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.

Structural Determinants of Virion Assembly and Release in the C Terminus of the Mason-Pfizer Monkey Virus Capsid Protein.

The transition from an immature to a fully infectious mature retrovirus particle is associated with molecular switches that trigger dramatic conformational changes in the structure of the Gag proteins. A dominant maturation switch that stabilizes the immature capsid (CA) lattice is located downstream of the CA protein in many retroviral Gags. The HIV-1 Gag protein contains a stretch of 5 amino acid residues termed the "clasp motif," important for the organization of the hexameric subunits that provide stability to the overall immature HIV-1 shell. Sequence alignment of the CA C-terminal domains (CTDs) of HIV-1 and Mason-Pfizer monkey virus (M-PMV) highlighted a spacer-like domain in M-PMV that may provide a comparable function. The importance of the sequences spanning the CA-nucleocapsid (NC) cleavage has been demonstrated by mutagenesis, but the specific requirements for the clasp motif in several steps of M-PMV particle assembly and maturation have not been determined in detail. In the present study, we report an examination of the role of the clasp motif in the M-PMV life cycle. We generated a series of M-PMV Gag mutants and assayed for assembly of the recombinant proteins in vitro and for the assembly, maturation, release, genomic RNA packaging, and infectivity of the mutant viruses in vivo. The mutants revealed major defects in virion assembly and release in HEK 293T and HeLa cells and even larger defects in infectivity. Our data identify the clasp motif as a fundamental contributor to CA-CTD interactions necessary for efficient retroviral infection. IMPORTANCE The C-terminal domain of the capsid protein of many retroviruses has been shown to be critical for virion assembly and maturation, but the functions of this region of M-PMV are uncertain. We show that a short "clasp" motif in the capsid domain of the M-PMV Gag protein plays a key role in M-PMV virion assembly, genome packaging, and infectivity.