Strand transfer events during HIV-1 reverse transcription.

Human immunodeficiency virus type 1 (HIV-1) and other retroviruses replicate through reverse transcription, a process in which the single stranded RNA of the viral genome is converted to a double stranded DNA. The virally encoded reverse transcriptase (RT) mediates reverse transcription through DNA polymerase and RNase H activities. Conversion of the plus strand RNA to plus/minus strand RNA/DNA hybrid involves a transfer of the growing DNA strand from one site on the genomic RNA to another. This is called minus strong-stop DNA transfer. Later synthesis of the second or plus DNA strand involves a second strand transfer, involving a similar mechanism as the minus strand transfer. A basic feature of the strand transfer mechanism is the use of the RT RNase H to remove segments of the RNA template strand from the growing DNA strand, freeing a single stranded region to anneal to the second site. Viral nucleocapsid protein (NC) functions to promote transfer by facilitating this strand exchange process. Two copies of the RNA genomes, sometimes non-identical, are co-packaged in the genomes of retroviruses. The properties of the reverse transcriptase allow a transfer of the growing DNA strand between these genomes to occur occasionally at any point during reverse transcription, producing recombinant viral progeny. Recombination promotes structural diversity of the virus that helps it to survive host immunity and drug therapy. Recombination strand transfer can be forced by a break in the template, or can occur at sites where folding structure of the template pauses the RT, allowing a concentration of RNase H cleavages that promote transfers. Transfer can be a simple one-step process, or can proceed by a complex multi-step invasion mechanism. In this latter process, the second RNA template interacts with the growing DNA strand well behind the DNA 3'-terminus. The newly formed RNA-DNA hybrid expands by branch migration and eventually catches the elongating DNA primer 3'-terminus to complete the transfer. Transfers are also promoted by interactions between the two RNA templates, which accelerate transfer by a proximity effect. Other details of the role of strand transfers in reverse transcription and the biochemical features of the transfer reaction are discussed.

Apparent defects in processive DNA synthesis, strand transfer, and primer elongation of Met-184 mutants of HIV-1 reverse transcriptase derive solely from a dNTP utilization defect.

The 2',3'-dideoxy-3'-thiacytidine drug-resistant M184I HIV-1 reverse transcriptase (RT) has been shown to synthesize DNA with decreased processivity compared with the wild-type RT. M184A displays an even more severe processivity defect. However, the basis of this decreased processivity has been unclear, and both primer-template binding and dNTP interaction defects have been proposed to account for it. In this study, we show that the altered properties of the M184I and M184A RT mutants that we have measured, including decreased processivity, a slower rate of primer extension, and increased strand transfer activity, can all be explained by a defect in dNTP utilization. These alterations are observed only at low dNTP concentration and vanish as the dNTP concentration is raised. The mutant RTs exhibit a normal dissociation rate from a DNA primer-RNA template while paused during synthesis. Slower than normal synthesis at physiological dNTP concentration, coupled with normal dissociation from the primer-template, results in the lowered processivity. The mutant RTs exhibit normal DNA 3'-end-directed and RNA 5'-end-directed ribonuclease H activity. The reduced rate of DNA synthesis causes an increase in the ratio of ribonuclease H to polymerase activity thereby promoting increased strand transfer. These latter results are consistent with an observed higher rate of recombination by HIV-1 strains with Met-184 mutations.

Evidence that creation of invasion sites determines the rate of strand transfer mediated by HIV-1 reverse transcriptase.

Strand transfer during reverse transcription can produce genetic recombination in human immunodeficiency virus type 1 (HIV-1) when two genomic RNAs, that are not identical, are co-packaged in the virus. Strand transfer was measured in vitro, in reactions involving primer switching from a donor to acceptor RNA template. The transfer product appeared with much slower kinetics than full-length synthesis on the donor template. The goal of this study was to learn more about the transfer mechanism by defining the steps that limit its rate. We previously proposed transfer to include the steps of acceptor invasion, hybrid propagation, terminus transfer, and re-initiation of synthesis on the acceptor template. Unexpectedly, with our templates increasing acceptor concentration increased the transfer efficiency but had no effect on the rate of transfer. Templates with a short region of homology limiting hybrid propagation exhibited a slow accumulation of transfer products, suggesting that for tested long homology templates hybrid propagation was not rate limiting. Substituting a DNA acceptor and adding Klenow polymerase accelerated re-initiation and extension exclusively on the DNA acceptor. This lead to a small rate increase due to faster extension on the acceptor, suggesting re-initiation of synthesis on the tested RNA acceptors was not rate limiting. A substrate was designed in which the 5' end of the primer was single stranded, and complimentary to the acceptor, i.e. having a pre-made invasion site. With this substrate, increasing concentrations of acceptor increased the rate of transfer. Together these data suggest that RNase H cleavage, and dissociation of RNA fragments creating an invasion site was rate limiting on most tested templates. When an accessible invasion site was present, acceptor interaction at that site influence the rate.

Effects of donor and acceptor RNA structures on the mechanism of strand transfer by HIV-1 reverse transcriptase.

Template switching during reverse transcription contributes to recombination in human immunodeficiency virus type 1 (HIV-1). Our recent studies suggest that the process can occur through a multi-step mechanism involving RNase H cleavage, acceptor invasion, branch migration, and finally primer terminus transfer. In this study, we analyzed the effects of reverse transcriptase (RT)-pausing, RNase H cleavages and template structure on the transfer process. We designed a series of donor and acceptor template pairs with either minimal pause sites or with pause sites at various locations along the template. Restriction sites within the region of homology allowed efficient mapping of the location of primer terminus transfer. Blocking oligomers were used to probe the acceptor invasion site. Introduction of strong pause sites in the donor increased transfer efficiency. However, the new pauses were not necessarily associated with effective invasion. In this system, the primary invasion occurred at a region of donor cleavage associated with weak pausing. These results together with acceptor structure predictions indicated that a potential invasion site is used only in conjunction with a favorable acceptor structure. Stabilizing acceptor structure at the predicted invasion region lowered the transfer efficiency, supporting this conclusion. Differing from previous studies, terminus transfer occurred at a short distance from the invasion site. Introduction of structure into the acceptor template shifted the location of terminus transfer. Nucleocapsid protein, which can improve cDNA-acceptor interactions, increased transfer efficiency with some shift of terminus transfer closer to the invasion site. Overall results support that the acceptor structure has a major influence on the efficiency and position of the invasion and terminus transfer steps.