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

Tarun Mishra; Stacia Phillips; Yutao Zhao; Bethany Wilms; Chuan He; Li Wu

doi:10.1128/mbio.02214-24

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. 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. 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.

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

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