Structural features in the HIV-1 repeat region facilitate strand transfer during reverse transcription

By frequently rearranging large regions of the genome, genetic recombination is a major determinant in the plasticity of the human immunodeficiency virus type I (HIV-1) population. In retroviruses, recombination mostly occurs by template switching during reverse transcription. The generation of retroviral vectors provides a means to study this process after a single cycle of infection of cells in culture. Using HIV-1-derived vectors, we present here the first characterization and estimate of the strength of a recombination hot spot in HIV-1 in vivo. In the hot spot region, located within the C2 portion of the gp120 envelope gene, the rate of recombination is up to ten times higher than in the surrounding regions. The hot region corresponds to a previously identified RNA hairpin structure. Although recombination breakpoints in vivo cluster in the top portion of the hairpin, the bias for template switching in this same region appears less marked in a cell-free system. By modulating the stability of this hairpin we were able to affect the local recombination rate both in vitro and in infected cells, indicating that the local folding of the genomic RNA is a major parameter in the recombination process. This characterization of reverse transcription products generated after a single cycle of infection provides insights in the understanding of the mechanism of recombination in vivo and suggests that specific regions of the genome might be prompted to yield different rates of evolution due to the presence of circumscribed recombination hot spots.

Section: Discussionmentioning

confidence: 99%

The Structure of HIV-1 Genomic RNA in the gp120 Gene Determines a Recombination Hot Spot in Vivo

Galetto¹,

Moumen²,

Giacomoni³

et al. 2004

“…The reason why some retroviruses have evolved a shorter homologous region for ϪsssDNA transfer is not known, but the effects of differences in homology length may be compensated by a myriad of factors. Berkhout et al (24) proposed that in addition to homology, structural features of the HIV-1 repeat regions facilitate minus strand transfer. They propose that "kissing interactions" between the anti-TAR and antipoly(A) hairpins in the ϪsssDNA and the TAR and poly(A) of the 3Ј-untranslated region (acceptor) initiate the first interactions between the cDNA and acceptor.…”

Section: Discussionmentioning

confidence: 99%

“…In murine leukemia virus, an R region with as little as 12 or 14 nt does not affect transfer significantly (21,22). In addition to homology, RNA structure has also been implicated in the minus strand transfer mechanism (17,23,24).The viral NC protein has been shown to stimulate minus strand transfer (see Ref. 1 and references therein and Ref.…”

mentioning

confidence: 99%

Mechanism of Minus Strand Strong Stop Transfer in HIV-1 Reverse Transcription

Chen¹,

Balakrishnan²,

Roques

et al. 2003

Retrovirus minus strand strong stop transfer (minus strand transfer) requires reverse transcriptase-associated RNase H, R sequence homology, and viral nucleocapsid protein. The minus strand transfer mechanism in human immunodeficiency virus-1 was examined in vitro with purified protein and substrates. Blocking donor RNA 5 -end cleavage inhibited transfers when template homology was 19 nucleotides (nt) or less. Cleavage of the donor 5 -end occurred prior to formation of transfer products. This suggests that when template homology is short, transfer occurs through a primer terminus switch-initiated mechanism, which requires cleavage of the donor 5 terminus. On templates with 26-nt and longer homology, transfer occurred before cleavage of the donor 5 terminus. Transfer was unaffected when donor 5 -end cleavages were blocked but was reduced when internal cleavages within the donor were restricted. Based on the overall data, we conclude that in human immunodeficiency virus-1, which contains a 97-nt R sequence, minus strand transfer occurs through an acceptor invasion-initiated mechanism. Transfer is initiated at internal regions of the homologous R sequence without requiring cleavage at the donor 5 -end. The acceptor invades at gaps created by reverse transcriptase-RNase H in the donor-cDNA hybrid. The fragmented donor is eventually strand-displaced by the acceptor, completing the transfer.Reverse transcription, during which the single-stranded viral genomic RNA is converted into the double-stranded DNA, is an essential step in viral replication (see Ref. 1 and reference therein). This process is catalyzed by the virus-encoded enzyme reverse transcriptase (RT). 1 RT has two enzymatic activities, the DNA polymerase and the RNase H activities. Reverse transcription is initiated by the partial annealing between the cellular tRNA Lys3 primer and the viral primer binding site, which is located near the 5Ј-end of the viral RNA. Synthesis proceeds to the 5Ј-end of the genomic RNA, creating the minus strand strong stop DNA (ϪsssDNA), which comprises all of the U5 and R sequences from the 5Ј-untranslated region. During minus strand synthesis, RT simultaneously cleaves the copied RNA using its RNase H activity. This frees the ϪsssDNA, enabling its transfer to the homologous R sequence in the 3Ј-untranslated region and continuation of minus strand synthesis. This step is called minus strand strong stop transfer (minus strand transfer), and it is obligatory for reverse transcription and viral replication. Extensive studies have identified the RT-RNase H activity, R sequence homology, and viral nucleocapsid (NC) protein as important components for minus strand transfer (see Ref. 2 and references therein).Viruses with defective RNase H fail to complete reverse transcription, particularly the minus strand transfer step, indicating that RNase H is required for the transfer event (3-8). HIV-1 RT has been shown to partially cleave the template RNA during polymerization, leaving RNA fragments annealed to the cDNA (9, 10). Since there a...

mentioning

confidence: 99%

“…In parallel, studies carried out on RNA templates containing hairpin regions have suggested that such structures could favor template switching by RTs (13,16,17). In these cases it was proposed that the hairpin structures enhance the probability of strand transfer by mediating an interaction between donor and acceptor RNA that increases their spatial proximity (13,16,17). Extensive random searches for the occurrence of recombination hot spots during in vitro reverse transcription by HIV-1 RT had revealed the correlation between the location of these hot spots and the presence of predicted hairpin regions in the RNA template (14,18).…”

mentioning

confidence: 99%

Evidence for a Mechanism of Recombination during Reverse Transcription Dependent on the Structure of the Acceptor RNA

Moumen

Polomack

Unge

et al. 2003

Genetic recombination is a major force driving retroviral evolution. In retroviruses, recombination proceeds mostly through copy choice during reverse transcription. Using a reconstituted in vitro system, we have studied the mechanism of strand transfer on a major recombination hot spot we previously identified within the genome of HIV-1. We show that on this model sequence the frequency of copy choice is strongly influenced by the folding of the RNA template, namely by the presence of a stable hairpin. This structure must be specifically present on the acceptor template. We previously proposed that strand transfer follows a two-step process: docking of the nascent DNA onto the acceptor RNA and strand invasion. The frequency of recombination under copy choice conditions was not dependent on the concentration of the acceptor RNA, in contrast with strand transfer occurring at strong arrests of reverse transcription. During copy choice strand transfer, the docking step is not rate limiting. We propose that the hairpin present on the acceptor RNA could mediate strand transfer following a mechanism reminiscent of branch migration during DNA recombination.