J Virol 87:4005C4016. The functions of K8 and associated T1.4 RNA were investigated in detail, and the results showed that T1.4 mediates the binding of K8 to ori-Lyt DNA. The T1.4-K8 complex physically bound to KSHV ori-Lyt DNA and recruited other proteins and cofactors to Oclacitinib maleate assemble a replication complex. Depletion of T1.4 abolished DNA replication in primary infection. These findings provide mechanistic insights into the role of K8 in coordination with T1.4 RNA in regulating KSHV DNA replication during infection. IMPORTANCE Genomewide analyses of the mammalian transcriptome revealed that a large proportion of sequence previously annotated as noncoding regions is actually transcribed and gives rise to stable RNAs. The emergence of a large number of noncoding RNAs suggests that functional RNA-protein complexes, e.g., ribosomes or spliceosomes, are not ancient relics of the last ribo-organism but would be well adapted to a regulatory role in biology. K8 has been puzzling because of its unique characteristics, such as multiple regulatory roles in SERK1 gene expression and DNA replication without DNA binding capability. This study reveals the mechanism underlying its regulatory role by demonstrating that K8 is an RNA binding protein that binds to DNA and initiates DNA replication in coordination with a noncoding RNA. It is suggested that many K8 functions, if not all, are carried out through its associated RNAs. infection of fresh cells are essential for viral pathogenesis and tumorigenesis (6). During infection, a set of viral genes are expressed and participate in immune evasion, epigenetic modification, and transcriptional reprogramming before the latent infection is established (10,C12). Short-term viral DNA replication occurs in the period between virus infection and latency establishment, which allows the KSHV genome to be amplified from Oclacitinib maleate 1 to 50 to 100 copies. This phase of viral DNA replication is termed abortive lytic replication as, unlike lytic replication, it does not produce infectious virions. We and others have found that viral abortive replication is sensitive to phosphonoacetic acid (PAA), a specific inhibitor of herpesvirus DNA polymerase (11, 13), suggesting that the DNA replication in this phase is different from latent replication, which is cellular DNA polymerase dependent but utilizes lytic DNA replication machinery. However, the mechanism that controls abortive replication during infection is elusive. KSHV DNA replication initiates at an origin (such as ori-Lyt) and requires infection (13). In addition to its role in viral DNA replication, K8 has been shown to be a global gene expression repressor, inhibiting both viral and cellular gene expression (22, 23). Several studies have uncovered linkages between K8 and epigenetic regulation, such as inhibiting the H3K9me3 demethylase JMJD2A and mediating SUMO-2/3 modification of the KSHV chromosome (24, 25). In addition, K8 was reported to inhibit the activity of CDK2, which plays a pivotal role in cell cycle progression. Inhibition of CDK2 activity by K8 resulted in a prolonged G1 phase with concomitant induction of p21, P27, and C/EBP (26, 27). To elucidate the biological functions of K8 in the KSHV life cycle and the mechanisms underlying these actions, we attempted to identify components that interact with K8 in the belief that knowing the K8 binding partners would provide clues to reveal K8 function and its underlying mechanism. In this effort, we found that K8 is a novel RNA binding protein and that its RNA binding ability is essential to its role in viral DNA replication during KSHV infection. Furthermore, a viral RNA, namely, T1.4 or ori-Lyt-associated RNA, was found to mediate the interaction between K8 and ori-Lyt DNA and to be absolutely required for viral DNA replication. Through this investigation, a novel mechanism of viral DNA replication by coordinates actions of K8 protein and viral noncoding RNA was revealed. RESULTS K8 predominately interacts with RNA binding proteins. In order to elucidate the mechanisms underlying the K8 actions in viral DNA replication and other events in the viral life cycle, a proteomics approach was undertaken to identify the proteins that interact with K8 in KSHV-infected cells during lytic replication. BCBL-1 cells were induced for reactivation with tetradecanoyl phorbol acetate (TPA) for 48 h and subjected to Oclacitinib maleate coimmunoprecipitation (co-IP) with an antibody against K8. The resultant proteins were resolved on SDS-PAGE and revealed by Coomassie blue staining (Fig..

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