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Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) is a known regulatory transcription factor that has been shown to interact with histone deacetylase 1 (HDAC1) when cotransfected in human cell lines and by in vitro binding experiments. Previous studies have shown that EBNA3C interacts with p300 and prothymosin alpha (ProTα) in EBV-infected cells and may be involved in recruiting acetyltransferases to the chromatin for acetylation of histones and transcriptional activation. EBNA3C has also been shown to function as a repressor of transcription when directed to promoters. In this report, we show that EBNA3C complexed with ProTα can also recruit deacetylase activity and associates in a complex that includes HDAC1 and HDAC2 in human B cells. A complex of EBNA3C and ProTα coimmunoprecipitated with HDAC1 and HDAC2 in cell lines stably expressing EBNA3C. Additionally, this complex associated with the mSin3A and NCoR corepressors in EBNA3C-expressing cell lines and may function in a complex with additional transcription factors known to be repressors of transcription. EBNA3C in complex with ProTα recruited deacetylase activity in cell lines stably expressing EBNA3C, and this activity was shown to be partially sensitive to trichostatin A (TSA). This suggests an association with other deacetylases that are insensitive to the general inhibitory effects of TSA, as the entire activity was not abolished in multiple assays. The association between EBNA3C and the corepressors as well as HDACs is likely to depend on the presence of ProTα in the complex. Immunoprecipitation with anti-ProTα antibody immunoprecipitated EBNA3C and the other repressors, whereas immunoprecipitation with anti-EBNA3C antibody resulted in little or no association with these molecules associated with transcription repression. Clearly, EBNA3C functions as a component of a number of dynamic complexes which function in repression and activation of transcription.

Previous studies have demonstrated the interaction between the Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) and the metastatic suppressor Nm23-H1 both in vitro and in vivo (C. Subramanian, M. A. Cotter II, and E. S. Robertson, Nat. Med. 7:350-355, 2001). EBNA3C can reverse the ability of Nm23-H1 to suppress migration of Burkitt's lymphoma and breast carcinoma cell lines in vitro. EBNA3C contributes to EBV-associated human cancers by regulating transcription of a number of cellular and viral promoters and by targeting and altering the transcription activities of the metastasis suppressor Nm23-H1. Cyclo-oxygenase-2 (COX-2), an inducible enzyme important in inflammation, is overexpressed in a variety of cancers and can influence cell migration. In this report we show that Nm23-H1 and EBNA3C can modulate expression of COX-2 in the context of EBV infection and transformation. The levels of COX-2 were consistently higher in EBV-positive cells than in EBV-negative cells. Additionally, we show that Nm23-H1 can upregulate the COX-2 promoter element in luciferase reporter assays, whereas EBNA3C alone did not affect the level of response but clearly contributed to an additive increase when coexpressed with Nm23-H1. The downstream effect of COX-2 expression was also evaluated and showed that prostaglandin E2 levels increased with Nm23-H1 and that there was some level of cooperativity in the presence of EBNA3C. The majority of this response was mediated through the cyclic AMP response element and NF-κB sites. These studies suggest a potential role for COX-2 in EBV-associated human cancers.

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes 12 pre-microRNAs (pre-miRNAs). Current studies have shown that these miRNAs are involved in regulation of viral and host gene expression, implicating a role in the maintenance of viral latency and suppression of antiviral innate immunity. However, the functions of these miRNAs remain largely unknown. On the basis of the sequence homology between oncogenic miR-155 and KSHV-encoded miR-K12-11, we hypothesized that miR-K12-11 could attenuate transforming growth factor β (TGF-β) signaling, facilitating viral infection and tumorigenesis. In the present study, we demonstrated that ectopic expression of miR-K12-11 in Ramos, a TGF-β-sensitive cell line, downregulated TGF-β signaling and facilitated cell proliferation upon TGF-β treatment by directly targeting SMAD5, an important mediator in TGF-β signaling. In addition, the downregulation of SMAD5 by miR-K12-11 was further confirmed in a de novo KSHV infection system or latently infected KSHV-positive B-lymphoma cell lines. More importantly, repression of miR-K12-11 by a specific sponge inhibitor restored the expression of SMAD5 in both de novo-infected and latently infected cells. Finally, we found that restoration of SMAD5, in addition to the TGF-β type II receptor, which was epigenetically silenced by the latent viral protein latency-associated nuclear antigen, sensitized BC3 cells to the cytostatic effect of TGF-β signaling. Taken together, our findings highlight a novel mechanism in which miR-K12-11 downregulates TGF-β signaling and suggest that viral miRNAs and proteins may exert a dichotomy regulation in virus-induced oncogenesis by targeting the same signaling pathway.

