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Kaposi's sarcoma-associated herpesvirus (KSHV) is tightly linked to at least two lymphoproliferative disorders, primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD). However, the development of KSHV-mediated lymphoproliferative disease is not fully understood. Here, we generated two recombinant KSHV viruses deleted for the first RBP-Jκ binding site (RTA1st) and all three RBP-Jκ binding sites (RTAall) within the RTA promoter. Our results showed that RTA1st and RTAall recombinant viruses possess increased viral latency and a decreased capability for lytic replication in HEK 293 cells, enhancing colony formation and proliferation of infected cells. Furthermore, recombinant RTA1st and RTAall viruses showed greater infectivity in human peripheral blood mononuclear cells (PBMCs) relative to wt KSHV. Interestingly, KSHV BAC36 wt, RTA1st and RTAall recombinant viruses infected both T and B cells and all three viruses efficiently infected T and B cells in a time-dependent manner early after infection. Also, the capability of both RTA1st and RTAall recombinant viruses to infect CD19+ B cells was significantly enhanced. Surprisingly, RTA1st and RTAall recombinant viruses showed greater infectivity for CD3+ T cells up to 7 days. Furthermore, studies in Telomerase-immortalized human umbilical vein endothelial (TIVE) cells infected with KSHV corroborated our data that RTA1st and RTAall recombinant viruses have enhanced ability to persist in latently infected cells with increased proliferation. These recombinant viruses now provide a model to explore early stages of primary infection in human PBMCs and development of KSHV-associated lymphoproliferative diseases. [ABSTRACT FROM AUTHOR]

Cytokine-mediated JAK/STAT signaling controls numerous important biologic responses like immune function, cellular growth, and differentiation. Inappropriate activation of this signaling pathway is associated with a range of malignancies. Kaposi's sarcoma-associated herpesvirus (KSHV) is the infectious viral agent associated with Kaposi's sarcoma and may also contribute to B-cell disorders, which include primary effusion lymphoma (PEL) and multicentric Castleman's disease. However, regulation of cytokine-mediated lymphocytic immune response by KSHV is not fully understood. In this report, we demonstrate that KSHV suppresses the interleukin-4 (IL-4)-stimulated immune response of B-lymphocyte activation and cell proliferation. Moreover, we show that the latency-associated nuclear antigen (LANA) encoded by KSHV is essential for viral blocking of IL-4-induced signaling. LANA reduces phosphorylation of the signal transducers and activators of transcription 6 (STAT6) on Y-641 and concomitantly its DNA binding ability. Importantly, knockdown of endogenous STAT6 dramatically increases the sensitivity of PEL cells to low-serum stress or chemical-mediated cellular apoptosis and reactivation of KSHV from latent replication. Thus, these findings suggest that the IL-4/STAT6 signaling network is precisely controlled by KSHV for survival, maintenance of latency, and suppression of the host cytokine immune response of the virus-infected cells. [ABSTRACT FROM AUTHOR]

TRIM32 is often aberrantly expressed in many types of cancers. Kaposi’s sarcoma-associated herpesvirus (KSHV) is linked with several human malignancies, including Kaposi’s sarcoma and primary effusion lymphomas (PELs). Increasing evidence has demonstrated the crucial role of KSHV lytic replication in viral tumorigenesis. However, the role of TRIM32 in herpesvirus lytic replication remains unclear. Here, we reveal that the expression of TRIM32 is upregulated by KSHV in latency, and reactivation of KSHV lytic replication leads to the inhibition of TRIM32 in PEL cells. Strikingly, RTA, the master regulator of lytic replication, interacts with TRIM32 and dramatically promotes TRIM32 for degradation via the proteasome systems. Inhibition of TRIM32 induces cell apoptosis and in turn inhibits the proliferation and colony formation of KSHV-infected PEL cells and facilitates the reactivation of KSHV lytic replication and virion production. Thus, our data imply that the degradation of TRIM32 is vital for the lytic activation of KSHV and is a potential therapeutic target for KSHV-associated cancers. IMPORTANCE TRIM32 is associated with many cancers and viral infections; however, the role of TRIM32 in viral oncogenesis remains largely unknown. In this study, we found that the expression of TRIM32 is elevated by Kaposi’s sarcoma-associated herpesvirus (KSHV) in latency, and RTA (the master regulator of lytic replication) induces TRIM32 for proteasome degradation upon viral lytic reactivation. This finding provides a potential therapeutic target for KSHV-associated cancers. [ABSTRACT FROM AUTHOR]

