Characterizing the Functional Consequences of Zinc Finger Antiviral Protein (ZAP) Binding to RNA.

PROJECT SUMMARY Alphaviruses are mosquito-borne viruses that cause chronic arthritis and fatal encephalitis in humans. Upon detection of the virus by the innate immune system, host cells produce interferons (IFN) that stimulate the expression of IFN-stimulated genes (ISGs) with antiviral activities. Zinc finger antiviral protein (ZAP) is an ISG and RNA-binding protein that binds viral RNA predominantly at CG-rich sequences and blocks viral replication through viral RNA degradation or translation suppression. ZAP also binds host mRNAs and regulates various host transcripts of functionally distinct cellular pathways, although the connection between ZAP's antiviral activity and its host regulatory function is poorly understood. Interestingly, alphaviruses have similar genomic CG levels yet display a range of sensitivities to the antiviral activity of ZAP, where Sindbis virus (SINV) is strongly inhibited by ZAP, while the highly pathogenic chikungunya virus (CHIKV) is poorly inhibited. Our lab has used RNA binding assays and bioinformatic approaches to demonstrate CG content is not sufficient to explain differential ZAP binding to alphaviral RNA, and that there are differences in ZAP-mediated host transcript binding during SINV and CHIKV infection. Therefore, we hypothesize that direct inhibition of viral RNA and the indirect regulation of host mRNA transcripts by ZAP both contribute to the overall antiviral activity of ZAP. We will first functionally characterize the sequence and structural determinants within ZAP RNA binding sites we identified in the SINV genome (Aim 1). We will then interrogate the role of differentially expressed genes with transcripts bound by ZAP during SINV and CHIKV infection (Aim 2). This work will elucidate ZAP-RNA interactions that explain innate immune evasion by ZAP-resistant viruses such as CHIKV, providing a foundation for therapeutic research. Under the fellowship training plan, I will advance my abilities in experimental design and science communication by executing, presenting, and publishing the results of this project, as well as through coursework and teaching as required by my doctoral program. My research training will take place in a rigorous and multidisciplinary scientific environment at the University of California, Los Angeles, where I have access to all the materials and expertise necessary for the completion of this project.