A novel method for screening viral interferon-resistance genes
Author(s)
Clarke, DTW
Irving, AT
Lambley, EH
Payne, E
McMillan, NAJ
Year published
2004
Metadata
Show full item recordAbstract
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-α/Jak-Stat signaling ...
View more >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-α/Jak-Stat signaling pathway. Expression cloning of viral IFN-blocking genes into 2fTGH and consequent selection with IFN-α and 6-thioguanine result in the outgrowth of cells that are no longer responsive to IFN-α. 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-α 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-α signaling inhibition.
View less >
View more >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-α/Jak-Stat signaling pathway. Expression cloning of viral IFN-blocking genes into 2fTGH and consequent selection with IFN-α and 6-thioguanine result in the outgrowth of cells that are no longer responsive to IFN-α. 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-α 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-α signaling inhibition.
View less >
Journal Title
Journal of Interferon & Cytokine Research
Volume
24
Issue
8
Subject
Virology
Biological Sciences
Agricultural and Veterinary Sciences
Medical and Health Sciences