Establishment of a High-Throughput Assay to Monitor Influenza A Virus RNA Transcription and Replication

PLoS One. 2015 Jul 21;10(7):e0133558. doi: 10.1371/journal.pone.0133558. eCollection 2015.

Abstract

Influenza A virus (IAV) poses significant threats to public health because of the recent emergence of highly pathogenic strains and wide-spread resistance to available anti-influenza drugs. Therefore, new antiviral targets and new drugs to fight influenza virus infections are needed. Although IAV RNA transcription/replication represents a promising target for antiviral drug development, no assay ideal for high-throughput screening (HTS) application is currently available to identify inhibitors targeting these processes. In this work, we developed a novel HTS assay to analyze the transcription and replication of IAV RNA using an A549 cell line stably expressing IAV RNA-dependent RNA polymerase (RdRp) complex, NP and a viral mini-genomic RNA. Both secreted Gaussia luciferase (Gluc) and blasticidin resistance gene (Bsd) were encoded in the viral minigenome and expressed under the control of IAV RdRp. Gluc serves as a reporter to monitor the activity of IAV RdRp, and Bsd is used to maintain the expression of all foreign genes. Biochemical studies and the statistical analysis presented herein demonstrate the high specificity, sensitivity and reproducibility of the assay. This work provides an ideal HTS assay for the identification of inhibitors targeting the function of IAV RdRp and a convenient reporting system for mechanism study of IAV RNA transcription / replication.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line, Tumor
  • Cell Survival
  • Drug Resistance, Viral / genetics
  • Genes, Reporter
  • Genome
  • Genome, Viral
  • HEK293 Cells
  • Humans
  • Influenza A virus / genetics*
  • Influenza A virus / physiology
  • Influenza, Human / virology
  • Luciferases / metabolism
  • Pyrrolidinones
  • RNA, Small Interfering / metabolism
  • RNA, Viral / genetics*
  • RNA-Dependent RNA Polymerase / genetics
  • Reproducibility of Results
  • Transcription, Genetic*
  • Virus Replication / drug effects

Substances

  • Pyrrolidinones
  • RNA, Small Interfering
  • RNA, Viral
  • blasticidin A
  • Luciferases
  • RNA-Dependent RNA Polymerase

Grants and funding

This work was supported by China 973 programs 2012CB911102 (SC), China Mega-Project for Infectious Disease 2013ZX10004601-002 (SC), The National S&T Major Special Project on Major New Drug Innovation 2012ZX09301-002-004 (SC), and the Xiehe Scholar (SC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.