N6-methyladenosine (m6A) is a prevalent RNA modification that plays a key role in regulating eukaryotic cellular mRNA functions. RNA m6A modification is regulated by two groups of cellular proteins, writers and erasers that add or remove m6A, respectively. HIV-1 RNA contains m6A modifications that modulate viral infection and gene expression in CD4+ T cells. However, it remains unclear whether m6A modifications of HIV-1 RNA modulate innate immune responses in myeloid cells that are important for antiviral immunity. Here we show that m6A 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 m6A-modification significantly reduced IFN-I mRNA expression relative to their unmodified RNA counterparts. We generated HIV-1 with altered m6A levels of RNA by manipulating the expression of the m6A erasers (FTO and ALKBH5) or pharmacological inhibition of m6A 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 m6A levels enhanced IFN-I expression, whereas HIV-1 RNA with increased m6A modifications had opposite effects. Our mechanistic studies indicated that m6A 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 m6A modifications of HIV-1 RNA evade innate immune sensing in myeloid cells.

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 cells. However, it remains unclear whether m 6 A modifications of HIV-1 RNA modulate innate immune responses in cells or HIV-1-infected individuals. Here we show that m 6 A modification of HIV-1 RNA suppresses the expression of antiviral cytokine type-I interferon (IFN-I) in human monocytic cells. Transfection of differentiated monocytic 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 RNA m 6 A levels by manipulating the expression of the m 6 A erasers or pharmacological inhibition of m 6 A addition in virus-producing cells. RNA transfection and viral infection of differentiated monocytic cells 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 revealed that m 6 A of HIV-1 RNA escaped the RIG-I-mediated RNA sensing and activation of the transcription factors IRF3 and IRF7 that drive IFN-I gene expression. Moreover, RNA of peripheral blood mononuclear cells from HIV-1 viremic patients showed increased m 6 A levels that correlated with increased IFN-I mRNA expression compared to levels from HIV-1-suppressed patients on antiretroviral therapy. Together, our results suggest that RNA m 6 A modifications regulate viral replication and antiviral innate immune responses in HIV-1-infected individuals. Author Summary HIV-1 is known as a weak inducer of antiviral cytokines including IFN-I, but it is unclear how HIV-1 evades innate immunity. Different types of RNA modifications including m 6 A within the HIV-1 genome modulate viral replication; however, the role of m 6 A modifications of HIV-1 RNA in regulating innate immune responses remains elusive. In this study, we found that HIV-1 RNA modified with m 6 A suppresses the expression of IFN-I in differentiated monocytic cells by avoiding innate immune detection of viral RNA mediated by RIG-I, an RNA sensor in host cells. We also observed significantly increased RNA m 6 A modifications of peripheral blood mononuclear cells from HIV-1 viremic patients compared to virally suppressed patients on combined antiretroviral therapy, suggesting a functional link between m 6 A modifications and antiretroviral treatment. Investigating the functions of m 6 A modifications of HIV-1 RNA in regulating innate immune sensing and IFN-I induction in monocytic cells can help understand the mechanisms of HIV-1 persistence.