The HIV-1 repeated sequence R as a robust hot-spot for copy-choice recombination.

Template switching during reverse transcription is crucial for retroviral replication. While strand transfer on the terminal repeated sequence R is essential to achieve reverse transcription, template switching from internal regions of the genome (copy choice) leads to genetic recombination. We have developed an experimental system to study copy-choice recombination in vitro along the HIV-1 genome. We identify here several genomic regions, including the R sequence, where copy choice occurred at high rates. The frequency of copy choice occurring in a given region of template was strongly influenced by the surrounding sequences, an observation that suggests a pivotal role of the folding of template RNA in the process. The sequence R, instead, constituted an exception to this rule since it was a strong hot-spot for copy choice in the different sequence contexts tested. We suggest therefore that the structure of this region has been optimised during viral evolution to ensure efficient template switching independently from the sequences that might surround it.

DNA synthesis by HIV-1 reverse transcriptase at the central termination site: a kinetic study.

Human immunodeficiency virus, type 1 (HIV-1) reverse transcriptase (RT) terminates plus-strand DNA synthesis at the center of the HIV-1 genome, a process important for HIV-1 infectivity. The central termination sequence contains two termination sites (Ter1 and Ter2) located at the 3'-end of A(n)T(m) motifs, and the narrowing of the DNA minor groove generated by these motifs is responsible for termination. Kinetic data associated with the binding of RT and its ability to elongate in vitro various DNA duplexes and triplexes surrounding the Ter2 terminator were analyzed using a simple kinetic scheme. At Ter2, RT still displays a reasonable affinity for the corresponding DNA, but the binding of the next nucleotide and above all its incorporation rate are markedly hampered. Features affecting the width of the minor groove act directly at this last step. The constraint exerted against elongation by the A(n)T(m) tract persists at two positions downstream of the terminator.

Structures of complexes formed by HIV-1 reverse transcriptase at a termination site of DNA synthesis.

This study presents structural parameters associated with termination of human immunodeficiency virus, type 1 (HIV-1) reverse transcriptase (RT) at Ter2, the major termination site located in the center of the HIV-1 genome. DNA footprinting studies of various elongation complexes formed by RT around wild type and mutant Ter2 sites have revealed two major structural transformations of these complexes when the enzyme gets closer to Ter2. First, the interactions between RT and the DNA duplex are less extended, although the global affinity of the enzyme for this duplex is only decreased by 2-fold. Second, there is an atypical positioning of the RT RNase H domain on the DNA duplex. We interpret our data as indicating that the A(n)T(m) motif located upstream of Ter2 prevents a classical positioning of the enzyme on the double-stranded part of the DNA duplex at some precise positions of elongation downstream of this motif. Instead, novel species of binary and/or ternary complexes, characterized by atypical footprints, are formed. The new rate-limiting step of the reaction, characterized in the preceding paper (Lavigne, M., Polomack, L., and Buc, H. (2001) J. Biol. Chem. 276, 31429-31438), would be a transition leading from these new species to a catalytically competent ternary complex.