Epitranscriptomic m(6)A modifications during reactivation of HIV-1 latency in CD4(+) T cells.

Despite effective antiretroviral therapy reducing HIV-1 viral loads to undetectable levels, the presence of latently infected CD4(+) T cells poses a major barrier to HIV-1 cure. N(6)-methyladenosine (m(6)A) modification of viral and cellular RNA has a functional role in regulating HIV-1 infection. m(6)A modification of HIV-1 RNA can affect its stability, translation, and splicing in cells and suppresses type-I interferon induction in macrophages. However, the function of m(6)A modification in regulating HIV-1 latency reactivation remains unknown. We used the Jurkat T cell line-derived HIV-1 latency model (J-Lat cells) to investigate changes in m(6)A levels of cellular RNA in response to latency reversal. We observed a significant increase in m(6)A levels of total cellular RNA upon reactivation of latent HIV-1 in J-Lat cells. This increase in m(6)A levels was transient and returned to steady-state levels despite continued high levels of viral gene expression in reactivated cells compared to control cells. Upregulation of m(6)A levels occurred without significant changes in the protein expression of m(6)A writers or erasers that add or remove m(6)A, respectively. Knockdown of m(6)A writers in J-Lat cells significantly reduced HIV-1 reactivation. Treatment with an m(6)A writer inhibitor reduced cellular RNA m(6)A levels, along with a reduction in HIV-1 reactivation. Furthermore, using m(6)A-specific sequencing, we identified cellular RNAs that are differentially m(6)A-modified during HIV-1 reactivation in J-Lat cells. Knockdown of identified m(6)A-modified RNA validates these results with an established primary CD4(+) T cell model of HIV-1 latency. These results show the importance of m(6)A RNA modification in HIV-1 latency reversal. IMPORTANCE: RNA m(6)A modification is important for regulating gene expression and innate immune responses to HIV-1 infection. However, the functional significance of m(6)A modification during HIV-1 latency reactivation is unknown. To address this important question, in this study, we used established cellular models of HIV-1 latency, m(6)A-specific sequencing at single-base resolution, and functional assays. We demonstrate that HIV-1 latency reversal leads to increased levels of cellular m(6)A modification, correlates with cellular m(6)A levels, and is dependent on the catalytic activity of the m(6)A methyltransferase enzyme. We also identified cellular genes that are differentially m(6)A-modified during HIV-1 reactivation, as well as the sites of m(6)A within HIV-1 RNA. Our novel findings point toward a significant role for m(6)A modification in HIV-1 latency reversal.