The Role of ZAP and TRIM25 RNA Binding in Restricting Viral Translation

Abstract

The innate immune response controls the acute phase of virus infections; critical to this response is the induction of type I interferon (IFN) and resultant IFN-stimulated genes to establish an antiviral environment. One such gene, zinc finger antiviral protein (ZAP), is a potent antiviral factor that inhibits replication of diverse RNA and DNA viruses by binding preferentially to CpG-rich viral RNA. ZAP restricts alphaviruses and the flavivirus Japanese encephalitis virus (JEV) by inhibiting translation of their positive-sense RNA genomes. While ZAP residues important for RNA binding and CpG specificity have been identified by recent structural studies, their role in viral translation inhibition has yet to be characterized. Additionally, the ubiquitin E3 ligase tripartite motif-containing protein 25 (TRIM25) has recently been uncovered as a critical co-factor for ZAP’s suppression of alphavirus translation. While TRIM25 RNA binding is required for efficient TRIM25 ligase activity, its importance in the context of ZAP translation inhibition remains unclear. Here, we characterized the effects of ZAP and TRIM25 RNA binding on translation inhibition in the context of the prototype alphavirus Sindbis virus (SINV) and JEV. To do so, we generated a series of ZAP and TRIM25 RNA binding mutants, characterized loss of their binding to SINV genomic RNA, and assessed their ability to interact with each other and to suppress SINV replication, SINV translation, and JEV translation. We found that mutations compromising general RNA binding of ZAP and TRIM25 impact their ability to restrict SINV replication, but mutations specifically targeting ZAP CpG-mediated RNA binding have a greater effect on SINV and JEV translation inhibition. Interestingly, ZAP-TRIM25 interaction is a critical determinant of JEV translation inhibition. Taken together, these findings illuminate the contribution of RNA binding and co-factor interaction to the synergistic inhibition of viral translation by ZAP and TRIM25.

Transcriptome Regulation by PARP13 in Basal and Antiviral States in Human Cells

“…To assess the degree to which PARP13 RNA binding contributes to its well-documented association with TRIM25 (Choudhury et al 2017; Zheng et al 2017; Li et al 2017; Yang et al 2022; Gonçalves-Carneiro et al 2021), we performed co-immunoprecipitation experiments. We transfected GFP-tagged PARP13.1, PARP13.2, and PARP13.2(Q) mutant, which is RNA-binding-deficient due to quintuple mutations (Todorova et al 2014), into WT cells and immunoprecipitated using GFP antibodies.…”

“…A limitation of applying spatial correlation methods to assess TRIM25 and PARP13 co-localization is that it is impossible to discern whether all PARP13 sites are bound simultaneously, only that they are regularly spaced relative to TRIM25 binding sites and each other. A recent study suggests that PARP13 RNA binding may even compete with its binding to TRIM25 (Yang et al 2022). Our data cannot inform us whether TRIM25 binds different target transcripts post-viral infection, so it is possible that TRIM25 binding in 3p-RNA-treated cells may have a different relationship with PARP13 binding than what we are observing.…”

“…Binding to common transcripts reinforces TRIM25:PARP13 interactions The ISG15 E3 ligase TRIM25 is also a primary co-factor of PARP13 for immune regulation. Both proteins require their RNA-binding domains to perform their roles in the innate antiviral response (Gao et al 2002;Guo et al 2004;Sanchez et al 2018;Yang et al 2022). To interrogate whether the requirement of these proteins for RNA binding is related to their interaction with one another, we used CLIP-seq data to identify their shared targets of TRIM25 and PARP13.…”

“…TRIM25 binding to PARP13 requires the SPRY domain and Nterminus, respectively (Zheng et al 2017;Li et al 2017;Choudhury et al 2017;Gonçalves-Carneiro et al 2021). Antiviral activity requires TRIM25 and PARP13 colocalization at membranes and the RNA-binding domains of both proteins (Kmiec et al 2021;Li et al 2017;Choudhury et al 2017;Sanchez et al 2018;Gao et al 2002;Guo et al 2004;Yang et al 2022).…”

“…TRIM25 binding to PARP13 requires the SPRY domain and N-terminus, respectively (Zheng et al 2017; Li et al 2017; Choudhury et al 2017; Gonçalves-Carneiro et al 2021). Antiviral activity requires TRIM25 and PARP13 colocalization at membranes and the RNA-binding domains of both proteins (Kmiec et al 2021; Li et al 2017; Choudhury et al 2017; Sanchez et al 2018; Gao et al 2002; Guo et al 2004; Yang et al 2022). Ablation of TRIM25’s putative RNA-binding domain in its SPRY domain decreases PARP13 interaction (Choudhury et al 2017), suggesting the interaction may be mediated via protein-RNA interaction and indicating the possibility of co-regulation of shared targets.…”