Epitranscriptomic Enhancement of Influenza A Virus Gene Expression and Replication

Abstract

Summary Many viral RNAs are modified by methylation of the N6 position of adenosine (m6A). m6A is thought to regulate RNA splicing, stability, translation and secondary structure. Influenza A virus (IAV) expresses m6A-modified RNAs but the effects of m6A on this segmented RNA virus remain unclear. We demonstrate that global inhibition of m6A addition inhibits IAV gene expression and replication. In contrast, overexpression of the cellular m6A “reader” protein YTHDF2 increases IAV gene expression and replication. To address whether m6A residues modulate IAV RNA function in cis, we mapped m6A residues on the IAV plus (mRNA) and minus (vRNA) strands and used synonymous mutations to ablate m6A on both strands of the hemagglutinin (HA) segment. These mutations inhibited HA mRNA and protein expression, while leaving other IAV mRNAs and proteins unaffected, and also resulted in reduced IAV pathogenicity in mice. Thus, m6A residues in IAV transcripts enhance viral gene expression.

Acetylation of cytidine residues boosts HIV-1 gene expression by increasing viral RNA stability

“…We have previously used photo-assisted (PA) crosslinking of modification-specific antibodies to 4-thiouridine (4SU)-labelled RNA, followed by RNase footprinting of antibody-bound RNA and deep sequencing of bound RNA fragments, to map both m 6 A (PA-m 6 A-seq) and m 5 C (PA-m 5 C-seq) residues on the gRNAs and mRNAs encoded by HIV-1 and other viruses 3,5,6,10 . As an ac4C-specific antibody recently became available, we asked if a similar approach (PA-ac4C-seq) might also allow us to map ac4C modification sites on HIV-1 transcripts 17 .…”

“…Previously, we and others have reported the detection and mapping of several epitranscriptomic modifications on HIV-1 transcripts 3,4,10,15 . These modifications include methylation of the N 6 position of adenosine (m 6 A), of the C 5 position of cytidine (m 5 C) and of the ribose moiety of all four ribonucleotides (2’O-methylation, collectively N m ). All three of these epitranscriptomic modifications have now been shown to boost HIV-1 replication in cis .…”

Extensive epitranscriptomic methylation of A and C residues on murine leukemia virus transcripts enhances viral gene expression

“…PA-m 6 A-seq and PA-m 5 C-seq were performed as previously described (8–10). Briefly, 3T3 cells were infected with MLV as described above.…”

“…After a further 24 h, total cellular RNA was extracted from the MLV-infected 3T3 cells using TRIzol, while MLV gRNA was extracted from virions that were collected by ultracentrifugation of the supernatant media through a 20% sucrose cushion. Total cellular poly(A)+ RNA was purified using oligo-dT magnetic beads (AM1922; Invitrogen) and 10 μg of poly(A)+ RNA or virion gRNA was then used following the previously reported PA-m 6 A-seq protocol (10, 24) using either an m 6 A-specific (202111; Synaptic Systems) or m 5 C-specific (C15200081; Diagenode) polyclonal antibody.…”

“…At least one characteristic T to C mutation, resulting from 4SU incorporation and crosslinking to the antobody used, were required in both mouse and viral aligned reads. All data was processed using in-house Perl scripts and Samtools (34), and visualized with IGV, as previously described (10). The raw sequencing data obtained by RNA-seq have been submitted to the NCBI expression omnibus and are available under GenBank accession number GE (in process)…”