The role of HIV-1 Rev during allosteric inhibition of integrase

Abstract

The HIV-1 integration step is a pertinent process in the HIV-1 replication cycle that integrates the viral genome into the host chromosome. HIV-1 integrase is crucial in facilitating the integration step. Integration is dependent on many co-factors and viral- as well as host accessory proteins. Lens epithelium-derived growth factor (LEDGF/p75) is a host protein that binds to the hydrophobic pocket of integrase formed by a dimer of integrase. The function of LEDGF/p75 is to tether integrase to the host chromosome for successful integration, and consequently provides a drug target for a novel class of HIV-1 inhibitors. The latest studies on the HIV-1 integration process revealed that the HIV-1 regulatory factor, Rev, plays a cardinal role in the regulation of the integration step. Previous studies have demonstrated that Rev and LEDGF/p75 compete for the hydrophobic pocket located on integrase where Rev is able to dissociate the integrase-LEDGF/p75 complex and subsequently form integrase-Rev complexes and LEDGF/p75-Rev complexes. Interestingly, the latest class of HIV-1 inhibitors known as allosteric integrase inhibitors (ALLINIs), compete with LEDGF/p75 and Rev for the same hydrophobic site located on integrase. Further to this, ALLINIs and Rev inhibit the integration step in the early phase of the replication cycle by inducing an integrase oligomerization shift. Consequently, inactive multimers of integrase are formed that are unable to bind to the host chromosome thus preventing integration. Due to the similarities between Rev and ALLINIs in the mechanism of inhibiting integration and the shared binding site on integrase, this study postulated that ALLINIs interfere with the interaction between Rev and integrase. The first objective was to determine whether ALLINIs, Mut 101 and CX05168, had an effect on HIV-1 subtype C Rev and integrase through in vitro selection resistant studies followed by genotypic- as well as phenotypic analysis. This study reports the first mutations against Mut 101 within the HIV-1 subtype C IN gene. The integrase mutations identified include: H171L for the 05ZAFV3 (FV 3) isolate and Q164L, L172I, and L172I/H171Q for the 05ZAFV26 (FV 26) isolate. In vitro phenotypic inhibition studies demonstrated the phenotypic significance of the mutations through a shift in EC50 values (0.53 ± 0.01µM for drug naive; 0.21 ± 0.11µM for Q164L mutated isolate; 2.67 ± 0.95µM for L172I mutated isolate) and the infectious ability of the isolates (FV 3 H171L and FV 26 L172I/H171Q). Molecular docking studies further confirmed the effects of the mutations by predicting the interaction between integrase harbouring the mutations and Mut 101. An increase in the binding energies of the predicted models suggests that the presence of mutations are responsible for weaker interactions between integrase harbouring the identified mutations and Mut 101. Data for in vitro resistance studies selected by CX05168 could not be generated as CX05168 failed to inhibit viral replication of the FV 3 and FV 26 isolates at its reported EC50 for HIV-1 subtype B isolates. This prompted in vitro phenotypic inhibition studies to determine the EC50 values of CX05168 against subtype C isolates (FV 3 = 4.3 ± 0.4µM and FV 26 = 40.1 ± 0.8µM) which was significantly higher than the reported EC50 of CX05168 against HIV-1 subtype B (2.35 ± 0.28µM). Furthermore, genotypic analysis of the rev gene was not achieved as the amplification thereof faced continuous challenges. The second objective was to delineate how Rev affect the oligomerization of integrase in the presence of Mut 101 and CX05168. AlphaScreen dimerization assays, BS3 crosslinking studies and dynamic light scattering experiments confirmed the multimerization of integrase in the presence of Rev, Mut 101 and CX05168. However, the aberrant multimerization of integrase induced by the ALLINIs was significantly reduced in the presence of Rev. This finding suggests that Rev and the ALLINIs have distinct pathways to induce integrase multimerization and that Rev indeed modulates the multimerization mechanism of integrase induced by ALLINIs. The third objective aimed to investigate the integrase-Rev interaction in the presence of Mut 101 and CX05168. Through an ELISA method, the dissociation constant (Kd) of integrase-Rev was calculated at 461 ± 5.65nM. Order of addition ELISA experiments demonstrated that ALLINIs do not interfere with the integrase-Rev interaction once Rev is already bound to integrase. However, when ALLINIs are added to integrase before the addition of Rev, a significant shift in Kd was observed. The Kd increased to 850 ± 70.71nM in the presence of Mut 101 and 1010 ± 127.27nM in the presence of CX05168. The integrase-Rev interaction was confirmed through isothermal titration calorimetry where the Kd (419nM) obtained significantly correlated with the Kd calculated through ELISA studies. This study is the first to report the thermodynamic properties of the integrase-Rev interaction. The interaction elicited an exothermic reaction with an enthalphy change (∆H) = -5350J/mol, a temperature entropy change (T∆S) = 103.9J/mol.K, stoichiometry of 1 and free energy (∆G) = -5453.9J/mol which indicated that the reaction was enthalpically driven. Thermodynamics of the integrase-Rev interaction could not be established in the presence of Mut 101 and CX05168 suggesting that multimerization of integrase could have intercepted the reaction. Overall, this study reports novel data and provides insight on the hypothesis of the study which establishes that ALLINIs distort the interaction between Rev and integrase and has downstream effects on integrase multimerization. The data presented in this study may lead to a better understanding of ALLINIs in the replication process, especially in HIV-1 subtype C isolates, and may add value to the development process of future ALLINIs.