Hepatitis B, which caused by hepatitis B virus (HBV) infection, remains a major health threat worldwide. Hepatic injury and regeneration from chronic inflammation are the main driving factors of liver fibrosis and cirrhosis in chronic hepatitis B. Proinflammatory tumor necrosis factor alpha (TNF-α) has been implicated as a major inducer of liver cell death during viral hepatitis. Here, we report that in hepatoma cell lines and in primary mouse and human hepatocytes, expression of hepatitis B virus core (HBc) protein made cells susceptible to TNF-α-induced apoptosis. We found by tandem affinity purification and mass spectrometry that receptor of activated protein kinase C 1 (RACK1) interacted with HBc. RACK1 was recently reported as a scaffold protein that facilitates the phosphorylation of mitogen-activated protein kinase kinase 7 (MKK7) by its upstream activators. Our study showed that HBc abrogated the interaction between MKK7 and RACK1 by competitively binding to RACK1, thereby downregulating TNF-α-induced phosphorylation of MKK7 and the activation of c-Jun N-terminal kinase (JNK). In line with this finding, specific knockdown of MKK7 increased the sensitivity of hepatocytes to TNF-α-induced apoptosis, while overexpression of RACK1 counteracted the proapoptotic activity of HBc. Capsid particle formation was not obligatory for HBc proapoptotic activity, as analyzed using an assembly-defective HBc mutant. In conclusion, the expression of HBc sensitized hepatocytes to TNF-α-induced apoptosis by disrupting the interaction between MKK7 and RACK1. Our study is thus the first indication of the pathogenic effects of HBc in liver injury during hepatitis B. IMPORTANCE Our study revealed a previously unappreciated role of HBc in TNF-α-mediated apoptosis. The proapoptotic activity of HBc is important for understanding hepatitis B pathogenesis. In particular, HBV variants associated with severe hepatitis may upregulate apoptosis of hepatocytes through enhanced HBc expression. Our study also found that MKK7 is centrally involved in TNF-α-induced hepatocyte apoptosis and revealed a multifaceted role for JNK signaling in this process.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a human gammaherpesvirus with latent and lytic reactivation cycles. The mechanism by which KSHV evades the innate immune system to establish latency has not yet been precisely elucidated. Toll-like receptors (TLRs) are the first line of defense against viral infections. Myeloid differentiation factor 88 (MyD88) is a key adaptor that interacts with all TLRs except TLR3 to produce inflammatory factors and type I interferons (IFNs), which are central components of innate immunity against microbial infection. Here, we found that KSHV replication and transcription activator (RTA), which is an immediate-early master switch protein of viral cycles, downregulates MyD88 expression at the protein level by degrading MyD88 through the ubiquitin (Ub)-proteasome pathway. We identified the interaction between RTA and MyD88 in vitro and in vivo and demonstrated that RTA functions as an E3 ligase to ubiquitinate MyD88. MyD88 also was repressed at the early stage of de novo infection as well as in lytic reactivation. We also found that RTA inhibited lipopolysaccharide (LPS)-triggered activation of the TLR4 pathway by reducing IFN production and NF-κB activity. Finally, we showed that MyD88 promoted the production of IFNs and inhibited KSHV LANA-1 gene transcription. Taken together, our results suggest that KSHV RTA facilitates the virus to evade innate immunity through the degradation of MyD88, which might be critical for viral latency control. IMPORTANCE MyD88 is an adaptor for all TLRs other than TLR3, and it mediates inflammatory factors and IFN production. Our study demonstrated that the KSHV RTA protein functions as an E3 ligase to degrade MyD88 through the ubiquitin-proteasome pathway and block the transmission of TLRs signals. Moreover, we found that KSHV inhibited MyD88 expression during the early stage of de novo infection as well as in lytic reactivation. These results provide a potential mechanism for the virus to evade innate immunity.

