title: Consensus HIV-1 subtype A integrase and its raltegravir-resistant variants: design and characterization of the enzymatic properties.
abstract: Model studies of the subtype B and non-subtype B integrases are still required to compare their susceptibility to antiretroviral drugs, evaluate the significance of resistance mutations and identify the impact of natural polymorphisms on the level of enzymatic reactivity. We have therefore designed the consensus integrase of the HIV-1 subtype A strain circulating in the former Soviet Union territory (FSU-A) and two of its variants with mutations of resistance to the strand transfer inhibitor raltegravir. Their genes were synthesized, and expressed in E coli; corresponding His-tagged proteins were purified using the affinity chromatography. The enzymatic properties of the consensus integrases and their sensitivity to raltegravir were examined in a series of standard in vitro reactions and compared to the properties of the integrase of HIV-1 subtype B strain HXB2. The consensus enzyme demonstrated similar DNA-binding properties, but was significantly more active than HXB-2 integrase in the reactions of DNA cleavage and integration. All integrases were equally susceptible to inhibition by raltegravir and elvitegravir, indicating that the sporadic polymorphisms inherent to the HXB-2 enzyme have little effect on its susceptibility to drugs. Insensitivity of the mutated enzymes to the inhibitors of strand transfer occurred at a cost of a 30-90% loss of the efficacies of both 3'-processing and strand transfer. This is the first study to describe the enzymatic properties of the consensus integrase of HIV-1 clade A and the effects of the resistance mutations when the complex actions of sporadic sequence polymorphisms are excluded.
authors.affiliation: Lomonosov Moscow State University, Belozersky Institute of Physical Chemical Biology and Chemistry Department, Leninskie gory 1/40, 119991 Moscow, Russia.
authors.forename: Olga
authors.initials: O
authors.lastname: Shadrina
authors.affiliation: Engelhardt Institute of Molecular Biology, Vavilov str 32, 119991 Moscow, Russia; Ivanovsky Institute of Virology, Gamaleja str 16, 123098 Moscow, Russia; Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Nobels väg 16, 17177 Stockholm, Sweden.
authors.forename: Olga
authors.initials: O
authors.lastname: Krotova
authors.affiliation: Lomonosov Moscow State University, Belozersky Institute of Physical Chemical Biology and Chemistry Department, Leninskie gory 1/40, 119991 Moscow, Russia.
authors.forename: Julia
authors.initials: J
authors.lastname: Agapkina
authors.affiliation: Lomonosov Moscow State University, Belozersky Institute of Physical Chemical Biology and Chemistry Department, Leninskie gory 1/40, 119991 Moscow, Russia.
authors.forename: Ekaterina
authors.initials: E
authors.lastname: Knyazhanskaya
authors.affiliation: Lomonosov Moscow State University, Belozersky Institute of Physical Chemical Biology and Chemistry Department, Leninskie gory 1/40, 119991 Moscow, Russia.
authors.forename: Sergey
authors.initials: S
authors.lastname: Korolev
authors.affiliation: Engelhardt Institute of Molecular Biology, Vavilov str 32, 119991 Moscow, Russia; Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Nobels väg 16, 17177 Stockholm, Sweden.
authors.forename: Elizaveta
authors.initials: E
authors.lastname: Starodubova
authors.affiliation: Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Nobels väg 16, 17177 Stockholm, Sweden.
authors.forename: Alecia
authors.initials: A
authors.lastname: Viklund
authors.affiliation: Ivanovsky Institute of Virology, Gamaleja str 16, 123098 Moscow, Russia; Department of Medical Microbiology, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Lukashov
authors.affiliation: Department of Biomolecular Science, University of Urbino "Carla Bo", Via Saffi, 2, 61029 Urbino, Italy.
authors.forename: Mauro
authors.initials: M
authors.lastname: Magnani
authors.affiliation: Department of Laboratory Medicine, Lund University, Sölvegatan 19, SE-205 02 Malmö, Sweden.
authors.forename: Patrik
authors.initials: P
authors.lastname: Medstrand
authors.affiliation: Engelhardt Institute of Molecular Biology, Vavilov str 32, 119991 Moscow, Russia.
authors.forename: Vadim
authors.initials: V
authors.lastname: Karpov
authors.affiliation: Lomonosov Moscow State University, Belozersky Institute of Physical Chemical Biology and Chemistry Department, Leninskie gory 1/40, 119991 Moscow, Russia. Electronic address: gottikh@belozersky.msu.ru.
authors.forename: Marina
authors.initials: M
authors.lastname: Gottikh
authors.affiliation: Ivanovsky Institute of Virology, Gamaleja str 16, 123098 Moscow, Russia; Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Nobels väg 16, 17177 Stockholm, Sweden. Electronic address: maria.issagouliantis@ki.se.
authors.forename: Maria
authors.initials: M
authors.lastname: Isaguliants
mesh_terms: D044966:Anti-Retroviral Agents / Q000627:therapeutic use; D004279:DNA, Viral / Q000235:genetics; D024882:Drug Resistance, Viral / Q000235:genetics; D015658:HIV Infections / Q000188:drug therapy / Q000235:genetics / Q000821:virology*; D019427:HIV Integrase / Q000737:chemistry* / Q000235:genetics; D019428:HIV Integrase Inhibitors / Q000737:chemistry; D015497:HIV-1 / Q000737:chemistry* / Q000201:enzymology / Q000472:pathogenicity; D006801:Humans; D008956:Models, Chemical; D008962:Models, Theoretical; D009154:Mutation; D011760:Pyrrolidinones / Q000627:therapeutic use; D000068898:Raltegravir Potassium
chemical_list: D044966:Anti-Retroviral Agents; D004279:DNA, Viral; D019428:HIV Integrase Inhibitors; D011760:Pyrrolidinones; D000068898:Raltegravir Potassium; D019427:HIV Integrase
keywords: Catalytic activity; Consensus; Drug resistance; HIV-1 integrase; Raltegravir; Subtype A