Macveigh-Fierro, D. | bioRxiv Science

bioRxiv2021

The m6A reader YTHDC2 is essential for escape from KSHV SOX-induced RNA decay

The role m6A modifications have increasingly been associated with diverse set of roles in modulating viruses and influencing the outcomes of viral infection. Here we report that the landscape of m6A deposition is drastically shifted during KSHV (Kaposi Sarcoma Associated herpesvirus) lytic infection for both viral and host transcripts. In line with previous reports, we also saw an overall decrease in host methylation in favor of viral mRNA along with 5 hypomethylation and 3 hypermethylation. During KSHV lytic infection, a major shift in overall mRNA abundance is driven by the viral endoribonuclease SOX, which induces the decay of greater than 70% of transcripts. Here, we reveal that Interlukin-6 (IL-6) mRNA, a well-characterized SOX-resistant transcript, is m6A modified during lytic infection. Furthermore, we show that this modification falls within the IL-6 SOX Resistance Element (SRE), an RNA element in IL-6 3 UTR that was previously shown to be sufficient for protection from SOX cleavage. We show that the presence of this m6A modification is essential to confer SOX resistance to the IL-6 mRNA. We next show that this modification recruits the m6A reader YTHDC2 and found that YTHDC2 is necessary for the escape of the IL-6 transcript. These results shed light on how the host cell has evolved to use RNA modifications to circumvent viral manipulation of RNA fate during KSHV infection.

microbiology↗

bioRxiv2020

The KSHV ORF20 Protein Interacts With The Viral Processivity Factor ORF59 And Promote Viral Reactivation.

AO_SCPLOWBSTRACTC_SCPLOWUpon KSHV lytic reactivation, rapid and widespread amplification of viral DNA (vDNA) triggers significant nuclear reorganization. As part of this striking shift in nuclear architecture, viral replication compartments are formed as sites of lytic vDNA production along with remarkable spatial remodeling and relocalization of cellular and viral proteins. These viral replication compartments house several lytic gene products that coordinate viral gene expression, vDNA replication, and nucleocapsid assembly. The viral proteins and mechanisms that regulate this overhaul of the nuclear landscape during KSHV replication remain largely unknown. KSHVs ORF20 is a widely conserved lytic gene among all herpesviruses suggesting it may have a fundamental contribution to the progression of herpesviral infection. Here, we utilized a promiscuous biotin ligase proximity labeling method to identify the proximal interactome of ORF20, which includes several replication-associated viral proteins, one of which is ORF59, the KSHV DNA processivity factor. Using co-immunoprecipitation and immunofluorescence assays, we confirmed the interaction between ORF20 and ORF59 and tracked the localization of both proteins to KSHV replication compartments. To further characterize the function of ORF20, we generated an ORF20-deficient KSHV and compared its replicative fitness relative to wild type virus. Virion production was significantly diminished in the ORF20-deficient virus as observed by supernatant transfer assays. Additionally, we tied this defect in viable virion formation to a reduction in viral late gene expression. Lastly, we observed an overall reduction in vDNA replication in the ORF20-deficient virus implying a key role for ORF20 in the regulation of lytic replication. Taken together, these results capture the essential role of KSHV ORF20 in progressing viral lytic infection by regulating vDNA replication alongside other crucial lytic proteins within KSHV replication compartments.

microbiology↗