Kaposi's sarcoma-associated herpesvirus (KSHV), which belongs to the Gammaherpesviridae , typically displays two different phases in its life cycle, the latent phase and the lytic phase. Latency-associated nuclear antigen (LANA), the primary viral product during latency, has been reported to bind to a series of cellular gene promoters to modulate gene transcription. To systemically elucidate the cellular genes regulated by LANA, we identified genome-wide LANA binding sites by chromatin immunoprecipitation coupled with sequencing (ChIP-seq). We stratified ChIP-seq data and found that LANA might be involved in the macromolecule catabolic process. Specifically, we found and verified that LANA could directly bind to the promoter of the SUMO/sentrin-specific peptidase 6 (SENP6) gene in vivo and in vitro . LANA could repress SENP6 promoter activity in a dose-dependent manner in a reporter gene assay. LANA expression was sufficient to inhibit endogenous SENP6 expression at both the RNA and protein levels. Moreover, SENP6 overexpression in KSHV-infected cells reduced LANA at the protein level. Mechanistically, we found that SENP6 could interact with LANA and reduce the formation of sumoylated LANA, which relies on the desumoylation ability of SENP6. During de novo infection, SENP6 overexpression would decrease the abundance of LANA and enhance viral gene expression, which would hamper the establishment of latency. Taken together, these data suggest that KSHV-encoded LANA could inhibit SENP6 expression to regulate the abundance of itself, which may play an important role in controlling the establishment of latency. IMPORTANCE LANA, as a key latent protein produced by KSHV, is responsible for episome persistence and regulates viral reactivation. In the present study, our results demonstrated that LANA could bind to the promoter region of the SENP6 gene and inhibit SENP6 expression while the regulated SENP6 could in turn modulate the abundance of LANA through desumoylation. This delicate regulation may provide important insights to explain the abundance of LANA during KSHV latency.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a typical gammaherpesvirus that establishes persistent lifelong infection in host cells. In order to establish successful infection, KSHV has evolved numerous immune evasion strategies to bypass or hijack the host immune system. However, host cells still produce immune cytokines abundantly during primary KSHV infection. Whether the immune effectors produced are able to inhibit viral infection and how KSHV successfully conquers these immune effectors remain largely unknown. The guanylate-binding protein 1 (GBP1) gene is an interferon-stimulated gene and exerts antiviral functions on several RNA viruses; however, its function in DNA virus infection is less well understood. In this study, we found that KSHV infection increases both the transcriptional and protein levels of GBP1 at the early stage of primary infection by activating the NF-κB pathway. The overexpression of GBP1 significantly inhibited KSHV infection, while the knockdown of GBP1 promoted KSHV infection. The GTPase activity and dimerization of GBP1 were demonstrated to be responsible for its anti-KSHV activity. Furthermore, we found that GBP1 inhibited the nuclear delivery of KSHV virions by disrupting the formation of actin filaments. Finally, we demonstrated that replication and transcription activator (RTA) promotes the degradation of GBP1 through a proteasome pathway. Taken together, these results provide a new understanding of the antiviral mechanism of GBP1, which possesses potent anti-KSHV activity, and suggest the critical role of RTA in the evasion of the innate immune response during primary infection by KSHV. IMPORTANCE GBP1 can be induced by various cytokines and exerts antiviral activities against several RNA viruses. Our study demonstrated that GBP1 can exert anti-KSHV function by inhibiting the nuclear delivery of KSHV virions via the disruption of actin filaments. Moreover, we found that KSHV RTA can promote the degradation of GBP1 through a proteasome-mediated pathway. Taken together, our results elucidate a novel mechanism of GBP1 anti-KSHV activity and emphasize the critical role of RTA in KSHV evasion of the host immune system during primary infection.

