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.