Pre-Steady-State Kinetic Characterization of Wild Type and 3‘-Azido-3‘-deoxythymidine (AZT) Resistant Human Immunodeficiency Virus Type 1 Reverse Transcriptase:  Implication of RNA Directed DNA Polymerization in the Mechanism of AZT Resistance†

Biochemistry ◽  
1997 ◽  
Vol 36 (46) ◽  
pp. 14064-14070 ◽  
Author(s):  
Stephen G. Kerr ◽  
Karen S. Anderson
Keyword(s):  
Human Immunodeficiency Virus ◽  
Kinetic Characterization ◽  
Wild Type ◽  
Dna Polymerization ◽  
Steady State Kinetic ◽  
Immunodeficiency Virus ◽  
Azt Resistance ◽  
State Kinetic

scholarly journals Mechanisms by Which the G333D Mutation in Human Immunodeficiency Virus Type 1 Reverse Transcriptase Facilitates Dual Resistance to Zidovudine and Lamivudine

2007 ◽  
Vol 52 (1) ◽  
pp. 157-163 ◽  
Author(s):  
Shannon Zelina ◽  
Chih-Wei Sheen ◽  
Jessica Radzio ◽  
John W. Mellors ◽  
Nicolas Sluis-Cremer
Keyword(s):  
Human Immunodeficiency Virus ◽  
Steady State ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Steady State Kinetic ◽  
Immunodeficiency Virus ◽  
State Kinetic ◽  
Hiv 1

ABSTRACT Recent studies have identified a role for mutations in the connection and RNase H domains of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) resistance to nucleoside analog RT inhibitors (NRTI). To provide insight into the biochemical mechanism(s) involved, we investigated the effect of the G333D mutation in the connection domain of RT on resistance to zidovudine (AZT) and lamivudine (3TC) in enzymes that contain both M184V and thymidine analog mutations (TAMs; M41L, L210W, and T215Y). Our results from steady-state kinetic, pre-steady-state kinetic, and thermodynamic analyses indicate that G333D facilitates dual resistance to AZT and 3TC in two ways. First, in combination with M184V, G333D increased the ability of HIV-1 RT to effectively discriminate between the normal substrate dCTP and 3TC-triphosphate. Second, G333D enhanced the ability of RT containing TAMs and M184V to bind template/primer terminated by AZT-monophosphate (AZT-MP), thereby restoring ATP-mediated excision of AZT-MP under steady-state assay conditions. This study is the first to elucidate a molecular mechanism whereby a mutation in the connection domain of RT can affect NRTI susceptibility at the enzyme level.

scholarly journals Mechanism of Inhibition of Human Immunodeficiency Virus Type 1 Reverse Transcriptase by a Stavudine Analogue, 4′-Ethynyl Stavudine Triphosphate

2008 ◽  
Vol 52 (6) ◽  
pp. 2035-2042 ◽  
Author(s):  
Guangwei Yang ◽  
Jimin Wang ◽  
Yao Cheng ◽  
Ginger E. Dutschman ◽  
Hiromichi Tanaka ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Steady State ◽  
Reverse Transcriptase ◽  
Structural Basis ◽  
Mechanism Of Inhibition ◽  
Steady State Kinetic ◽  
Immunodeficiency Virus ◽  
State Kinetic ◽  
Hiv 1

