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

Abstract

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.

Post-Transcriptional Regulation of Viral RNA through Epitranscriptional Modification

“…Through preventing the addition of 2′-O-methyl marks to HIV-1 gRNA by siRNA mediated FTSJ3 knockdown, the authors show that incoming gRNA induces IFN-α and IFN-β expression. Since this work was published four further studies exploring innate immune sensing of viral RNA were published by Lu et al [ 37 ], Chen et al [ 35 ], Kim et al [ 34 ] and Lu et al [ 42 ] demonstrating, in a similar manner to Ringeard et al, that HMPV, HIV-1, HBV/HCV, VSV, MeV and SeV RNAs are also modified to avoid detection by the host cell. In each of these cases, the viral RNA of each virus is m 6 A modified and this is found to prevent recognition by RIG-I, in validation of the phenomenon described previously by Durbin et al These studies perform experiments to reduce the modification of virally encoded adenosines using methods such as mutation of the viral genome or treatment with 3-deazaadenosine (DAA), an inhibitor of S-Adenosylhomocysteine (SAH) hydrolase and find that this in turn increases the cellular type 1 interferon response to infection.…”

Dodging the Host Interferon-Stimulated Gene Mediated Innate Immunity by HIV-1: A Brief Update on Intrinsic Mechanisms and Counter-Mechanisms

“…2-O methylated HIV-1 RNA is not sensed effectively by the cytosolic RNA sensors Melanoma-Differentiation-Associated Gene 5 (MDA5) and RIG-I. In HIV-1-infected dendritic cells and macrophages, the adenosine methylation (N6-methyladenosine) of HIV-1 genomic RNA correlated with resistance against IFN-mediated immune responses ( 39 ). The cellular writers, such as methyltransferase-like METTL3 and METTL14, add a methyl group to adenosine and erasers, such as fat mass and obesity-associated protein (FTO) and α-ketoglutarate dependent dioxygenase AlkB homolog-5 (ALKBH5), remove the methyl groups from mRNA.…”

“…The cellular writers, such as methyltransferase-like METTL3 and METTL14, add a methyl group to adenosine and erasers, such as fat mass and obesity-associated protein (FTO) and α-ketoglutarate dependent dioxygenase AlkB homolog-5 (ALKBH5), remove the methyl groups from mRNA. The HIV-1 produced by HEK293T cells overexpressing FTO or ALKBH5 enhanced induction of IFNs in the target cells by decreasing adenosine methylation in its genome and thus preventing the activation of IRF3 and IRF7, suggesting the importance of host-directed HIV-1 RNA modification in the immune evasion strategy ( 39 ).…”

The Landscape of IFN/ISG Signaling in HIV-1-Infected Macrophages and Its Possible Role in the HIV-1 Latency

“…Additionally, m6A reader proteins (YTHDF1-3) can both positively and negatively affect different steps in the life cycle of the virus [ 5 , 164 , 165 , 166 ]. A recent study demonstrated that in myeloid cells (monocytes and macrophages) the m6A modification in HIV-1 RNA can suppress Type I IFN expression, and when the m6A modification is altered/defective, the affected RNA is sensed by RIG-1 [ 128 ]. However, to date, no studies have directly linked the IFN/ISG response and the m6A modification in macrophages that serve as replication-competent latent HIV-1 reservoirs.…”