The HIV-1 Pr55 precursor specifically selects genomic RNA (gRNA) from a large variety of cellular and spliced viral RNAs (svRNAs), however the molecular mechanisms of this selective recognition remains poorly understood. To gain better understanding of this process, we analyzed the interactions between Pr55 and a large panel of viral RNA (vRNA) fragments encompassing the main packaging signal (Psi) and its flanking regions by fluorescence spectroscopy. We showed that the gRNA harbors a high affinity binding site which is absent from svRNA species, suggesting that this site might be crucial for selecting the HIV-1 genome. Our stoichiometry analysis of protein/RNA complexes revealed that few copies of Pr55 specifically associate with the 5' region of the gRNA. Besides, we found that gRNA dimerization significantly impacts Pr55 binding, and we confirmed that the internal loop of stem-loop 1 (SL1) in Psi is crucial for specific interaction with Pr55. Our analysis of gRNA fragments of different length supports the existence of a long-range tertiary interaction involving sequences upstream and downstream of the Psi region. This long-range interaction might promote optimal exposure of SL1 for efficient Pr55 recognition. Altogether, our results shed light on the molecular mechanisms allowing the specific selection of gRNA by Pr55 among a variety of svRNAs, all harboring SL1 in their first common exon.
Targeting of the human immunodeficiency virus type 1 (HIV-1) Gag precursor Pr55 gag to the plasma membrane, the site of virus assembly, is primarily mediated by the N-terminal matrix (MA) domain. N-myristylation of MA is essential for the stable association of Pr55 gag with membranes and for virus assembly. We now show that single amino acid substitutions near the N terminus of MA can dramatically impair assembly without compromising myristylation. Subcellular fractionation demonstrated that Gag membrane binding was compromised to a similar extent as in the absence of the myristyl acceptor site, indicating that the myristyl group was not available for membrane insertion. Remarkably, the effects of the N-terminal modifications could be completely suppressed by second-site mutations in the globular core of MA. The compensatory mutations enhanced Gag membrane binding and increased viral particle yields above wild-type levels, consistent with an increase in the exposure of the myristyl group. Our results support a model in which the compact globular core of MA sequesters the myristyl group to prevent aberrant binding to intracellular membranes, while the N terminus is critical to allow the controlled exposure of the myristyl group for insertion into the plasma membrane.
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