Simultaneous singlecell in situ analysis of human adenovirus type 5 DNA and mRNA expression patterns in lytic and persistent infection

Tomasz Krzywkowski, Sibel Ciftci, Farzaneh Assadian, Mats Nilsson, Tanel Punga

Research output: Contribution to journalArticlepeer-review

10 Scopus citations

Abstract

An efficient adenovirus infection results in high-level accumulation of viral DNA and mRNAs in the infected cell population. However, the average viral DNA and mRNA content in a heterogeneous cell population does not necessarily reflect the same abundance in individual cells. Here, we describe a novel padlock probe-based rolling-circle amplification technique that enables simultaneous detection and analysis of human adenovirus type 5 (HAdV-5) genomic DNA and virusencoded mRNAs in individual infected cells. We demonstrate that the method is applicable for detection and quantification of HAdV-5 DNA and mRNAs in short-term infections in human epithelial cells and in long-term infections in human B lymphocytes. Single-cell evaluation of these infections revealed high heterogeneity and unique cell subpopulations defined by differential viral DNA content and mRNA expression. Further, our single-cell analysis shows that the specific expression pattern of viral E1A 13S and 12S mRNA splice variants is linked to HAdV-5 DNA content in the individual cells. Furthermore, we show that expression of a mature form of the HAdV-5 histone-like protein VII affects virus genome detection in HAdV-5-infected cells. Collectively, padlock probes combined with rolling-circle amplification should be a welcome addition to the method repertoire for the characterization of the molecular details of the HAdV life cycle in individual infected cells.

Original languageEnglish (US)
Article numbere00166-17
JournalJournal of virology
Volume91
Issue number11
DOIs
StatePublished - Jun 1 2017

Keywords

  • Adenovirus
  • Lytic infection
  • Persistent infection
  • Rolling-circle amplification
  • Single-cell analysis

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