Abstract.
HIV-1 integrase (IN) is an important target for contemporary antiretroviral drug design research. Historically, efforts at inactivating the enzyme have focused upon blocking its active site. However, it has become apparent that new classes of allosteric inhibitors will be necessary to advance the antiretroviral field in light of the emergence of viral strains resistant to contemporary clinically used IN drugs. In this study we have characterized the importance of a close network of IN residues, distant from the active site, as important for the obligatory multimerization of the enzyme and viral replication as a whole. Specifically, we have determined that the configuration of six residues within a highly symmetrical region at the IN dimerization interface, composed of a four-tiered aromatic interaction flanked by two salt bridges, significantly contributes to proper HIV-1 replication.

Additionally, we have utilized a quantitative luminescence assay to examine IN oligomerization and have determined that there is a very low tolerance for amino acid substitutions along this region. Even conservative residue substitutions negatively impacted IN multimerization, resulting in an inactive viral enzyme and a non-replicative virus. We have shown that there is a very low tolerance for amino acid variation at the symmetrical dimeric interface region characterized in this study, and therefore drugs designed to target the amino acid network detailed here could be expected to yield a significantly reduced number of drug-resistant escape mutations compared to contemporary clinically-evaluated antiretrovirals.

Introduction.
HIV-1 integrase (IN) is an enzyme essential for viral replication. After more than a decade of intensive research, the first IN drug - raltegravir – was approved by the FDA in October of 2007. This advance has been a major achievement, but like other HIV-1 antiretroviral drugs targeting reverse transcriptase (RT) and protease, considerable resistance has already emerged following clinical use [1], [2], [3], [4]. IN is critical for the viral life cycle, as it acts to integrate the viral DNA into the host cell chromosomal material. The resulting integrated provirus is invulnerable to current antiretrovirals, and upon upregulation of certain cellular transcription factors, the provirus can be replicated by host cell machinery to generate progeny virus [5], [6], [7]. The provirus can also remain dormant for years in memory T-cells, greatly contributing to the difficulty in eradicating viral infection. In fact, it has been shown that very low levels of HIV-1 transcription can persist in peripheral blood mononuclear cells in patients receiving antiretroviral therapy, exacerbating the problem of emerging drug-resistant viral strains [8], [9], [10], [11].

IN exists as a monomer, dimer, and higher oligomers in solution, and multimerization is essential for its catalytic activity [12], [13], [14]. The amino acid network at the IN dimeric interface is extensive, and it is stabilized by both hydrophobic and electrostatic interactions between four α -helices (α1, α3, α5, and α6) from each monomer, and an additional subunit interface interaction donated by a β-strand from each monomer [5], [15], [16]. It contains three distinct domains: an N-terminal domain (residues 1–50) that binds zinc, a catalytic core domain (residues 50–212) that contains the active site DD(35)E motif and many residues essential for dimerization, and a C-terminal domain (residues 213–288) that possesses nonspecific DNA affinity and is important for IN tetramerization [17], [18], [19]. After viral entry into the host cell, IN associates with RT, the viral RNA genome, and multiple other viral and cellular proteins in a large nucleoprotein complex termed the reverse transcription complex [20], [21]. After reverse transcription is completed, IN cleaves a dinucleotide from the 3′ end of the newly-formed viral DNA at a conserved CA sequence to yield a reactive hydroxyl moiety via a cytosolic reaction termed 3′-processing [22]. IN, in complex with the processed viral DNA and viral and host proteins, forms another large nucleoprotein assembly termed the preintegration complex (PIC) [23]. The PIC enters the nucleus through the nuclear pore, and IN then adheres to the host cell chromatin with the assistance of the cellular cofactor LEDGF/p75 [24], [25]. Once tethered to the host cell chromatin, IN utilizes the free 3′-hydroxyl group of the viral DNA in a nucleophilic attack upon the host genome largely within transcriptionally active regions [26] to stably integrate the proviral DNA, a reaction termed strand transfer [26], [27], [28], [29]. IN uses the same active site to catalyze both the 3′-processing and strand transfer reactions by coordinating two Mg2+ ions with three critical acidic residues (Asp64, Asp116, and Glu152) within the active site (DD(35)E motif) [30], [31]. Rational drug design efforts have thus far been mainly directed toward developing compounds that bind to the Mg2+-coordinating active site, but it has become apparent that new classes of allosteric inhibitors that disrupt IN-cofactor interactions [32], [33] or IN multimerization [34] will be necessary to advance the antiretroviral field in light of the emergence of viral strains resistant to contemporary clinically used IN drugs.

Although there have been scant structural studies focusing directly on the IN dimeric interface [35], [36], [37], and a handful of studies aimed at abrogating or modulating multimerization using peptidic or small-molecule compounds [34], [38], [39], [40], [41], [42], [43], [44], [45], relatively little is currently known about the inhibition of IN catalysis through blocking its oligomerization. Traditional small molecule drug design programs aimed at disrupting protein-protein interactions have been hindered by the belief that most drug-like small molecules do not provide a high enough binding energy to bind and disrupt large interfacial protein hotspots. However, great advances have been made in both antiretroviral and cancer drug design targeting protein-protein hotspots [32], [46]. Previously, our group identified an allosteric site of inhibition at the dimeric interface of IN [47]. Specifically, we found that a photoaffinity-labeled coumarin compound disrupted proper IN multimerization and, therefore, delivered inhibition of IN catalytic activities via an allosteric inhibitory mechanism. In a follow up study to further characterize the IN interfacial dimeric region, we identified a highly symmetric amino acid network composed of a four-tiered aromatic interaction flanked by two charged centers, with contributions from both monomers of the IN dimer [48]. In the current study, we aimed to further analyze the importance of each amino acid involved in this IN hotspot for oligomerization. Through site-directed mutagenesis, we determined that all but one substitution made at the dimeric interface abolished in vitro enzymatic activity of IN. Furthermore, we used a quantitative AlphaScreen®-based assay [49] to study the contribution of substituted residues to IN multimerization – a platform that has already proved potentially viable for future identification of IN multimerization-disrupting inhibitors [45]. Using this assay we validated our enzymatic activity results by showing that only the active IN mutant was able to functionally oligomerize. Many of the IN mutants detailed in this study proved difficult to purify and concentrate through traditional biochemical techniques, and thus the present study utilized IN inclusion bodies to analyze the strand transfer activity of the mutant proteins in the presence of Mn2+. Furthermore, we show that IN mutants exhibiting defective oligomerization can exhibit some degree of strand transfer in vitro. We went further to generate IN mutants in the NL4.3 viral clone and showed that, although exogenous RT activity mostly resembled WT levels, dimeric interface mutant viruses were non-infectious. We conclude that the structural conformation of the amino acid network detailed in this study is important for HIV-1 replication, and even conservative mutations at this region are not tolerable.