Identify and Characterize Novel Interferon-Stimulated Genes during West Nile Virus infection

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

ABSTRACT OF DISSERTATION Identify and Characterize Novel Interferon-Stimulated Gene During West Nile Virus Infection by Jian Qing Li Doctor of Philosophy in Biology and Biomedical Sciences, Molecular Microbiology and Microbial Pathogenesis Washington University in Saint Louis, 2013 Dr. Michael S. Diamond, Chairperson The innate immune system is responsible for transiently inhibiting infection and priming adaptive response. Although many immune effectors and signaling molecules have been described, there are few systematic approaches for identifying these functional molecules, especially under biologically relevant condition. These studies feature the development of an shRNA-based high throughput screen platform with an intact interferon (IFN) response to identify novel antiviral factors and negative regulators, and characterize their mechanism of action. To identify novel antiviral ISGs restricting WNV infection, these studies have applied a lentviral-based screen platform using high throughput flow cytometry analysis. We screened 813 shRNA constructs against 245 different human ISG for effects on West Nile Virus (WNV) infection after IFN pretreatment. The primary screen yielded 62 `hits' including proteins involved in pattern recognition (TLR3 and MAVS), IFN signaling (STAT2, JAK2 and IRF9), and known IFN effector functions against RNA viruses (PKR, OAS1, and IFIT2). Validation studies confirmed that silencing of 30 different ISGs resulted in increased WNV infection relative to control shRNA (3 to 114-fold, P < 0.05). The list of functional antiviral ISGs includes both established viral restricting factors and genes not previously implicated in cell-intrinsic control of WNV infectivity, such as DDX24, IFI44L, IFI6, IFRD1, IL13RA1, MAFK, PAK3, SAMD9L, and SC4MOL. Among those genes, IFI6 and SC4MOL also significantly inhibited WNV infection within an ectopically expression assay, suggesting they are both necessary and sufficient to confer antiviral effects. In the late phases of an immune response, after viral infection is cleared, counter-regulatory mechanisms must be activated to prevent excessive tissue damage. During the course of our screen, we also established that ASCC3 is required to taper the expression of multiple ISG induced by IFNb;. Silencing of ASCC3 resulted in elevated antiviral responses against several unrelated viruses whereas ectopic expression of ASCC3 facilitated the growth of WNV. We verified that the integrity of the NF-kB activation pathway was not essential for ASCC3-dependent effects on infection. Rather, we established its functional dependency of ASCC3 on the transcription factors IRF-3 and IRF-7. Collectively, our results suggest a model in which IFNb induces expression of antiviral, pro-inflammatory, and counter-regulatory ISGs, the latter of which include proteins such as ASCC3. Expression of ASCC3 dampens the type I IFN-dependent signals likely by modulating activity of IRF-3 and IRF-7 pathways, and prevents extensive inflammation and injury to cells.