Date Published: January 6, 2017
Publisher: Public Library of Science
Author(s): Melody M. H. Li, Zerlina Lau, Pamela Cheung, Eduardo G. Aguilar, William M. Schneider, Leonia Bozzacco, Henrik Molina, Eugen Buehler, Akinori Takaoka, Charles M. Rice, Dan P. Felsenfeld, Margaret R. MacDonald, Ana Fernandez-Sesma.
The host factor and interferon (IFN)-stimulated gene (ISG) product, zinc-finger antiviral protein (ZAP), inhibits a number of diverse viruses by usurping and intersecting with multiple cellular pathways. To elucidate its antiviral mechanism, we perform a loss-of-function genome-wide RNAi screen to identify cellular cofactors required for ZAP antiviral activity against the prototype alphavirus, Sindbis virus (SINV). In order to exclude off-target effects, we carry out stringent confirmatory assays to verify the top hits. Important ZAP-liaising partners identified include proteins involved in membrane ion permeability, type I IFN signaling, and post-translational protein modification. The factor contributing most to the antiviral function of ZAP is TRIM25, an E3 ubiquitin and ISG15 ligase. We demonstrate here that TRIM25 interacts with ZAP through the SPRY domain, and TRIM25 mutants lacking the RING or coiled coil domain fail to stimulate ZAP’s antiviral activity, suggesting that both TRIM25 ligase activity and its ability to form oligomers are critical for its cofactor function. TRIM25 increases the modification of both the short and long ZAP isoforms by K48- and K63-linked polyubiquitin, although ubiquitination of ZAP does not directly affect its antiviral activity. However, TRIM25 is critical for ZAP’s ability to inhibit translation of the incoming SINV genome. Taken together, these data uncover TRIM25 as a bona fide ZAP cofactor that leads to increased ZAP modification enhancing its translational inhibition activity.
The recent re-emergence and spread of viruses beyond their normal geographic distribution have affected countries worldwide. Understanding the biology of host factors with broad antiviral activity is crucial to vaccine and drug development efforts to counteract existing and emerging viral infections.
We reported in this study that ZAP requires multiple host factors for its maximal antiviral activity. We identified novel partners of ZAP that are normally important for a range of cellular processes, such as membrane ion permeability, innate immune signaling, and post-translational protein modification. Among the hits, JAK1 is a kinase important for signaling of the type I IFN receptor and can potentially act by augmenting the stimulatory effects of ZAP on the RIG-I pathway. On the other hand, KCNH5 is an outward rectifying potassium channel and it is not clear how it can stimulate ZAP’s function. It has been shown that reduction of the intracellular K+ concentration can activate the NLRP3 inflammasome, linking ion efflux to innate immunity . It is interesting to note that known interacting partners of ZAP, such as RIG-I or components of the exosome complex, were not hits in the screen, although that is likely largely dependent on the type of assay used and the basal expression levels of genes that are knocked down. Moreover, although MAP3K14 was previously found to be synergistic with ZAP in an ISG overexpression screen , it was not a hit in our confirmatory screen. It is likely that synergistic effects are less pronounced in our screen where endogenous levels of proteins are being interrogated and the effect from silencing of one gene might be compensated by a homologous gene or another ZAP partner.