HIV-1 envelope proteins up-regulate N6-methyladenosine levels of cellular RNA independently of viral replication

Nagaraja Tirumuru,Li Wu

doi:10.1074/jbc.ra118.005608

Nagaraja Tirumuru, Li Wu

Open Access

https://doi.org/10.1074/jbc.ra118.005608

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Abstract

N6-methyladenosine (m6A) modification of HIV-1 RNA regulates viral replication and protein expression. The m6A modification is regulated by two groups of cellular proteins named writers and erasers that add or remove m6A, respectively. HIV-1 infection of CD4+ T-cells increases m6A levels of cellular mRNA, but the underlying mechanism is unknown. Here, we show that HIV-1 infection of CD4+ primary T-cells or Jurkat cells significantly increases m6A levels of cellular RNA independently of viral replication. Compared with HIV-1-infected CD4+ T-cells, similar m6A up-regulation was detected in total RNA from HIV-1-infected cells treated with a reverse-transcriptase inhibitor or with heat-inactivated HIV-1. Compared with mock controls, significantly increased m6A levels were detected in total RNA from Jurkat cells infected by single-cycle HIV-1 pseudotyped with an HIV-1 envelope (Env) glycoprotein, but not with vesicular stomatitis virus glycoprotein G (VSV-G). Overexpression of HIV-1 Env in HEK293T cells did not affect m6A levels of cellular RNA, suggesting that de novo synthesis of Env is not required for m6A up-regulation. Interestingly, treatment of Jurkat cells with recombinant gp120 of HIV-1 Env significantly increased m6A levels of cellular RNA, which was reduced by a gp120-neutralizing antibody. Preincubation of Jurkat cells with a CD4 receptor-neutralizing antibody blocked HIV-1-induced up-regulation of m6A levels in cellular RNA. Moreover, HIV-1 infection or gp120 treatment did not alter the protein expression of m6A writers and erasers in cells. Our findings suggest that HIV-1 gp120 binding to the CD4 receptor is required for m6A up-regulation in cells.

Highlights

N6-methyladenosine (m6A) modification of HIV-1 RNA regulates viral replication and protein expression
To systematically analyze the effects of HIV-1 infection on cellular RNA m6A levels, CD4ϩ Jurkat T-cells were infected with replication-competent HIV-1NL4–3 using a range (0.5, 1, and 5) of multiplicity of infection (MOI)
Total cellular RNA was stained with methylene blue (MB) as a loading control to normalize relative m6A levels

Summary

To whom correspondence should be addressed

N6-methyladenosine (m6A) modification of cellular mRNAs is the most abundant and important in controlling the cellular gene expression and physiological functions (4 –6). Methyltransferase-like 3, 14, and 16 (METTL3, 14, and 16) are m6A writers that catalyze the addition of methyl group at sixth position in adenosine of mRNA [8] Another group of proteins named m6A erasers include fat mass and obesity-associated protein (FTO) and AlkB family member 5 (AlkBH5) that remove m6A. These two groups of proteins (writers and erasers) orchestrate the mRNA fate and cells function through m6A modifications (4, 6 – 8). Exposure of soluble gp120 exerts significant immunological and neurotoxic effects on different target cells [20, 21] It is unknown whether HIV-1 Env binding to its receptors can alter m6A levels of cellular RNA. Our findings suggest that HIV-1 gp120 interaction with the CD4 receptor is critical for HIV-1–induced m6A up-regulation of cellular RNA in CD4ϩ T-cells

Results

Discussion

Experimental procedures