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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

Kenneth Curr^1,

¹ Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA
² Department of Biochemistry and Molecular Biology, UMDNJ – New Jersey Medical School, Newark, NJ 07103, USA
³ Emory University School of Medicine, Veterans Affairs Medical Center, Decatur, GA 30033, USA
⁴ HIV Resistance Response Database Initiative, Cambridge, UK

Correspondence
Vinayaka R. Prasad
prasad{at}aecom.yu.edu

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 lacZ 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.

Present address: Gladstone Institute of Virology and Immunology, University of California San Francisco, San Francisco, CA, USA.