N(6)-methyladenosine of HIV-1 RNA regulates viral infection and HIV-1 Gag protein expression

The internal N6-methyladenosine (m6A) methylation of eukaryotic nuclear RNA controls post-transcriptional gene expression, which is regulated by methyltransferases (writers), demethylases (erasers), and m6A-binding proteins (readers) in cells. The YTH domain family proteins (YTHDF1–3) bind to m6A-modified cellular RNAs and affect RNA metabolism and processing. Here, we show that YTHDF1–3 proteins recognize m6A-modified HIV-1 RNA and inhibit HIV-1 infection in cell lines and primary CD4+ T-cells. We further mapped the YTHDF1–3 binding sites in HIV-1 RNA from infected cells. We found that the overexpression of YTHDF proteins in cells inhibited HIV-1 infection mainly by decreasing HIV-1 reverse transcription, while knockdown of YTHDF1–3 in cells had the opposite effects. Moreover, silencing the m6A writers decreased HIV-1 Gag protein expression in virus-producing cells, while silencing the m6A erasers increased Gag expression. Our findings suggest an important role of m6A modification of HIV-1 RNA in viral infection and HIV-1 protein synthesis. DOI: http://dx.doi.org/10.7554/eLife.15528.001
eLife digest HIV infection is a global health challenge. The antiviral drugs that are currently available limit the ability of the virus to multiply in infected individuals, but they rarely eliminate the virus entirely. A better understanding of how HIV behaves in the cell would help researchers to find a cure for persistent HIV infection. When HIV enters an immune cell, its genetic material – in the form of molecules of ribonucleic acid (RNA) – is used as a template to make molecules of DNA. This viral DNA can integrate into the host cell’s DNA, where it is used as a template to make more viral RNA molecules, which are then used to make viral proteins. Some of the viral RNAs are also packaged into new virus particles. In cells, RNA molecules are often subject to a chemical modification called adenosine methylation, which regulates how that RNA is translated into proteins. Specific enzymes add molecules called methyl tags to particular locations on the RNA, while other enzymes remove them. A family of proteins called YTHDF1–3 recognize and bind to these methyl tags on the RNA and influence how much protein is produced from the target RNA. There is evidence to suggest that the cell can add methyl tags to HIV RNA. However, the extent to which this happens, and what effects this modification has on HIV replication and viral protein production, are not clear. Tirumuru et al. addressed these questions by analyzing how changing the levels of YTHDF1–3 proteins and the enzymes that add or remove methyl tags in human cells affected HIV infection. The experiments show that YTHDF1–3 proteins inhibited HIV infection in immune cells called T-lymphocytes by recognizing HIV RNA that had been methylated, mainly by targeting the step where the viral RNA is copied into DNA. Altering the levels of the enzymes that add or remove methyl tags in the cells can change the amount of methyl tags attached to RNA molecules, which alters the amount of HIV protein produced. For example, when more RNA molecules had methyl tags, the cells produced more HIV proteins. These findings suggest that adenosine methylation plays an important role in regulating the ability of HIV to thrive and multiply in T-lymphocytes, which are an important target for HIV. Since the RNAs of other human viruses, such as influenza virus, can also be modified by adenosine methylation, drugs that target this pathway could have the potential to be used to fight a variety of viral illnesses. DOI: http://dx.doi.org/10.7554/eLife.15528.002