N6-methyladenosine Binding Proteins Negatively Regulate HIV-1 Infectivity and Viral Production

The internal N6-methyladenosine (m6A) modification of cellular mRNA regulates post-transcriptional gene expression. The YTH domain family proteins (YTHDF1-3, or Y1-3 for short) bind to m6A-modified cellular mRNA and modulate its metabolism and processing, thereby affecting protein translation in cells. We previously reported that HIV-1 RNA contains m6A modification and that Y1-3 proteins inhibit HIV-1 infection by decreasing HIV-1 reverse transcription. Here we extended our studies to better understand the mechanisms of Y1-3-mediated inhibition of HIV-1 infection and viral production. Overexpression of Y1-3 proteins in target cells decreased HIV-1 genome RNA (gRNA) levels and inhibited early and late reverse transcription. Purified recombinant Y1-3 proteins preferentially bound to the m6A-modified 5' leader sequence of gRNA compared with its unmodified RNA counterpart, consistent with the strong binding of Y1-3 to HIV-1 gRNA in infected cells. HIV-1 mutants with two altered m6A modification sites in the 5' leader sequence of gRNA demonstrated significantly lower infectivity compared with wild-type HIV-1, suggesting that these sites are important for viral infection. Interestingly, HIV-1 produced from cells with knockdown of endogenous Y1 and Y3 proteins had increased viral infectivity, while HIV-1 produced from cells with overexpression of Y1-3 proteins showed reduced viral infectivity. We demonstrated that Y1-3 proteins and HIV-1 Gag formed a complex with RNA in cells. Furthermore, HIV-1 produced from cells treated with an inhibitor to block m6A modification showed a 3-fod decrease in m6A level of HIV-1 RNA, but a 3-fold increase in viral infectivity compared to HIV-1 produced from mock-treated cells. These results suggest the inhibitory effects of Y1-3 proteins on HIV-1 infection and provide new insight into the mechanisms of m6A modification of HIV-1 RNA in regulating viral replication, which clarify some discrepancies in the previously published studies in this area.