scholarly journals The Glutamine Side Chain at Position 91 on the β5a−β5b Loop of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Is Required for Stabilizing the dNTP Binding Pocket

Biochemistry ◽  
2011 ◽  
Vol 50 (37) ◽  
pp. 8067-8077 ◽  
Author(s):  
Nootan Pandey ◽  
Chaturbhuj A. Mishra ◽  
Dinesh Manvar ◽  
Alok K. Upadhyay ◽  
Tanaji T. Talele ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Binding Pocket ◽  
Side Chain ◽  
Immunodeficiency Virus ◽  
Dntp Binding

scholarly journals Nonnucleoside Inhibitor Binding Affects the Interactions of the Fingers Subdomain of Human Immunodeficiency Virus Type 1 Reverse Transcriptase with DNA

2004 ◽  
Vol 78 (7) ◽  
pp. 3387-3397 ◽  
Author(s):  
Elena N. Peletskaya ◽  
Alex A. Kogon ◽  
Steven Tuske ◽  
Edward Arnold ◽  
Stephen H. Hughes
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Active Site ◽  
Cross Linking ◽  
Immunodeficiency Virus ◽  
Dntp Binding ◽  
Hiv 1

ABSTRACT Site-directed photoaffinity cross-linking experiments were performed by using human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) mutants with unique cysteine residues at several positions (i.e., positions 65, 67, 70, and 74) in the fingers subdomain of the p66 subunit. Since neither the introduction of the unique cysteine residues into the fingers nor the modification of the SH groups of these residues with photoaffinity cross-linking reagents caused a significant decrease in the enzymatic activities of RT, we were able to use this system to measure distances between specific positions in the fingers domain of RT and double-stranded DNA. HIV-1 RT is quite flexible. There are conformational changes associated with binding of the normal substrates and nonnucleoside RT inhibitors (NNRTIs). Cross-linking was used to monitor intramolecular movements associated with binding of an NNRTI either in the presence or in the absence of an incoming deoxynucleoside triphosphate (dNTP). Binding an incoming dNTP at the polymerase active site decreased the efficiency of cross-linking but caused only modest changes in the preferred positions of cross-linking. This finding suggests that the fingers of p66 are closer to an extended template in the “open” configuration of the enzyme with the fingers away from the active site than in the closed configuration with the fingers in direct contact with the incoming dNTP. NNRTI binding caused increased cross-linking in experiments with diazirine reagents (especially with a diazirine reagent with a longer linker) and a moderate shift in the preferred sites of interaction with the template. Cross-linking occurred closer to the polymerase active site for RTs modified at positions 70 and 74. The effects of NNRTI binding were more pronounced in the absence of a bound dNTP; pretreatment of HIV-1 RT with an NNRTI reduced the effect of dNTP binding. These observations can be explained if the binding of NNRTI causes a decrease in the flexibility in the fingers subdomain of RT-NNRTI complex and a decrease in the distance from the fingers to the template extension.

scholarly journals Influence of naturally occurring insertions in the fingers subdomain of human immunodeficiency virus type 1 reverse transcriptase on polymerase fidelity and mutation frequencies in vitro

2006 ◽  
Vol 87 (2) ◽  
pp. 419-428 ◽  
Author(s):  
Kenneth Curr ◽  
Snehlata Tripathi ◽  
Johan Lennerstrand ◽  
Brendan A. Larder ◽  
Vinayaka R. Prasad
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Nucleoside Analogue ◽  
Binding Pocket ◽  
Resistance Mutations ◽  
Immunodeficiency Virus

The fingers subdomain of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is a hotspot for nucleoside analogue resistance mutations. Some multi-nucleoside analogue-resistant variants contain a T69S substitution along with dipeptide insertions between residues 69 and 70. This set of mutations usually co-exists with classic zidovudine-resistance mutations (e.g. M41L and T215Y) or an A62V mutation and confers resistance to multiple nucleoside analogue inhibitors. As insertions lie in the vicinity of the dNTP-binding pocket, their influence on RT fidelity was investigated. Commonly occurring insertion mutations were selected, i.e. T69S-AG, T69S-SG and T69S-SS alone, in combination with 3′-azido-2′,3′-deoxythymidine-resistance mutations M41L, L210W, R211K, L214F, T215Y (LAGAZ and LSGAZ) or with an alternate set where A62V substitution replaces M41L (VAGAZ, VSGAZ and VSSAZ). Using a lacZα gapped duplex substrate, the forward mutation frequencies of recombinant wild-type and mutant RTs bearing each of the above sets of mutations were measured. All of the mutants displayed significant decreases in mutation frequencies. Whereas the dipeptide insertions alone showed the least decrease (4·0- to 7·5-fold), the VAG series showed an intermediate reduction (5·0- to 11·4-fold) and the LAG set showed the largest reduction in mutation frequencies (15·3- and 16·3-fold for LAGAZ and LSGAZ, respectively). Single dNTP exclusion assays for mutants LSGAZ and LAGAZ confirmed their large reduction in misincorporation efficiencies. The increased in vitro fidelity was not due to excision of the incorrect nucleotide via ATP-dependent removal. There was also no direct correlation between increased fidelity and template–primer affinity, suggesting a change in the active site that is conducive to better discrimination during dNTP insertion.

scholarly journals Drug Resistance Mutations in the Nucleotide Binding Pocket of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Differentially Affect the Phosphorolysis-Dependent Primer Unblocking Activity in the Presence of Stavudine and Zidovudine and Its Inhibition by Efavirenz

2005 ◽  
Vol 49 (1) ◽  
pp. 342-349 ◽  
Author(s):  
Emmanuele Crespan ◽  
Giada A. Locatelli ◽  
Reynel Cancio ◽  
Ulrich Hübscher ◽  
Silvio Spadari ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Drug Resistance ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Nucleotide Binding ◽  
Binding Pocket ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) derivatives with D113E, Y115F, F116Y, Q151E/N, and M184V mutations were studied for their phosphorolysis-mediated resistance to the nucleoside RT inhibitors (NRTIs) zidovudine and stavudine and for their inhibition by the nonnucleoside analogs (NNRTIs) efavirenz and nevirapine. The results presented here indicate that these single amino acid substitutions within the nucleotide binding pocket of the viral RT can independently affect different enzymatic properties, such as catalytic efficiency, drug binding, and phosphorolytic activity. Moreover, small local alterations of the physicochemical properties of the microenvironment around the active site can have profound effects on some NRTIs while hardly affecting other ones. In conclusion, even though different mutations within the nucleotide binding pocket of HIV-1 RT can result in a common phenotype (i.e., drug resistance), the molecular mechanisms underlying this phenotype can be very different. Moreover, the same mutation can give rise to different phenotypes depending on the nature of the substrates and/or inhibitors.

A Role for dNTP Binding of Human Immunodeficiency Virus Type 1 Reverse Transcriptase in Viral Mutagenesis†

Biochemistry ◽  
2004 ◽  
Vol 43 (15) ◽  
pp. 4490-4500 ◽  
Author(s):  
Kellie K. Weiss ◽  
Renxiang Chen ◽  
Mark Skasko ◽  
Holly M. Reynolds ◽  
Kwi Lee ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Immunodeficiency Virus ◽  
Viral Mutagenesis ◽  
Dntp Binding
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