Comparative biochemical analysis of HIV-1 subtype B and C integrase enzymes

Abstract

Background: Integrase inhibitors are currently being incorporated into highly active antiretroviral therapy (HAART). Due to high HIV variability, integrase inhibitor efficacy must be evaluated against a range of integrase enzymes from different subtypes.

Methods: This study compares the enzymatic activities of HIV-1 integrase from subtypes B and C as well as susceptibility to various integrase inhibitors in vitro. The catalytic activities of both enzymes were analyzed in regard to each of 3' processing and strand transfer activities both in the presence and absence of the integrase inhibitors raltegravir (RAL), elvitegravir (EVG), and MK-2048.

Results: Our results show that integrase function is similar with enzymes of either subtype and that the various integrase strand transfer inhibitors (INSTIs) that were employed possessed similar inhibitory activity against both enzymes.

Conclusion: This suggests that the use of integrase inhibitors against HIV-1 subtype C will result in comparable outcomes to those obtained against subtype B infections.

Figure 1

Purification of recombinant subtype B and C integrase enzymes. N-terminal His tags of the enzymes were removed from purified subtype B and C recombinant proteins by thrombin cleavage. Lane 1, protein ladder (10-250 kDa) (New England Biolabs); INB, subtype B integrase; INC, subtype C integrase.

Figure 2

3' Processing assay. One representative reaction (out of five reactions) is illustrated. Recombinant enzyme was incubated at 37°C with templates (radiolabeled double stranded oligonucleotide INT1/2) for the indicated times up to 120 minutes. The 21-mer substrate and 19-mer 3' processing products are indicated.

Figure 3

Strand transfer assay. One representative reaction (out of five reactions) is depicted. Recombinant integrase enzyme was incubated at 37°C for 3 minutes for the initial 3' processing reaction. T35/SK70, double stranded oligonucleotide substrate, was added and reaction tubes were incubated at 37°C for the indicated times up to 120 minutes. The 21-mer substrate, 19-mer 3' processing, and strand transfer products are indicated.

Figure 4

Disintegration assay. One representative reaction (out of five reactions) is portrayed. Recombinant enzyme was incubated at 37°C for the indicated times up to 120 minutes with disintegration template (radiolabeled oligonucleotide D). Disintegration template and product are indicated. (C-), Negative control lane without integrase enzyme. Top panel, subtype B integrase; bottom panel, subtype C integrase.

Figure 5

Inhibition of 3' processing as a function of increasing RAL concentration. Subtype B and C 3' processing activity (presented as relative percentage) in relation to increasing RAL concentration. This graph was prepared with GraphPad Prism 4.0, the combined result of quantification and analyses of at least 3 independent experiments.

Figure 6

Inhibition of 3' processing as a function of increasing MK-2048 concentration. Subtype B and C 3' processing activity (presented as relative percentage) in relation to increasing MK-2048 concentration. This graph was prepared with GraphPad Prism 4.0, the combined result of quantification and analyses of at least 3 independent experiments.

Figure 7

Inhibition of 3' processing as a function of increasing EVG concentration. Subtype B and C 3' processing activity (presented as relative percentage) in relation to increasing EVG concentration. This graph was prepared with GraphPad Prism 4.0, the combined result of quantification and analyses of at least 3 independent experiments.

Figure 8

Inhibition of strand transfer as a function of increasing RAL concentration. Subtype B and C strand transfer activity (presented as relative percentage) in relation to increasing RAL concentration. This graph was prepared with GraphPad Prism 4.0, the combined result of quantification and analyses of at least 3 independent experiments.

Figure 9

Inhibition of strand transfer as a function of increasing EVG concentration. Subtype B and C strand transfer activity (presented as relative percentage) in relation to increasing EVG concentration. This graph was prepared with GraphPad Prism 4.0, the combined result of quantification and analyses of at least 3 independent experiments.

Figure 10

Inhibition of strand transfer as a function of increasing MK-2048 concentration. Subtype B and C strand transfer activity (presented as relative percentage) in relation to increasing MK-2048 concentration. This graph was prepared with GraphPad Prism 4.0, the combined result of quantification and analyses of at least 3 independent experiments.

Figure 11

Inhibition of disintegration as a function of increasing RAL concentration. Subtype B and C disintegration activity (presented as relative percentage) in reaction to increasing RAL concentration. This graph was prepared with GraphPad Prism 4.0, the combined result of quantification and analyses of 3 independent experiments.

Figure 12

Inhibition of disintegration as a function of increasing MK-2048 concentration. Subtype B and C disintegration activity (presented as relative percentage) in reaction to increasing MK-2048 concentration. This graph was prepared with GraphPad Prism 4.0, the combined result of quantification and analyses of 3 independent experiments.

Figure 13

Inhibition of disintegration as a function of increasing EVG concentration. Subtype B and C disintegration activity (presented as relative percentage) in reaction to increasing EVG concentration. This graph was prepared with GraphPad Prism 4.0, the combined result of quantification and analyses of 3 independent experiments.