellular labile iron pool. TfR and the iron pool are increased early during infection and then return to mock levels at the late stages of infection. We identified the cellular ubiquitin ligase MARCH1 as an important regulator of TfR. MARCH1 plays a proviral role during infection, as its knockdown leads to a decrease in infectious titers. Knockdown of MARCH1 also leads to an increase in ROS, lipid peroxidation, and mitochondrial dysfunction. Inhibiting an early increase in TfR expression during infection also decreases virus production. These findings indicate the importance of tightly regulating iron metabolism during HCMV infection to facilitate efficient virus production. IMPORTANCE Iron is essential for cells, playing important roles in energy generation, DNA replication, and gene expression. During infection, HCMV alters many cellular processes to aid its replication. We found that iron levels are tightly regulated during infection and that dysregulation of iron levels alters the ability to produce infectious virions. We also found that HCMV inactivates many of the cellular safeguards put in place to deal with excess iron. Thus, infected cells become more susceptible to variations in iron levels, which could be exploited as a therapeutic strategy for dealing with HCMV infections.

Human Cytomegalovirus Decreases Major Histocompatibility Complex Class II by Regulating Class II Transactivator Transcript Levels in a Myeloid Cell Line.

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that encodes many proteins to modulate the host immune response. Extensive efforts have led to the elucidation of multiple strategies employed by HCMV to effectively block NK cell targeting of virus-infected cells and the major histocompatibility complex (MHC) class I-primed CD8+ T cell response. However, viral regulation of the MHC class II-mediated CD4+ T cell response is understudied in endogenous MHC class II-expressing cells, largely because the popular cell culture systems utilized for studying HCMV do not endogenously express MHC class II. Of the many cell types infected by HCMV in the host, myeloid cells, such as monocytes, are of particular importance due to their role in latency and subsequent dissemination throughout the host. We investigated the impact of HCMV infection on MHC class II in Kasumi-3 cells, a myeloid-progenitor cell line that endogenously expresses the MHC class II gene, HLA-DR. We observed a significant reduction in the expression of surface and total HLA-DR at 72 h postinfection (hpi) and 120 hpi in infected cells. The decrease in HLA-DR expression was independent of the expression of previously described viral genes that regulate the MHC class II complex or the unique short (US) region of HCMV, a region expressing many immunomodulatory genes. The altered surface level of HLA-DR was not a result of increased endocytosis and degradation but was a result of a reduction in HLA-DR transcripts due to a decrease in the expression of the class II transactivator (CIITA).IMPORTANCE Human cytomegalovirus (HCMV) is an opportunistic herpesvirus that is asymptomatic for healthy individuals but that can lead to severe pathology in patients with congenital infections and immunosuppressed patients. Thus, it is important to understand the modulation of the immune response by HCMV, which is understudied in the context of endogenous MHC class II regulation. Using Kasumi-3 cells as a myeloid progenitor cell model endogenously expressing MHC class II (HLA-DR), this study shows that HCMV decreases the expression of HLA-DR in infected cells by reducing the transcription of HLA-DR transcripts early during infection independently of the expression of previously implicated genes. This is an important finding, as it highlights a mechanism of immune evasion utilized by HCMV to decrease the expression of MHC class II in a relevant cell system that endogenously expresses the MHC class II complex.

UL88 Mediates the Incorporation of a Subset of Proteins into the Virion Tegument.

Little is known about the human cytomegalovirus (HCMV) tegument protein UL88. Large-scale genomic studies have reported disparate results for UL88-null viruses, reporting both no phenotype and a >1-log decrease in virus titers. UL88 has also been reported to interact with UL69 and UL48, but the functional relevance of this interaction is unknown. Here, we report that UL88, which is conserved among different viral strains, is dispensable for production of infectious HCMV virions in multiple HCMV strains and cell types. However, the specific infectivity of HCMV virions suffers in the absence of UL88, as more genomes are required per PFU. This may be a result of altered virion tegument protein composition, as Western blot analysis shows a significant reduction in the tegument levels of pp71, UL47, and UL48 in viruses lacking UL88. While an interaction between UL88 and UL48 has previously been reported, we show that UL88 can interact with UL47; however, UL88 does not appear to be part of a stable complex consisting of UL47 and UL48. These findings identify an important role for UL88 in incorporating the viral proteins UL47 and UL48 into the virion tegument layer.IMPORTANCE A better understanding of the role and functions of tegument proteins in HCMV, many of which remain uncharacterized, will contribute to our understanding of the biology of HCMV. The virus has a large genome, greater than 230 kb, and functional annotation of these genes is important for identifying novel targets for improving therapeutic intervention. This study identifies a role for a viral tegument protein with unknown function, UL88, in maintaining the proper tegument composition of HCMV virions. Virions produced in the absence of UL88 exhibit decreased fitness and require more genomes per infectious unit.

The Role of RNA Sensors in Regulating Innate Immunity to Gammaherpesviral Infections.

