Riplet Binds the Zinc Finger Antiviral Protein (ZAP) and Augments ZAP-Mediated Restriction of HIV-1

The zinc finger antiviral protein (ZAP) is an interferon-stimulated gene (ISG) with potent intrinsic antiviral activity. ZAP inhibits the replication of retroviruses, including murine leukemia virus (MLV) and HIV-1, as well as alphaviruses, filoviruses, and hepatitis B virus, and also the retrotransposition of LINE-1 and Alu retroelements. ZAP operates posttranscriptionally to reduce the levels of viral transcripts available for translation in the cytoplasm, although additional functions might be involved. Recent studies have shown that ZAP preferentially binds viral mRNAs containing clusters of CpG dinucleotides via its four CCCH-type zinc fingers. ZAP lacks enzymatic activity and utilizes other cellular proteins to suppress viral replication. Tripartite motif 25 (TRIM25) and the nuclease KHNYN have been identified as ZAP cofactors. In this study, we identify Riplet, a protein known to play a central role in the activation of the retinoic acid-inducible gene I (RIG-I), as a novel ZAP cofactor. Overexpression of Riplet acts to strongly augment ZAP's antiviral activity. Riplet is an E3 ubiquitin ligase containing three domains, an N-terminal RING finger domain, a central coiled-coil domain, and a C-terminal P/SPRY domain. We show that Riplet interacts with ZAP via its P/SPRY domain and that the ubiquitin ligase activity of Riplet is not required to stimulate ZAP-mediated virus inhibition. Moreover, we show that Riplet interacts with TRIM25, suggesting that both Riplet and TRIM25 may operate as a complex to augment ZAP activity. IMPORTANCE The ZAP is a potent restriction factor inhibiting replication of many RNA viruses by binding directly to viral RNAs and targeting them for degradation. We here identify RIPLET as a cofactor that stimulates ZAP activity. The finding connects ZAP to other innate immunity pathways and suggests oligomerization as a common theme in sensing pathogenic RNAs. The ZAP is a potent restriction factor inhibiting replication of many RNA viruses by binding directly to viral RNAs and targeting them for degradation. We here identify RIPLET as a cofactor that stimulates ZAP activity. The finding connects ZAP to other innate immunity pathways and suggests oligomerization as a common theme in sensing pathogenic RNAs.