Epitranscriptomic mA modifications during reactivation of HIV-1 latency in CD4 T cells.

UNLABELLED

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. -methyladenosine (mA) modification of viral and cellular RNA has a functional role in regulating HIV-1 infection. mA 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 mA 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 mA levels of cellular RNA in response to latency reversal. We observed a significant increase in mA levels of total cellular RNA upon reactivation of latent HIV-1 in J-Lat cells. This increase in mA 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 mA levels occurred without significant changes in the protein expression of mA writers or erasers that add or remove mA, respectively. Knockdown of mA writers in J-Lat cells significantly reduced HIV-1 reactivation. Treatment with an mA writer inhibitor reduced cellular RNA mA levels, along with a reduction in HIV-1 reactivation. Furthermore, using mA-specific sequencing, we identified cellular RNAs that are differentially mA-modified during HIV-1 reactivation in J-Lat cells. Knockdown of identified mA-modified RNA validates these results with an established primary CD4 T cell model of HIV-1 latency. These results show the importance of mA RNA modification in HIV-1 latency reversal.

IMPORTANCE

RNA mA modification is important for regulating gene expression and innate immune responses to HIV-1 infection. However, the functional significance of mA 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, mA-specific sequencing at single-base resolution, and functional assays. We demonstrate that HIV-1 latency reversal leads to increased levels of cellular mA modification, correlates with cellular mA levels, and is dependent on the catalytic activity of the mA methyltransferase enzyme. We also identified cellular genes that are differentially mA-modified during HIV-1 reactivation, as well as the sites of mA within HIV-1 RNA. Our novel findings point toward a significant role for mA modification in HIV-1 latency reversal.