Kaposi's sarcoma-associated herpesvirus (KSHV) and the Epstein-Barr virus (EBV) are double-stranded DNA oncogenic gammaherpesviruses. These two viruses are associated with multiple human malignancies, including both B and T cell lymphomas, as well as epithelial- and endothelial-derived cancers. KSHV and EBV establish a life-long latent infection in the human host with intermittent periods of lytic replication. Infection with these viruses induce the expression of both viral and host RNA transcripts and activates several RNA sensors including RIG-I-like receptors (RLRs), Toll-like receptors (TLRs), protein kinase R (PKR) and adenosine deaminases acting on RNA (ADAR1). Activation of these RNA sensors induces the innate immune response to antagonize the virus. To counteract this, KSHV and EBV utilize both viral and cellular proteins to block the innate immune pathways and facilitate their own infection. In this review, we summarize how gammaherpesviral infections activate RNA sensors and induce their downstream signaling cascade, as well as how these viruses evade the antiviral signaling pathways to successfully establish latent infection and undergo lytic reactivation.

The regulation of KSHV lytic reactivation by viral and cellular factors.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic herpesvirus that exhibits two distinct phases of infection in the host-latent and lytic. The quiescent latent phase is defined by limited expression of a subset of viral proteins and microRNAs, and an absence of virus production. KSHV periodically reactivates from latency to undergo active lytic replication, leading to production of new infectious virions. This switch from the latent to the lytic phase requires the viral protein regulator of transcription activator (RTA). RTA, along with other virally encoded proteins, is aided by host factors to facilitate this transition. Herein, we highlight the key host proteins that are involved in mediating RTA activation and KSHV lytic replication and discuss the cellular processes in which they function. We will also focus on the modulation of viral reactivation by the innate immune system, and how KSHV influences key immune signaling pathways to aid its own lifecycle.

Hepatitis B Virus Immunopathology, Model Systems, and Current Therapies.

Most people develop acute hepatitis B virus (HBV)-related hepatitis that is controlled by both humoral and cellular immune responses following acute infection. However, a number of individuals in HBV-endemic areas fail to resolve the infection and consequently become chronic carriers. While a vaccine is available and new antiviral drugs are being developed, elimination of persistently infected cells is still a major issue. Standard treatment in HBV infection includes IFN-α, nucleoside, or nucleotide analogs, which has direct antiviral activity and immune modulatory capacities. However, immunological control of the virus is often not durable. A robust T-cell response is associated with control of HBV infection and liver damage; however, HBV-specific T cells are deleted, dysfunctional, or become exhausted in chronic hepatitis patients. As a result, efforts to restore virus-specific T-cell immunity in chronic HBV patients using antiviral therapy, immunomodulatory cytokines, or therapeutic vaccination have had little success. Adoptive cell transfer of T cells with specificity for HBV antigen+ cells represents an approach aiming to ultimately eliminate residual hepatocytes carrying HBV covalently closed circular DNA (cccDNA). Here, we discuss recent findings describing HBV immunopathology, model systems, and current therapies.

c-Myc-Induced Survivin Is Essential for Promoting the Notch-Dependent T Cell Differentiation from Hematopoietic Stem Cells.

Notch is indispensable for T cell lineage commitment, and is needed for thymocyte differentiation at early phases. During early stages of T cell development, active Notch prevents other lineage potentials including B cell lineage and myeloid cell (e.g., dendritic cell) lineage. Nevertheless, the precise intracellular signaling pathways by which Notch promotes T cell differentiation remain unclear. Here we report that the transcription factor c-Myc is a key mediator of the Notch signaling-regulated T cell differentiation. In a well-established in vitro differentiation model of T lymphocytes from hematopoietic stem cells, we showed that Notch1 and 4 directly promoted c-Myc expression; dominant-negative (DN) c-Myc inhibited early T cell differentiation. Moreover, the c-Myc expression activated by Notch signaling increased the expression of survivin, an inhibitor of apoptosis (IAP) protein. We further demonstrated that over-expression of c-Myc increased the abundance of survivin and the T cell differentiation thereof, whereas dn c-Myc reduced survivin levels and concomitantly retarded the differentiation. The c-Myc-dependent survivin induction is functionally germane, because Notch-dependent T cell differentiation was canceled by the depletion of survivin. These results identify both c-Myc and survivin as important mediators of the Notch signaling-regulated differentiation of T lymphocytes from hematopoietic stem cells.

Development and characterization of naive single-type tumor antigen-specific CD8+ T lymphocytes from murine pluripotent stem cells.

