Sequence and structural determinants required for priming of plus-strand DNA synthesis by the human immunodeficiency virus type 1 polypurine tract

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.of short RNA-DNA hybrids, we find that bases at the 3Ј end of the PPT are essential for priming activity. The helical structure of the hybrid may play a role in primer recognition by RT, but it is not the sole determinant of activity. MATERIALS AND METHODS Materials. Restriction enzymes, Taq polymerase, T4 ligase, and T4 DNA polymerase were purchased from Boehringer Mannheim (Indianapolis, Ind.). T4 polynucleotide kinase, RNasin, and RNase ONE, an RNase which degrades RNA without base specificity, were obtained from Promega Biotec (Madison, Wis.). HIV-1 RT was purchased from Worthington Biochemical Corp. (Freehold, N.J.). Radioactively labeled nucleotides [␣-32 P]dATP (3,000 Ci/mmol), [␥-32 P]ATP (3,000 Ci/mmol), and [␣-35 S]dATP␣S were purchased from Amersham Life Science Inc. (Arlington Heights, Ill.). RNA oligonucleotides were purchased from Oligos Etc., Inc. (Wilsonville, Oreg.). Sequence ladders were prepared with reagents from a Sequenase kit (U.S. Biochemicals Corp.) according to the manufacturer's instructions.Preparation of minus-strand single-stranded DNA. Circular minus-strand single-stranded DNA templates (3,352 nt in length), which contain a 185-nt region complementary to the PPT and the surrounding region, were prepared from an HIV-1 LAI clone (pKP8115). This clone was derived from a plasmid containing the HIV-1 LAI 3Ј long terminal repeat and part of nef (gift of Keith Peden, Center for Biologics Evaluation and Research, Food and Drug Administration) and contains nt 8587 to 8772 (60, numbering according to GenBank accession number K02013) inserted between the EcoRI and XbaI sites of the pGEM 3Zf(ϩ) vector (Promega Biotec). The PPT is lo...

Identification of a sequence element immediately upstream of the polypurine tract that is essential for replication of simian immunodeficiency virus

“…While comparing several SIV and HIV sequences, we noted a uridine‐rich sequence located immediately upstream of the PPT. Powell and Levin (1996) have previously noted this sequence in various strains of HIV‐1. We have found a similar U‐rich sequence in the vast majority of other retroviruses and retroviral elements.…”

“…It is believed that this enables the short stretch of purines to serve as a primer for the synthesis of a plus strand of DNA using the previously synthesized minus‐strand DNA as a template (Sorge and Hughes, 1982; Finston and Champoux, 1984; Omer et al ., 1984; Resnick et al ., 1984; Smith et al ., 1984a,b; Repaske et al ., 1989; Huber and Richardson, 1990; Luo et al ., 1990; Pullen and Champoux, 1990; Charneau et al ., 1992; Heyman et al ., 1995; Lauermann et al ., 1995; for reviews see Coffin, 1990; Champoux, 1993; Telesnitsky and Goff, 1993). The specificity of PPT excision and the role of its various nucleotides in directing the proper 3′‐cut and subsequent DNA synthesis initiation have been convincingly demonstrated in a number of retroviral systems (Finston and Champoux, 1984; Smith et al ., 1984b; Repaske et al ., 1989; Huber and Richardson, 1990; Luo et al ., 1990; Pullen and Champoux, 1990; Wöhrl and Moelling, 1990; Gopalakrishnan et al ., 1992; Pullen et al ., 1992, 1993; DeStefano et al ., 1993; Hottiger et al ., 1994; DeStefano, 1995; Fuentes et al ., 1995; Lauermann et al ., 1995; Palaniappan et al ., 1996; Powell and Levin, 1996; Wilhelm et al ., 1997). However, neither the mechanism of PPT protection from the seemingly non‐specific degradation by RNase H nor the requirements for its 5′‐end cut have been established (Oyama et al ., 1989; Huber and Richardson, 1990; Luo et al ., 1990; Pullen and Champoux, 1990; Wöhrl and Moelling, 1990; Pullen et al ., 1992, 1993; DeStefano et al ., 1993; Fuentes et al ., 1995; Lauermann et al ., 1995; Hughes et al ., 1996; Palaniappan et al...…”

“…The specificity of PPT excision and the role of its various nucleotides in directing the proper 3′‐cut and subsequent DNA synthesis initiation have been convincingly demonstrated in a number of retroviral systems (Finston and Champoux, 1984; Smith et al ., 1984b; Repaske et al ., 1989; Huber and Richardson, 1990; Luo et al ., 1990; Pullen and Champoux, 1990; Wöhrl and Moelling, 1990; Gopalakrishnan et al ., 1992; Pullen et al ., 1992, 1993; DeStefano et al ., 1993; Hottiger et al ., 1994; DeStefano, 1995; Fuentes et al ., 1995; Lauermann et al ., 1995; Palaniappan et al ., 1996; Powell and Levin, 1996; Wilhelm et al ., 1997). However, neither the mechanism of PPT protection from the seemingly non‐specific degradation by RNase H nor the requirements for its 5′‐end cut have been established (Oyama et al ., 1989; Huber and Richardson, 1990; Luo et al ., 1990; Pullen and Champoux, 1990; Wöhrl and Moelling, 1990; Pullen et al ., 1992, 1993; DeStefano et al ., 1993; Fuentes et al ., 1995; Lauermann et al ., 1995; Hughes et al ., 1996; Palaniappan et al ., 1996; Powell and Levin, 1996; for review see Champoux, 1993).…”

Ribonuclease H: properties, substrate specificity and roles in retroviral reverse transcription

“…5) which is a substrate for the viral integrase. Although cleavage at the PPT site is very efficient in the internal cleavage mode for M‐MLV, HIV‐1 reverse transcriptase is less efficient in this mode and cleavage may instead occur through the DNA 3′‐end‐directed mode at a pause site during HIV‐1 minus‐strand synthesis [21,48,52,66,86–93]. A possible explanation for the reduced efficiency of cleavage by the HIV‐1 enzyme is that although the M‐MLV PPT sequence conforms to the preferred nucleotide pattern for internal cleavage described above (Fig.…”

“…Removal of the PPT primer appears to occur by an internal cleavage event precisely at the RNA–DNA junction [48,51,52,90,91,101]. Apparently the same sequence features responsible for PPT primer generation determine the site of primer removal and override the natural tendency of the RNase H to cleave one ribonucleotide away from an RNA–DNA junction.…”