The presence of m6A within the 5 ?-UTR of the HIV-1 genomic RNA defines its use as mRNA or as the packaged genome

Abstract

The human immunodeficiency virus type 1 (HIV-1) is the etiological agent of the acquired immunodeficiency syndrome (AIDS). Their genome consists of a positive sense RNA (genomic RNA or gRNA), which is found as a dimer within the viral particle. During the late stages of viral replication, the gRNA plays to major roles by acting as an mRNA encoding the structural proteins Gag and Gag-Pol, and is the genome packaged into the newly produced viral particles. Although it is known that the 5 ́-untranslated region (5 ́UTR) is key in the regulation on the transition from translation to packaging, the molecular mechanisms involved in this regulation are not well understood. It was recently reported that HIV-1 gene expression is post-transcriptionally regulated by the presence of N6- methyladenosine (m6A) along the gRNA. The addition of this modification is mediated by the methyltransferases METTL3/14 and is removed by the demethylases FTO and ALKBH5. Here, we show that the hypermethylation of the gRNA through the overexpression of the METTL3/14 methyltransferase complex induces a strong decrease of the gRNA packaged into released viral particles. We also observed that this effect was dynamic since overexpression of the m6A demethylase FTO (but not ALKBH5) induced an increase in the packaged gRNA. Interestingly, m6A-seq analyses revealed that the 5 ́UTR of the gRNA is methylated within the cell, but not in the viral particle, indicating that the absence of m6A within the 5 ́UTR is probably necessary for the packaging of the gRNA. When we analyzed in detail, we identified A198 and the A242 as the potential methylated residues. We are currently investigating the impact of m6A within the 5 ́-UTR on the interaction with Gag as well as the dimerization of the gRNA and their structure in order to understand the molecular mechanism involved in this regulation. FONDECYT 1160176; ANILLO ACT-1408.