| Popis: |
Many viruses have evolved mechanisms to antagonize the interferon (IFN) system, targeting all the major components involved in receptor binding and signalling. Although a number of these viral 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 signalling 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 signalling pathway are lost. To show the utility of our system, we have used a known suppressor of IFN signalling, 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 signalling 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 signalling inhibition. |