ABSTRACT 2′,3′-Didehydro-3′-deoxy-4′-ethynylthymidine (4′-Ed4T), a recently discovered nucleoside reverse transcriptase (RT) inhibitor, exhibits 5- to 10-fold-higher activity against human immunodeficiency virus type 1 (HIV-1) and less cytotoxicity than does its parental compound d4T (stavudine). Using steady-state kinetic approaches, we have previously shown that (i) 4′-ethynyl-d4T triphosphate (4′-Ed4TTP) inhibits HIV-1 RT more efficiently than d4TTP does and (ii) its inhibition efficiency toward the RT M184V mutant is threefold less than that toward wild-type (wt) RT. In this study we used pre-steady-state kinetic approaches in an attempt to understand its mechanism of inhibition. With wt and the M184V mutant RTs, 4′-Ed4TTP has three- to fivefold-lower Kd (dissociation constant) values than d4TTP, while d4TTP has up to eightfold-higher Kd values than dTTP. Inhibition is more effective in DNA replication with RNA template than with DNA template. In general, the M184V mutant exhibits poorer binding for all three nucleoside triphosphates than does wt RT. The structural basis for the lower binding affinity of d4TTP than of dTTP could be the lack of hydrogen bonds from the missing 3′-hydroxyl group in d4TTP to the backbone amide of Y115 and also to the side chain of Q151. The structural basis for the higher binding affinity of 4′-Ed4TTP than of d4TTP could be the additional binding of the 4′-ethynyl group in a preformed hydrophobic pocket by A114, Y115, M184, F160, and part of D185.

scholarly journals Subtype-Specific Differences in the Human Immunodeficiency Virus Type 1 Reverse Transcriptase Connection Subdomain of CRF01_AE Are Associated with Higher Levels of Resistance to 3′-Azido-3′-Deoxythymidine

2009 ◽  
Vol 83 (17) ◽  
pp. 8502-8513 ◽  
Author(s):  
Krista A. Delviks-Frankenberry ◽  
Galina N. Nikolenko ◽  
Frank Maldarelli ◽  
Saiki Hase ◽  
Yutaka Takebe ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Rnase H ◽  
Wild Type ◽  
Dna Templates ◽  
Subtype B ◽  
Immunodeficiency Virus ◽  
Azt Resistance ◽  
Hiv 1 ◽  
Rna And Dna

ABSTRACT We previously shown that mutations in the connection (CN) subdomain of human immunodeficiency virus type 1 (HIV-1) subtype B reverse transcriptase (RT) increase 3′-azido-3′-deoxythymidine (AZT) resistance in the context of thymidine analog mutations (TAMs) by affecting the balance between polymerization and RNase H activity. To determine whether this balance affects drug resistance in other HIV-1 subtypes, recombinant subtype CRF01_AE was analyzed. Interestingly, CRF01_AE containing TAMs exhibited 64-fold higher AZT resistance relative to wild-type B, whereas AZT resistance of subtype B containing the same TAMs was 13-fold higher, which in turn correlated with higher levels of AZT-monophosphate (AZTMP) excision on both RNA and DNA templates. The high level of AZT resistance exhibited by CRF01_AE was primarily associated with the T400 residue in wild-type subtype AE CN subdomain. An A400T substitution in subtype B enhanced AZT resistance, increased AZTMP excision on both RNA and DNA templates, and reduced RNase H cleavage. Replacing the T400 residue in CRF01_AE with alanine restored AZT sensitivity and reduced AZTMP excision on both RNA and DNA templates, suggesting that the T400 residue increases AZT resistance in CRF01_AE at least in part by directly increasing the efficiency of AZTMP excision. These results show for the first time that CRF01_AE exhibits higher levels of AZT resistance in the presence of TAMs and that this resistance is primarily associated with T400. Our results also show that mixing the RT polymerase, CN, and RNase H domains from different subtypes can underestimate AZT resistance levels, and they emphasize the need to develop subtype-specific genotypic and phenotypic assays to provide more accurate estimates of clinical drug resistance.

scholarly journals Kinetic Characterization of Human Immunodeficiency Virus Type-1 Protease-resistant Variants

1996 ◽  
Vol 271 (30) ◽  
pp. 17979-17985 ◽  
Author(s):  
S. Pazhanisamy ◽  
Cameron M. Stuver ◽  
Aine B. Cullinan ◽  
Nara Margolin ◽  
B. G. Rao ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Kinetic Characterization ◽  
Immunodeficiency Virus
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