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

Abstract

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.IMPORTANCERNA m6A modification is important for regulating gene expression and innate immune responses to HIV-1 infection. However, the functional significance of m6A 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, m6A-specific sequencing at single-base resolution, and functional assays. We demonstrate that HIV-1 latency reversal leads to increased levels of cellular m6A modification, correlates with cellular m6A levels, and is dependent on the catalytic activity of the m6A methyltransferase enzyme. We also identified cellular genes that are differentially m6A-modified during HIV-1 reactivation, as well as the sites of m6A within HIV-1 RNA. Our novel findings point toward a significant role for m6A modification in HIV-1 latency reversal.

Keywords

• RNA m6A modification
• HIV-1 latency reversal
• J-Lat cells
• primary CD4+T cells
• POLR3H
• TUG1