Impact of N6-methyladenosine (m6A) machinery on HIV-1 replication in primary CD4+ T cells.

N6-methyladenosine (m6A) is the most prevalent internal modification of cellular and viral RNA and is critical to the regulation of its localization, stability, and translation. Previous studies on the role of m6A during HIV-1 replication have produced conflicting results. Since m6A function can vary dramatically by cell type and state, here we aimed to clarify the role of the m6A machinery during HIV-1 replication in primary CD4+ T cells. Using CRISPR-Cas9 we targeted 46 cellular genes implicated in m6A or 5-methylcytosine (m5C) regulation and measured subsequent HIV-1 replication in primary CD4+ T cells. Only knockout of the m6A writer complex auxiliary proteins VIRMA and WTAP, and the m6A reader YTHDF2 were validated as significantly decreasing HIV-1 replication. In contrast, knockout of METTL3 or METTL14, which form the catalytic core of the writer complex, resulted in only marginal changes in HIV-1 infection, despite significant decreases in total cellular m6A levels. Chemical inhibition of METTL3 led to a dose-dependent decrease in HIV-1 infection, coupled with an increase in protein levels of METTL3 and other writer complex members. Expression of writer proteins was also co-dependent, revealing complex regulatory feedback mechanisms. Overall, these results clarify the role of epitranscriptomic machinery during HIV-1 replication in primary CD4+ T cells and suggest regulation by auxiliary members of the m6A writer complex is more influential than the function of the catalytic core itself on HIV-1 infection in primary CD4+ T cells.