KSHV ORF59 and PAN RNA Recruit Histone Demethylases to the Viral Chromatin during Lytic Reactivation.

Kaposi's sarcoma-associated herpesvirus (KSHV) causes multiple malignancies in immunocompromised individuals. KSHV primarily establishes a lifelong latency in infected humans during which only a subset of viral genes is expressed while most of the viral genome remains transcriptionally silent with condensed chromatin. However, during the lytic phase, the viral genome undergoes dramatic changes in chromatin landscape leading to a transcriptionally active state with the expression of most of the viral genes and production of progeny virions. Multiple cellular and viral factors influence the epigenetic gene regulation and transitioning of virus from latency to the lytic state. We have earlier shown that KSHV ORF59, viral processivity factor, binds to a protein arginine methyl transferase 5 (PRMT5) to alter the histone arginine methylation during reactivation. Additionally, ORF59 has been shown to interact with most abundantly expressed KSHV long noncoding polyadenylated nuclear RNA (PAN RNA), which associates with the viral epigenome during reactivation. Interestingly, PAN RNA interacts with UTX and JMJD3, cellular H3K27me3 demethylases, and removes the repressive marks on the chromatin. In this study, we report that the recruitment of histone demethylases to the viral chromatin is facilitated by the expression of ORF59 protein and PAN RNA. Using biochemical and localization assays including co-immunoprecipitation and immunofluorescence, we demonstate ORF59 localizes with UTX and JMJD3. Our results confirm that PAN RNA enhances the interaction of ORF59 with the chromatin modifying enzymes UTX and JMJD3.

Kaposi's Sarcoma-Associated Herpesvirus Deregulates Host Cellular Replication during Lytic Reactivation by Disrupting the MCM Complex through ORF59.

Minichromosome maintenance proteins (MCMs) play an important role in DNA replication by binding to the origins as helicase and recruiting polymerases for DNA synthesis. During the S phase, MCM complex is loaded to limit DNA replication once per cell cycle. We identified MCMs as ORF59 binding partners in our protein pulldown assays, which led us to hypothesize that this interaction influences DNA replication. ORF59's interactions with MCMs were confirmed in both endogenous and overexpression systems, which showed its association with MCM3, MCM4, MCM5, and MCM6. Interestingly, MCM6 interacted with both the N- and C-terminal domains of ORF59, and its depletion in BCBL-1 and BC3 cells led to an increase in viral genome copies, viral late gene transcripts, and virion production compared to the control cells following reactivation. MCMs perform their function by loading onto the replication competent DNA, and one means of regulating chromatin loading/unloading, in addition to enzymatic activity of the MCM complex, is by posttranslational modifications, including phosphorylation of these factors. Interestingly, a hypophosphorylated form of MCM3, which is associated with reduced loading onto the chromatin, was detected during lytic reactivation and correlated with its inability to associate with histones in reactivated cells. Additionally, chromatin immunoprecipitation showed lower levels of MCM3 and MCM4 association at cellular origins of replication and decreased levels of cellular DNA synthesis in cells undergoing reactivation. Taken together, these findings suggest a mechanism in which KSHV ORF59 disrupts the assembly and functions of MCM complex to stall cellular DNA replication and promote viral replication.IMPORTANCE KSHV is the causative agent of various lethal malignancies affecting immunocompromised individuals. Both lytic and latent phases of the viral life cycle contribute to the progression of these cancers. A better understanding of how viral proteins disrupt functions of a normal healthy cell to cause oncogenesis is warranted. One crucial lytic protein produced early during lytic reactivation is the multifunctional ORF59. In this report, we elucidated an important role of ORF59 in manipulating the cellular environment conducive for viral DNA replication by deregulating the normal functions of the host MCM proteins. ORF59 binds to specific MCMs and sequesters them away from replication origins in order to sabotage cellular DNA replication. Blocking cellular DNA replication ensures that cellular resources are utilized for transcription and replication of viral DNA.

KSHV LANA upregulates the expression of epidermal growth factor like domain 7 to promote angiogenesis.

