Distinct roles of Kaposi's sarcoma-associated herpesvirus-encoded viral interferon regulatory factors in inflammatory response and cancer.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent associated with Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman disease (MCD). Similar to other herpesviruses, KSHV has two life cycles, latency and lytic replication. In latency, the KSHV genome persists as a circular episome in the nucleus of the host cell and only a few viral genes are expressed. In this review, we focus on oncogenic, antiapoptotic, and immunomodulating properties of KSHV-encoded homologues of cellular interferon regulatory factors (IRFs)--viral IRF1 (vIRF1) to vIRF4--and their possible role in the KSHV-mediated antiviral response, apoptosis, and oncogenicity.

Kaposi sarcoma-associated herpesvirus vIRF-3 protein binds to F-box of Skp2 protein and acts as a regulator of c-Myc protein function and stability.

The Kaposi sarcoma-associated herpesvirus (KSHV) has been linked to Kaposi sarcoma, body cavity-based lymphoma, and Castleman disease. vIRF-3 is a KSHV latent gene that is critical for proliferation of KSHV-positive lymphoid cells. Furthermore, vIRF-3 contributes to KSHV-associated pathogenesis by stimulating c-Myc transcription activity. Here we show that vIRF-3 can associate with Skp2, a key component of the SCF(skp2) ubiquitin ligase complex. Skp2 is a transcriptional co-factor for c-Myc that was shown to regulate the stability of c-Myc protein as well as c-Myc-dependent transcription. In this study, we show that vIRF-3 binds to the F-box of Skp2 and recruits it to c-Myc-regulated promoters to activate c-Myc-dependent transcription. Additionally, cells overexpressing vIRF-3 exhibit higher levels of c-Myc ubiquitylation, suggesting that ubiquitylation is necessary for c-Myc-mediated transcription. Moreover, vIRF-3 can stabilize the c-Myc protein by increasing its half-life. Collectively, these results indicate that vIRF-3 can effectively manipulate c-Myc stability and function and thus contribute to c-Myc-induced KSHV-associated lymphomagenesis.

Biogenesis of hepatitis B virus e antigen is driven by translocon-associated protein complex and regulated by conserved cysteine residues within its signal peptide sequence.

Hepatitis B virus uses e antigen (HBe), which is dispensable for virus infectivity, to modulate host immune responses and achieve viral persistence in human hepatocytes. The HBe precursor (p25) is directed to the endoplasmic reticulum (ER), where cleavage of the signal peptide (sp) gives rise to the first processing product, p22. P22 can be retro-translocated back to the cytosol or enter the secretory pathway and undergo a second cleavage event, resulting in secreted p17 (HBe). Here, we report that translocation of p25 to the ER is promoted by translocon-associated protein complex. We have found that p25 is not completely translocated into the ER; a fraction of p25 is phosphorylated and remains in the cytoplasm and nucleus. Within the p25 sp sequence, we have identified three cysteine residues that control the efficiency of sp cleavage and contribute to proper subcellular distribution of the precore pool.

ATM-Dependent Phosphorylation of Hepatitis B Core Protein in Response to Genotoxic Stress.

Chronic hepatitis caused by infection with the Hepatitis B virus is a life-threatening condition. In fact, 1 million people die annually due to liver cirrhosis or hepatocellular carcinoma. Recently, several studies demonstrated a molecular connection between the host DNA damage response (DDR) pathway and HBV replication and reactivation. Here, we investigated the role of Ataxia-telangiectasia-mutated (ATM) and Ataxia telangiectasia and Rad3-related (ATR) PI3-kinases in phosphorylation of the HBV core protein (HBc). We determined that treatment of HBc-expressing hepatocytes with genotoxic agents, e.g., etoposide or hydrogen peroxide, activated the host ATM-Chk2 pathway, as determined by increased phosphorylation of ATM at Ser1981 and Chk2 at Thr68. The activation of ATM led, in turn, to increased phosphorylation of cytoplasmic HBc at serine-glutamine (SQ) motifs located in its C-terminal domain. Conversely, down-regulation of ATM using ATM-specific siRNAs or inhibitor effectively reduced etoposide-induced HBc phosphorylation. Detailed mutation analysis of S-to-A HBc mutants revealed that S170 (S168 in a 183-aa HBc variant) is the primary site targeted by ATM-regulated phosphorylation. Interestingly, mutation of two major phosphorylation sites involving serines at positions 157 and 164 (S155 and S162 in a 183-aa HBc variant) resulted in decreased etoposide-induced phosphorylation, suggesting that the priming phosphorylation at these serine-proline (SP) sites is vital for efficient phosphorylation of SQ motifs. Notably, the mutation of S172 (S170 in a 183-aa HBc variant) had the opposite effect and resulted in massively up-regulated phosphorylation of HBc, particularly at S170. Etoposide treatment of HBV infected HepG2-NTCP cells led to increased levels of secreted HBe antigen and intracellular HBc protein. Together, our studies identified HBc as a substrate for ATM-mediated phosphorylation and mapped the phosphorylation sites. The increased expression of HBc and HBe antigens in response to genotoxic stress supports the idea that the ATM pathway may provide growth advantage to the replicating virus.