It remains crucial to develop a laboratory model for studying hepatitis B virus (HBV) chronic infection. We hereby produced a recombinant covalently closed circular DNA (rcccDNA) in view of the key role of cccDNA in HBV persistence. A loxP -chimeric intron was engineered into a monomeric HBV genome in a precursor plasmid (prcccDNA), which was excised using Cre/ loxP -mediated DNA recombination into a 3.3-kb rcccDNA in the nuclei of hepatocytes. The chimeric intron was spliced from RNA transcripts without interrupting the HBV life cycle. In cultured hepatoma cells, cotransfection of prcccDNA and pCMV-Cre (encoding Cre recombinase) resulted in accumulation of nuclear rcccDNA that was heat stable and epigenetically organized as a minichromosome. A mouse model of HBV infection was developed by hydrodynamic injection of prcccDNA. In the presence of Cre recombinase, rcccDNA was induced in the mouse liver with effective viral replication and expression, triggering a compromised T-cell response against HBV. Significant T-cell hyporesponsiveness occurred in mice receiving 4 μg prcccDNA, resulting in prolonged HBV antigenemia for up to 9 weeks. Persistent liver injury was observed as elevated alanine transaminase activity in serum and sustained inflammatory infiltration in the liver. Although a T-cell dysfunction was induced similarly, mice injected with a plasmid containing a linear HBV replicon showed rapid viral clearance within 2 weeks. Collectively, our study provides an innovative approach for producing a cccDNA surrogate that established HBV persistence in immunocompetent mice. It also represents a useful model system in vitro and in vivo for evaluating antiviral treatments against HBV cccDNA. IMPORTANCE (i) Unlike plasmids that contain a linear HBV replicon, rcccDNA established HBV persistence with sustained liver injury in immunocompetent mice. This method could be a prototype for developing a mouse model of chronic HBV infection. (ii) An exogenous intron was engineered into the HBV genome for functionally seamless DNA recombination. This original approach could be also extended to other viral studies. (iii) rcccDNA was substantially induced in the nuclei of hepatocytes and could be easily distinguished by its exogenous intron using PCR. This convenient model system affords the opportunity to test antivirals directly targeting HBV cccDNA.

Kaposi's sarcoma-associated herpesvirus (KSHV) typically displays two different phases in its life cycle, the default latent phase and the lytic phase. There is a short period of lytic gene expression in the early stage of KSHV primary infection. The factors involved in the shutdown process of lytic gene expression are poorly identified. It has been shown that the latency-associated nuclear antigen (LANA) encoded by KSHV plays an important role in the establishment of viral latency. In screening, we identified a host protein, Krüppel-associated box domain-associated protein 1 (KAP1), that bound to LANA. We validated the interaction between LANA and KAP1 in vivo and in vitro , as well as their colocalization in the nucleus. We mapped out that LANA interacted with both the N- and C-terminal domains of KAP1. Based on the interface of LANA-KAP1 interaction determined, we proved that LANA recruited KAP1 to the RTA promoter region of the KSHV genome. We revealed that KAP1 was involved in transcriptional repression by LANA. We found multiple cooccupation sites of LANA and KAP1 on the whole KSHV genome by chromatin immunoprecipitation for sequencing (ChIP-seq) and demonstrated that LANA-recruited KAP1 played a critical role in the shutdown of lytic gene expression during the early stage of KSHV primary infection. Taken together, our data suggest that LANA interacts with KAP1 and represses lytic gene expression to facilitate the establishment of KSHV latency. IMPORTANCE Our study revealed the mechanism of transcriptional repression by LANA during KSHV primary infection, providing new insights into the process of KSHV latency establishment.

We detected three avian influenza hemagglutinin (HA) subtypes (H7, H9, and H5) and two neuraminidase (NA) subtypes (N9 and N2), as well as H7N9-related H9N9 reassortant intermediates, cocirculating among poultry in Huzhou, China, during April 2013. The results of our study reveal not only that Huzhou is one of the geographic origins of the novel H7N9 virus but also that cocirculation poses a potential threat to humans in the future.

We previously reported a proof-of-concept study for curing chronic hepatitis B virus (HBV) infection using a foreign-antigen recombinant HBV (rHBV) as a gene therapy vector. Targeted elimination of wild-type HBV (wtHBV)-infected cells could be achieved by functionally activating an in situ T-cell response against the foreign antigen. However, as chronic HBV infection spreads to all hepatocytes, specific targeting of virus-infected cells is thought to be less critical. It is also feared that rHBV may not induce active immunization in a setting resembling natural infection. For this immunotherapeutic approach to be practically viable, in the present study, we used a recombinant adenovirus (rAd) vector for rHBV delivery. The rAd vector allowed efficient transduction of wtHBV-producing HepG2 cells, with transferred rHBV undergoing dominant viral replication. Progeny rHBV virions proved to be infectious, as demonstrated in primary tupaia hepatocytes. These results greatly expanded the antiviral capacity of the replication-defective rAd/rHBV in wtHBV-infected liver tissue. With prior priming in the periphery, transduction with rAd/rHBV attracted a substantial influx of the foreign-antigen-specific T-effector cells into the liver. Despite the fully activated T-cell response, active expression of rHBV was observed for a prolonged time, which is essential for rHBV to achieve sustained expansion. In a mouse model of HBV persistence established by infection with a recombinant adeno-associated virus carrying the wtHBV genome, rAd/rHBV-based immunotherapy elicited a foreign-antigen-specific T-cell response that triggered effective viral clearance and subsequent seroconversion to HBV. It therefore represents an efficient strategy to overcome immune tolerance, thereby eliminating chronic HBV infection. IMPORTANCE Adenovirus-delivered rHBV activated a foreign-antigen-specific T-cell response that abrogated HBV persistence in a mouse model. Our study provides further evidence of the potential of foreign-antigen-based immunotherapy for the treatment of chronic HBV infection.

