Pre-existing distortions in nucleic acid structure aid polypurine tract selection by HIV-1 reverse transcriptase.
Precise cleavage at the polypurine tract (PPT)/U3 junction by human immunodeficiency virus type 1 (HIV-1) reverse transcriptase RNase H is critical for generating a correct viral DNA end for subsequent integration. Using potassium permanganate (KMnO(4)) modification, we have identified a significant distortion in the nucleic acid structure at the HIV-1 PPT/U3 junction in the absence of trans-acting factors. Unusually high reactivity of template thymine +1 is detected when the PPT primer is extended by DNA or RNA at its 3' terminus. Chemical footprinting suggests that the extent of base unstacking in the wild-type species is comparable when the +1 A:T base pair is replaced by a C:T mismatch. However, reactivity of this template base is diminished after alterations to upstream (rA)(4):(dT)(4) or (rG)(6):(dC)(6) tracts. Importantly, there is a correlation between the structural deformation at base pair +1 and precise cleavage at the PPT/U3 junction by HIV-1 reverse transcriptase/RNase H. KMnO(4) modification also revealed unusually high reactivity for one of two (dT)(4):(rA)(4) duplexes upstream of the PPT/U3 junction, suggesting a significant structural distortion within the PPT itself in the absence of the retroviral polymerase. Structural abnormalities in this region are not only essential for resistance of the PPT to hydrolysis but also significantly impact the conformation of the PPT/U3 junction. Our data collectively suggest that the entire PPT sequence contributes to the structural distortion at the PPT/U3 junction, potentially providing a mechanism for its selective processing.
Kvaratskhelia
Mamuka
M
Reverse Transcriptase Biochemistry Section, Resistance Mechanisms Laboratory, HIV Drug Resistance Program, NCI-Frederick, National Institutes of Health, Frederick, Maryland 21702, USA.
Budihas
Scott R
SR
Le Grice
Stuart F J
SF
D020137:Base Pair Mismatch; D001483:Base Sequence; D004279:DNA, Viral / Q000737:chemistry* / Q000378:metabolism; D054303:HIV Reverse Transcriptase / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009154:Mutation; D011196:Potassium Permanganate / Q000494:pharmacology; D011485:Protein Binding; D011687:Purines / Q000737:chemistry*; D012367:RNA, Viral / Q000737:chemistry* / Q000378:metabolism; D012190:Retroviridae / Q000378:metabolism; D016914:Ribonuclease H / Q000378:metabolism
D004279:DNA, Viral; D011687:Purines; D012367:RNA, Viral; D011196:Potassium Permanganate; D054303:HIV Reverse Transcriptase; D016914:Ribonuclease H