Single-base m⁶A epitranscriptomics reveals novel HIV-1 host interaction targets in primary CD4⁺ T cells

N ⁶-methyladenosine (m⁶A) is the most prevalent cellular mRNA modification and plays a critical role in regulating RNA stability, localization, and gene expression. m⁶A modification plays a vital role in modulating the expression of viral and cellular genes during HIV-1 infection. HIV-1 infection increases cellular RNA m⁶A levels in many cell types, which facilitates HIV-1 replication and infectivity in target cells. However, the function of m⁶A modification in regulating HIV-1 infection of primary CD4⁺ T cells remains unclear. Here, we demonstrate that HIV-1 infection of Jurkat CD4⁺ T cells and primary CD4⁺ T cells promotes the interaction between the m⁶A writer complex subunits methyltransferase-like 3 and 14 (METTL3/METTL14). Using single-base m⁶A-specific RNA sequencing, we identified several differentially m⁶A-modified cellular mRNAs, including perilipin 3 (PLIN3), during HIV-1 infection in primary CD4⁺ T cells. Interestingly, HIV-1 infection increased PLIN3 mRNA level by enhancing its stability, but PLIN3 protein level was decreased. Knocking down PLIN3 in primary CD4⁺ T cells reduced HIV-1 production but enhanced virion infectivity. In contrast, in Jurkat cells, PLIN3 mRNA and protein expression levels were unaffected by HIV-1 infection, and knocking out PLIN3 did not impact HIV-1 production or infectivity. These results indicate that the interplay between HIV-1 and PLIN3 is cell-type specific and only observed in primary CD4⁺ T cells. Overall, our results highlight the importance of m⁶A RNA modification in HIV-1-infected primary CD4⁺ T cells and suggest its significance as a regulatory mechanism in HIV-1 infection.