UCLA

Alphaviruses are arthropod-borne viruses that infect mammals and cause arthritogenic and/or encephalitic disease. The risk of an alphavirus outbreak increases as urbanization and global warming alter the habitats of alphaviruses’ vectors and host species. Zinc finger antiviral protein (ZAP) is an interferon-stimulated gene that is upregulated to combat alphavirus infections. While the functions of individual ZAP domains in viral infection are known, it is unclear if and how they—along with other cofactors—may work together to create a strong defense against alphaviruses. This dissertation aims to understand the anti-alphaviral mechanism of ZAP using evolutionary approaches and molecular biology.To determine the sites that have functional importance in ZAP throughout its evolutionary history with viruses, we harnessed the power of positive selection analysis. Using complementary computational approaches that model codon substitution rates, we identified the sites on ZAP that have been the targets of positive selection in the host-virus arms race. We found seven positively selected sites distributed across the ZAP gene. When one of the positively selected sites is mutated to an alanine, we observed that the mutant is almost 10-fold better at inhibiting alphaviruses. This improvement is not due to differences in ZAP’s interaction with viral RNA or its cofactor tripartite motif containing 25 (TRIM25), but is associated with a reduced ability to bind to poly(ADP-ribose). Next, to understand how TRIM25 cofactors contribute to ZAP-mediated antiviral activity, we followed up on poly(A) binding protein cytoplasmic 4 (PABPC4) and investigated its role in regulating viral RNA translation. We found that TRIM25 ubiquitination bolsters PABPC4’s enhancement of alphavirus inhibition. We also demonstrated that PABPC4 binds to alphaviral RNA and blocks its translation early on in infection. The work presented here shows that ZAP’s interactions with ancient viruses and host factors have shaped its antiviral activity, and implicates translational regulation and modification as key components of the host defense against alphaviruses.