Toll-like receptor dual-acting agonists are potent inducers of PBMC-produced cytokines that inhibit hepatitis B virus production in primary human hepatocytes.

Recombinant interferon-α (IFN-α) treatment functionally cures chronic hepatitis B virus (HBV) infection in some individuals and suppresses virus replication in hepatocytes infected in vitro. We studied the antiviral effect of conditioned media (CM) from peripheral blood mononuclear cells (PBMCs) stimulated with agonists of Toll-like receptors (TLRs) 2, 7, 8 and 9. We found that CM from PBMCs stimulated with dual-acting TLR7/8 (R848) and TLR2/7 (CL413) agonists were more potent drivers of inhibition of HBe and HBs antigen secretion from HBV-infected primary human hepatocytes (PHH) than CM from PBMCs stimulated with single-acting TLR7 (CL264) or TLR9 (CpG-B) agonists. Inhibition of HBV in PHH did not correlate with the quantity of PBMC-produced IFN-α, but it was a complex function of multiple secreted cytokines. More importantly, we found that the CM that efficiently inhibited HBV production in freshly isolated PHH via various cytokine repertoires and mechanisms did not reduce covalently closed circular (ccc)DNA levels. We confirmed our data with a cell culture model based on HepG2-NTCP cells and the plasmacytoid dendritic cell line GEN2.2. Collectively, our data show the importance of dual-acting TLR agonists inducing broad cytokine repertoires. The development of poly-specific TLR agonists provides novel opportunities towards functional HBV cure.

Hepatitis B Core Protein Is Post-Translationally Modified through K29-Linked Ubiquitination.

Hepatitis B virus (HBV) core protein (HBc) plays many roles in the HBV life cycle, such as regulation of transcription, RNA encapsidation, reverse transcription, and viral release. To accomplish these functions, HBc interacts with many host proteins and undergoes different post-translational modifications (PTMs). One of the most common PTMs is ubiquitination, which was shown to change the function, stability, and intracellular localization of different viral proteins, but the role of HBc ubiquitination in the HBV life cycle remains unknown. Here, we found that HBc protein is post-translationally modified through K29-linked ubiquitination. We performed a series of co-immunoprecipitation experiments with wild-type HBc, lysine to arginine HBc mutants and wild-type ubiquitin, single lysine to arginine ubiquitin mutants, or single ubiquitin-accepting lysine constructs. We observed that HBc protein could be modified by ubiquitination in transfected as well as infected hepatoma cells. In addition, ubiquitination predominantly occurred on HBc lysine 7 and the preferred ubiquitin chain linkage was through ubiquitin-K29. Mass spectrometry (MS) analyses detected ubiquitin protein ligase E3 component N-recognin 5 (UBR5) as a potential E3 ubiquitin ligase involved in K29-linked ubiquitination. These findings emphasize that ubiquitination of HBc may play an important role in HBV life cycle.

Induction of MHC class I molecule cell surface expression and epigenetic activation of antigen-processing machinery components in a murine model for human papilloma virus 16-associated tumours.

Epigenetic events play an important role in tumour progression and also contribute to escape of the tumour from immune surveillance. In this study, we investigated the up-regulation of major histocompatibility complex (MHC) class I surface expression on tumour cells by epigenetic mechanisms using a murine tumour cell line expressing human E6 and E7 human papilloma virus 16 (HPV16) oncogenes and deficient in MHC class I expression, as a result of impaired antigen-presenting machinery (APM). Treatment of the cells with the histone deacetylase inhibitor Trichostatin A, either alone or in combination with the DNA demethylating agent 5-azacytidine, induced surface re-expression of MHC class I molecules. Consequently, the treated cells became susceptible to lysis by specific cytotoxic T lymphocytes. Further analysis revealed that epigenetic induction of MHC class I surface expression was associated with the up-regulation of APM genes [transporter associated with antigen processing 1 (TAP-1), TAP-2, low-molecular-mass protein 2 (LMP-2) and LMP-7]. The results demonstrate that expression of the genes involved in APM are modulated by epigenetic mechanisms and suggest that agents modifying DNA methylation and/or histone acetylation have the potential to change the effectiveness of antitumour immune responses and therapeutically may have an impact on immunological output.