Hypoxia-inducible factor 1 (HIF-1) is a ubiquitously expressed transcriptional regulator involved in induction of numerous genes associated with angiogenesis and tumor growth. Kaposi's sarcoma, associated with increased angiogenesis, is a highly vascularized, endothelial cell-derived tumor. Previously, we have shown that the latency-associated nuclear antigen (LANA) encoded by Kaposi's sarcoma-associated herpesvirus (KSHV) targets the HIF-1 α suppressors von Hippel-Lindau protein and p53 for degradation via its suppressor of cytokine signaling-box motif, which recruits the EC5S ubiquitin complex. Here we further show that HIF-1 α was aberrantly accumulated in KSHV latently infected primary effusion lymphoma (PEL) cells, as well as HEK293 cells infected with KSHV, and also show that a potential α-helical amino-terminal domain of LANA was important for HIF-1 α nuclear accumulation in normoxic conditions. Moreover, we have now determined that this association was dependent on the residues 46 to 89 of LANA and the oxygen-dependent degradation domain of HIF-1 α. Introduction of specific small interfering RNA against LANA into PEL cells also resulted in a diminished nuclear accumulation of HIF-1 α. Therefore, these data show that LANA can function not only as an inhibitor of HIF-1 α suppressor proteins but can also induce nuclear accumulation of HIF-1 α during KSHV latent infection. [ABSTRACT FROM AUTHOR]

Hypoxia can induce lytic replication of Kaposi's sarcoma-associated herpesvirus (KSHV) in primary effusion lymphoma (PEL) cells. However, the molecular mechanism of lytic reactivation of KSHV by hypoxia remains unclear. Here we show that the latency-associated nuclear antigen (LANA), which plays a crucial role in modulating viral and cellular gene expression, directly associated with a low oxygen responder, hypoxia-inducible factor-1α (HIF-1α). LANA enhanced not only the transcriptional activities of HIF-1α but also its mRNA level. Coimmunoprecipitation and immunofluorescence studies documented a physical interaction between LANA and HIF-1α in transiently transfected 293T cells as well as in PEL cell lines during hypoxia. Through sequence analysis, several putative hypoxia response elements (HRE-1 to -6) were identified in the essential lytic gene Rta promoter. Reporter assays showed that HRE-2 (-1130 to -1123) and HRE-5 and HRE-6 (+234 to +241 and +812 to +820, respectively, within the intron sequence) were necessary and sufficient for the LANA-mediated HIF-1α response. Electrophoretic mobility shift assays showed HIF-1α-dependent binding of a LANA protein complex specifically to the HRE-2, -5, and -6 motifs within the promoter regulatory sequences. This study demonstrates that hypoxia-induced KSHV lytic replication is mediated at least in part through cooperation of HIF-1α with LANA bound to the HRE motifs of the Rta promoter. [ABSTRACT FROM AUTHOR]

Kaposi's sarcoma-associated herpesvirus (KSHV; or human herpesvirus 8 [HHV8]) is implicated in the pathogenesis of many human malignancies including Kaposi's sarcoma (KS), multicentric Castleman's disease (MCD), and primary effusion lymphoma (PEL). KSHV infection displays two alternative life cycles, referred to as the latent and lytic or productive cycle. Previously, we have reported that the replication and transcription activator (RTA), a major lytic cycle transactivator, contributes to the development of KSHV latency by inducing latency-associated nuclear antigen (LANA) expression during early stages of infection by targeting RBP-Jκ, the master regulator of the Notch signaling pathway. Here, we generated a bacterial artificial chromosome (BAC) KSHV recombinant virus with a deletion of the RBP-Jκ site within the LANA promoter to evaluate the function of the RBP-Jκ cognate site in establishing primary latent infection. The results showed that genetic disruption of the RBP-Jκ binding site within the KSHV LANA promoter led to enhanced expression of the KSHV-encoded immediate early RTA, resulting in an increase in lytic replication during primary infection of human peripheral blood mononuclear cells (PBMCs). This system provides a powerful tool for use in indentifying additional cellular and viral molecules involved in LANA-mediated latency maintenance during the early stages of KSHV infection. [ABSTRACT FROM AUTHOR]

