Decreasing ZAP Poly(ADP-ribose) Binding Enhances Antiviral Activity Against Alphaviruses

Zinc finger antiviral protein (ZAP), also known as PARP13, is an antiviral factor effective against several virus families. ZAP contains three major protein domains: N-terminal zinc fingers, central WWE domains, and a C-terminal inactive ADP-ribosyltransferase domain, which is present only in the longer isoform ZAPL. While both the zinc finger and ADP-ribosyltransferase domains inhibit viral replication, the role of the WWE domains remains unclear. WWE domains bind poly(ADP-ribose) (PAR). In this study, we focus on ZAPS, the shorter isoform comprised of the minimal unit that confers antiviral activity and lacking the ADP-ribosyltransferase domain, to investigate how PAR-binding contributes to antiviral defense against alphaviruses. We identified the Y659 residue on the second WWE domain as essential for PAR-binding both in vitro and in cells. When infected with Chikungunya or Sindbis virus, cells with the Y659A mutant of ZAPS had reduced formation of replication complexes, decreased levels of viral genomic and subgenomic transcripts, and fewer infectious virions released compared to cells with the unmutated ZAPS, suggesting enhanced antiviral activity of the PAR-binding mutant. These findings suggest that inhibiting PAR-binding in ZAPS could potentiate host antiviral functions, offering a novel therapeutic strategy against alphavirus infections.