Dynamics of the human and viral m6A RNA methylomes during HIV-1 infection of T cells

Abstract

N6-methyladenosine (m6A) is the most prevalent internal modification of eukaryotic mRNA. Very little is known of the function of m6A in the immune system or its role in host–pathogen interactions. Here we investigated the topology, dynamics, and bidirectional influences of the viral–host RNA methylomes during HIV-1 infection of human CD4 T cells. We show that viral infection triggers a massive increase in m6A in both host and viral mRNAs. In HIV-1 mRNA, we identified 14 methylation peaks in coding and noncoding regions, splicing junctions, and splicing regulatory sequences. We also identified a set of 56 human gene transcripts that were uniquely methylated in HIV-1-infected T cells and were enriched for functions in viral gene expression. The functional relevance of m6A for viral replication was demonstrated by silencing of the m6A writer or the eraser enzymes, which decreased or increased HIV-1 replication, respectively. Furthermore, methylation of two conserved adenosines in the stem loop II region of HIV-1 Rev Response Element (RRE) RNA enhanced binding of HIV-1 Rev protein to the RRE in vivo and influenced nuclear export of RNA. Our results identify a new mechanism for the control of HIV-1 replication and its interaction with the host immune system.

Viral Epitranscriptomics

“…2), one might anticipate that, if viral RNAs are indeed m 6 A modified, this would primarily or exclusively occur for RNAs generated by nuclear DNA or RNA viruses. In fact, analyses of three DNA viruses (adenovirus, herpes simplex virus type 1, and SV40), four retroviruses (the closely related avian sarcoma virus and Rous sarcoma virus as well as HIV-1 and feline leukemia virus), and the orthomyxovirus influenza A virus (IAV) have revealed m 6 A residues present at levels that are at least as high as the number of m 6 A residues detected on cellular mRNAs (4)(5)(6)(7)(8)(9)(10)(11)(43)(44)(45)(46). Moreover, in HIV-1, where m 6 A residues have been mapped at near single-nucleotide resolution, the consensus m 6 A addition sites that are utilized are highly conserved across HIV-1 isolates (10).…”

“…We note that m 6 A addition has been proposed to affect mRNA splicing (24)(25)(26), stability (27,47), and translation (47)(48)(49)(50), to modify RNA structure (51), and to inhibit the recognition of viral RNAs by Toll-like receptors and RIG-I (52, 53), and so m 6 A could positively regulate several aspects of the viral life cycle. Indeed, knockdown of the METTL3 and/or METTL14 m 6 A writers using RNA interference (RNAi) has been reported to inhibit HIV-1 replication up to 5-fold, while knockdown of the ALKBH5 m 6 A demethylase enhanced HIV-1 replication up to 8-fold (11,14). Similarly, in CD4-positive T cells, overexpression 6 A reader proteins YTHDF1, YTHDF2, and YTHDF3 (upper panels) and of the writer components METTL3, METTL14, and WTAP (lower panels), and were then subjected to immunofluorescence using an anti-FLAG antibody.…”

“…Of these, by far the most prevalent internal modified base found on mRNAs is m 6 A, and recent work has now begun to reveal how m 6 A affects mRNA function and how to precisely map the m 6 A residues present on mRNAs (1)(2)(3). m 6 A is also highly prevalent on a wide range of different viral RNA species (4)(5)(6)(7)(8)(9)(10)(11)(12)(13), and recently, the first reports demonstrating a significant phenotypic effect of these m 6 A modifications have been published (10)(11)(12)(13)(14). Therefore, we will focus this review entirely on m 6 A and how this particular modification might affect different aspects of the viral life cycle.…”

Publisher's Note

“…Reducing methylation at this site by knocking down METTL3/METTL14 or by mutating the site substantially inhibits viral RNA export into the cytoplasm, an essential step in the HIV-1 replication cycle. 50 Interestingly, Tirumuru et al report a binding site for the m 6 A-binding protein YTHDF1 in this region. The proteins YTHDF1, YTHDF2 and YTHDF3, all established m 6 A-binding proteins, appear to negatively regulate HIV-1 viral reverse transcription and mRNA transcription in this study.…”

“…[49][50][51] Though each study took different approaches and reaches different conclusions in some cases, they do converge in many respects. They find that HIV-1 RNA carries multiple m 6 A peaks that appear to be important in infection, and that the cellular host responds with infection-specific methylation of multiple transcripts potentially involved in the response to viral infection.…”