The Kissing Hairpin Sequence Promotes Recombination within the HIV-I 5′ Leader Region

Abstract

The role of RNA-RNA template interactions in facilitating recombination during reverse transcription of minus strand DNA has been examined. The tested hypothesis is that template switching by reverse transcriptase is promoted at sites where homologous regions of two RNAs are brought in close proximity via stable intertemplate interactions. Frequency and distribution of template switching between homologous donor and acceptor RNAs were examined within the human immunodeficiency virus type I (HIV-I) 5-untranslated region (UTR) containing the dimer initiation sequence (DIS). Results were compared with control nondimerizing templates from the pol region. The dimerizing UTR templates displayed a 4-fold higher transfer efficiency than the control. A striking 53% of transfers in the UTR mapped near the DIS, of which two-thirds occurred immediately 5 to this sequence. In the UTR template, deletion of the DIS hairpin disrupted template dimerization and caused a 4-fold drop in transfer efficiency. Insertion of the DIS within the pol template increased both dimerization and transfer efficiency. Transfer distributions revealed that in both sets of templates, DISinduced dimerization increased the efficiency of transfers across the whole template, with the transfers peaking around the dimerization site. Overall, these results suggest that template dimerization facilitated by the unique geometry of the DIS-promoted kissing interactions effectively promotes recombination within the HIV-I 5-UTR.Retroviruses are characterized by a diploid, positive sense RNA genome. After entry into the host cell, the viral reverse transcriptase (RT) 1 copies it into double-stranded DNA by a process utilizing both DNA polymerase and RNase H activities. Completion of synthesis of both minus and plus strand DNA involves template switching events in which the strong stop DNA is transferred from the original template to a complementary sequence at the 3Ј end of either the same or a second homologous copy of the template (1). In addition to these obligatory transfers, strand transfers involving internal regions of the RNA or complementary DNA have also been demonstrated to occur during replication (2-4). These include both intrastrand (resulting in deletions or duplications) and interstrand transfers (resulting in recombination).The presence of two RNA copies provides a functional advantage during reverse transcription, allowing RT to switch templates should it encounter a break or damage within the RNA strand being copied. The forced copy-choice model of recombination (5) predicts that the process occurs during minus strand DNA synthesis. However, because template switching also occurs in the absence of breaks in the RNA and may not always be forced, the process fits the more generalized copy-choice model (6). Such interstrand template switching involving two co-packaged nonidentical RNAs would then result in a recombinant provirus. Recombination during plus strand synthesis is described by the strand displacement-assimilation model (7,8) which propo...

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

“…It was first suggested that the hairpins formed by these regions could favor template switching by increasing the spatial proximity between the donor and the acceptor RNAs through an intermolecular interac- tion occurring within the hairpin region (13,16,17). Our results do not support such an interpretation because here the hairpin structure is not required on the donor RNA and, therefore, cannot act by mediating an interaction with the acceptor RNA.…”

“…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).…”

“…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).…”

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

“…Recent studies in our laboratory have shown evidence for an acceptor invasion-initiated mechanism for internal transfer events that lead to recombination (56,78). Transfers occur through a two-step "dock and lock" mechanism, where the acceptor interacts (docks) with the donor-cDNA complex at regions where RNase H cleavage is enhanced (76,78).…”

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

“…Several in vitro works have suggested an implication of hairpin structures in promoting copy choice by bringing donor and acceptor RNAs into close proximity (33,34) or by favoring pausing of reverse transcription at the base of the hairpin followed by strand transfer within the hairpin region (20). Based on an in vitro study of the same C2 sequence studied here, we previously proposed that strand transfer would begin by the docking of the nascent DNA onto the acceptor RNA in the loop of the hairpin and would then proceed through a process of strand exchange similar to branch migration occurring during DNA-DNA recombination (10).…”