N6-methyladenosine modification of HIV-1 RNA suppresses type-I interferon induction in differentiated monocytic cells and primary macrophages

N 6 -methyladenosine (m 6 A) is a prevalent RNA modification that plays a key role in regulating eukaryotic cellular mRNA functions. RNA m 6 A modification is regulated by two groups of cellular proteins, writers and erasers that add or remove m 6 A, respectively. HIV-1 RNA contains m 6 A modifications that modulate viral infection and gene expression in CD4 + T cells. However, it remains unclear whether m 6 A modifications of HIV-1 RNA modulate innate immune responses in myeloid cells that are important for antiviral immunity. Here we show that m 6 A modification of HIV-1 RNA suppresses the expression of antiviral cytokine type-I interferon (IFN-I) in differentiated human monocytic cells and primary monocyte-derived macrophages. Transfection of differentiated monocytic U937 cells with HIV-1 RNA fragments containing a single m 6 A-modification significantly reduced IFN-I mRNA expression relative to their unmodified RNA counterparts. We generated HIV-1 with altered m 6 A levels of RNA by manipulating the expression of the m 6 A erasers (FTO and ALKBH5) or pharmacological inhibition of m 6 A addition in virus-producing cells, or by treating HIV-1 RNA with recombinant FTO in vitro . HIV-1 RNA transfection or viral infection of differentiated U937 cells and primary macrophages demonstrated that HIV-1 RNA with decreased m 6 A levels enhanced IFN-I expression, whereas HIV-1 RNA with increased m 6 A modifications had opposite effects. Our mechanistic studies indicated that m 6 A of HIV-1 RNA escaped retinoic acid-induced gene I (RIG-I)-mediated RNA sensing and activation of the transcription factors IRF3 and IRF7 that drive IFN-I gene expression. Together, these findings suggest that m 6 A modifications of HIV-1 RNA evade innate immune sensing in myeloid cells.