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

N⁶-methyladenosine (m⁶A) plays a critical role in regulating RNA Ostability, localization, and gene expression. m⁶A modification is also important for modulating the expression of viral and cellular genes during HIV-1 infection. 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 activated 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 differentially m⁶A-modified cellular mRNAs in HIV-1-infected primary CD4⁺ T cells, including perilipin 3 (PLIN3). We also identified 30 m⁶A sites in HIV-1 RNA from infected primary CD4⁺ T cells. HIV-1 infection increased PLIN3 mRNA level and nuclear accumulation but decreased PLIN3 protein expression in primary CD4⁺ T cells. Polysome profiling revealed that PLIN3 mRNA was less actively translated during HIV-1 infection of primary CD4⁺ T cells. Furthermore, PLIN3 knockdown in primary CD4⁺ T cells significantly reduced HIV-1 release but enhanced virion infectivity. Our results highlight the importance of m⁶A RNA modification during HIV-1 infection and suggest PLIN3 as a regulatory protein of HIV-1 replication in primary CD4⁺ T cells.IMPORTANCEm⁶A is a common chemical modification on mRNA that regulates RNA stability, localization, and gene expression. m⁶A modification of viral and cellular RNA is important for HIV-1 infection. We found that HIV-1 infection of primary CD4⁺ T cells promotes the interaction between the m⁶A writer complex subunits that add m⁶A modification. Using m⁶A-specific RNA sequencing, we identified several cellular mRNAs with altered m⁶A modifications during HIV-1 infection, including PLIN3. Interestingly, HIV-1 infection increased PLIN3 mRNA levels and nuclear localization but reduced PLIN3 protein expression in primary CD4⁺ T cells. When we knocked down PLIN3 in primary CD4⁺ T cells, it decreased HIV-1 release but made the HIV-1 more infectious. Our findings show the importance of m⁶A RNA modification in HIV-1 infection by regulating host genes like PLIN3 and suggest a unique regulatory mechanism in HIV-1-infected primary CD4⁺ T cells.