Epitranscriptomic m6A modifications during reactivation of HIV-1 latency in CD4+ T cells

Abstract

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. N6-methyladenosine (m6A) modification of viral and cellular RNA has a functional role in regulating HIV-1 infection. m6A 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 m6A 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 m6A levels of cellular RNA in response to latency reversal. We observed a significant increase in m6A levels of total cellular RNA upon reactivation of latent HIV-1 in J-Lat cells. This increase in m6A 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 m6A levels occurred without significant changes in the protein expression of m6A writers or erasers that add or remove m6A, respectively. Knockdown of m6A writers in J-Lat cells significantly reduced HIV-1 reactivation. Treatment with an m6A writer inhibitor reduced cellular RNA m6A levels, along with a reduction in HIV-1 reactivation. Furthermore, using m6A-specific sequencing, we identified cellular RNAs that are differentially m6A-modified during HIV-1 reactivation in J-Lat cells. Knockdown of identified m6A-modified RNA validates these results with an established primary CD4+ T cell model of HIV-1 latency. These results show the importance of m6A RNA modification in HIV-1 latency reversal.