Virus-host interactions are frequently studied in bulk cell populations, obscuring cell-to-cell variation. Here we investigate endogenous herpesvirus gene expression at the single-cell level, combining a sensitive and robust fluorescent in situ hybridization platform with multiparameter flow cytometry, to study the expression of gammaherpesvirus non-coding RNAs (ncRNAs) during lytic replication, latent infection and reactivation in vitro. This method allowed robust detection of viral ncRNAs of murine gammaherpesvirus 68 (γHV68), Kaposi’s sarcoma associated herpesvirus and Epstein-Barr virus, revealing variable expression at the single-cell level. By quantifying the inter-relationship of viral ncRNA, viral mRNA, viral protein and host mRNA regulation during γHV68 infection, we find heterogeneous and asynchronous gene expression during latency and reactivation, with reactivation from latency identified by a distinct gene expression profile within rare cells. Further, during lytic replication with γHV68, we find many cells have limited viral gene expression, with only a fraction of cells showing robust gene expression, dynamic RNA localization, and progressive infection. Lytic viral gene expression was enhanced in primary fibroblasts and by conditions associated with enhanced viral replication, with multiple subpopulations of cells present in even highly permissive infection conditions. These findings, powered by single-cell analysis integrated with automated clustering algorithms, suggest inefficient or abortive γHV infection in many cells, and identify substantial heterogeneity in viral gene expression at the single-cell level.

RNA 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 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 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 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 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 non-coding RNA expression within the infected cell.

24Virus-host interactions are frequently studied in bulk cell populations, obscuring 25 cell-to-cell variation. Here we investigate endogenous herpesvirus gene expression at 26 the single-cell level, combining a sensitive and robust fluorescent in situ hybridization 27 platform with multiparameter flow cytometry, to study the expression of 28 gammaherpesvirus non-coding RNAs (ncRNAs) during lytic replication, latent infection 29 and reactivation in vitro. This method allowed robust detection of viral ncRNAs of 30 murine gammaherpesvirus 68 (γHV68), Kaposi's sarcoma associated herpesvirus and 31 Epstein-Barr virus, revealing variable expression at the single-cell level. By quantifying 32 the inter-relationship of viral ncRNA, viral mRNA, viral protein and host mRNA 33 regulation during γHV68 infection, we find heterogeneous and asynchronous gene 34 expression during latency and reactivation, with reactivation from latency identified by a 35 distinct gene expression profile within rare cells. Further, during lytic replication with 36 γHV68, we find many cells have limited viral gene expression, with only a fraction of 37 cells showing robust gene expression, dynamic RNA localization, and progressive 38 infection. Lytic viral gene expression was enhanced in primary fibroblasts and by 39 conditions associated with enhanced viral replication, with multiple subpopulations of 40 cells present in even highly permissive infection conditions. These findings, powered by 41 single-cell analysis integrated with automated clustering algorithms, suggest inefficient 42 or abortive γHV infection in many cells, and identify substantial heterogeneity in viral 43 gene expression at the single-cell level. 44 45 3 AUTHOR SUMMARY 46 The gammaherpesviruses are a group of DNA tumor viruses that establish 47 The Herpesviridae are a family of large dsDNA viruses that include multiple 63 prominent human and animal pathogens [1]. Although these viruses infect different cell 64 types, and are associated with diverse pathologies, they share conserved genes and 65 two fundamental phases of infection: lytic replication and latent infection [1]. Lytic 66 replication is characterized by a cascade of viral gene expression, active viral DNA 67 replication and the production of infectious virions. Conversely, latency is characterized 68 4 by limited viral gene expression and the absence of de novo viral replication. While 69 latent infection is a relatively quiescent form of infection, the herpesviruses can 70 reactivate from latency, to reinitiate lytic replication. 71 Among the herpesviruses, the gammaherpesviruses (γHV) are lymphotropic 72 viruses that include the human pathogens Epstein-Barr virus (EBV) [2] and Kaposi's 73 sarcoma associated herpesvirus (KSHV) [3]. Murine gammaherpesvirus 68 (γHV68, or 74 MHV-68; ICTV nomenclature Murid herpesvirus 4, MuHV-4), is a well-described small 75 animal model for the γHVs [4]. While these viruses establish a lifelong infection that is 76 often clinically inapparent, immune-suppressed individuals are part...