HIV-1 Nucleocapsid Protein Increases Strand Transfer Recombination by Promoting Dimeric G-quartet Formation

Abstract

A preferred site for HIV-1 recombination was identified in vivo and in vitro surrounding the beginning of the HIV-1 gag gene. This G-rich gag hotspot for recombination contains three evenly spaced G-runs that stalled reverse transcriptase. Disruption of the G-runs suppressed both the associated pausing and strand transfer in vitro. Significantly, this same gag sequence was able to fold into a G-quartet monomer, dimer, and tetramer, depending on the cations employed. The pause band at the G-run (nucleotide (nt) 405-409), which was predicted to be involved in forming a G-quartet monomer, diminished with increased HIV-1 nucleocapsid (NC) protein. More NC induced stronger pauses at other G-runs (nt 363-367 and nt 382-384), a region that forms a G-quartet dimer, adhering the two RNA templates. We hypothesized that NC induces the unfolding of the monomeric G-quartet but stabilizes the dimeric interaction. We tested this by inserting a known G-quartet formation sequence, 5-(UGGGGU) 4 -3, into a relatively structure-free template from the HIV-1 pol gene. Strand transfer assays were performed with cations that either encourage (K ؉ ) or discourage (Li ؉ ) G-quartet formation with or without NC. Strikingly, a G-quartet monomer was observed without NC, whereas a G-quartet dimer was observed with NC, both only in the presence of K ؉ . Moreover, the transfer efficiency of the dimerized template (with K ؉ and NC) reached about 90%, approximately 2.5-fold of that of the non-dimerized template. Evidently, template dimerization induced by NC creates a proximity effect, leading to the unique high peak of transfer at the gag recombination hotspot.

Mechanism of HIV-1 RNA Dimerization in the Central Region of the Genome and Significance for Viral Evolution

“…However, dimerization of templates through DIS and G-quartets in gag was previously shown to stimulate strand transfer (24,44). Moreover, in the context of whole genomes, the opportunity for multiple contact points should favor dimeric over monomeric G-quartet formation.…”

“…Switching by the RT-We previously showed that a major recombination hot spot in the HIV-1 gag region near DIS correlates with sequences rich in G residues that can form G-quartet structures (23,24,30). We showed enhanced strand transfer resulting from G-quartet dimer formation that holds the templates in close proximity and/or G-quartet monomer or dimer formation that increases frequency of RT RNase H by pausing the RT (Fig.…”

“…This structure adheres the two RNA genomes; thus, it represents a different mode of genome dimerization in retroviruses from that of the DIS hairpin interaction. Formation of dimeric G-quartet structure correlates with hot spots of recombination, exhibiting an increased rate of template switching (23,24).…”

Recombination in viruses: Mechanisms, methods of study, and evolutionary consequences

“…Under the second hypothesis, which does not exclude the former, there might be a direct link between enhanced recombination rates and the formation of secondary RNA structure, because these RNA structures induce stops, stalling or delays in DNA synthesis, thus favouring strand invasion (Simon-Loriere and Holmes, 2011). Interestingly, the presence or absence of the viral chaperone nucleocapsid modulates RNA secondary structure, revealing an evolutionary role for these proteins (Negroni and Buc, 2000); this effect is related to HIV-1 nucleocapsid promoting dimeric G-quartet formation (Shen et al, 2011).…”

High-Frequency Illegitimate Strand Transfers of Nascent DNA Fragments During Reverse Transcription Result in Defective Retrovirus Genomes

“…24,31,32 To investigate if these patterns could be identified with our sequence data, we determined frequencies of the bases at which nascent DNA fragments paused. Only small percentages (12%) of nascent cDNA fragments paused at homopolymer sites (3-5 bases).…”