It is widely held that any given virus uses only one type of nucleic acid for genetic information storage. However, this consensus has been challenged slightly by several recent studies showing that many RNA species are present within a range of DNA viruses that include Kaposi's sarcoma-associated herpesvirus (KSHV). RNAs extracted from purified DNA virus particles exhibit great diversity in terms of length, abundance, temporal expression, cellular localization, and coding capacity during viral infection. In addition to known RNA species, the current study showed that small regulatory RNAs were present in KSHV virions. A large number of viral and cellular microRNAs (miRNAs), as well as unusual small RNAs (usRNAs), were detected in KSHV virions by using deep sequencing. Both viral and host miRNAs detected in small RNAs extracted from KSHV virions were further shown to colocalize with KSHV virions directly by in situ hybridization (ISH)-electron microscopy (EM) (ISH-EM). Some of these miRNAs were differentially present in the host cells and KSHV virions, suggesting that they are not randomly present in KSHV virions. The virional miRNAs could be transported into host cells, and they are biologically functional during de novo viral infection. Our study revealed miRNAs and usRNAs as a novel group of components in KSHV virions.

Latency-associated nuclear antigen 1 (LANA-1) of Kaposi’s sarcoma-associated herpesvirus (KSHV) is the major viral latent protein and functions as a multifaceted protein. Here, we report that LANA-1 attenuates the endothelial response to tumor necrosis factor alpha (TNF-) stimulation and inhibits consequent neutrophil chemotaxis. Reporter assays showed that LANA-1 constantly repressed nuclear factor (NF)-B transactivity upon TNF- stimulation. We also found that LANA-1 decreased nuclear p65 protein levels through enhancement of polyubiquitylation-mediated p65 degradation and that an elongin B/elongin C–cullin 5–LANA-1–p65 complex assembled by LANA-1 was responsible for this enhanced p65 degradation. In telomerase-immortalized human umbilical vein endothelial cells, LANA-1 was demonstrated to repress interleukin-8 expression, which was involved in neutrophil recruitment to the inflammatory site. Through an in vitro transmigration assay, we determined a suppressive effect of LANA-1 on neutrophil chemotaxis. Our work suggests that KSHV LANA-1 is a negative modulator of acute inflammation and sheds light on a new mechanism by which KSHV during the latent life cycle evades the host innate immune response. Kaposi’s sarcoma-associated herpesvirus (KSHV) is a recently discovered DNA tumor virus (15, 16), which has been identified as the etiological agent of three malignancies: Kaposi’s sarcoma, multicentric Castleman’s disease, and primary effusion lymphoma (14, 89). Like other herpesviruses, KSHV displays dual-phase, lytic and latent life cycles. During the lytic

Epstein-Barr virus (EBV) was the first human DNA virus to be associated with cancer. Its oncogenic potential was further demonstrated by its ability to transform primary B lymphocytes in vitro. EBV nuclear antigen 3C (EBNA3C) is one of a small subset of latent antigens critical for the transformation of human primary B lymphocytes. Although EBNA3C has been shown to modulate several cellular functions, additional targets involved in cellular transformation remain to be explored. EBNA3C can recruit key components of the SCFSkp2ubiquitin ligase complex. In this report, we show that EBNA3C residues 130 to 190, previously shown to bind to the SCFSkp2complex, also can strongly associate with the c-Myc oncoprotein. Additionally, the interaction of EBNA3C with c-Myc was mapped to the region of c-Myc that includes the highly conserved Skp2 binding domain. Skp2 has been shown to regulate c-Myc stability and also has been shown to function as a coactivator of transcription for c-Myc target genes. We now show that the EBV latent oncoprotein EBNA3C can stabilize c-Myc and that the recruitment of both c-Myc and its cofactor Skp2 to c-Myc-dependent promoters can enhance c-Myc-dependent transcription. This same region of EBNA3C also recruits and modulates the activity of retinoblastoma and p27, both major regulators of the mammalian cell cycle. The inclusion of c-Myc in the group of cellular targets modulated by this domain further accentuates the importance of these critical residues of EBNA3C in bypassing the cell cycle checkpoints.

