ABSTRACTRNA polymerase III (pol III) transcribes multiple non-coding (nc) RNAs that are essential for cellular function. Pol III-dependent transcription is also engaged during certain viral infections, including 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-dependent transcriptional activity was regulated during gammaherpesvirus infection, using the murine gammaherpesvirus 68 (γHV68) system. To compare the transcriptional regulation of host and viral pol III-dependent ncRNAs, we analyzed a series of pol III-dependent promoters using a newly-generated luciferase reporter optimized to measure pol III activity. We measured promoter activity from these constructs at the translation level via luciferase activity and at the transcription level via 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 transcriptional activity of multiple host and viral pol III-dependent promoters, and further identified the ability of accessory sequences to influence both baseline and inducible promoter activity after infection. These studies highlight how lytic gammaherpesvirus infection alters the transcriptional landscape of host cells to increase pol III-derived transcription, a process that may further modify cellular function and enhance viral gene expression and pathogenesis.IMPORTANCEGammaherpesviruses 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 non-coding 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 its use of multiple promoter types and diverse RNAs with variable stability and processing requirements. Here, we established a reporter system to directly study RNA polymerase III-dependent promoter 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 non-coding RNA expression within the infected cell.
Identification of Novel MicroRNA-Like Molecules Generated from Herpesvirus and Host tRNA Transcripts