Functional analysis of host and viral determinants for ZAP inhibition

PROJECT SUMMARY/ABSTRACT Emerging viruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have devastated the world. We are desperately in need of broadly acting antivirals that can curb the spread of the next emerging virus. However, a major knowledge gap exists due to our incomplete understanding of the cellular pathways that control or facilitate viral replication. Type I interferon (IFN) is the principal host innate immune response to viral infection and regulates the expression of an array of IFN-stimulated genes (ISGs). Zinc finger antiviral protein (ZAP) is one of the most potent ISGs that blocks the replication of diverse RNA and DNA viruses. ZAP attenuates alphavirus production by up to 8 logs and is indispensable for IFN efficacy against alphaviruses. Moreover, increased virulence of human immunodeficiency virus 1 (HIV-1) and SARS-CoV-2 associates with viral evasion from ZAP recognition. Importantly, highly pathogenic alphaviruses such as chikungunya virus (CHIKV) have developed resistance to ZAP, highlighting ZAP as a critical driver of viral pathogenesis. ZAP is proposed to act through viral translational inhibition (alphavirus and flavivirus) and viral RNA degradation (other viruses), however, how ZAP blocks viral replication and how viruses evade from ZAP recognition are still poorly understood. We recently made two important advances towards this goal: First, we showed that ZAP viral translational inhibition requires the host factor TRIM25, an E3 ubiquitin ligase. TRIM25-mediated ZAP antiviral mechanism is innovative, and independent of RIG-I, IRF3, and IFN. Using a novel "substrate trapping" approach we have now identified numerous cellular proteins with exciting roles in translation and RNA processes to be TRIM25 interactors and potential substrates. These preliminary findings connect the ubiquitination process to viral translational suppression by ZAP for the first time and provide cellular targets for antiviral therapy. Second, we identified differences in alphavirus sensitivity to ZAP. Highly virulent alphaviruses such as CHIKV that have infected millions of people in recent epidemics can evade from or counteract ZAP recognition through their viral non-structural gene region. Discovery of viral strategies for evasion and antagonism will identify druggable viral targets and inform development of vaccine strains with weakened ability to counteract ZAP. Taken together, our results have led us to hypothesize that ZAP recruits TRIM25 to ubiquitinate and modulate cellular factors leading to viral translational inhibition, and highly pathogenic alphaviruses have evolved strategies to escape and/or antagonize ZAP antiviral activity. We propose to elucidate the mechanism of TRIM25-mediated ZAP translational inhibition (Aim 1) and determine the mechanisms of viral resistance to ZAP (Aim 2). Success of these Aims will advance our understanding of how the IFN pathway co-opts cellular processes to block viral replication and drive viral pathogenesis, and provide promising host and viral targets for therapeutic intervention of alphavirus and other virus infections.