A novel method for screening viral interferon-resistance genes.
Many viruses have evolved mechanisms to antagonize the interferon (IFN) system, targeting all the major components involved in receptor binding and signaling. Although a number of these vital proteins are homologous to cellular proteins involved in IFN downregulation (e.g., viral IFN regulatory factors [vIRFs]), many share little resemblance to known proteins. To determine the IFN-blocking properties of these proteins, functional assays are required. Here, we present a new and rapid functional screening method, based on the 2fTGH cell line, which is able to determine viral gene products that inhibit the IFN-alpha/Jak-Stat signaling pathway. Expression cloning of viral IFN-blocking genes into 2fTGH and consequent selection with IFN-alpha and 6-thioguanine result in the outgrowth of cells that are no longer responsive to IFN-alpha. We also demonstrate that selection occurs if members of the Jak-Stat signaling pathway are lost. To show the utility of our system, we have used a known suppressor of IFN signaling, the human papillomavirus (HPV) E7 gene. Expression of E7 causes the loss of ability of 2fTGH cells to respond to IFN-alpha treatment because of a functional disruption of the signaling pathway. This approach offers a new strategy for identifying novel viral genes or new functions of already described viral genes that have a role in IFN-alpha signaling inhibition.
Centre for Immunology and Cancer Research, Princess Alexandra Hospital, University of Queensland, Brisbane, Australia.
Daniel T W
DT
Clarke
Aaron T
AT
Irving
Eleanore H
EH
Lambley
Elizabeth
E
Payne
Nigel A J
NA
McMillan
D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism; D004198:Disease Susceptibility; D005814:Genes, Viral / Q000235:genetics*; D005820:Genetic Testing / Q000379:methods*; D006801:Humans; D050832:Interferon-Stimulated Gene Factor 3; D050834:Interferon-Stimulated Gene Factor 3, gamma Subunit; D016898:Interferon-alpha / Q000276:immunology* / Q000378:metabolism; D027383:Papillomaviridae / Q000235:genetics / Q000276:immunology; D015398:Signal Transduction; D014157:Transcription Factors / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics; D014777:Virus Diseases / Q000276:immunology* / Q000821:virology*; D014780:Viruses / Q000235:genetics / Q000276:immunology
D004268:DNA-Binding Proteins; C494227:IRF9 protein, human; D050832:Interferon-Stimulated Gene Factor 3; D050834:Interferon-Stimulated Gene Factor 3, gamma Subunit; D016898:Interferon-alpha; D014157:Transcription Factors; D014764:Viral Proteins