TRIM25 Enhances the Antiviral Action of Zinc-Finger Antiviral Protein (ZAP)

Abstract

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.

TRIM25 Is Required for the Antiviral Activity of Zinc Finger Antiviral Protein

“…5). During the course of this work, Li et al reported that TRIM25-mediated ubiquitination was essential for ZAP (33). Our results reported here are consistent with theirs.…”

“…Very recently, Li et al reported that TRIM25 mediated ZAP ubiquitination and synergized with ZAP to inhibit Sindbis virus replication (33). In the present study, we confirmed their results that TRIM25 is required for the antiviral activity of ZAP.…”

“…1A). We failed to analyze the interactions between the endogenous TRIM25 and endogenous ZAP, although their interactions were shown by Li et al (33). Our failure could be accounted for by the fact that our antibodies against TRIM25 and ZAP were not good enough for immunoprecipitation assays.…”

“…Another possible explanation is that some unidentified lysine residues are the ubiquitination sites of ZAP that are important for its activity. Loss of ubiquitination of the mutant ZAP in the paper by Li et al could not exclude the possibility that ubiquitination of the mutant ZAP was below the detection limit (33). There are 13 lysine residues in NZAP, with 4 (K76, K89, K107, and K151) located in the putative RNA-binding cleft and 9 (K12, K119, K122, K131, K154, K197, K222, K226, and K248) outside the cleft (12).…”

“…TRIM25 ubiquitinates NZAP in vitro. In the paper by Li et al, seven lysine residues (K226R, K296R, K314R, K401R, K416R, K448R, and K629R) were identified to be ubiquitinated (33). Mutation of all of these residues abolished ZAP ubiquitination but did not affect its antiviral activity.…”