K1 is the first open reading frame encoded by Kaposi's sarcoma-associated herpesvirus (KSHV) and lies positionally to the immediate right of the terminal repeats. K1 is a transmembrane glycoprotein having a functional immunoreceptor tyrosine-based activation motif (ITAM) capable of activating B-cell receptor signaling. K1 is expressed mostly during the lytic cycle of the virus and its promoter lies within the terminal repeat which contains the binding sites for latency-associated nuclear antigen (LANA). The K1 promoter (K1p) having LANA binding sites assayed by reporter assay demonstrated that LANA is capable of down-regulating K1 promoter transcriptional activity. However, the KSHV replication transcription activator RTA up-regulates K1p transcriptional activity. The promoter deleted of LANA binding sites showed loss in LANA-mediated down-regulation but was unaffected for RTA-mediated up-regulation. Increasing amounts of RTA rescued LANA-mediated repression of K1p transcriptional activity in cotransfection experiments. Reporter assay data suggest that LANA binding to its cognate sequence is critical for LANA-mediated repression of K1p as a LANA construct lacking the DNA binding domain was unable to repress K1p transcription. Additionally, KSHV primary infection experiments suggest that K1 is expressed during early infection but is repressed on the establishment of latency and so follows an expression profile similar to that of RTA during infection. Analysis of the promoter sequence revealed the presence of Oct-1 transcription factor binding sites within the −116 to +76 region. Mutational analysis of the Oct-1 sites abolished RTA-mediated transcriptional activation, suggesting that RTA up-regulates K1p transcription through binding to this transcription factor.

Epstein-Barr virus latent protein EBNA3C has been shown to bind Nm23-H1, a known suppresser of cell migration and metastasis and a regulator of the guanine exchange factor Tiam-1. This interaction results in cellular translocation of Nm23-H1 to the nucleus and suppression of the antimigratory effect in vitro. Furthermore, these proteins can synergistically increase transcription of a basal promoter when targeted to DNA by fusion to a Gal4 DNA binding domain. In this report, we show that EBNA3C and Nm23-H1 can cooperate to upregulate expression of MMP-9, known to be expressed in aggressive forms of lymphomas. This upregulation resulted in increased levels of MMP-9 mRNA, as well as a detectable increase in MMP-9 gelatinolytic activity. Specific mutations in the MMP-9 promoter showed that the Ap1 and NFκB binding sites are important for upregulation by the proteins. Additionally, it was shown for the first time that EBNA3C and Nm23-H1 can bind subunits of these transcription factors. This suggests that the ability of EBNA3C to reverse the antimigratory effects of Nm23-H1 is likely to be in part through the synergistic upregulation of MMP-9, mediated through interactions with the AP1 and NFκB transcription factors.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent contributing to development of Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman desease. Following primary infection, latency is typically established. However, the mechanism by which KSHV establishes latency is not understood. We have reported that the latency-associated nuclear antigen (LANA) can repress RTA (for replication and transcription activator) expression by down-regulating its promoter. In this study, we show that RTA is associated with the virion particle. We also show that RTA can activate the LANA promoter and induce LANA expression in transient reporter assays. Additionally, the transcription of RTA correlates with LANA expression in the early stages of de novo infection of KSHV, and induction of LANA transcription is responsive to induction of RTA with an inducible system. This induction in LANA transcription was dependent on recombination signal sequence binding protein Jκ (RBP-Jκ), as a RBP-Jκ-deficient cell line was significantly delayed and inefficient in LANA transcription with expression of RTA. These studies suggest that RTA contributes to establishment of KSHV latency by activating LANA expression in the early stages of infection by utilizing the major effector of the Notch signaling pathway RBP-Jκ. This describes a feedback mechanism by which LANA and RTA can regulate each other and is likely to be a key event in the establishment of KSHV latency.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the major biological cofactor contributing to development of Kaposi's sarcoma. KSHV establishes a latent infection in human B cells expressing the latency-associated nuclear antigen (LANA), a critical factor in the regulation of viral latency. LANA controls KSHV latent infection through repression of RTA, an activator of many lytic promoters. RTA activates the expression of several lytic viral genes by interacting with recombination signal sequence-binding protein Jκ (RBP-Jκ), a transcriptional repressor and the target of the Notch signaling pathway. The recognition that a number of KSHV lytic gene promoters, including RTA, contain RBP-Jκ binding sites raised the possibility that RBP-Jκ-mediated repression may be central to the establishment of latency. Here, we tested this hypothesis by examining the regulation of RTA by LANA through binding to RBP-Jκ. This study demonstrates that LANA physically associates with RBP-Jκ in vitro and in KSHV-infected cells, with the complex formed capable of binding to RBP-Jκ cognate sequences. RBP-Jκ binding sites within the RTA promoter have been found to be critical for LANA-mediated repression. Our study describes a novel mechanism through which LANA maintains KSHV latency by targeting a major downstream effector of the Notch signaling pathway.

