Nontemplated base addition by HIV-1 RT can induce nonspecific strand transfer in vitro.


After minus-strand strong-stop DNA (-sssDNA) synthesis, the RNA ...
After minus-strand strong-stop DNA (-sssDNA) synthesis, the RNA template is degraded by the RNase H activity of reverse transcriptase (RT), generating a single-stranded DNA. The genomes of some retroviruses contain sequences that could lead to self-priming of their minus signsssDNA. Self-priming was prevented by annealing a DNA oligonucleotide to the 3' end of model DNAs that corresponded to the 3' ends of the -sssDNAs (-R ssDNA) from human immunodeficiency virus type 1 (HIV-1), type 2 (HIV-2), and human T-cell leukemia virus type 1 (HTLV-1) but nonspecific strand transfer to ssDNA molecules in solution was induced in vitro (Golinelli and Hughes, 2001). This nonspecific strand transfer involved the addition of a nontemplated base to the 3' end of -R ssDNAs that was part of a blunt-ended duplex. In the case of HIV-2 -R ssDNA, A and C were added more efficiently than G and T. Strand transfer to ssDNA in solution occurred only if the nontemplated base could form a basepair with the last base at the 3' end of the ssDNA. If there was a mismatch, strand transfer did not occur. There was no detectable strand transfer to internal sites in the target ssDNA except to the second position from the 3' end of the DNA acceptor when the sequences at the 3' ends of the two DNAs allowed the formation of two basepairs. The nontemplated base addition and the one-basepair strand transfer were both affected by the salt concentration in the reaction, the nature of the reverse transcriptase (HIV-1 versus Moloney murine leukemia virus), and the sequence at the 3' end of -R ssDNA. NC reduced the efficiency of nonspecific strand transfer in vitro, suggesting that NC may have a role in reducing nonspecific strand transfer in vivo.




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