UCLA

Humanity is plagued by myriad viruses, most of which have no vaccines nor antiviral therapeutics. In order to address this pressing need and stave off future viral pandemics, basic science is required to illuminate both virulence determinants and host defenses. Alphaviruses are arthropod-borne, positive-sense RNA viruses that can cause febrile rashes, debilitating arthritis, and encephalomyelitis, which can prove fatal. The acute phase of their infection is controlled by the type I interferon (IFN) response, wherein host detection of invading pathogens induces expression of IFN and subsequent IFN-stimulated genes (ISGs) to establish an antiviral environment. In this dissertation, I present a study on the roles of ubiquitination and RNA binding in antiviral mechanisms utilized by two ISGs, the ubiquitin E3 ligase tripartite motif-containing protein 25 (TRIM25) and the RNA CpG sensor zinc finger antiviral protein (ZAP), to restrict alphavirus translation. While both TRIM25 and ZAP are potent antiviral factors individually, whether through direct or indirect means of viral inhibition, attention has turned in recent years to their cooperative inhibition of varied viruses. Previous work done by our lab suggests that TRIM25-mediated ubiquitination is essential for ZAP inhibition of alphavirus translation, but the exact substrates that mediate this antiviral activity are unknown. Moreover, not only do both TRIM25 and ZAP bind RNA, but also RNA binding is critical to their cellular function. For TRIM25, RNA binding stimulates its ligase activity; for ZAP, RNA binding is required for its detection and inhibition of viral RNA. However, the requirement for ZAP and TRIM25 RNA binding in their synergistic inhibition of viral translation remains unknown. Here, we utilized a “substrate trapping” approach to elucidate TRIM25 ubiquitination substrates involved in diverse and antiviral cellular processes. We generated a point mutation in the TRIM25 RING catalytic domain that abolishes TRIM25 ligase activity and traps substrates, enabling us to identify and characterize bona fide TRIM25 substrates involved in translation and nucleic acid metabolism. Moreover, our results suggest that several of these substrates contribute to TRIM25 antiviral activity. Together, these findings lay the groundwork for understanding how TRIM25-mediated ubiquitination of diverse substrates may modulate translation, nucleic acid metabolism, and antiviral activity. We also characterized the effects of ZAP and TRIM25 RNA binding on inhibiting viral translation. We demonstrate that mutations affecting ZAP RNA binding to CpG motifs significantly impact its ability to restrict viral replication and translation, and that the ability of ZAP to bind viral RNA is significantly negatively correlated with its ability to associate with TRIM25. These results suggest that ZAP RNA binding and interaction with TRIM25 may form two distinct determinants for ZAP antiviral mechanisms dependent on the viral context. Finally, I conclude with our collaborative work in characterizing the ability of novel antifusion peptides induce positive Gaussian curvature, thus remodeling membranes and restricting alphavirus replication. The work presented in this dissertation represents a multifaceted examination of the multiple determinants of TRIM25 and ZAP antiviral activity against alphaviruses, and includes a forward-looking development of antiviral therapeutics.