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes 12 pre-microRNAs (pre-miRNAs). Current studies have shown that these miRNAs are involved in regulation of viral and host gene expression, implicating a role in the maintenance of viral latency and suppression of antiviral innate immunity. However, the functions of these miRNAs remain largely unknown. On the basis of the sequence homology between oncogenic miR-155 and KSHV-encoded miR-K12-11, we hypothesized that miR-K12-11 could attenuate transforming growth factor β (TGF-β) signaling, facilitating viral infection and tumorigenesis. In the present study, we demonstrated that ectopic expression of miR-K12-11 in Ramos, a TGF-β-sensitive cell line, downregulated TGF-β signaling and facilitated cell proliferation upon TGF-β treatment by directly targeting SMAD5, an important mediator in TGF-β signaling. In addition, the downregulation of SMAD5 by miR-K12-11 was further confirmed in a de novo KSHV infection system or latently infected KSHV-positive B-lymphoma cell lines. More importantly, repression of miR-K12-11 by a specific sponge inhibitor restored the expression of SMAD5 in both de novo -infected and latently infected cells. Finally, we found that restoration of SMAD5, in addition to the TGF-β type II receptor, which was epigenetically silenced by the latent viral protein latency-associated nuclear antigen, sensitized BC3 cells to the cytostatic effect of TGF-β signaling. Taken together, our findings highlight a novel mechanism in which miR-K12-11 downregulates TGF-β signaling and suggest that viral miRNAs and proteins may exert a dichotomy regulation in virus-induced oncogenesis by targeting the same signaling pathway.

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 geneRtapromoter. 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 theRtapromoter.

Previous studies have demonstrated the interaction between the Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) and the metastatic suppressor Nm23-H1 both in vitro and in vivo (C. Subramanian, M. A. Cotter II, and E. S. Robertson, Nat. Med. 7: 350-355, 2001). EBNA3C can reverse the ability of Nm23-H1 to suppress migration of Burkitt's lymphoma and breast carcinoma cell lines in vitro. EBNA3C contributes to EBV-associated human cancers by regulating transcription of a number of cellular and viral promoters and by targeting and altering the transcription activities of the metastasis suppressor Nm23-H1. Cyclo-oxygenase-2 (COX-2), an inducible enzyme important in inflammation, is overexpressed in a variety of cancers and can influence cell migration. In this report we show that Nm23-H1 and EBNA3C can modulate expression of COX-2 in the context of EBV infection and transformation. The levels of COX-2 were consistently higher in EBV-positive cells than in EBV-negative cells. Additionally, we show that Nm23-H1 can upregulate the COX-2 promoter element in luciferase reporter assays, whereas EBNA3C alone did not affect the level of response but clearly contributed to an additive increase when coexpressed with Nm23-H1. The downstream effect of COX-2 expression was also evaluated and showed that prostaglandin E 2 levels increased with Nm23-H1 and that there was some level of cooperativity in the presence of EBNA3C. The majority of this response was mediated through the cyclic AMP response element and NF-κB sites. These studies suggest a potential role for COX-2 in EBV-associated human cancers.

