Influence of Drug Resistance Mutations on the Activity of HIV-1 Subtypes A and B Integrases: a Comparative Study

Abstract

Integration of human immunodeficiency virus (HIV-1) DNA into the genome of an infected cell is one of the key steps in the viral replication cycle. The viral enzyme integrase (IN), which catalyzes the integration, is an attractive target for the development of new antiviral drugs. However, the HIV-1 therapy often results in the IN gene mutations inducing viral resistance to integration inhibitors. To assess the impact of drug resistance mutations on the activity of IN of HIV-1 subtype A strain FSU-A, which is dominant in Russia, variants of the consensus IN of this subtype containing the primary resistance mutations G118R and Q148K and secondary compensatory substitutions E138K and G140S were prepared and characterized. Comparative study of these enzymes with the corresponding mutants of IN of HIV-1 subtype B strains HXB-2 was performed. The mutation Q148K almost equally reduced the activity of integrases of both subtypes. Its negative effect was partially compensated by the secondary mutations E138K and G140S. Primary substitution G118R had different influence on the activity of proteins of the subtypes A and B, and the compensatory effect of the secondary substitution E138K also depended on the viral subtype. Comparison of the mutants resistance to the known strand transfer inhibitors raltegravir and elvitegravir, and a new inhibitor XZ-259 (a dihydro-1H-isoindol derivative), showed that integrases of both subtypes with the Q148K mutation were insensitive to raltegravir and elvitegravir but were effectively inhibited by XZ-259. The substitution G118R slightly reduced the efficiency of IN inhibition by raltegravir and elvitegravir and caused no resistance to XZ_259.

Consensus Integrase of a New HIV-1 Genetic Variant CRF63_02A1

“…IN is involved in two successive reactions during viral replication: 3’-end processing (in which it catalyzes the cleavage of the GT dinucleotide from both 3’-ends of viral DNA) and strand transfer (insertion of the processed viral DNA into the cell DNA). Both of these reactions can be studied in vitro using standard protocols [25]. We used a standard synthetic DNA duplex U5B/ U5A mimicking the U5 region of HIV-1 DNA LTR for the 3’-processing reaction.…”

“…5 ). Of note, the profiles of the strand transfer products were different for IN_A and IN_B [25]. The profiles of the integration products can vary if the modes of complex formation between IN and target DNA differ.…”

“…Consensus IN_CRF and IN_A proteins and those with the mutations Q148K/G140S and G118R/E138K were expressed in Escherichia coli strain Rosetta (DE3) (Novagen) and purified according to [24, 25]. The proteins were analyzed by 12% Laemmli PAGE, followed by staining with SimplyBlueTM SafeStain (Invitrogen, USA).…”

“…The radioactive 32 P-label was inserted at the 5’-end of the U5B and U5B-2 oligonucleotides, and DNA substrates were formed as described in [25].…”

“…For the 3’-end processing reaction, 5 nM U5B/U5A duplex (with 32P-labeled U5B-chain) was incubated with 100 nM IN in buffer A as described in [25]. The reaction products were precipitated and analyzed by electrophoresis in 20% polyacrylamide/7 M urea gel in TBE buffer.…”