Multidimensional analysis of Gammaherpesvirus RNA expression reveals unexpected heterogeneity of gene expression

Lauren M. Oko, Abigail K. Kimball, Rachael E. Kaspar, Ashley N. Knox, Carrie B. Coleman, Rosemary Rochford, Tim Chang, Benjamin Alderete, Linda F. van Dyk, Eric T. Clambey

Research output: Contribution to journalArticlepeer-review

4 Scopus citations

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.

Original languageEnglish (US)
Article numbere1007849
JournalPLoS pathogens
Volume15
Issue number6
DOIs
StatePublished - 2019
Externally publishedYes

Bibliographical note

Funding Information:
This research was funded by National Institutes of Health grants R01CA103632 and R01CA168558 to L.F.V.D., R21AI134084 to E.T.C. and L.F.V.D., and by an American Heart Association National Scientist Development grant (#13SDG14510023), a Colorado Clinical and Translational Sciences Initiative Novel methods development grant, and funding from the University of Colorado Dept. of Anesthesiology to E.T.C.. The Colorado CTSI is supported by NIH/ NCATS Colorado CTSA Grant Number UL1 TR002535. The Flow Cytometry Shared Resource of the University of Colorado Cancer Center receives direct funding support from the National Cancer Institute through Cancer Center Support Grant P30CA046934. Contents are the authors? sole responsibility and do not necessarily represent official NIH views. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Publisher Copyright:
© 2019 Oko et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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