Ekram W. Abd El- Wahab, Redmond P. Smyth, Elodie Mailler, Serena Bernacchi, Valérie Vivet-Boudou, Marcel Hijnen, Fabrice Jossinet, Johnson Mak, Jean-Christophe Paillart and Roland Marquet
Tropical Health Department, High Institute of Public Health, Alexandria University, Egypt
During assembly, HIV-1 must select its genomic RNA (gRNA) from a variety of cellular and viral spliced RNAs. Despite a large number of studies, there is no consensus on how the Pr55Gag precursor achieves this selection. These studies were limited by the expression and purification of intact full-length Pr55Gag protein. Here, we purified soluble full-length Pr55Gag and we investigated the specific determinants of the selective binding of Pr55Gag to HIV-1 gRNA using RNA binding and footprinting assays. Our results revealed that Pr55Gag exhibits a higher binding affinity for gRNA than for spliced vRNA species. Importantly, we demonstrate that the primary Pr55Gag binding site consists of the internal loop and the lower part of stem-loop 1 (SL1), the upper part of which initiates gRNA dimerization. Further analyses on viral RNA fragments of different length spanning the Psi and/or its flanking regions are in favor of a long-distance tertiary interaction involving sequences upstream of SL1 and downstream of SL4, which promotes the optimal binding of Pr55Gag to gRNA. Altogether our data shed light on the importance of a proper gRNA conformation that regulates its specific binding to Pr55Gag, and could result in the competent selection and packaging of the genome. We propose a new model to explain how Pr55Gag discriminates and specifically selects gRNA from cellular RNAs and viral spliced vRNAs that also harbor functional SL1 in their first common exon. A double regulation ensures specific binding of Pr55Gag to the gRNA despite the fact that SL1 is also present in spliced viral RNAs. The region upstream of SL1, which is present in all viral RNAs, prevents binding to SL1, but this negative effect is counteracted by sequences downstream of SL4, which are unique to the gRNA.
Keywords: HIV-1, genomic RNA, specific recognition, Gag precursor.
This research was published in nature communications, 2014.
Ekram W. Abd El-Wahab, Redmond P. Smyth, Serena Bernacchi, Marcel Hijnen, Fabrice Jossinet, Johnson Mak, Jean- Christophe Paillart & Roland Marquet. Specific Recognition of the HIV-1 genomic RNA by the Gag precursor. Nat Commun. 2014 Jul 2;5:4304. doi: 10.1038/ncomms5304.