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is expressed in the majority of latency programs in EBV-infected cells and is critical for the maintenance of EBV episomes in the infected cells. EBNA1 is also known to be involved in transcriptional activation and regulates expression of the EBV latent genes, including the EBNAs and LMP1. Thus, EBNA1 is a multifunctional protein with critical functions required for the persistence of the viral genome over successive generations, producing new daughter cells from the infected cell. We identify EBNA1 here as an interacting EBNA with the known suppressor of metastasis and cell migration, Nm23-H1. Nm23-H1 inhibits cell migration when expressed in cancer cells. We show that EBNA1 associates with Nm23-H1 in EBV-infected cells in vitro, as well as in lymphoblastoid cell lines (LCLs). Nm23-H1 predominantly localizes to the cytoplasm in BJAB and 293T cells; however, upon expression of EBNA1, Nm23-H1 is translocated to the nucleus in similar compartments to EBNA1, suggesting a potential functional role that is linked to EBNA1. Convincingly, in EBV-transformed LCLs Nm23-H1 is localized predominantly to the nucleus and colocalizes to similar compartment as EBNA1. Further, we tested the effects of EBNA1 on Nm23-H1-mediated suppression of cell migration and showed that EBNA1 rescues the suppression of cell migration mediated by Nm23-H1. These in vitro studies suggest that EBNA1 plays a critical role in regulating the activities of Nm23-H1, including cell migration, through a mechanism which involves direct interaction of this major regulator in EBV-infected cells.

Like other herpesviruses, Kaposi's sarcoma-associated herpesvirus (KSHV, also designated human herpesvirus 8) can establish a latent infection in the infected host. During latency a small number of genes are expressed. One of those genes encodes latency-associated nuclear antigen (LANA), which is constitutively expressed in cells during latent as well as lytic infection. LANA has previously been shown to be important for the establishment of latent episome maintenance through tethering of the viral genome to the host chromosomes. Under specific conditions, KSHV can undergo lytic replication, with the production of viral progeny. The immediate-early Rta, encoded by open reading frame 50 of KSHV, has been shown to play a critical role in switching from viral latent replication to lytic replication. Overexpression of Rta from a heterologous promoter is sufficient for driving KSHV lytic replication and the production of viral progeny. In the present study, we show that LANA down-modulates Rta's promoter activity in transient reporter assays, thus repressing Rta-mediated transactivation. This results in a decrease in the production of KSHV progeny virions. We also found that LANA interacts physically with Rta both in vivo and in vitro. Taken together, our results demonstrate that LANA can inhibit viral lytic replication by inhibiting expression as well as antagonizing the function of Rta. This suggests that LANA may play a critical role in maintaining latency by controlling the switch between viral latency and lytic replication.

Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) is a known regulatory transcription factor that has been shown to interact with histone deacetylase 1 (HDAC1) when cotransfected in human cell lines and by in vitro binding experiments. Previous studies have shown that EBNA3C interacts with p300 and prothymosin alpha (ProTα) in EBV-infected cells and may be involved in recruiting acetyltransferases to the chromatin for acetylation of histones and transcriptional activation. EBNA3C has also been shown to function as a repressor of transcription when directed to promoters. In this report, we show that EBNA3C complexed with ProTα can also recruit deacetylase activity and associates in a complex that includes HDAC1 and HDAC2 in human B cells. A complex of EBNA3C and ProTα coimmunoprecipitated with HDAC1 and HDAC2 in cell lines stably expressing EBNA3C. Additionally, this complex associated with the mSin3A and NCoR corepressors in EBNA3C-expressing cell lines and may function in a complex with additional transcription factors known to be repressors of transcription. EBNA3C in complex with ProTα recruited deacetylase activity in cell lines stably expressing EBNA3C, and this activity was shown to be partially sensitive to trichostatin A (TSA). This suggests an association with other deacetylases that are insensitive to the general inhibitory effects of TSA, as the entire activity was not abolished in multiple assays. The association between EBNA3C and the corepressors as well as HDACs is likely to depend on the presence of ProTα in the complex. Immunoprecipitation with anti-ProTα antibody immunoprecipitated EBNA3C and the other repressors, whereas immunoprecipitation with anti-EBNA3C antibody resulted in little or no association with these molecules associated with transcription repression. Clearly, EBNA3C functions as a component of a number of dynamic complexes which function in repression and activation of transcription.