HLA-I EXPRESSION AND IFN-GAMMA SIGNALING IN IFN-? RESISTANT HCV REPLICON CELLS

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 我们已经开发了多种稳定的细胞系，含有亚基因组HCV RNA，对干扰素α（IFN-γ）治疗有抗性。这些IFN-γ抗性复制子细胞的表征显示，由于缺陷的Jak-STAT途径，STAT 1和STAT 2蛋白的磷酸化和核转位存在缺陷。在这项研究中，我们开发了一种替代策略，通过改善细胞内STAT 1信号转导来克服细胞培养模型中的干扰素耐药性。在STAT 1的Src同源2（SH 2）结构域（Ala-656和Asn-658处）中具有双半胱氨酸取代的工程化STAT 1-CC分子以IFN-γ依赖性方式有效磷酸化并易位到IFN抗性细胞的细胞核。与野生型STAT 1和STAT 3相比，转染含有STAT 1-CC的质粒克隆显著激活GAS启动子。工程化的STAT 1-CC的活性依赖于酪氨酸残基701的磷酸化，因为在位置701处具有取代的苯丙氨酸残基的构建体（STAT 1-CC-Y 701 F）不能激活复制子细胞中的GAS启动子。在IFN-γ处理后的耐药细胞系中，细胞内STAT 1-CC蛋白的表达表现为磷酸化和核转位。将STAT 1-CC质粒克隆瞬时转染到干扰素抗性细胞系中导致以IFN-γ依赖性方式抑制病毒复制和清除病毒。此外，与野生型和Y至F突变体对照相比，用STAT 1-CC构建体转染的抗性复制子细胞显著上调表面HLA-1表达。这些结果表明，STAT 1分子的SH 2结构域的修饰允许通过增加STAT 1磷酸化、核转位、HLA-1表面表达和延长干扰素抗病毒基因活化来改善IFN-γ信号传导。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. We have developed multiple stable cell lines containing subgenomic HCV RNA that are resistant to treatment with interferon alpha (IFN-¿). Characterization of these IFN-¿ resistant replicon cells showed defects in the phosphorylation and nuclear translocation of STAT1 and STAT2 proteins due to a defective Jak-STAT pathway. In this study, we have developed an alternative strategy to overcome interferon resistance in a cell culture model by improving intracellular STAT1 signaling. An engineered STAT1-CC molecule with double cysteine substitutions in the Src-homology 2 (SH2) domains of STAT1 (at Ala-656 and Asn-658) efficiently phosphorylates and translocates to the nucleus of IFN-resistant cells in an IFN-¿ dependent manner. Transfection of a plasmid clone containing STAT1-CC significantly activated the GAS promoter compared to wild type STAT1 and STAT3. The activity of the engineered STAT1-CC is dependent upon the phosphorylation of tyrosine residue 701, since the construct with a substituted phenylalanine residue at position 701 (STAT1-CC-Y701F) failed to activate GAS promoter in the replicon cells. Intracellular expression of STAT1-CC protein showed phosphorylation and nuclear translocation in the resistant cell line after IFN-¿ treatment. Transient transfection of STAT1-CC plasmid clone into an interferon resistant cell line resulted in inhibition of viral replication and viral clearance in an IFN-¿ dependent manner. Furthermore, the resistant replicon cells transfected with STAT1-CC constructs significantly up regulated surface HLA-1 expression when compared to the wild type and Y to F mutant controls. These results suggest that modification of the SH2 domain of the STAT1 molecule allows for improved IFN-¿ signaling through increased STAT1 phosphorylation, nuclear translocation, HLA-1 surface expression, and prolonged interferon antiviral gene activation.