KSHV ORF59 and PAN RNA Recruit Histone Demethylases to the Viral Chromatin during Lytic Reactivation

Abstract

Kaposi’s sarcoma-associated herpesvirus (KSHV) causes multiple malignancies in immunocompromised individuals. KSHV primarily establishes a lifelong latency in infected humans during which only a subset of viral genes is expressed while most of the viral genome remains transcriptionally silent with condensed chromatin. However, during the lytic phase, the viral genome undergoes dramatic changes in chromatin landscape leading to a transcriptionally active state with the expression of most of the viral genes and production of progeny virions. Multiple cellular and viral factors influence the epigenetic gene regulation and transitioning of virus from latency to the lytic state. We have earlier shown that KSHV ORF59, viral processivity factor, binds to a protein arginine methyl transferase 5 (PRMT5) to alter the histone arginine methylation during reactivation. Additionally, ORF59 has been shown to interact with most abundantly expressed KSHV long noncoding polyadenylated nuclear RNA (PAN RNA), which associates with the viral epigenome during reactivation. Interestingly, PAN RNA interacts with UTX and JMJD3, cellular H3K27me3 demethylases, and removes the repressive marks on the chromatin. In this study, we report that the recruitment of histone demethylases to the viral chromatin is facilitated by the expression of ORF59 protein and PAN RNA. Using biochemical and localization assays including co-immunoprecipitation and immunofluorescence, we demonstate ORF59 localizes with UTX and JMJD3. Our results confirm that PAN RNA enhances the interaction of ORF59 with the chromatin modifying enzymes UTX and JMJD3.

The m6A landscape of polyadenylated nuclear (PAN) RNA and its related methylome in the context of KSHV replication

“…2), we revealed that PAN RNA can carry up to 5 m 6 A sites. These modifications localize within PAN RNA secondary structure motifs that we have previously shown to display low SHAPE and low Shannon entropy (12), which are characteristic of biologically significant RNA motifs (94)(95)(96)(97). We noted that PAN m 6 A status is dynamic and changes during the progression of KSHV replication, achieving the highest level during the late lytic stages.…”

The m6A landscape of polyadenylated nuclear (PAN) RNA and its related methylome in the context of KSHV replication

“…In our study, we provide the first comprehensive insight into the m 6 A status of KSHV-encoded PAN RNA, the key regulator of viral lytic reactivation ( Rossetto et al 2013 ; Withers et al 2018 ; Hiura et al 2020 ). Using two independent mapping approaches, that is, the 4SedTTP RT with next-generation sequencing and direct RNA sequencing, we revealed that PAN RNA can carry up to 5 m 6 A sites.…”

“…This process might operate as a fine-tuning mechanism, coordinating the function and biology of PAN RNA, as it has been demonstrated for other transcripts ( Liu et al 2015 ; Park et al 2019 ; Zhou et al 2019 ; Lee et al 2020 ). PAN RNA is a highly multifunctional transcript involved in the interactions with viral ( Rossetto et al 2013 ; Campbell et al 2014a ) and cellular proteins the ( Rossetto and Pari 2012 ), modulating the chromatin and episomal DNA state during viral lytic reactivation ( Strahan et al 2017 ; Hiura et al 2020 ), and facilitating the nuclear export of late viral mRNAs ( Withers et al 2018 ). The dynamic m 6 A modifications to PAN RNA might provide the means to orchestrate its functions with the immediate need of the pathogen.…”

Regulation of KSHV Latency and Lytic Reactivation

“…Polyadenylated nuclear (PAN) RNA, a highly-expressed non-coding KSHV lytic RNA, facilitates interaction between ORF59, histone demethylases ubiquitously transcribed tetratricopeptide repeat X chromosome (UTX) and Jumonji-domain-containing 3 (JMJD3), and the viral genome. The subsequent reduction in H3K27 trimethylation, particularly at the ORF50 promoter, promotes robust lytic gene expression [ 98 , 99 ]. Conversely, KSHV lytic protein K8 acts as a modulator of KSHV lytic reactivation by recruiting HDACs to the RTA promoter to repress transcriptional activity, potentially serving as a brake on runaway lytic activity [ 100 ].…”