Abstract
BackgroundThe recent discovery of the role of m6A methylation in the regulation of HIV-1 replication unveiled a novel layer of regulation for HIV gene expression. This epitranscriptomic modification of HIV-1 RNAs is under the dynamic control of specific writers and erasers. In addition, cytoplasmic readers of the m6A mark are recruited to the modified viral RNAs and regulate HIV-1 replication. Yet, little is known about the effects of m6A writers and readers on the biogenesis of HIV-1 RNAs.ResultsWe showed that the METTL3/14 m6A methyltransferase complex and the m6A YTHDF2 cytoplasmic reader down regulates the abundance of HIV-1 RNAs in infected cells. We also identified the m6A nuclear reader YTHDC1 as a novel regulator of HIV-1 transcripts. In HIV-1 producer cells, we showed that knocking down YTHDC1 increases the levels of unspliced and incompletely spliced HIV-1 RNAs, while levels of multiply spliced transcripts remained unaffected. In addition, we observed that depletion of YTHDC1 has no effect on the nuclear cytoplasmic distribution of viral transcripts. YTHDC1 binds specifically to HIV-1 transcripts in a METTL3-dependent manner. Knocking down YTHDC1 reduces the expression of Env and Vpu viral proteins in producer cells and leads to the incorporation of unprocessed Env gp160 in virus particles, resulting in the decrease of their infectivity.ConclusionsOur findings indicate that, by controlling HIV-1 RNA biogenesis and protein expression, the m6A nuclear reader YTHDC1 is required for efficient production of infectious viral particles.Graphical
Highlights
The recent discovery of the role of m6A methylation in the regulation of Human Immunodeficiency Virus 1 (HIV-1) replication unveiled a novel layer of regulation for HIV gene expression
HeLa cells were infected with a VSVg-pseudotyped NL4-3 virus and newly synthesized HIV-1 RNAs were measured by RTqPCR using primers specific for US, incompletely spliced (IS) and multiply spliced (MS) HIV-1 transcripts (Additional file 1: Fig. S1)
We observed that US transcripts levels increased by 2- to fourfold upon depletion of Methyltransferase-like 3 (METTL3), WTAP, VIRMA and RBM15 while an increase of IS and MS mRNA levels was only seen upon depletion of some of the subunits (Fig. 1B)
Summary
The recent discovery of the role of m6A methylation in the regulation of HIV-1 replication unveiled a novel layer of regulation for HIV gene expression. This epitranscriptomic modification of HIV-1 RNAs is under the dynamic control of specific writers and erasers. The core of the methyltransferase complex responsible for the methylation of N6-adenosine is composed of the heterodimer Methyltransferase-like 3 (METTL3) and METTL14 Within this complex, METTL3 is the catalytically active subunit while METTL14 plays a structural role and is involved in RNA recognition [3,4,5]. m6A methylation regulates different steps of RNA biogenesis including stability, splicing, nuclear export and translation [13]. The nuclear reader YTHDC1, which has been shown to regulate splicing, alternative polyadenylation and nuclear export decreases the abundance of cellular transcripts [24,25,26]
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