Rodriguez, W. | Search bioRxiv

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↗

bioRxiv2018

C19ORF66 broadly escapes viral-induced endonuclease cleavage and restricts Kaposi Sarcoma Associated Herpesvirus (KSHV)

One striking characteristic of certain herpesviruses is their ability to induce rapid and widespread RNA decay in order to gain access to host resources. This phenotype is induced by viral endoribonucleases, including SOX in KSHV, muSOX in MHV68, BGLF5 in EBV and vhs in HSV-1. Here, we performed comparative RNA-seq upon expression of these herpesviral endonucleases in order to characterize their effect on the host transcriptome. Consistent with previous reports, we found that approximately two thirds of transcripts are downregulated in cells expressing any of these viral endonucleases. Among transcripts spared from degradation, we uncovered a cluster of transcripts that systematically escape degradation from all tested endonucleases. Among these escapees, we identified C19ORF66 and reveal that like the previously identified escapees, this transcript is protected from degradation by its 3UTR. We then show that C19ORF66, a known anti-viral protein, is a potent KSHV restriction factor, suggesting that its ability to escape viral cleavage may be an important component of the host response to viral infection. Collectively, our comparative approach is a powerful tool to pinpoint key regulators of the viral-host interplay and led us to uncover a novel KSHV regulator.

microbiology↗