Lytic infection with murine gammaherpesvirus 68 activates host and viral RNA polymerase III promoters and enhances non-coding RNA expression

Abstract

218 words Importance: 141 words Text: 5,479 words γHV68 infection activates pol III-dependent promoters 2 ABSTRACT 1RNA polymerase III (pol III) transcribes multiple non-coding (nc) RNAs that are essential for cellular function. Pol 2 III-dependent transcription is also engaged during certain viral infections, including the gammaherpesviruses 3 (HVs), where pol III-dependent viral ncRNAs promote pathogenesis. Additionally, several host ncRNAs are 4 upregulated during HV infection and play integral roles in pathogenesis by facilitating viral establishment and 5 gene expression. Here we sought to investigate how pol III-dependent transcriptional activity was regulated 6 during gammaherpesvirus infection, using the murine gammaherpesvirus 68 (HV68) system. To compare the 7 transcriptional regulation of host and viral pol III-dependent ncRNAs, we analyzed a series of pol III-dependent 8 promoters using a newly-generated luciferase reporter optimized to measure pol III activity. We measured 9 promoter activity from these constructs at the translation level via luciferase activity and at the transcription level 10 via RT-qPCR. We further measured endogenous ncRNA expression at single cell-resolution by flow cytometry. 11These studies demonstrated that lytic infection with γHV68 increased the transcriptional activity of multiple host 12 and viral pol III-dependent promoters, and further identified the ability of accessory sequences to influence both 13 baseline and inducible promoter activity after infection. These studies highlight how lytic gammaherpesvirus 14 infection alters the transcriptional landscape of host cells to increase pol III-derived transcription, a process that 15 may further modify cellular function and enhance viral gene expression and pathogenesis. 16 IMPORTANCE 17Gammaherpesviruses are a prime example of how viruses can alter the host transcriptional landscape to 18 establish infection. Despite major insights into how these viruses modify RNA polymerase II-dependent 19 generation of messenger RNAs, how these viruses influence the activity of host RNA polymerase III remains 20 much less clear. Small non-coding RNAs produced by RNA polymerase III are increasingly recognized to play 21 critical regulatory roles in cell biology and virus infection. Studies of RNA polymerase III dependent transcription 22 are complicated by its use of multiple promoter types and diverse RNAs with variable stability and processing 23 requirements. Here, we established a reporter system to directly study RNA polymerase III-dependent promoter 24 responses during gammaherpesvirus infection and utilized single-cell flow cytometry-based methods to reveal 25 that gammaherpesvirus lytic replication broadly induces pol III activity to enhance host and viral non-coding RNA 26 expression within the infected cell. 27 γHV68 infection activates pol III-dependent promoters