N6-Methyladenosine–binding proteins suppress HIV-1 infectivity and viral production

Wuxun Lu,Nagaraja Tirumuru,Corine St Gelais,Pratibha C Koneru,Chang Liu,Mamuka Kvaratskhelia,Chuan He,Li Wu

doi:10.1074/jbc.ra118.004215

Abstract

The internal N6-methyladenosine (m6A) modification of cellular mRNA regulates post-transcriptional gene expression. The YTH domain family proteins (YTHDF1-3 or Y1-3) bind to m6A-modified cellular mRNAs and modulate their metabolism and processing, thereby affecting cellular protein translation. We previously reported that HIV-1 RNA contains the m6A modification and that Y1-3 proteins inhibit HIV-1 infection by decreasing HIV-1 reverse transcription activity. Here, we investigated the mechanisms of Y1-3-mediated inhibition of HIV-1 infection in target cells and the effect of Y1-3 on viral production levels in virus-producing cells. We found that Y1-3 protein overexpression in HIV-1 target cells decreases viral genomic RNA (gRNA) levels and inhibits both early and late reverse transcription. Purified recombinant Y1-3 proteins preferentially bound to the m6A-modified 5' leader sequence of gRNA compared with its unmodified RNA counterpart, consistent with the strong binding of Y1-3 proteins to HIV-1 gRNA in infected cells. HIV-1 mutants with two altered m6A modification sites in the 5' leader sequence of gRNA exhibited significantly lower infectivity than WT, replication-competent HIV-1, confirming that these sites alter viral infection. HIV-1 produced from cells in which endogenous Y1, Y3, or Y1-3 proteins were knocked down singly or together had increased viral infectivity compared with HIV-1 produced in control cells. Interestingly, we found that Y1-3 proteins and HIV-1 Gag protein formed a complex with RNA in HIV-1-producing cells. Overall, these results indicate that Y1-3 proteins inhibit HIV-1 infection and provide new insights into the mechanisms by which the m6A modification of HIV-1 RNA affects viral replication.

Highlights

The internal N6-methyladenosine (m6A) modification of cellular mRNA regulates post-transcriptional gene expression
To delineate at which stage of HIV-1 replication, post-entry, the YTHDF proteins might be acting on inhibition, we examined whether Y1–3 proteins alter the levels of incoming HIV-1 genomic RNA (gRNA) at 1, 3, and 6 h post-infection
The 5Ј untranslated regions (UTRs) is critical for HIV-1 reverse transcription, genome packaging, and viral infectivity [23, 28], and we demonstrated that m6A modifications in this region were important for Y1–3 binding to HIV-1 RNA and that overexpression of Y1–3 proteins reduces HIV-1 infection by inhibition of reverse transcription [15]

Summary

Introduction

The internal N6-methyladenosine (m6A) modification of cellular mRNA regulates post-transcriptional gene expression. We found that Y1–3 protein overexpression in HIV-1 target cells decreases viral genomic RNA (gRNA) levels and inhibits both early and late reverse transcription. We found that Y1–3 proteins and HIV-1 Gag protein formed a complex with RNA in HIV-1–producing cells Overall, these results indicate that Y1–3 proteins inhibit HIV-1 infection and provide new insights into the mechanisms by which the m6A modification of HIV-1 RNA affects viral replication. In addition to YTH domain-containing proteins, the host proteins FMR1 and LRPPRC have been identified as m6A readers [6, 7] These reader proteins modulate m6A-modified mRNA stability and translation, thereby playing an important role in regulating post-transcriptional gene expression (8 –12). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. M6A-binding proteins suppress HIV-1 infectivity ever, the underlying mechanisms of Y1–3–mediated inhibition of HIV-1 infection remain unclear

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