Epigenetic factor siRNA screen during primary KSHV infection identifies novel host restriction factors for the lytic cycle of KSHV

Abstract

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.

Epigenetic Regulation of Kaposi’s Sarcoma-Associated Herpesvirus Latency

“…Recently, this group performed a comprehensive epigenome analysis of the KSHV genome and found the CpG motif as a cis-acting sequence for KDM2B, which consequently recruits Polycomb repressive complexes (PRCs) (Figure 1G; Gunther et al, 2019). Interestingly, a recent report from Naik et al (2020) describes siRNA screening and time course ChIP experiments showing that early deposition of KDM2B on the KSHV genome limits the enrichment of the active marks H3K4me3 and H3K36me2 and helps in the maintenance of viral latency (Figure 1H). The binding of KDM2B prior to PRC-regulated heterochromatin supports the potential role of KDM2B in the recruitment of PRCs to viral DNA following de novo initial infection.…”

“…In addition to viral proteins, cellular factors may also be involved in recruitment. The direct binding of the PRC component KDM2B to CpG islands may also help in the recruitment of PRCs to the KSHV genome (Gunther et al, 2019;Naik et al, 2020).…”

Clinical Manifestations and Epigenetic Regulation of Oral Herpesvirus Infections

“…However, in some cases, specific KSHV proteins, the viral long non-coding PAN RNA, and the unmethylated CpG motifs in the viral DNA have been implicated in the regulation of the recruitment of host epigenetic factors to the viral episome [ 152 , 154 , 155 ]. A recent siRNA screen of host epigenetic factors during de novo KSHV infection identified several new players in the formation of the KSHV epigenome and establishment of latency beyond PRC1 and PRC2, which includes the histone demethlyase KDM2B, NuRD and Tip60 repressive complexes among many others [ 156 ]. It is likely that the binding of all of these host epigenetic factors contribute to the different epigenetic layers of the KSHV episome, which can support the establishment and maintenance of the chromatin structure of latent viral episomes.…”

“…Moreover, a recent siRNA epigenetic screen identified histone demethylase KDM2B as a key restriction factor of lytic gene transcription not only in latent infection but also in the lytic infection of primary gingival epithelial cells [ 156 ]. KDM2B is a histone demethylase associated with the removal of activating H3K4me3, H3K36me2 and H3K79me2 marks, and has also been shown to play a role in PRC1 recruitment to specific regions in the host genome [ 157 , 158 , 159 , 160 ].…”

“…KDM2B is a histone demethylase associated with the removal of activating H3K4me3, H3K36me2 and H3K79me2 marks, and has also been shown to play a role in PRC1 recruitment to specific regions in the host genome [ 157 , 158 , 159 , 160 ]. The viral gene expression inhibitory function of KDM2B was linked to its histone demethylase and DNA-binding activity [ 156 ]. KDM2B protein levels can be dysregulated by different environmental stimuli such as hypoxia, which increases KDM2B expression, and this can affect the outcome of KSHV infection [ 161 , 162 ].…”