UCLA

As the potential for viral epidemics grows, so does the need to understand host antiviral mechanisms and the ways in which viruses evade these defenses. Alphaviruses are positive-sense single-stranded RNA viruses that cause serious disease in humans and whose geographic distributions continue to expand. The type I interferon (IFN) response controls the acute phase of alphavirus infection by stimulating expression of antiviral effectors including zinc finger antiviral protein (ZAP). ZAP restricts certain alphaviruses by translational inhibition. While the ability of ZAP to bind RNA is critical for its antiviral activity against a broad spectrum of viruses, it is unclear how ZAP RNA binding is linked to its ability to inhibit viral RNA translation. The precise determinants of ZAP recognition of viral and host RNA also remain uncertain. In this dissertation, we aimed to elucidate the principles and functional consequences of ZAP RNA recognition during alphavirus infection. First, we characterized how RNA binding by ZAP and its co-factor TRIM25 affects their abilities to inhibit viral RNA translation. We demonstrated that mutations targeting the ability of ZAP to bind CpG dinucleotides also compromises its ability to restrict viral replication and translation. Additionally, we found a negative correlation between the ability of ZAP to bind RNA and its ability to interact with TRIM25, suggesting that these interactions may form distinct determinants for ZAP antiviral activity against different viruses. Next, we leveraged a panel of related alphaviruses with different sensitivities to ZAP to investigate the molecular determinants of how some alphaviruses can evade ZAP restriction. Previous work from our lab found that Ross River virus (RRV) and Sindbis virus (SINV) are sensitive to ZAP inhibition, while o’nyong’nyong virus (ONNV) and the particularly pathogenic chikungunya virus (CHIKV) are resistant to ZAP. We identified a 500-bp window within the genomes of these viruses where in vitro ZAP CpG-specific binding correlates with ZAP sensitivity. However, upon testing ZAP binding to other windows with CpG contents correlating to ZAP sensitivity, we found that ZAP preferentially binds CpG dinucleotides only in certain alphavirus RNA contexts. Our results suggest a potential strategy of immune evasion by ZAP-resistant alphaviruses by suppression or masking of CpG dinucleotides in regions important for ZAP recognition and/or inhibition. Finally, we investigated ZAP binding to virus and host RNA in a cellular context during infection with a ZAP-sensitive alphavirus virus, SINV, and a ZAP-resistant alphavirus, CHIKV. We found that ZAP binds distinct regions of the SINV and CHIKV genomes and that ZAP binding sites on host mRNAs also shift during infection with these two viruses. These findings provide a basis for evaluating strategies by which a highly pathogenic virus like CHIKV may evade recognition and remodel the host cellular environment during infection. The work presented in this dissertation broadens our understanding of the molecular mechanisms underlying immune recognition of alphaviruses, as well as how some alphaviruses have developed strategies to subvert this recognition. Our results will provide targets for the development of antivirals to reinforce weak points in the immune response.