Optimal approaches to differentiate tumor antigen-specific cytotoxic T lymphocytes (CTLs) from pluripotent stem cells (PSCs) remain elusive. In the current study, we showed that combination of in vitro priming through Notch ligands and in vivo development facilitated the generation of tumor Ag-specific CTLs that effectively inhibited tumor growth. We co-cultured the murine induced PSCs (iPSCs) genetically modified with tyrosinase-related protein 2 (TRP2)-specific T cell receptors with OP9 cell line expressing both Notch ligands Delta-like 1 and 4 (OP9-DL1/DL4) for a week before adoptively transferred into recipient C67BL/6 mice. Three weeks later, B16 melanoma cells were inoculated subcutaneously, and the antitumor activity of the iPSC-derived T cells was assessed. We observed the development of the TRP2-specific iPSC-CD8+ T cells that responded to Ag stimulation and infiltrated into melanoma tissues, significantly inhibited the tumor growth, and improved the survival of the tumor-bearing mice. Thus, this approach may provide a novel effective strategy to treatment of malignant tumors.

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity.

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

Melanoma Immunotherapy in Mice Using Genetically Engineered Pluripotent Stem Cells.

Adoptive cell transfer (ACT) of antigen (Ag)-specific CD8(+) cytotoxic T lymphocytes (CTLs) is a highly promising treatment for a variety of diseases. Naive or central memory T-cell-derived effector CTLs are optimal populations for ACT-based immunotherapy because these cells have a high proliferative potential, are less prone to apoptosis than terminally differentiated cells, and have the higher ability to respond to homeostatic cytokines. However, such ACT with T-cell persistence is often not feasible due to difficulties in obtaining sufficient cells from patients. Here we present that in vitro differentiated HSCs of engineered PSCs can develop in vivo into tumor Ag-specific naive CTLs, which efficiently suppress melanoma growth. Mouse-induced PSCs (iPSCs) were retrovirally transduced with a construct encoding chicken ovalbumin (OVA)-specific T-cell receptors (TCRs) and survival-related proteins (i.e., BCL-xL and survivin). The gene-transduced iPSCs were cultured on the delta-like ligand 1-expressing OP9 (OP9-DL1) murine stromal cells in the presence of murine recombinant cytokines (rFlt3L and rIL-7) for a week. These iPSC-derived cells were then intravenously adoptively transferred into recipient mice, followed by intraperitoneal injection with an agonist α-Notch 2 antibody and cytokines (rFlt3L and rIL-7). Two weeks later, naive OVA-specific CD8(+) T cells were observed in the mouse peripheral lymphatic system, which were responsive to OVA-specific stimulation. Moreover, the mice were resistant to the challenge of B16-OVA melanoma induction. These results indicate that genetically modified stem cells may be used for ACT-based immunotherapy or serve as potential vaccines.

C-Myc regulation by costimulatory signals modulates the generation of CD8+ memory T cells during viral infection.

The signalling mechanisms of costimulation in the development of memory T cells remain to be clarified. Here, we show that the transcription factor c-Myc in CD8(+) T cells is controlled by costimulatory molecules, which modulates the development of memory CD8(+) T cells. C-Myc expression was dramatically reduced in Cd28(-/-) or Ox40(-/-) memory CD8(+) T cells, and c-Myc over-expression substantially reversed the defects in the development of T-cell memory following viral infection. C-Myc regulated the expression of survivin, an inhibitor of apoptosis, which promoted the generation of virus-specific memory CD8(+) T cells. Moreover, over-expression of survivin with bcl-xL, a downstream molecule of NF-κB and intracellular target of costimulation that controls survival, in Cd28(-/-) or Ox40(-/-) CD8(+) T cells, reversed the defects in the generation of memory T cells in response to viral infection. These results identify c-Myc as a key controller of memory CD8(+) T cells from costimulatory signals.

Stem cell-derived tissue-associated regulatory T cells ameliorate the development of autoimmunity.

Pluripotent stem cells (PSCs) have the potential to produce almost all of the cells in the body, including regulatory T cells (Tregs). However, the exact conditions required for the development of antigen (Ag)-specific Tregs from PSCs (i.e., PSC-Tregs) are not well delineated. Ag-specific PSC-Tregs can be tissue/organ-associated and migrate to local inflamed tissues/organs to suppress the autoimmune response after adoptive transfer, thereby avoiding potential overall immunosuppression from non-specific Tregs. In this study, we developed a new approach to generate functional Ag-specific Tregs from induced PSCs (iPSCs), i.e., iPSC-Tregs, which had the ability to generate an Ag-specific immunosuppressive response in a murine model of arthritis. We retrovirally transduced murine iPSCs with a construct containing genes of Ag-specific T cell receptor (TCR) and the transcriptional factor FoxP3. We differentiated the iPSCs into Ag-specific iPSC-Tregs using in vitro or in vivo Notch signaling, and demonstrated that adoptive transfer of such Tregs dramatically suppressed autoimmunity in a well-established Ag-induced arthritis model, including the inflammation, joint destruction, cartilage prostaglandin depletion, osteoclast activity, and Th17 production. Our results indicate that PSCs can be used to develop Ag-specific Tregs, which have a therapeutic potential for Treg-based therapies of autoimmune disorders.