Consensus Integrase of a New HIV-1 Genetic Variant CRF63_02A1

Abstract

The high genetic variability of the human immunodeficiency virus (HIV-1) leads to a constant emergence of new genetic variants, including the recombinant virus CRF63_02A1, which is widespread in the Siberian Federal District of Russia. We studied HIV-1 CRF63_02A1 integrase (IN_CRF) catalyzing the incorporation of viral DNA into the genome of an infected cell. The consensus sequence was designed, recombinant integrase was obtained, and its DNA-binding and catalytic activities were characterized. The stability of the IN_CRF complex with the DNA substrate did not differ from the complex stability for subtype A and B integrases; however, the rate of complex formation was significantly higher. The rates and efficiencies of 3'-processing and strand transfer reactions catalyzed by IN_CRF were found to be higher, too. Apparently, all these distinctive features of IN_CRF may result from specific amino acid substitutions in its N-terminal domain, which plays an important role in enzyme multimerization and binding to the DNA substrate. It was also found that the drug resistance mutations Q148K/G140S and G118R/E138K significantly reduce the catalytic activity of IN_CRF and its sensitivity to the strand transfer inhibitor raltegravir. Reduction in sensitivity to raltegravir was found to be much stronger in the case of double-mutation Q148K/G140S.

Keywords: CRF63_02A1 genetic variant; drug resistance mutations; human immunodeficiency virus; integrase; strand transfer inhibitor.

Fig. 1

Amino acid sequences of IN_CRF, IN_A, and IN_B. The amino acids specific to IN_CRF are highlighted in bold and underlined; amino acids specific to other subtypes are underlined; amino acids whose mutations lead to drug resistance of the virus are shown with rectangles; amino acids of the catalytic domain are shown in red

Fig. 2

SDS-PAGE analysis of purified consensus IN_CRF and its mutant forms G118R/E138K and Q148K/ G140S. Lane 1 – IN_CRF; lane 2 – IN_A; lane 3 – IN_CRF (G118R/E138K); lane 4 – IN_CRF (Q148K/G140S); MW – molecular weight marker

Fig. 3

Characterization of the DNA-binding activity of IN_CRF as compared to that of IN_A. The average values of at least three independent measurements for which the standard deviation did not exceed 15% are given. A – The dependence between the IN/DNA-substrate complex concentration and IN concentration. B – The kinetics of DNA-substrate fluorescence polarization after DNA binding to IN_CRF and IN_A

Fig. 4

3’-Processing efficiency as a function of time

Fig. 5

Characteristics of the strand transfer reaction catalyzed by IN_CRF and IN_A. The average values of at least three independent measurements for which the standard deviation did not exceed 15% are given. A – Strand transfer kinetics. B – The products of the strand transfer reaction for IN_CRF and IN_A (electrophoretic analysis of the reaction products after 300 and 360 min)

Fig. 6

Electrophoretic analysis of the strand transfer reaction products for IN_CRF (lane 2) and its mutants G118R/ E138K (lane 3) and Q148K/G140S (lane 4) (reaction time 300 min). Lane 1 – DNA control without IN added. The reaction efficiency is shown above the gel