Lytic Infection with Murine Gammaherpesvirus 68 Activates Host and Viral RNA Polymerase III Promoters and Enhances Noncoding RNA Expression.

RNA polymerase III (pol III) transcribes multiple noncoding RNAs (ncRNAs) that are essential for cellular function. Pol III-dependent transcription is also engaged during certain viral infections, including those of the gammaherpesviruses (γHVs), where pol III-dependent viral ncRNAs promote pathogenesis. Additionally, several host ncRNAs are upregulated during γHV infection and play integral roles in pathogenesis by facilitating viral establishment and gene expression. Here, we sought to investigate how pol III promoters and transcripts are regulated during gammaherpesvirus infection using the murine gammaherpesvirus 68 (γHV68) system. To compare the transcription of host and viral pol III-dependent ncRNAs, we analyzed a series of pol III promoters for host and viral ncRNAs using a luciferase reporter optimized to measure pol III activity. We measured promoter activity from the reporter gene at the translation level via luciferase activity and at the transcription level via reverse transcription-quantitative PCR (RT-qPCR). We further measured endogenous ncRNA expression at single-cell resolution by flow cytometry. These studies demonstrated that lytic infection with γHV68 increased the transcription from multiple host and viral pol III promoters and further identified the ability of accessory sequences to influence both baseline and inducible promoter activity after infection. RNA flow cytometry revealed the induction of endogenous pol III-derived ncRNAs that tightly correlated with viral gene expression. These studies highlight how lytic gammaherpesvirus infection alters the transcriptional landscape of host cells to increase pol III-derived RNAs, a process that may further modify cellular function and enhance viral gene expression and pathogenesis. IMPORTANCE Gammaherpesviruses are a prime example of how viruses can alter the host transcriptional landscape to establish infection. Despite major insights into how these viruses modify RNA polymerase II-dependent generation of messenger RNAs, how these viruses influence the activity of host RNA polymerase III remains much less clear. Small noncoding RNAs produced by RNA polymerase III are increasingly recognized to play critical regulatory roles in cell biology and virus infection. Studies of RNA polymerase III-dependent transcription are complicated by multiple promoter types and diverse RNAs with variable stability and processing requirements. Here, we characterized a reporter system to directly study RNA polymerase III-dependent responses during gammaherpesvirus infection and utilized single-cell flow cytometry-based methods to reveal that gammaherpesvirus lytic replication broadly induces pol III activity to enhance host and viral noncoding RNA expression within the infected cell.

Citation

Ashley N Knox, Alice Mueller, Eva M Medina, Eric T Clambey, Linda F van Dyk. Lytic Infection with Murine Gammaherpesvirus 68 Activates Host and Viral RNA Polymerase III Promoters and Enhances Noncoding RNA Expression. Journal of virology. 2021 Jun 24;95(14):e0007921

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PMID: 33910955

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