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Multidimensional analysis of Gammaherpesvirus RNA expression reveals unexpected heterogeneity of gene expression
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 cytomet...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6576797/ https://www.ncbi.nlm.nih.gov/pubmed/31166996 http://dx.doi.org/10.1371/journal.ppat.1007849 |
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author | Oko, Lauren M. Kimball, Abigail K. Kaspar, Rachael E. Knox, Ashley N. Coleman, Carrie B. Rochford, Rosemary Chang, Tim Alderete, Benjamin van Dyk, Linda F. Clambey, Eric T. |
author_facet | Oko, Lauren M. Kimball, Abigail K. Kaspar, Rachael E. Knox, Ashley N. Coleman, Carrie B. Rochford, Rosemary Chang, Tim Alderete, Benjamin van Dyk, Linda F. Clambey, Eric T. |
author_sort | Oko, Lauren M. |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-6576797 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-65767972019-06-28 Multidimensional analysis of Gammaherpesvirus RNA expression reveals unexpected heterogeneity of gene expression Oko, Lauren M. Kimball, Abigail K. Kaspar, Rachael E. Knox, Ashley N. Coleman, Carrie B. Rochford, Rosemary Chang, Tim Alderete, Benjamin van Dyk, Linda F. Clambey, Eric T. PLoS Pathog Research Article 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. Public Library of Science 2019-06-05 /pmc/articles/PMC6576797/ /pubmed/31166996 http://dx.doi.org/10.1371/journal.ppat.1007849 Text en © 2019 Oko et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article Oko, Lauren M. Kimball, Abigail K. Kaspar, Rachael E. Knox, Ashley N. Coleman, Carrie B. Rochford, Rosemary Chang, Tim Alderete, Benjamin van Dyk, Linda F. Clambey, Eric T. Multidimensional analysis of Gammaherpesvirus RNA expression reveals unexpected heterogeneity of gene expression |
title | Multidimensional analysis of Gammaherpesvirus RNA expression reveals unexpected heterogeneity of gene expression |
title_full | Multidimensional analysis of Gammaherpesvirus RNA expression reveals unexpected heterogeneity of gene expression |
title_fullStr | Multidimensional analysis of Gammaherpesvirus RNA expression reveals unexpected heterogeneity of gene expression |
title_full_unstemmed | Multidimensional analysis of Gammaherpesvirus RNA expression reveals unexpected heterogeneity of gene expression |
title_short | Multidimensional analysis of Gammaherpesvirus RNA expression reveals unexpected heterogeneity of gene expression |
title_sort | multidimensional analysis of gammaherpesvirus rna expression reveals unexpected heterogeneity of gene expression |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6576797/ https://www.ncbi.nlm.nih.gov/pubmed/31166996 http://dx.doi.org/10.1371/journal.ppat.1007849 |
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