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. ImportanceMayaro virus (MAYV) is a neglected, mosquito-borne tropical virus that causes debilitating pathologies, such as chronic joint pain that can last from months to years. Currently, MAYV transmissions are endemic in sylvatic and peri-urban regions in Central and South America and the Caribbean. However, MAYV has been detected in urban-adapted mosquitos like Aedes aegypti (Ae. aegypti) and is a concern for potential global spread. Consequently, investigating the mechanisms of MAYV infection is critical to uncover opportunities for antiviral drug development. In this study, we report that MAYV infection requires host fatty acid synthase (FASN) derived palmitate for palmitoylation of the viral non-structural protein 1 (nsP1). In addition, we report that inhibiting FASN with the clinically advanced small molecule TVB-2640 significantly reduced MAYV infection and nsP1 palmitoylation. This study highlights FASN as an essential host factor for MAYV replication and establishes it as a promising therapeutic target for MAYV and related alphaviruses.