Kaposi's sarcoma (KS) is a highly-vascularized tumor characterized by inflammation and extensive neo-angiogenesis. The KS tumor microenvironment is rich in inflammatory and pro-angiogenic cytokines. Here, we report that the expression of Epidermal growth factor-like domain 7 (EGFL7) is upregulated in Kaposi's sarcoma-associated herpes virus (KSHV) infected cells. EGFL7 is a secreted pro-angiogenic cytokine that has been implicated in angiogenesis and the proliferation of endothelial cells during many pathological conditions. Our data show that KS tumors as well as primary effusion lymphoma cells have increased levels of EGFL7 compared to the uninfected cells. We determined that the expression of a KSHV latent protein, LANA (latency-associated nuclear antigen), is the main viral factor responsible for this upregulation. The modulation of EGFL7 expression by LANA involves sequestration of death domain-associated protein 6 (Daxx) from the EGFL7 promoter. Daxx acts as a suppressor of promoter activity by binding to the avian erythroblastosis virus E26 oncogene homolog 1 (Ets-1), which is the core transcription factor required for the expression of EGFL7. We additionally show that the upregulation of EGFL7 by LANA contributes to the promotion of angiogenesis since siRNA-mediated knockdown of EGFL7 reduced in vitro tubulogenesis in LANA-expressing HUVEC cells. EGFL7 promotes angiogenesis through autocrine as well as paracrine mechanisms as the supernatant from LANA expressing cells depleted of EGFL7 showed reduced tubulogenesis. This study for the first time demonstrates EGFL7 to be an important angiogenic molecule secreted during KSHV infection that could be exploited for blocking KSHV associated malignancies in conjugation with other anti-angiogenic therapies.

Quantifying Symmetrically Methylated H4R3 on the Kaposi's Sarcoma-associated Herpesvirus (KSHV) Genome by ChIP-Seq.

Post-translational modifications to histone tails contribute to the three-dimensional structure of chromatin and play an important role in detegrmining the relative expression of nearby genes. One such modification is symmetric di-methylation of arginine residues, which may exhibit different effects on gene expression including blocking the binding of transcriptional activators, or recruiting repressive effector molecules. Recent ChIP-Seq studies have demonstrated the importance of cross-talk between different histone modifications in gene regulation. Thus, to acquire a comprehensive understanding of the combined efforts of these epigenetic marks, ChIP-Seq must be utilized for identifying specific enrichment on the chromatin. Tumorigenic herpesvirus KSHV, employs epigenetic mechanisms for gene regulation, and by evaluating relative abundance of multiple histone modifications in a thorough, unbiased way, using ChIP-Seq, we can get a superior insight concerning the complex mechanisms of viral replication and pathogenesis.

KSHV encoded ORF59 modulates histone arginine methylation of the viral genome to promote viral reactivation.

Kaposi's sarcoma associated herpesvirus (KSHV) persists in a highly-ordered chromatin structure inside latently infected cells with the majority of the viral genome having repressive marks. However, upon reactivation the viral chromatin landscape changes into 'open' chromatin through the involvement of lysine demethylases and methyltransferases. Besides methylation of lysine residues of histone H3, arginine methylation of histone H4 plays an important role in controlling the compactness of the chromatin. Symmetric methylation of histone H4 at arginine 3 (H4R3me2s) negatively affects the methylation of histone H3 at lysine 4 (H3K4me3), an active epigenetic mark deposited on the viral chromatin during reactivation. We identified a novel binding partner to KSHV viral DNA processivity factor, ORF59-a protein arginine methyl transferase 5 (PRMT5). PRMT5 is an arginine methyltransferase that dimethylates arginine 3 (R3) of histone H4 in a symmetric manner, one hallmark of condensed chromatin. Our ChIP-seq data of symmetrically methylated H4 arginine 3 showed a significant decrease in H4R3me2s on the viral genome of reactivated cells as compared to the latent cells. Reduction in arginine methylation correlated with the binding of ORF59 on the viral chromatin and disruption of PRMT5 from its adapter protein, COPR5 (cooperator of PRMT5). Binding of PRMT5 through COPR5 is important for symmetric methylation of H4R3 and the expression of ORF59 competitively reduces the association of PRMT5 with COPR5, leading to a reduction in PRMT5 mediated arginine methylation. This ultimately resulted in a reduced level of symmetrically methylated H4R3 and increased levels of H3K4me3 marks, contributing to the formation of an open chromatin for transcription and DNA replication. Depletion of PRMT5 levels led to a decrease in symmetric methylation and increase in viral gene transcription confirming the role of PRMT5 in viral reactivation. In conclusion, ORF59 modulates histone-modifying enzymes to alter the chromatin structure during lytic reactivation.

