N1-Methyladenosine Regulates EIF5A mRNA Stability and Translation to Promote HIV-1 Infection

N1-methyladenosine (m1A) is a reversible RNA modification that participates in diverse physiological and pathological processes, including viral infection. Objective: To investigate the regulatory role of m1A in host dependency factors required for human immunodeficiency virus type 1 (HIV-1) infection. Methods: MeRIP-Seq, m1A RIP-qPCR, and SELECT were employed to identify and validate m1A modification sites on EIF5A mRNA. Site-directed mutagenesis, polysome profiling, and chemical inhibition were used to dissect the regulatory mechanism. Moreover, functional assays, including qRT-PCR and Western blot analysis, were conducted to evaluate the role of m1A-mediated regulation of EIF5A in viral infection. We found that m1A promoted HIV-1 infection by upregulating the expression of eukaryotic translation initiation factor 5A (EIF5A). Mechanistically, YTHDF1 bound to m1A-methylated EIF5A mRNA and positively regulated both its stability and translation. We identified A205 within the coding sequence of EIF5A as a key m1A site, its mutation reduced EIF5A expression, mRNA stability, translation, and YTHDF1 binding. Finally, inhibition of m1A methylation decreased EIF5A mRNA stability and translation, and suppressed HIV-1 infection. Collectively, these findings reveal that m1A-mediated regulation of EIF5A promotes HIV-1 infection and suggest that targeting m1A methylation could be a potential antiviral strategy.