UNLABELLED: Viruses have evolved mechanisms to inhibit the innate immune response to infection. The aim of this study was to develop an efficient screening method to identify viral proteins and their ability to block Jak-Stat signaling using hepatitis C virus (HCV) as an example. The 2FTGH cell assay system was used in combination with transient transfection of HCV proteins in this study. Using 1000U/ml IFN and 30mM 6-TG to treat 2FTGH cells, it was established that transient protein expression in this cell system yielded 39% and 0% cell survival for the positive (HPV E7) and negative controls (GFP expression) respectively. Transient expression of HCV Core-p7 resulted in 22% cell survival, consistent with previous reports, while expression of the HCV serine protease NS3/4a resulted in 54% cell survival. NS3/4a was subsequently shown to inhibit phosphorylation of Stat-1 at the serine residue 727. CONCLUSION: the 2FTGH cell assay system can be adapted for transient screening to examine the ability of viral proteins or other potential inhibitors to block the Jak-Stat signaling pathway. We show that HCV NS3/4a is able to block this pathway at the stage of Stat-1 serine 727 phosphorylation.
UNLABELLED: Viruses have evolved mechanisms to inhibit the innate immune response to infection. The aim of this study was to develop an efficient screening method to identify viral proteins and their ability to block Jak-Stat signaling using hepatitis C virus (HCV) as an example. The 2FTGH cell assay system was used in combination with transient transfection of HCV proteins in this study. Using 1000U/ml IFN and 30mM 6-TG to treat 2FTGH cells, it was established that transient protein expression in this cell system yielded 39% and 0% cell survival for the positive (HPV E7) and negative controls (GFP expression) respectively. Transient expression of HCV Core-p7 resulted in 22% cell survival, consistent with previous reports, while expression of the HCVserine protease NS3/4a resulted in 54% cell survival. NS3/4a was subsequently shown to inhibit phosphorylation of Stat-1 at the serine residue 727. CONCLUSION: the 2FTGH cell assay system can be adapted for transient screening to examine the ability of viral proteins or other potential inhibitors to block the Jak-Stat signaling pathway. We show that HCVNS3/4a is able to block this pathway at the stage of Stat-1serine 727 phosphorylation.
Authors: Ashley D Frazier; Chun L Zhang; Lei Ni; Cheng J Ma; Ying Zhang; Xiao Y Wu; Antwan N Atia; Zhi Q Yao; Jonathan P Moorman Journal: Viral Immunol Date: 2010-10 Impact factor: 2.257