Productive Mayaro Virus Infection Requires Host Fatty Acid Synthase for nsP1 S-palmitoylation

To date, twenty-seven pathogenic human viruses require host-catalyzed de novo fatty acid biosynthesis for replication. This pathway is driven by fatty acid synthase (FASN), which produces palmitate. Palmitate is a precursor for various functions during viral infection, including lipid droplet formation for assembly, beta-oxidation for ATP generation, and post-translational modification of proteins. Whether Mayaro virus (MAYV), an emerging alphavirus that causes debilitating arthritogenic disease, required FASN for infection was unknown. Using genetic and pharmacological approaches in a human cell line and primary cell model, we found that MAYV requires FASN-dependent palmitate synthesis for virion production. To determine how palmitate contributes to infection, we pharmacologically inhibited pathways downstream of FASN and found that only 2-bromopalmitate (2-BP), a protein palmitoylation inhibitor, led to a 94% reduction in MAYV infection. S-palmitoylation is a post-translational modification in which palmitate is attached to sulfur atoms in cysteine residues. In chikungunya virus, a related alphavirus, FASN-dependent palmitoylation of nonstructural protein 1 (nsP1) is essential for membrane association and replication. Consequently, we hypothesized that MAYV nsP1 is palmitoylated in a FASN-dependent manner. Using an alkyne acetate analog, Alk-4, metabolized by FASN into alkyne palmitate, we observed specific labeling of wild-type nsP1 at conserved cysteine residues (C417-419), but not of a cysteine-to-alanine triple mutant. Treatment with TVB-2640 or 2-BP abrogated Alk-4 labeling of wild-type nsP1 during active infection, reinforcing that MAYV protein palmitoylation is a FASN-dependent process. Our findings reveal a conserved mechanism of FASN-dependent protein palmitoylation in alphaviruses and highlight FASN as a potential anti-viral target.