Role of glutamine 151 of human immunodeficiency virus type-1 reverse transcriptase in substrate selection as assessed by site-directed mutagenesis.
A natural mutation at codon 151 (Gln --> Met; Q151M) of HIV-1 RT has been shown to confer resistance to the virus against dideoxy nucleoside analogues [Shirasaka, T., et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 2398], suggesting that Gln 151 may be involved in conferring sensitivity to nucleoside analogues. To understand its functional implication, we generated two mutant derivatives of this residue (Q151M and Q151N) and examined their sensitivities to ddNTPs and their ability to discriminate against rNTPs versus dNTP substrates on natural U5-PBS HIV-1 RNA template. We found that Q151M was highly discriminatory against all four ddNTPs but was able to incorporate rNTPs as efficiently as the wild type enzyme. In contrast, the Q151N mutant was only moderately resistant to ddNTPs but exhibited a higher level of discrimination against rNTPs. The fidelity of misinsertion was found to be highest for the Q151N mutant followed by Q151M and the wild type enzyme. These results point toward the importance of the amino acid side chain at position 151 in influencing the ability of the enzyme in recognition and discrimination against the sugar moieties of nucleotide substrates.
Kaushik
N
N
Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry-New Jersey Medical School, 185 South Orange Avenue, Newark, New Jersey 07103, USA.
Talele
T T
TT
Pandey
P K
PK
Harris
D
D
Yadav
P N
PN
Pandey
V N
VN
D001216:Asparagine / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites / Q000187:drug effects / Q000235:genetics; D003848:Deoxyguanine Nucleotides / Q000494:pharmacology; D015224:Dideoxynucleosides / Q000494:pharmacology; D004352:Drug Resistance, Microbial; D005973:Glutamine / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008715:Methionine / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary / Q000235:genetics; D012367:RNA, Viral / Q000378:metabolism; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity / Q000235:genetics; D013698:Templates, Genetic
D003848:Deoxyguanine Nucleotides; D015224:Dideoxynucleosides; D012367:RNA, Viral; D018894:Reverse Transcriptase Inhibitors; D005973:Glutamine; D001216:Asparagine; C029603:deoxyguanosine triphosphate; D008715:Methionine; D054303:HIV Reverse Transcriptase