The small ubiquitin-like modifier (SUMO) is a protein that regulates a wide variety of cellular processes by covalent attachment of SUMO moieties to a diverse array of target proteins. Sumoylation also plays an important role in the replication of many viruses. Previously, we showed that Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a SUMO-ligase, K-bZIP, which catalyzes sumoylation of host and viral proteins. We report here that this virus also encodes a gene that functions as a SUMO-targeting ubiquitin-ligase (STUbL) which preferentially targets sumoylated proteins for degradation. K-Rta, the major transcriptional factor which turns on the entire lytic cycle, was recently found to have ubiquitin ligase activity toward a selected set of substrates. We show in this study that K-Rta contains multiple SIMs (SUMO interacting motif) and binds SUMOs with higher affinity toward SUMO-multimers. Like RNF4, the prototypic cellular STUbL, K-Rta degrades SUMO-2/3 and SUMO-2/3 modified proteins, including promyelocytic leukemia (PML) and K-bZIP. PML-NBs (nuclear bodies) or ND-10 are storage warehouses for sumoylated proteins, which negatively regulate herpesvirus infection, as part of the intrinsic immune response. Herpesviruses have evolved different ways to degrade or disperse PML bodies, and KSHV utilizes K-Rta to inhibit PML-NBs formation. This process depends on K-Rta's ability to bind SUMO, as a K-Rta SIM mutant does not effectively degrade PML. Mutations in the K-Rta Ring finger-like domain or SIM significantly inhibited K-Rta transactivation activity in reporter assays and in the course of viral reactivation. Finally, KSHV with a mutation in the Ring finger-like domain or SIM of K-Rta replicates poorly in culture, indicating that reducing SUMO-conjugates in host cells is important for viral replication. To our knowledge, this is the first virus which encodes both a SUMO ligase and a SUMO-targeting ubiquitin ligase that together may generate unique gene regulatory programs.

Latent Kaposi's sarcoma-associated herpesvirus (KSHV) episomes are coated with viral latency-associated nuclear antigen (LANA). In contrast, LANA rapidly disassociates from episomes during reactivation. Lytic KSHV expresses polyadenylated nuclear RNA (PAN RNA), a long noncoding RNA (lncRNA). We report that PAN RNA promotes LANA-episome disassociation through an interaction with LANA which facilitates LANA sequestration away from KSHV episomes during reactivation. These findings suggest that KSHV may have evolved an RNA aptamer to regulate latent protein function. Kaposi's sarcoma-associated herpesvirus (KSHV; also called human herpesvirus 8) is a gammaherpesvirus linked to Kaposi's sarcoma (KS) and two lymphoproliferative disorders, primary effusion lymphoma (PEL; also called body cavity B lymphoma [BCBL]) and a subset of multicentric Castleman's disease (1). Pervasive transcription, which generates a wide variety of transcripts with little apparent protein-coding potential, has been observed on a genome-wide scale in beta-and gammaherpesviruses, including human cytomegalovirus (HCMV) (2) and KSHV (3, 4), during lytic growth. One class of noncoding RNAs, called long noncoding RNAs (lncRNAs), are defined as RNA polymerase II (Pol II)-transcribed noncoding RNAs greater than 200 nucleotides in length (5). KSHV encodes a viral lncRNA known as polyadenylated nuclear RNA (PAN RNA), an abundant early gene product. Although PAN RNA was first described 17 years ago (6), its discovery predated the widespread recognition of noncoding RNAs. However, recent studies have begun to focus on the role of PAN RNA in the KSHV life cycle. PAN RNA has been reported to play a role in KSHV gene expression, replication, and immune modulation (7-9). PAN RNA binds the transcription factor IRF4 (8), lysine demethylases UTX and JMJD3, and the lysine methyltransferase MLL2 (9), in support of the notion that, similar to cellular lncRNAs, PAN RNA interacts with transcriptional regulators and chromatin modifiers to modulate viral gene expression. Moreover, recent mapping studies have found widespread PAN RNA interaction sites on the KSHV episome as well as the host genome, and PAN RNA expression is required for optimal expression of the entire KSHV lytic gene expression program (10).Previously, we performed a large-scale coimmunoprecipitation (co-IP) analysis to identify latency-associated nuclear antigen (LANA)-interacting protein partners using stably LANA-expressing HeLa cells (11). In the experiment, RNA-binding factors such as hnRNPs, SF3B1, THRAP3, and DHX15 were found to be among LANA-interacting proteins. The result raised the questions of whether LANA possesses the property of RNA binding and whether LANA interacts with PAN RNA. To address this, the ability of LANA to interact with PAN RNA was evaluated in vitro and in vivo. In order to perform in vitro interaction analyses, fulllength LANA was expressed using recombinant baculovirus and purified by FLAG-M2 resin (Flag-LANA) (Fig. 1A). Purified Flag-LANA was incubated with PAN RNA that...

Kaposi's sarcoma-associated herpesvirus (KSHV) latent genomes are tethered to host histones to form a minichromosome also known as an "episome." Histones, which are core components of chromatin, are heavily modified by various histone-targeting enzymes. Posttranslational modifications of histones significantly influence accessibility of transcriptional factors and thus have profound effects on gene expression. Recent studies showed that epigenetic marks on the KSHV episome are well organized, exemplified by the absence of histone H3 lysine 9 (H3K9) methylation, a heterochromatic histone mark, from immediate early and latent gene promoters in naturally infected cells. The present study revealed a mechanistic insight into KSHV epigenome regulation via a complex consisting of LANA and the H3K9me1/2 histone demethylase JMJD1A/KDM3A. This complex was isolated from HeLa cell nuclear extracts stably expressing LANA and was verified by coimmunoprecipitation analyses and with purified proteins. LANA recruitment sites on the KSHV genome inversely correlated with H3K9me2 histone marks in naturally infected cells, and methylation of H3K9 significantly inhibited LANA binding to the histone H3 tail. Chromatin immunoprecipitation coupled with KSHV tiling arrays identified the recruitment sites of the complex, while depletion of LANA expression or overexpression of a KDM3A binding-deficient mutant decreased KDM3A recruitment to the KSHV genome. Finally, ablation of KDM3A expression from latently KSHV-infected cells significantly inhibited KSHV gene expression, leading to decreased KSHV replication during reactivation. Taken together, our results suggest that LANA may play a role in regulation of epigenetic marks on the KSHV genome, which is in part through association with the histone demethylase KDM3A.

Background: Post-translational modifications generate functional heterogeneity of viral regulatory factors. Results: Viral chromatin association by Kaposi sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) is modulated by protein arginine methyltransferase 1 (PRMT1)-directed methylation. Conclusion: Methylation of KSHV LANA antagonizes viral reactivation. Significance: Protein methylation contributes to the functional properties of viral regulatory proteins, including KSHV LANA.