Changes to the HIV long terminal repeat and to HIV integrase differentially impact HIV integrase assembly, activity, and the binding of strand transfer inhibitors.
Human immunodeficiency virus (HIV) integrase enzyme is required for the integration of viral DNA into the host cell chromosome. Integrase complex assembly and subsequent strand transfer catalysis are mediated by specific interactions between integrase and bases at the end of the viral long terminal repeat (LTR). The strand transfer reaction can be blocked by the action of small molecule inhibitors, thought to bind in the vicinity of the viral LTR termini. This study examines the contributions of the terminal four bases of the nonprocessed strand (G(2)T(1)C(-1)A(-2)) of the HIV LTR on complex assembly, specific strand transfer activity, and inhibitor binding. Base substitutions and abasic replacements at the LTR terminus provided a means to probe the importance of each nucleotide on the different functions. An approach is described wherein the specific strand transfer activity for each integrase/LTR variant is derived by normalizing strand transfer activity to the concentration of active sites. The key findings of this study are as follows. 1) The G(2):C(2) base pair is necessary for efficient assembly of the complex and for maintenance of an active site architecture, which has high affinity for strand transfer inhibitors. 2) Inhibitor-resistant enzymes exhibit greatly increased sensitivity to LTR changes. 3) The strand transfer and inhibitor binding defects of a Q148R mutant are due to a decreased affinity of the complex for magnesium. 4) Gln(148) interacts with G(2), T(1), and C(-1) at the 5' end of the viral LTR, with these four determinants playing important and overlapping roles in assembly, strand transfer catalysis and high affinity inhibitor binding.
Dicker
Ira B
IB
Department of Virology, Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, Connecticut 06492, USA. Ira.Dicker@BMS.com
Samanta
Himadri K
HK
Li
Zhufang
Z
Hong
Yang
Y
Tian
Yuan
Y
Banville
Jacques
J
Remillard
Roger R
RR
Walker
Michael A
MA
Langley
David R
DR
Krystal
Mark
M
D000241:Adenosine / Q000378:metabolism; D019943:Amino Acid Substitution; D020029:Base Pairing; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D002413:Cations, Divalent; D003596:Cytosine / Q000378:metabolism; D004279:DNA, Viral / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000235:genetics; D006151:Guanosine / Q000378:metabolism; D019427:HIV Integrase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019428:HIV Integrase Inhibitors / Q000494:pharmacology*; D016325:HIV Long Terminal Repeat / Q000235:genetics*; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000378:metabolism; D013941:Thymine / Q000378:metabolism; D014170:Transformation, Genetic; D016662:Virus Integration / Q000502:physiology
D002413:Cations, Divalent; D004279:DNA, Viral; D019428:HIV Integrase Inhibitors; D011994:Recombinant Proteins; D006151:Guanosine; D003596:Cytosine; D019427:HIV Integrase; D008274:Magnesium; D000241:Adenosine; D013941:Thymine