YTHDC1 regulates distinct post-integration steps of HIV-1 replication and is important for viral infectivity

Abstract

Background 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. 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. Results We showed that the METTL3/14 m6A methyltransferase complex and the m6A YTHDF2 cytoplasmic writer down regulates the abundance of HIV-1 RNAs in infected cells. We also identified the m6A nuclear writer 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. Conclusions Our 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 Abstract

m6A reader proteins: the executive factors in modulating viral replication and host immune response

“…Thus, DF2 binding can exert diametrically opposite effects on RNA stability, depending on the RNA sequence context ( Tsai et al., 2021 ). The finding of the role of DC1 in promoting viral infectivity was supported by another study ( N'Da Konan et al., 2022 ). For reader DF3, it was reported that DF3 was incorporated into HIV particles in a nucleocapsid-dependent manner and reduced viral infectivity in the next cycle of infection.…”

Single-molecule epitranscriptomic analysis of full-length HIV-1 RNAs reveals functional roles of site-specific m6As

“…The timing of m 6 A deposition (occurring before or after splicing) is key to understanding the connection, but it appears to be complex 52 – 55 , 70 – 73 . Notably, gene-specific or virus-specific investigations have established clearer links between m 6 As and alternative splicing, involving m 6 A writers 74 – 77 , the nuclear reader YTHDC1 9 , 10 , 78 and erasers 7 , 79 , 80 , as well as interactions with splicing regulatory elements 16 – 19 , 69 , 81 . However, the impact of YTHDC1 knockdown on HIV-1 alternative splicing appeared controversial in two recent studies 9 , 10 .…”

“…Notably, gene-specific or virus-specific investigations have established clearer links between m 6 As and alternative splicing, involving m 6 A writers 74 – 77 , the nuclear reader YTHDC1 9 , 10 , 78 and erasers 7 , 79 , 80 , as well as interactions with splicing regulatory elements 16 – 19 , 69 , 81 . However, the impact of YTHDC1 knockdown on HIV-1 alternative splicing appeared controversial in two recent studies 9 , 10 . G-quadruplexes (G4s), co-localized with the three major m 6 A sites, might also influence alternative splicing 82 , 83 .…”

Functional Impacts of Epitranscriptomic m6A Modification on HIV-1 Infection

“…The nuclear m 6 A reader YTHDC1 is known to be involved in RNA stability and splicing and the nuclear export of m 6 A-modified viral and cellular RNA [ 3 , 64 , 65 ]. Two independent studies assessed HIV-1 RNA splicing and nuclear export under conditions of overexpression or depletion of YTHDC1 during single-round infections [ 26 , 66 ]. Both studies found that YTHDC1 is required for not only maintaining overall viral RNA levels but also for the appropriate selection of splice sites.…”

“…This suggests that the observed changes in HIV-1 RNA upon YTHDC1 silencing or overexpression are due to the presence of m 6 A ( Figure 2 ). Overexpressed and endogenous YTHDC1 bind to spliced and unspliced HIV-1 RNA in infected cells in a METTL3-dependent manner [ 66 ] ( Figure 2 ). YTHDC1-binding sites were identified adjacent to HIV-1 splice acceptor (SA) sites 3 and 7 [ 60 ].…”