Zyxin restricts viral fusion and entry across multiple virus families

The entry of enveloped viruses into host cells requires fusion of the viral envelope with a host cell membrane. The composition and dynamics of cellular membranes are impacted by the underlying cytoskeleton. Zyxin, a cytosolic protein that bridges the actin cytoskeleton with adhesion receptor proteins, was recently identified as an antiviral factor that antagonizes herpes simplex virus 1 (HSV-1) entry. To determine if zyxin specifically interferes with membrane fusion, we examined its impact in a quantitative cell-cell fusion assay. HSV-1 entry proteins exhibited enhanced fusion activity with retinal pigment epithelial (RPE) cells when the cells were knocked out (KO) for zyxin. Zyxin-KO cells also showed enhanced fusion activity with pseudorabies virus (PRV), paramyxovirus, and rhabdovirus fusion proteins. Additionally, the size of plaques formed following infection with members of each viral family was increased in the absence of zyxin. Bulk RNA sequencing of wild-type (WT) and zyxin-KO RPE cells identified 18 genes that enrich into several ontology groups of interest, including regulation of membrane potential, herpes simplex virus 1 infection (CCL2 and ZNF14), NABA core matrisome (CCL2 and ZNF14), extracellular matrix organization (CTSK, SERPINE1, and TGFB2), cell-cell adhesion (RAC2 and TGFB2), anchoring fibril formation, and regulation of the MAPK cascade (ACKR3 and TGFB2). These results highlight zyxin as a broadly active antiviral factor and suggest potential therapeutic implications for viral infections.IMPORTANCEEnveloped viruses enter host cells by fusing their envelope with a cellular membrane. This process is triggered when a viral glycoprotein(s) engages with a cellular membrane receptor. Less well understood are the cytoplasmic factors that indirectly govern viral fusion and entry. We report that zyxin, a cellular protein that bridges cell-adhesion receptors with the underlying actin cytoskeleton, antagonizes the fusion and entry of several enveloped viruses.