Purine analog substitution of the HIV-1 polypurine tract primer defines regions controlling initiation of plus-strand DNA synthesis.
Despite extensive study, the mechanism by which retroviral reverse transciptases (RTs) specifically utilize polypurine tract (PPT) RNA for initiation of plus-strand DNA synthesis remains unclear. Three sequence motifs within or adjacent to the purine-rich elements are highly conserved, namely, a rU:dA tract region immediately 5' to the PPT, an rA:dT-rich sequence constituting the upstream portion of the PPT and a downstream rG:dC tract. Using an in vitro HIV-1 model system, we determined that the former two elements define the 5' terminus of the (+)-strand primer, whereas the rG:dC tract serves as the primary determinant of initiation specificity. Subsequent analysis demonstrated that G-->A or A-->G substitution at PPT positions -2, -4 and +1 (relative to the scissile phosphate) substantially reduces (+)-strand priming. We explored this observation further using PPT substrates substituted with a variety of nucleoside analogs [inosine (I), purine riboside (PR), 2-aminopurine (2-AP), 2,6-diaminopurine (2,6-DAP), isoguanine (iG)], or one of the naturally occurring bases at these positions. Our results demonstrate that for PPT positions -2 or +1, substituting position 2 of the purine was an important determinant of cleavage specificity. In addition, cleavage specificity was greatly affected by substituting -4G with an analog containing a 6-NH2 moiety.
Rausch
Jason W
JW
RT Biochemistry Section, Resistance Mechanisms Laboratory, HIV Drug Resistance Program, National Cancer Institute at Frederick, Frederick, MD 21702, USA.
Le Grice
Stuart F J
SF
D000225:Adenine / Q000737:chemistry; D001483:Base Sequence; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis*; D006147:Guanine / Q000737:chemistry; D015497:HIV-1 / Q000235:genetics*; D008957:Models, Genetic; D009154:Mutation; D011684:Purine Nucleosides / Q000737:chemistry*; D012313:RNA / Q000737:chemistry* / Q000378:metabolism; D012367:RNA, Viral / Q000737:chemistry* / Q000378:metabolism; D038621:Regulatory Sequences, Ribonucleic Acid; D016914:Ribonuclease H / Q000378:metabolism; D014498:Uracil / Q000737:chemistry
D004279:DNA, Viral; D011684:Purine Nucleosides; C034477:RNA primers; D012367:RNA, Viral; D038621:Regulatory Sequences, Ribonucleic Acid; D014498:Uracil; D006147:Guanine; D012313:RNA; D016914:Ribonuclease H; D000225:Adenine