SEQUENCE AND STRUCTURAL DETERMINANTS REQUIRED FOR PRIMING OF PLUS-STRAND DNA-SYNTHESIS BY THE HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 POLYPURINE TRACT

At the 3' end of all retroviral genomes there is a short, highly conse rved sequence known as the polypurine tract (PPT), which serves as the primer for plus-strand DNA synthesis. We have identified the determin ants 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 diffe rent nucleotide contexts, new priming sites are produced at the precis e 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 temp late is competent for initiation of plus-strand synthesis with HIV-1 r everse transcriptase. Thus, no cis-acting elements appear to be requir ed for priming activity. Changes at the 5' end of the PPT have no effe ct 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' en d functions like a wild-type PPT. A short hybrid having a similar heli cal 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 leve l. We conclude that helical structure as well as the presence of parti cular bases at the 3' end of the PPT is essential for PPT function.