Establishment of viral latency is not only essential for lifelong Kaposi's sarcoma-associated herpesvirus (KSHV) infection, but it is also a prerequisite of viral tumorigenesis. The latent viral DNA has a complex chromatin structure, which is established in a stepwise manner regulated by host epigenetic factors during de novo infection. However, despite the importance of viral latency in KSHV pathogenesis, we still have limited information about the repertoire of epigenetic factors that are critical for the establishment and maintenance of KSHV latency. Therefore, the goal of this study was to identify host epigenetic factors that suppress lytic KSHV genes during primary viral infection, which would indicate their role in latency establishment. We performed an siRNA screen targeting 392 host epigenetic factors during primary infection and analyzed which ones affect the expression of the viral replication and transcription activator (RTA) and/or the latency-associated nuclear antigen (LANA), which are viral genes essential for lytic replication and latency, respectively. As a result, we identified the Nucleosome Remodeling and Deacetylase (NuRD) complex, Tip60 and Tip60-associated co-repressors, and the histone demethylase KDM2B as repressors of KSHV lytic genes during both de novo infection and the maintenance of viral latency. Furthermore, we showed that KDM2B rapidly binds to the incoming viral DNA as early as 8 hpi, and can limit the enrichment of activating histone marks on the RTA promoter favoring the downregulation of RTA expression even prior to the polycomb proteins-regulated heterochromatin establishment on the viral genome. Strikingly, KDM2B can also suppress viral gene expression and replication during lytic infection of primary gingival epithelial cells, revealing that KDM2B can act as a host restriction factor of the lytic cycle of KSHV during both latent and lytic infections in multiple different cell types.

Dengue virus (DENV) nonstructural protein 4B (NS4B) is an endoplasmic reticulum (ER) membrane-associated protein, and mutagenesis studies have revealed its significance in viral genome replication. In this work, we demonstrated that NS4B is an N-glycosylated protein in virus-infected cells as well as in recombinant protein expression. NS4B is N glycosylated at residues 58 and 62 and exists in two forms, glycosylated and unglycosylated. We manipulated full-length infectious RNA clones and subgenomic replicons to generate N58Q, N62Q, and N58QN62Q mutants. Each of the single mutants had distinct effects, but the N58QN62Q mutation resulted in dramatic reduction of viral production efficiency without affecting secretion or infectivity of the virion in mammalian and mosquito C6/36 hosts. Real-time quantitative PCR (qPCR), subgenomic replicon, and trans-complementation assays indicated that the N58QN62Q mutation affected RNA replication possibly by the loss of glycans. In addition, four intragenic mutations (S59Y, S59F, T66A, and A137T) were obtained from mammalian and/or mosquito C6/36 cell culture systems. All of these second-site mutations compensated for the replication defect of the N58QN62Q mutant without creating novel glycosylation sites. In vivo protein stability analyses revealed that the N58QN62Q mutation alone or plus a compensatory mutation did not affect the stability of NS4B. Overall, our findings indicated that mutation of putative N-glycosylation sites affected the biological function of NS4B in the viral replication complex. IMPORTANCE This is the first report to identify and reveal the biological significance of dengue virus (DENV) nonstructural protein 4B (NS4B) posttranslation N-glycosylation to the virus life cycle. The study demonstrated that NS4B is N glycosylated in virus-infected cells and in recombinant protein expression. NS4B is modified by glycans atAsn-58 and Asn-62. Functional characterization implied that DENV NS4B utilizes the glycosylation machinery in both mammalian and mosquito hosts. Four intragenic mutations were found to compensate for replication and subsequent viral production deficiencies without creating novel N-glycosylation sites or modulating the stabilities of the protein, suggesting that glycans may be involved in maintaining the NS4B protein conformation. NS4B glycans may be necessary elements of the viral life cycle, but compensatory mutations can circumvent their requirement. This novel finding may have broader implications in flaviviral biology as the most likely glycan at Asn-62 of NS4B is conserved in DENV serotypes and in some related flaviviruses. D engue virus (DENV) belongs to the Flaviviridae family of theFlavivirus genus, exists in four serotypes (DENV1 to -4), and is transmitted to humans by Aedes mosquitoes. Incidences of dengue have grown dramatically around the globe in recent decades and are endemic to tropical and subtropical countries. According to the latest report of the World Health Organization (WHO) (February 2015), approximately 50% of th...