Nucleotide sequence context influences HIV replication fidelity by modulating reverse transcriptase binding and product release.
An RNA template/DNA primer (T/P) complex derived from the env gene of HIV-1 was used to examine the kinetic effects of specific basepair substitutions on dNTP incorporation and RNase H cleavage by HIV-1 reverse transcriptase (RT). Single basepair substitutions 2 or 6 nucleotides upstream from a defined polymerization site (denoted -2 and -6) were engineered by oligonucleotide synthesis to provide 7 T/P substrates for kinetic analysis. A -6 A/T substitution in the wild type sequence resulted in 14- and 7-fold increases in the apparent second order rate constants (k(2app)) for U/A and U/G basepair formation. The k(2app) for U/A formation was relatively unchanged for all other T/P basepair changes. The -6A/T substitution also uniquely lowered the RNase H cleavage rate by 3-fold. Combined kinetic and thermodynamic analyses indicated that these effects were due almost exclusively to increases in the KD (k(off)/k(on)) of initial binding of RT to the T/P and the rate of product release. The data suggest that certain sequence contexts may influence RT fidelity by modulating enzyme binding/dissociation rather than by altering dNTP binding affinity or the rate of the bond forming step.
Yamanaka
Rio
R
Division of Molecular Biology, Beckman Research Institute of the City of Hope Cancer Center, Duarte, CA, USA.
Termini
John
J
D001483:Base Sequence / Q000235:genetics / Q000502:physiology; D017931:DNA Primers / Q000235:genetics; D054303:HIV Reverse Transcriptase / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D011485:Protein Binding / Q000235:genetics / Q000502:physiology; D017433:Protein Structure, Secondary; D016914:Ribonuclease H / Q000378:metabolism; D014779:Virus Replication / Q000235:genetics* / Q000502:physiology
D017931:DNA Primers; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase; D016914:Ribonuclease H