The gag hotspot sequence folds into intermolecular G-quartets in vitro. A, sequences of the G-rich region of sWT and sGGG gag donor RNA templates. The three runs of G residues are underlined, whereas base substitutions made to disrupt the G-runs are indicated in bold. B, schematics of a dimeric and a tetrameric G-quartet. M+, a monovalent cation. C, non-denaturing gel assay of the sWT and sGGG gag donor RNA templates. Different templates and cations combinations are indicated at the top of the gel. Bands representing unfolded RNA, dimeric G-quartet, and tetrameric G-quartet are indicated.
Examples of the enhancement of intermolecular G-quartet folding by nucleic acid chaperone proteins are not rare. The β-subunit of the Oxytricha telomere-binding protein enhances the rate of folding of Oxytricha telomeric repeats (T4G4) into G-quartet dimers and tetramers by 105- to 106-fold (50). A prerequisite for intermolecular G-quartet formation is that either two or four strands of nucleic acids have to be brought into close proximity. The Oxytricha subunit is one of many basic proteins, including histone H1, cytochrome c, and poly-L-lysine that promote intermolecular G-quartet folding by facilitating the annealing of two complementary strands into a duplex (50). Because the presence of numerous basic amino acids is a feature even more conserved than the presence of zinc finger domains in retroviral NC proteins, it is evident why NC plays a positive role in G-quartet dimer and tetramer formation.
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To understand the influence of NC on G-quartet folding, we have to consider how this protein affects nucleic acid duplex stability. NC-nucleic acid interactions produce two distinct and opposite effects on duplex stability: a weak duplex-destabilizing effect resulting from the preferential binding of the zinc fingers of NC to single-stranded nucleic acids and a duplex-stabilizing effect depending on the nonspecific polyelectrolyte interactions between phosphates and the NC N-terminal basic domain (also called the cationic domain) (33). On one hand, as discussed above, the destabilization activity of NC on the G-quartet monomer depends solely on NC zinc fingers but not the basic domain, perhaps because of the preferential binding of NC to unpaired bases, which unstacks G-quartet structures. On the other hand, the basic domain of NC was reasoned to account for G-quartet dimer and tetramer stabilization, partially because its basic characteristic can neutralize negative charges on the nucleic acid backbone, thereby facilitating intermolecular association. However, we note that the intermolecular G-quartet stabilization effect of NC is certainly not simply caused only by its basicity because it was reported that spermidine, which also effectively neutralizes negative charges on the DNA backbone, was not found to have any effect on intermolecular G-quartet folding (50).
Because we have evidence that NC selectively stabilizes G-quartet dimers but not monomers and the dimerization of the viral RNAs appears to greatly enhance strand transfer, we wanted to know how all these factors interact to form the gag hotspot for recombination. We performed the strand transfer assay using the WT gag sequence with increasing amounts of NC and observed an RT pausing pattern indicating the enhancement of G-quartet dimer formation but unfolding of the G-quartet monomer. More significantly, we were able to recapitulate the gag hotspot for recombination in vitro when the RNA templates in the strand transfer assay were coated with an amount of NC at 8-fold excess for template coating. Approximately 57% of transfer events were mapped to the gag hotspot region in vitro, compared with slightly more than 60% observed in vivo. However, only 17.12% of the transfer events were located in the gag region in the absence of NC. It should also be noted that NC was not observed to substantially redistribute the general location of transfers with many substrates or alter the general mechanisms of strand transfer (51, 52). In addition, we have reported previously a 3.8-fold decrease in the number of the transfer events in the gag hotspot site when the G-runs involved in G-quartet formation were disrupted. All these data are consistent with the conclusions we drew from analysis of the pol-G-quartet substrate. Namely, both a template sequence that has the potential to fold into a G-quartet dimer and the presence of NC are critical for the formation of the gag hotspot.
On the basis of our investigation, we propose an NC-promoted G-quartet-derived mechanism that produces highly efficient strand transfer. During HIV-1 reverse transcription, both G-quartet monomers and dimers are able to stall RT synthesis. This induces RT-RNase H cleavage on the original donor RNA template, allowing strand invasion of a second acceptor RNA template to interact with the newly synthesized single-stranded DNA. The interaction propagates until transfer of the DNA strand to the acceptor RNA is complete. A key addition to this invasion mechanism is a proximity effect accomplished through a G-quartet dimer formed between donor and acceptor RNAs. Promoted by NC, the G-quartet dimer is stabilized, whereas the monomer is destabilized. This creates a physical connection between the two RNA templates. This proximity greatly enhances the efficiency of strand invasion at the point of joining and some distance into the surrounding sequence. Both mechanisms combine to create the major peak of strand transfer in the gag hotspot for recombination.
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