PRMT5: A novel regulator of Hepatitis B virus replication and an arginine methylase of HBV core.

In mammals, protein arginine methyltransferase 5, PRMT5, is the main type II enzyme responsible for the majority of symmetric dimethylarginine formation in polypeptides. Recent study reported that PRMT5 restricts Hepatitis B virus (HBV) replication through epigenetic repression of HBV DNA transcription and interference with encapsidation of pregenomic RNA. Here we demonstrate that PRMT5 interacts with the HBV core (HBc) protein and dimethylates arginine residues within the arginine-rich domain (ARD) of the carboxyl-terminus. ARD consists of four arginine rich subdomains, ARDI, ARDII, ARDIII and ARDIV. Mutation analysis of ARDs revealed that arginine methylation of HBc required the wild-type status of both ARDI and ARDII. Mass spectrometry analysis of HBc identified multiple potential ubiquitination, methylation and phosphorylation sites, out of which lysine K7 and arginines R150 (within ARDI) and R156 (outside ARDs) were shown to be modified by ubiquitination and methylation, respectively. The HBc symmetric dimethylation appeared to be linked to serine phosphorylation and nuclear import of HBc protein. Conversely, the monomethylated HBc retained in the cytoplasm. Thus, overexpression of PRMT5 led to increased nuclear accumulation of HBc, and vice versa, down-regulation of PRMT5 resulted in reduced levels of HBc in nuclei of transfected cells. In summary, we identified PRMT5 as a potent controller of HBc cell trafficking and function and described two novel types of HBc post-translational modifications (PTMs), arginine methylation and ubiquitination.

On the role of IRF in host defense.

Transcription factors of the interferon (IFN) regulatory factor (IRF) family have been shown to play an essential role in the regulated expression of type I IFN genes, IFN-stimulated genes (ISG), and other cytokines and chemokines. Three members of the IRF family, IRF-3, IRF-5, and IRF-7, have been identified as acting as direct transducers of virus-mediated signaling. In infected cells, these factors are activated by phosphorylation on the serine residues, transported to the nucleus, where they bind to the promoters of IFNA and IFNB genes and tether histone transacetylases to the transcription complex enhanceosome. IFNB and IFNA subtypes are expressed at different levels in infected cells. The ratio between the relative levels of IRF-3 and IRF-7 was shown to play an essential role in the inducible expression of type I IFN genes, whereas IRF-3 alone is sufficient for expression of the IFNB gene. IRF-5 was identified recently as another inducer of IFNA genes, which has two unique properties: (1) its activation is virus specific, and (2) the profile of IFNA genes induced by IRF-5 is distinct from that induced by IRF-7. Several viruses target functions of IRF to eliminate the early inflammatory response. Kaposi's sarcoma herpesvirus (KSHV) encodes a cluster of four genes with homology to cellular IRF. Three of these vIRF were shown to inhibit induction of IFN genes and ISG in infected cells and function as dominant negative mutants of cellular IRF. The unique properties of previously uncharacterized vIRF-2 and vIRF-3 are discussed.

p53 tumor suppressor protein stability and transcriptional activity are targeted by Kaposi's sarcoma-associated herpesvirus-encoded viral interferon regulatory factor 3.

Viruses have developed numerous strategies to counteract the host cell defense. Kaposi's sarcoma-associated herpesvirus (KSHV) is a DNA tumor virus linked to the development of Kaposi's sarcoma, Castleman's disease, and primary effusion lymphoma (PEL). The virus-encoded viral interferon regulatory factor 3 (vIRF-3) gene is a latent gene which is involved in the regulation of apoptosis, cell cycle, antiviral immunity, and tumorigenesis. vIRF-3 was shown to interact with p53 and inhibit p53-mediated apoptosis. However, the molecular mechanism underlying this phenomenon has not been established. Here, we show that vIRF-3 associates with the DNA-binding domain of p53, inhibits p53 phosphorylation on serine residues S15 and S20, and antagonizes p53 oligomerization and the DNA-binding affinity. Furthermore, vIRF-3 destabilizes p53 protein by increasing the levels of p53 polyubiquitination and targeting p53 for proteasome-mediated degradation. Consequently, vIRF-3 attenuates p53-mediated transcription of the growth-regulatory p21 gene. These effects of vIRF-3 are of biological relevance since the knockdown of vIRF-3 expression in KSHV-positive BC-3 cells, derived from PEL, leads to an increase in p53 phosphorylation, enhancement of p53 stability, and activation of p21 gene transcription. Collectively, these data suggest that KSHV evolved an efficient mechanism to downregulate p53 function and thus facilitate uncontrolled cell proliferation and tumor growth.

