Mutations within the Primer Binding Site of the Human Immunodeficiency Virus Type 1 Define Sequence Requirements Essential for Reverse Transcription

The primer binding site (PBS) is involved in two stages during the reverse transcription of the retroviral RNA genome. In the early stage, the PBS provides complementary sequences through which tRNALys,3binds the viral RNA genome to initiate minus-strand DNA synthesis; in the later stages, complemen...

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Bibliographic Details
Published in:Virology (New York, N.Y.) N.Y.), 1996-06, Vol.220 (2), p.290-298
Main Authors: WAKEFIELD, JOHN K., MORROW, CASEY D.
Format: Article
Language:English
Subjects:
AIDS/HIV
Animals
Base Sequence
Binding Sites
Cell Line, Transformed
Cercopithecus aethiops
DNA, Viral
HIV-1 - genetics
HIV-1 - physiology
human immunodeficiency virus 1
Humans
Molecular Sequence Data
Mutation
RNA - genetics
RNA, Transfer, Lys - genetics
RNA, Viral - genetics
Transcription, Genetic
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Summary:The primer binding site (PBS) is involved in two stages during the reverse transcription of the retroviral RNA genome. In the early stage, the PBS provides complementary sequences through which tRNALys,3binds the viral RNA genome to initiate minus-strand DNA synthesis; in the later stages, complementarity between the plus- and minus-strand copies of the PBS is required to facilitate the second template transfer needed to complete reverse transcription. We previously constructed a mutant HIV-1 proviral genome, designated as pHXB2PBS(pheC + 5) (now referred to as pheC + 5), which was used to identify regions of the PBS involved in the initiation and second template transfer steps of reverse transcription. To further define the sequence requirements of the PBS for the initiation of reverse transcription, we have made single nucleotide substitutions within the first six nucleotides of the pheC + 5 PBS. Our results demonstrate that mutations within the first five nucleotides of the PBS which disrupt base paring with tRNALys,3-PBS results in an noninfectious virus; a G-U base pair at position six of the tRNALys,3-PBS complex was tolerated. In contrast to the requirements for initiation, we found that complementary binding between only three base pairs of the plus- and minus-strand PBSs was required for the extension of plus-strand DNA during the second template transfer. Furthermore, regions of the minus-strand DNA of up to 24 nucleotides could be looped-out to facilitate the complementarity required for the completion of plus-strand DNA synthesis. Taken together, the results of our studies demonstrate that different features of the PBS with respect to RNA:RNA and DNA:DNA interactions are required for initiation of reverse transcription and the completion of plus-strand DNA synthesis, respectively.
ISSN:0042-6822
1096-0341
DOI:10.1006/viro.1996.0317