Biology subjects

Rodriguez, W.

Publications and source records attributed to Rodriguez, W..

3 recordsLinked to original sources

bioRxiv2022

Shiftless Restricts Viral Gene Expression and Influences RNA Granule Formation during KSHV lytic replication.

Herpesviral infection reflects thousands of years of co-evolution and the constant struggle between virus and host for control of cellular gene expression. During Kaposis sarcoma-associated herpesvirus (KSHV) lytic replication, the virus rapidly seizes control of host gene expression machinery by triggering a massive RNA decay event via a virally-encoded endoribonuclease, SOX. This virus takeover strategy decimates close to 80% of cellular transcripts, reallocating host resources toward viral replication. The host cell, however, is not entirely passive in this assault on RNA stability. A small pool of host transcripts that actively evade SOX cleavage has been identified over the years. One such "escapee", C19ORF66 (herein referred to as Shiftless - SHFL) encodes a potent anti-viral protein capable of restricting the replication of multiple DNA, RNA, and retroviruses including KSHV. Here, we show that SHFL restricts KSHV replication by targeting the expression of critical viral early genes, including the master transactivator protein, KSHV ORF50, and thus subsequently the entire lytic gene cascade. Consistent with previous reports, we found the SHFL interactome throughout KSHV infection is dominated by RNA-binding proteins that influence both translation and protein stability, including the viral protein ORF57, a crucial regulator of viral RNA fate. We next show that SHFL affects cytoplasmic RNA granule formation, triggering the disassembly of processing bodies. Taken together, our findings provide insights into the complex relationship between RNA stability, RNA granule formation, and the anti-viral response to KSHV infection. SignificanceIn the past five years, SHFL has emerged as a novel and integral piece of the innate immune response to viral infection. SHFL has been reported to restrict the replication of multiple viruses including several flaviviruses and the retrovirus HIV-1. However, to date, the mechanism(s) by which SHFL restricts DNA virus infection remains largely unknown. We have previously shown that following its escape from KSHV-induced RNA decay, SHFL acts as a potent anti-viral factor, restricting nearly every stage of KSHV lytic replication. In this study, we set out to determine the mechanism by which SHFL restricts KSHV infection. We demonstrate that SHFL impacts all classes of KSHV genes and found that SHFL restricts the expression of several key early genes, including KSHV ORF50 and ORF57. We then mapped the interactome of SHFL during KSHV infection and found several host and viral RNA-binding proteins that all play crucial roles in regulating RNA stability and translation. Lastly, we found that SHFL expression influences RNA granule formation both outside of and within the context of KSHV infection, highlighting its broader impact on global gene expression. Collectively, our findings highlight a novel relationship between a critical piece of the anti-viral response to KSHV infection and the regulation of RNA-protein dynamics.

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↗

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↗