Polymorphic substitution E157Q in HIV-1 integrase increases R263K-mediated dolutegravir resistance and decreases DNA binding activity

Abstract

Objectives: The E157Q substitution in HIV-1 integrase (IN) is a relatively common natural polymorphism associated with HIV resistance to IN strand transfer inhibitors (INSTIs). Although R263K is the most common resistance substitution for the INSTI dolutegravir, an INSTI treatment-experienced individual recently failed dolutegravir-based therapy, with E157Q being the only resistance-associated change reported. Given that different resistance pathways can sometimes synergize to confer high levels of resistance to antiretroviral drugs, we studied the effects of E157Q in association with R263K. Because Glu157 is thought to lie within the binding site of HIV IN DNA binding inhibitors such as FZ41, we also evaluated DNA binding activity and resistance to IN inhibitors in the presence of E157Q.

Methods: Purified recombinant IN proteins were assessed in cell-free assays for their strand transfer and DNA binding activities. NL4.3 viral stocks harbouring IN mutations were generated and characterized in the presence and absence of IN inhibitors in tissue culture.

Results: E157Q alone had little if any effect on the biochemical activity of IN, and partially restored the activity of R263K-containing IN. The E157Q/R263K double viral mutant displayed infectiousness in culture equivalent to WT, while increasing resistance to dolutegravir by 10-fold compared with lower-level resistance associated with R263K alone. None of the mutations tested showed significant resistance to either raltegravir or FZ41.

Conclusions: This study shows that E157Q may act as a compensatory mutation for R263K. Since E157Q is a natural polymorphism present in 1%-10% of HIV-positive individuals, it may be of particular importance for patients receiving INSTI therapy.

Figure 1.

Strand transfer activities of purified recombinant IN proteins. Biotinylated target DNA incorporation was measured by Eu–streptavidin fluorescence as a function of (a) protein concentration or (b) target DNA concentration, displayed as relative fluorescence units (RLU). (c) K_M values for each IN mutation were obtained from the curves in (b). (d) Enzyme proficiency was quantified by dividing V_max [taken from plateau in (b)] by K_M. Error bars display SEM. *P ≤ 0.05, **P ≤ 0.01 and ***P ≤ 0.001 by Student's t-test.

Figure 2.

Cell-free DNA binding activities of purified recombinant IN proteins. (a) Enzymatic activity as a function of increasing HIV LTR DNA concentration, displayed as relative fluorescence units (RLU). (b) K_M values for each mutation were obtained from the curves in (a). (c) Enzyme proficiency was quantified by dividing V_max [taken from plateau in (a)] by K_M. Error bars display SEM. *P ≤ 0.05 by Student's t-test.