Abstract
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.
Highlights
Kaposi’s sarcoma-associated herpesvirus (KSHV), known as human herpesvirus 8 (HHV8), is a member of the gammaherpesvirus family that is associated with Kaposi’s sarcoma (KS), Primary Effusion Lymphoma, a subset of Multicentric Castleman’s Disease, and KSHV Inflammatory Cytokine Syndrome [1,2,3,4]
We show the importance of dynamic epigenetic modifications on the viral chromatin in dictating whether KSHV displays the latent or lytic phase of its life cycle
Various chromatin-modifying enzymes are responsible for adding activating or repressive ‘marks’ on chromatin, one of these is a protein arginine methyl transferase 5 (PRMT5), which symmetrically dimethylates arginine 3 of histone H4 (H4R3me2s) and associates with condensed chromatin leading to restricted gene expression
Summary
Kaposi’s sarcoma-associated herpesvirus (KSHV), known as human herpesvirus 8 (HHV8), is a member of the gammaherpesvirus family that is associated with Kaposi’s sarcoma (KS), Primary Effusion Lymphoma, a subset of Multicentric Castleman’s Disease, and (in HIV-co-infected patients) KSHV Inflammatory Cytokine Syndrome [1,2,3,4]. The virus persists indefinitely in the infected host in a latent form during which time only a small fraction of regulatory viral proteins are expressed, most notably the latency-associated nuclear antigen protein [5,6,7]. The KSHV genome is maintained primarily in a heterochromatic conformation in which the genome is highly compact with restricted transcription of the viral genes [18, 19]. The compactness of KSHV chromatin during latency was confirmed by sequencing the nucleosomal depleted DNA in FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) assays, which revealed that only a small percentage of the viral genome, primarily the latency-associated regions, were in an active chromatin (euchromatin) state [18, 21, 22]
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