Major histocompatibility complex class II (MHC-II) molecules play a central role in adaptive antiviral immunity by presenting viral peptides to CD4+ T cells. Due to their key role in adaptive immunity, many viruses, including Kaposi's sarcoma-associated herpesvirus (KSHV), have evolved multiple strategies to inhibit the MHC-II antigen presentation pathway. The expression of MHC-II, which is controlled mainly at the level of transcription, is strictly dependent upon the binding of the class II transactivator (CIITA) to the highly conserved promoters of all MHC-II genes. The recruitment of CIITA to MHC-II promoters requires its direct interactions with a preassembled MHC-II enhanceosome consisting of cyclic AMP response element-binding protein (CREB) and nuclear factor Y (NF-Y) complex and regulatory factor X (RFX) complex proteins. Here, we show that KSHV-encoded latency-associated nuclear antigen (LANA) disrupts the association of CIITA with the MHC-II enhanceosome by binding to the components of the RFX complex. Our data show that LANA is capable of binding to all three components of the RFX complex, RFX-associated protein (RFXAP), RFX5, and RFX-associated ankyrin-containing protein (RFXANK), in vivo but binds more strongly with the RFXAP component in in vitro binding assays. Levels of MHC-II proteins were significantly reduced in KSHV-infected as well as LANA-expressing B cells. Additionally, the expression of LANA in a luciferase promoter reporter assay showed reduced HLA-DRA promoter activity in a dose-dependent manner. Chromatin immunoprecipitation assays showed that LANA binds to the MHC-II promoter along with RFX proteins and that the overexpression of LANA disrupts the association of CIITA with the MHC-II promoter. These assays led to the conclusion that the interaction of LANA with RFX proteins interferes with the recruitment of CIITA to MHC-II promoters, resulting in an inhibition of MHC-II gene expression. Thus, the data presented here identify a novel mechanism used by KSHV to downregulate the expressions of MHC-II genes. [ABSTRACT FROM AUTHOR]

Survivin is a master regulator of cell proliferation and cell viability and is highly expressed in most human tumors. The molecular network linked to survivin expression in tumors has not been completely elucidated. In this study, we show that latency-associated nuclear antigen (LANA), a multifunctional protein of Kaposi's sarcoma-associated herpesvirus (KSHV) that is found in Kaposi's sarcoma tumors, upregulates survivin expression and increases the proliferation of KSHV-infected B cells. Analysis of pathway-specific gene arrays showed that survivin expression was highly upregulated in BJAB cells expressing LANA. The mRNA levels of survivin were also upregulated in HEK 293 and BJAB cells expressing LANA. Similarly, protein levels of survivin were significantly higher in LANA-expressing, as well as KSHV-infected, cells. Survivin promoter activity assays identified GC/Sp1 and p53 cis-acting elements within the core promoter region as being important for LANA activity. Gel mobility shift assays revealed that LANA forms a complex with Sp1 or Sp1-like proteins bound to the GC/Sp1 box of the survivin promoter. In addition, a LANA/p53 complex bound to the p53 cis-acting element within the survivin promoter, indicating that upregulation of survivin expression can also occur through suppression of p53 function. Furthermore, immunohistochemistry analyses revealed that survivin expression was upregulated in KSHV-associated Kaposi's sarcoma tissue, suggesting that LANA plays an important role in the upregulation of survivin expression in KSHV-infected endothelial cells. Knockdown of survivin expression by lentivirus-delivered small hairpin RNA resulted in loss of cell proliferation in KSHV-infected cells. Therefore, upregulation of survivin expression in KSHV-associated human cells contributes to their proliferation. [ABSTRACT FROM AUTHOR]

Kaposi's sarcoma-associated herpesvirus (KSHV) genomes are tethered to the host chromosomes and partitioned faithfully into daughter cells with the host chromosomes. The latency-associated nuclear antigen (LANA) is important for segregation of the newly synthesized viral genomes to the daughter nuclei. Here, we report that the nuclear mitotic apparatus protein (NuMA) and LANA can associate in KSHV-infected cells. In synchronized cells, NuMA and LANA are colocalized in interphase cells and separate during mitosis at the beginning of prophase, reassociating again at the end of telophase and cytokinesis. Silencing of NuMA expression by small interfering RNA and expression of LGN and a dominant-negative of dynactin (P150-CC1), which disrupts the association of NuMA with microtubules, resulted in the loss of KSHV terminal-repeat plasmids containing the major latent origin. Thus, NuMA is required for persistence of the KSHV episomes in daughter cells. This interaction between NuMA and LANA is critical for segregation and maintenance of the KSHV episomes through a temporally controlled mechanism of binding and release during specific phases of mitosis. [ABSTRACT FROM AUTHOR]