HIV-1 accessory proteins VPR and Vif modulate antiviral response by targeting IRF-3 for degradation.

The activation of IRF-3 during the early stages of viral infection is critical for the initiation of the antiviral response; however the activation of IRF-3 in HIV-1 infected cells has not yet been characterized. We demonstrate that the early steps of HIV-1 infection do not lead to the activation and nuclear translocation of IRF-3; instead, the relative levels of IRF-3 protein are decreased due to the ubiquitin-associated proteosome degradation. Addressing the molecular mechanism of this effect we show that the degradation is independent of HIV-1 replication and that virion-associated accessory proteins Vif and Vpr can independently degrade IRF-3. The null mutation of these two genes reduced the capacity of the HIV-1 virus to down modulate IRF-3 levels. The degradation was associated with Vif- and Vpr-mediated ubiquitination of IRF-3 and was independent of the activation of IRF-3. N-terminal lysine residues were shown to play a critical role in the Vif- and Vpr-mediated degradation of IRF-3. These data implicate Vif and Vpr in the disruption of the initial antiviral response and point to the need of HIV-1 to circumvent the antiviral response during the very early phase of replication.

Stimulation of c-Myc transcriptional activity by vIRF-3 of Kaposi sarcoma-associated herpesvirus.

Kaposi sarcoma-associated herpesvirus is associated with two lymphoproliferative disorders, primary effusion lymphoma (PEL) and Castleman disease. In PEL, Kaposi sarcoma-associated herpesvirus is present in a latent form expressing only few viral genes. Among them is a viral homologue of cellular interferon regulatory factors, vIRF-3. To study the role of vIRF-3 in PEL lymphomagenesis, we analyzed the interaction of vIRF-3 with cellular proteins. Using yeast two-hybrid screen, we detected the association between vIRF-3 and c-Myc suppressor, MM-1alpha. The vIRF-3 and MM-1alpha interaction was also demonstrated by glutathione S-transferase pulldown assay and coimmunoprecipitation of endogenous vIRF-3 and MM-1alpha in PEL-derived cell lines. Overexpression of vIRF-3 enhanced the c-Myc-dependent transcription of the gene cdk4. Addressing the molecular mechanism of the vIRF-3-mediated stimulation, we demonstrated that the association between MM-1alpha and c-Myc was inhibited by vIRF-3. Furthermore, the recruitment of vIRF-3 to the cdk4 promoter and the elevated levels of the histone H3 acetylation suggest the direct involvement of vIRF-3 in the activation of c-Myc-mediated transcription. These findings indicate that vIRF-3 can effectively stimulate c-Myc function in PEL cells and consequently contribute to de-regulation of B-cell growth and differentiation.

Kaposi's sarcoma-associated herpesvirus-encoded vIRF-3 stimulates the transcriptional activity of cellular IRF-3 and IRF-7.

Kaposi's sarcoma-associated herpesvirus has been linked to Kaposi's sarcoma, body cavity-based lymphoma, and Castleman's disease. The Kaposi's sarcoma-associated herpesvirus genome contains a cluster of open reading frames encoding proteins (vIRFs) with homology to the cellular transcription factors of the interferon regulatory factor family. vIRF-3, also called LANA2, is a latently expressed nuclear protein. Here we demonstrate that vIRF-3 directly interacts with cellular interferon regulatory factor (IRF) IRF-3, IRF-7, and the transcriptional co-activator CBP/p300. The mapping of the vIRF-3 binding domain revealed that vIRF-3 associates with both IRF-3 and IRF-7 through its C-terminal region. The p300 domain, which interacts with vIRF-3, is distinct from the previously identified IBiD domain, to which both vIRF-1 and IRF-3 bind. Thus, in contrast to vIRF-1, vIRF-3 neither blocks the interaction between IRF-3 and p300 nor inhibits the histone acetylation. Although vIRF-3 is not a DNA-binding protein, it is recruited to the IFNA promoters via its interaction with IRF-3 and IRF-7. The presence of vIRF-3 in the enhanceosome assembled on the IFNA promoters increases binding of IRF-3, IRF-7, and acetylated histone H3 to this promoter region. Consequently, vIRF-3 stimulates the IRF-3- and IRF-7-mediated activation of type I interferon (IFNA and IFNB) genes and the synthesis of biologically active type I interferons in infected B cells. These studies illustrate that vIRF-3 and vIRF-1 have clearly distinct functions. In addition to its co-repressor activity, vIRF-3 can also act as a transcriptional activator on genes controlled by cellular IRF-3 and IRF-7.