| 1. |
- Meyer, Peter R, et al.
(författare)
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Effects of dipeptide insertions between codons 69 and 70 of human immunodeficiency virus type 1 reverse transcriptase on primer unblocking, deoxynucleoside triphosphate inhibition, and DNA chain elongation
- 2003
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Ingår i: Journal of Virology. - 0022-538X .- 1098-5514. ; 77:6, s. 3871-3877
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Tidskriftsartikel (refereegranskat)abstract
- Finger insertion mutations of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) (T69S mutations followed by various dipeptide insertions) have a multinucleoside resistance phenotype that can be explained by decreased sensitivity to deoxynucleoside triphosphate (dNTP) inhibition of the nucleotide-dependent unblocking activity of RT. We show that RTs with SG or AG (but not SS) insertions have three- to fourfold-increased unblocking activity and that all three finger insertion mutations have threefold-decreased sensitivity to dNTP inhibition. The additional presence of M41L and T215Y mutations increased unblocking activity for all three insertions, greatly reduced the sensitivity to dNTP inhibition, and resulted in defects in in vitro DNA chain elongation. The DNA chain elongation defects were partially repaired by additional mutations at positions 210, 211, and 214. These results suggest that structural communication between the regions of RT defined by these mutations plays a role in the multinucleoside resistance phenotype.
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| 2. |
- Curr, Kenneth, et al.
(författare)
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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
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Ingår i: Journal of General Virology. - : Microbiology Society. - 0022-1317 .- 1465-2099. ; 87, s. 419-428
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Tidskriftsartikel (refereegranskat)abstract
- 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 (LAG(AZ) and LSG(AZ)) or with an alternate set where A62V substitution replaces M41L (VAG(AZ), VSG(AZ) and VSS(AZ)). Using a lacZalpha 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 LAG(AZ) and LSG(AZ), respectively). Single dNTP exclusion assays for mutants LSG(AZ) and LAG(AZ) 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.
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