Affiliation:
1. Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, Bethesda, Maryland 20892, USA.
Abstract
At the 3' end of all retroviral genomes there is a short, highly conserved sequence known as the polypurine tract (PPT), which serves as the primer for plus-strand DNA synthesis. We have identified the determinants for in vitro priming by the human immunodeficiency virus type 1 (HIV-1) PPT. We show that when the PPT is removed and placed into different nucleotide contexts, new priming sites are produced at the precise 3' end of the PPT. In addition, we find that a hybrid consisting of a 15- or 20-nucleotide RNA primer annealed to a 35-nucleotide DNA template is competent for initiation of plus-strand synthesis with HIV-1 reverse transcriptase. Thus, no cis-acting elements appear to be required for priming activity. Changes at the 5' end of the PPT have no effect on primer function, whereas the identity of bases at the 3' end is crucial. A primer containing only the 6 G residues from the 3' end of the wild-type PPT sequence and 9 bases of random sequence at the 5' end functions like a wild-type PPT. A short hybrid having a similar helical structure but a primary sequence different from that of the PPT is cleaved imprecisely, resulting in initiation of synthesis at multiple sites; however, total primer extension is close to the wild-type level. We conclude that helical structure as well as the presence of particular bases at the 3' end of the PPT is essential for PPT function.
Publisher
American Society for Microbiology
Subject
Virology,Insect Science,Immunology,Microbiology
Reference64 articles.
1. RNase H activity of reverse transcriptases on substrates derived from the 5~ end of retroviral genome;Ben-Artzi H.;J. Biol. Chem.,1993
2. Boone L. R. and A. M. Skalka. 1993. Strand displacement synthesis by reverse transcriptase p. 119-133. In A. M. Skalka and S. P. Goff (ed.) Reverse transcriptase. Cold Spring Harbor Laboratory Press Cold Spring Harbor N.Y.
3. Efficient initiation and strand transfer of polypurine tract-primed plus-strand DNA prevent strand transfer of internally initiated plus-strand DNAs;Bowman E. H.;J. Virol.,1996
4. Sequence of simian immunodeficiency virus from macaque and its relationship to other human and simian retroviruses;Chakrabarti L.;Nature (London),1987
5. Champoux J. J. 1993. Roles of ribonuclease H in reverse transcription p. 103-117. In A. M. Skalka and S. P. Goff (ed.) Reverse transcriptase. Cold Spring Harbor Laboratory Press Cold Spring Harbor N.Y.
Cited by
85 articles.
订阅此论文施引文献
订阅此论文施引文献，注册后可以免费订阅5篇论文的施引文献，订阅后可以查看论文全部施引文献