Abstract
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.
Highlights
The Herpesviridae are a family of large dsDNA viruses that include multiple prominent human and animal pathogens [1]
To better define expression of γHV RNAs at the single cell level, we employed the PrimeFlow RNA assay [13] to study viral gene expression during murine gammaherpesvirus 68 infection, a small animal γHV [4, 13]. This method is a highly sensitive, extremely specific in situ hybridization assay, integrating Affymetrix-designed branched DNA technology with single-cell analysis powered by Single-cell heterogeneity of Gammaherpesvirus RNA expression multiparameter flow cytometry
We first tested the ability of PrimeFlow to measure multiple viral RNAs during lytic infection with γHV68, including small non-coding RNAs and mRNAs
Summary
The Herpesviridae are a family of large dsDNA viruses that include multiple prominent human and animal pathogens [1]. These viruses infect different cell types, and are associated with diverse pathologies, they share conserved genes and two fundamental phases of infection: lytic replication and latent infection [1]. Murine gammaherpesvirus 68 (γHV68, or MHV-68; ICTV nomenclature Murid herpesvirus 4, MuHV-4), is a well-described small animal model for the γHVs [4] While these viruses establish a lifelong infection that is often clinically inapparent, immune-suppressed individuals are at risk for γHV-associated malignancies [5]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