Manipulation of ubiquitin/SUMO pathways in human herpesviruses infection.

Post-translational modification of proteins with ubiquitin/small ubiquitin-like modifier (SUMO) molecules triggers multiple signaling pathways that are critical for many aspects of cellular physiology. Given that viruses hijack the biosynthetic and degradative systems of their host, it is not surprising that viruses encode proteins to manipulate the host's cellular machinery for ubiquitin/SUMO modification at multiple levels. Infection with a herpesvirus, among the most ubiquitous human DNA viruses, has been linked to many human diseases, including cancers. The interplay between human herpesviruses and the ubiquitylation/SUMOylation modification system has been extensively investigated in the past decade. In this review, we present an overview of recent advances to address how the ubiquitin/SUMO-modified system alters the latency and lytic replication of herpesvirus and how herpesviruses usurp the ubiquitin/SUMO pathways against the host's intrinsic and innate immune response to favor their pathogenesis.

The Modulation of Apoptotic Pathways by Gammaherpesviruses.

Apoptosis or programmed cell death is a tightly regulated process fundamental for cellular development and elimination of damaged or infected cells during the maintenance of cellular homeostasis. It is also an important cellular defense mechanism against viral invasion. In many instances, abnormal regulation of apoptosis has been associated with a number of diseases, including cancer development. Following infection of host cells, persistent and oncogenic viruses such as the members of the Gammaherpesvirus family employ a number of different mechanisms to avoid the host cell's "burglar" alarm and to alter the extrinsic and intrinsic apoptotic pathways by either deregulating the expressions of cellular signaling genes or by encoding the viral homologs of cellular genes. In this review, we summarize the recent findings on how gammaherpesviruses inhibit cellular apoptosis via virus-encoded proteins by mediating modification of numerous signal transduction pathways. We also list the key viral anti-apoptotic proteins that could be exploited as effective targets for novel antiviral therapies in order to stimulate apoptosis in different types of cancer cells.

Next-Generation Sequencing in the Understanding of Kaposi's Sarcoma-Associated Herpesvirus (KSHV) Biology.

Non-Sanger-based novel nucleic acid sequencing techniques, referred to as Next-Generation Sequencing (NGS), provide a rapid, reliable, high-throughput, and massively parallel sequencing methodology that has improved our understanding of human cancers and cancer-related viruses. NGS has become a quintessential research tool for more effective characterization of complex viral and host genomes through its ever-expanding repertoire, which consists of whole-genome sequencing, whole-transcriptome sequencing, and whole-epigenome sequencing. These new NGS platforms provide a comprehensive and systematic genome-wide analysis of genomic sequences and a full transcriptional profile at a single nucleotide resolution. When combined, these techniques help unlock the function of novel genes and the related pathways that contribute to the overall viral pathogenesis. Ongoing research in the field of virology endeavors to identify the role of various underlying mechanisms that control the regulation of the herpesvirus biphasic lifecycle in order to discover potential therapeutic targets and treatment strategies. In this review, we have complied the most recent findings about the application of NGS in Kaposi's sarcoma-associated herpesvirus (KSHV) biology, including identification of novel genomic features and whole-genome KSHV diversities, global gene regulatory network profiling for intricate transcriptome analyses, and surveying of epigenetic marks (DNA methylation, modified histones, and chromatin remodelers) during de novo, latent, and productive KSHV infections.