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AZT resistance alters enzymatic properties and creates an ATP-binding site in SFVmac reverse transcriptase

DOI zum Zitieren dieses Dokuments: https://doi.org/10.1186/s12977-015-0147-7

Title data

Schneider, Anna ; Schweimer, Kristian ; Rösch, Paul ; Wöhrl, Birgitta M.:
AZT resistance alters enzymatic properties and creates an ATP-binding site in SFVmac reverse transcriptase.
In: Retrovirology. Vol. 12 (2015) Issue 21 .
ISSN 1742-4690
DOI: https://doi.org/10.1186/s12977-015-0147-7

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Abstract

Background The replication of simian foamy virus from macaques can be inhibited by the nucleoside reverse transcriptase inhibitor azidothymidine (AZT, zidovudine). Four substitutions in the protease-reverse transcriptase (PR-RT) protein (K211I, I224T, S345T, E350K) are necessary to obtain highly AZT resistant and fully replication competent virus. AZT resistance is based on the excision of the incorporated AZTMP in the presence of ATP. I224T is a polymorphism which is not essential for AZT resistance per se, but is important for regaining efficient replication of the resistant virus. Results We constructed PR-RT enzymes harboring one to four amino acid substitutions to analyze them biochemically and to determine their ability to remove the incorporated AZTMP. S345T is the only single substitution variant exhibiting significant AZTMP excision activity. Although K211I alone showed no AZTMP excision activity, excision efficiency doubled when K211I was present in combination with S345T and E350K. K211I also decreased nucleotide binding affinity and increased fidelity. NMR titration experiments revealed that a truncated version of the highly AZT resistant mt4 variant, comprising only the fingers-palm subdomains was able to bind ATP with a K D -value of ca. 7.6 mM, whereas no ATP binding could be detected in the corresponding wild type protein. We could show by NMR spectroscopy that S345T is responsible for ATP binding, probably by making a tryptophan residue accessible. Conclusion Although AZT resistance in SFVmac is based on excision of the incorporated AZTMP like in HIV-1, the functions of the resistance substitutions in SFVmac PR-RT appear to be different. No mutation resulting in an aromatic residue like F/Y215 in HIV, which is responsible for π-π-stacking interactions with ATP, is present in SFVmac. Instead, S345T is responsible for creating an ATP binding site, probably by making an already existing tryptophan more accessible, which in turn can interact with ATP. This is in contrast to HIV-1 RT, in which an ATP binding site is present in the WT RT but differs from that of the AZT resistant enzyme.

Further data

Item Type: Article in a journal
Keywords: AZT resistance; ATP binding Reverse transcriptase; NMR; Foamy virus; HIV
DDC Subjects: 500 Science > 500 Natural sciences
500 Science > 540 Chemistry
Institutions of the University: Faculties > Faculty of Biology, Chemistry and Earth Sciences
Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry
Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Chair Biopolymers
Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Former Professors > Chair Biopolymers - Univ.-Prof. Dr. Paul Rösch
Profile Fields > Advanced Fields
Profile Fields > Advanced Fields > Polymer and Colloid Science
Faculties
Profile Fields
Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Former Professors
Language: English
Originates at UBT: Yes
Date Deposited: 01 Oct 2015 07:49
Last Modified: 08 Feb 2017 10:44
URI: https://epub.uni-bayreuth.de/id/eprint/3088

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