Structure-function analyses unravel distinct effects of allosteric inhibitors of HIV-1 integrase on viral maturation and integration

Abstract

Recently, a new class of HIV-1 integrase (IN) inhibitors with a dual mode of action, called IN-LEDGF/p75 allosteric inhibitors (INLAIs), was described. Designed to interfere with the IN-LEDGF/p75 interaction during viral integration, unexpectedly, their major impact was on virus maturation. This activity has been linked to induction of aberrant IN multimerization, while inhibition of the IN-LEDGF/p75 interaction accounts for weaker antiretroviral effect at integration. Since these dual activities result from INLAI binding to IN at a single binding site, we expected that these activities co-evolved together, driven by the affinity for IN. Using an original INLAI, MUT-A, and its activity on an Ala-125 (A125) IN variant, we found that these two activities on A125 IN can be fully dissociated: MUT-A-induced IN multimerization and the formation of eccentric condensates in viral particles, that are responsible for inhibition of virus maturation, were lost, while inhibition of the IN-LEDGF/p75 interaction and consequently integration, was fully retained. Hence the mere binding of INLAI to A125 IN is insufficient to promote the conformational changes of IN required for aberrant multimerization. By analyzing the Xray structures of MUT-A bound to the IN catalytic core domain (CCD) with or without the A125 polymorphism, we discovered that the loss of IN multimerization is due to stabilization of the A125 IN variant CCD dimer, highlighting the importance of the CCD dimerization energy for IN multimerization. Our study reveals that affinity for the LEDGF/p75-binding pocket is not sufficient to induce INLAI-dependent IN multimerization and the associated inhibition of viral maturation.The integrase (IN) protein of Human Immunodeficiency Virus type 1 (HIV-1) catalyzes the stable insertion of the viral cDNA genome into the host cell chromatin, a step of the viral life cycle that is required for efficient viral gene expression. Integration occurs via a two-step reaction where IN initially cleaves after a conserved CA dinucleotide at the 3' end of the viral cDNA genome to free a 3'-OH group (3' processing), which is next used to carry out a nucleophilic attack on cellular chromosomal DNA (strand transfer).IN is one of the preferred targets for the development of antiretroviral (ARV) drugs. However, given the high genetic variability of HIV-1, IN mutations conferring cross-resistance to the first generation INSTIs, RAL and EVG, were described in patients receiving INSTI-containing regimens (2). The second generation INSTI DTG has a higher genetic barrier and conserves good ARV activity against a number of RAL-and EVGresistant strains. Recent reports showed that Bictegravir, a second generation INSTI still in development from Gilead Sciences, has a resistance profile similar to DTG (3). Nevertheless, DTG and Bictegravir are sensitive to the most detrimental INSTI resistant mutations albeit at lower levels than first generation INSTIs (4). Therefore, the development of small molecule inhibitors impairing IN functions with dis...

Multifaceted HIV integrase functionalities and therapeutic strategies for their inhibition

“…242). Because Asn is often found at position 124 and A124D conferred significant resistance to the quinoline ALLINI BI-D (165), recent studies have tested antiviral activities of compounds against HIV-1 strains harboring representative 124/125 polymorphisms, such as Thr/Thr, Thr/ Ala, Ala/Thr, Ala/Ala, Asn/Thr, and Asn/Ala (200,207,215). Whereas Asn-124 and Ala-125 each conferred ϳ50-fold resistance to pyridine ALLINI compound 20 (200) and thiophene ALLINI MUT-A (215), respectively, naphthyridine ALLINI compound 23 remained active against such strains (207).…”

“…On the flip side, thiophene ALLINI MUT-A efficiently inhibited LEDGF/p75 binding to Ala-125containing IN yet in large part lost the ability to hypermultimerize this polymorphic variant. In this case, downstream interactions of the CCD-engaged ALLINI with the CTD of another IN oligomer seemed to underlie the loss of hypermultimerization activity (215). Thus, resistance to ALLINIs can be instilled by loss of direct IN binding contact, a shift in compound position within the CCD-binding pocket, or inability of bound compound to mediate downstream interactions.…”

Impact of LEDGIN treatment during virus production on residual HIV-1 transcription

“…Later it was found that LEDGINs also inhibit late stage replication (the so-called 'late effect' , Fig. 1) [63][64][65][66][67][68]. Viral particles produced in the presence of LEDGINs display morphological defects due to LEDGIN-induced IN multimerization [63][64][65][66][67].…”

Integrase-RNA interactions underscore the critical role of integrase in HIV-1 virion morphogenesis

“…A hallmark morphological defect of these viruses is the formation of aberrant viral particles with viral ribonucleoprotein (vRNP) complexes mislocalized outside of the conical CA lattice [11, 15, 21–23, 26–28]. Strikingly similar morphological defects are observed in virions produced from cells treated with allosteric integrase inhibitors (ALLINIs, also known as LEDGINs, NCINIs, INLAIs or MINIs) [26, 27, 48-55]. ALLINIs induce aberrant IN multimerization in virions by engaging the V-shaped pocket at the IN dimer interface, which also provides a principal binding site for the host integration targeting cofactor lens epithelium-derived growth factor (LEDGF)/p75 [50, 54, 56–60].…”

“…The structural basis for IN binding to RNA is not yet known; however, in vitro evidence indicates that IN binds RNA as lower-order multimers, and conversely RNA binding may prevent the formation of higher order IN multimers [28]. Notably, aberrant IN multimerization underlies the inhibition of IN-RNA interactions by ALLINIs [28] and subsequent defects in virion maturation [26-28, 48, 49, 51-55]. Therefore, it seems plausible that class II IN substitutions may exert their effect on virus replication by adversely affecting functional IN multimerization.…”