Abstract
The co-packaged RNA genomes of human immunodeficiency virus-1 recombine at a high rate. Recombination can mix mutations to generate viruses that escape immune response. A cell-culture-based system was designed previously to map recombination events in a 459-bp region spanning the primer binding site through a portion of the gag protein coding region. Strikingly, a strong preferential site for recombination in vivo was identified within a 112-nucleotide-long region near the beginning of gag. Strand transfer assays in vitro revealed that three pause bands in the gag hot spot each corresponded to a run of guanosine (G) residues. Pausing of reverse transcriptase is known to promote recombination by strand transfer both in vivo and in vitro. To assess the significance of the G runs, we altered them by base substitutions. Disruption of the G runs eliminated both the associated pausing and strand transfer. Some G-rich sequences can develop G-quartet structures, which were first proposed to form in telomeric DNA. G-quartet structure formation is highly dependent on the presence of specific cations. Incubation in cations discouraging G-quartets altered gel mobility of the gag template consistent with breakdown of G-quartet structure. The same cations faded G-run pauses but did not affect pauses caused by hairpins, indicating that quartet structure causes pausing. Moreover, gel analysis with cations favoring G-quartet structure indicated no structure in mutated templates. Overall, results point to reverse transcriptase pausing at G runs that can form quartets as a unique feature of the gag recombination hot spot.
Highlights
Multiple Pauses in Vitro Correlate with the Peak of Transfer in Vivo—To understand the factors that contribute to the peak of transfers in Human immunodeficiency virus type 1 (HIV-1) gag in vivo, we used a previously described strand transfer gag in vitro in which donor RNA (NL4-3) and acceptor RNA (JRCSF) harbor the same naturally occurring periodic sequence differences as in the cell culture-based system [28]
The primer could switch from the donor onto the acceptor followed by completion of synthesis on the acceptor to yield a 385-nt transfer product (TP), or the primer could complete the synthesis on the donor without transfer to yield a 353-nt donor extension product (DE) (Fig. 1A)
The locations of markers are indicated on the left of the gel, whereas the major reverse transcriptase (RT) pause sites are indicated on the right of the gel
Summary
These results highlight the contribution pausing sites in the direction of DNA synthesis, including the of runs of G residues in promoting the recombination in and DIS and SD, indicating the possibility of a shifting of the distri- around the hot spot region and demonstrate that bution of the transfer events to these downstream pause sites the overall transfer efficiency did not decrease very much as a (Fig. 2B). G-quartet Caused RT Pausing and Facilitated Transfers—To investigate the link between the special structure motif of the gag hot spot and its high frequency of crossovers, we performed strand transfer assays can be formed preferentially in the presence of specific monovalent cations [44].
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