Structural and Functional Insights into Foamy Viral Integrase

Successful integration of retroviral DNA into the host chromosome is an essential step for viral replication. The process is mediated by virally encoded integrase (IN) and orchestrated by 3''-end processing and the strand transfer reaction. In vitro reaction conditions, such as substrate specificity, cofactor usage, and cellular binding partners for such reactions by the three distinct domains of prototype foamy viral integrase (PFV-IN) have been described well in several reports. Recent studies on the three‑dimensional structure of the interacting complexes between PFV-IN and DNA, cofactors, binding partners, or inhibitors have explored the mechanistic details of such interactions and shown its utilization as an important target to develop anti-retroviral drugs. The presence of a potent, non-transferable nuclear localization signal in the PFV C-terminal domain extends its use as a model for investigating cellular trafficking of large molecular complexes through the nuclear pore complex and also to identify novel cellular targets for such trafficking. This review focuses on recent advancements in the structural analysis and in vitro functional aspects of PFV-IN.     

Treatment with suboptimal doses of raltegravir leads to aberrant HIV-1 integrations

Integration of the DNA copy of the HIV-1 genome into a host chromosome is required for viral replication and is thus an important target for antiviral therapy. The HIV-encoded enzyme integrase (IN) catalyzes two essential steps: 3′ processing of the viral DNA ends, followed by the strand transfer reaction, which inserts the viral DNA into host DNA. Raltegravir binds to IN and blocks the integration of the viral DNA. Using the Rous sarcoma virus-derived vector RCAS, we previously showed that mutations that cause one viral DNA end to be defective for IN-mediated integration led to abnormal integrations in which the provirus had one normal and one aberrant end, accompanied by rearrangements in the host genome. On the basis of these results, we expected that suboptimal concentrations of IN inhibitors, which could block one of the ends of viral integration, would lead to similar aberrant integrations. In contrast to the proviruses from untreated cells, which were all normal, ∼10–15% of the proviruses isolated after treatment with a suboptimal dose of raltegravir were aberrant. The aberrant integrations were similar to those seen in the RCAS experiments. Most of the aberrant proviruses had one normal end and one aberrant end and were accompanied by significant rearrangements in the host genome, including duplications, inversions, deletions and, occasionally, acquisition of sequences from other chromosomes. The rearrangements of the host DNA raise concerns that these aberrant integrations might have unintended consequences in HIV-1–infected patients who are not consistent in following a raltegravir-containing treatment regimen.     

A stable complex between integrase and viral DNA ends mediates human immunodeficiency virus integration in vitro.

Retroviral replication depends on integration of the viral genome into a chromosome of the host cell. The steps in this process are orchestrated in vivo by a large nucleoprotein complex and are catalyzed by the retroviral enzyme integrase. Several biochemical properties of the in vivo nucleoprotein complex were reproduced in vitro with purified integrase of human immunodeficiency virus type 1 and model viral DNA substrates. A stable complex between integrase and viral DNA was detected as an early intermediate in the integration reaction. After formation of this initial complex, the enzyme processively catalyzed the 3' end processing and strand transfer steps in the reaction. Complexes containing only purified integrase and the model viral DNA end were stable under a variety of conditions and efficiently used nonviral DNA molecules as integration targets. These complexes required a divalent cation for their formation, and their stability was highly dependent on the 5'-terminal dinucleotide of the viral DNA, for which no functional role has previously been defined. Thus, interactions between integrase and the extreme ends of the viral DNA molecule may be sufficient to account for the stability of the in vivo integration complex.     

Identification of a hexapeptide inhibitor of the human immunodeficiency virus integrase protein by using a combinatorial chemical library.

Integration of human immunodeficiency virus (HIV) DNA into the human genome requires the virus-encoded integrase (IN) protein, and therefore the IN protein is a suitable target for antiviral strategies. To find a potent HIV IN inhibitor, we screened a "synthetic peptide combinatorial library." We identified a hexapeptide with the sequence HCKFWW that inhibits IN-mediated 3'-processing and integration with an IC50 of 2 microM. The peptide is active on IN proteins from other retroviruses such as HIV-2, feline immunodeficiency virus, and Moloney murine leukemia virus, supporting the notion that a conserved region of IN is targeted. The hexapeptide was also tested in the disintegration reaction. This phosphoryl-transfer reaction can be carried out by the catalytic core of IN alone, and the peptide HCKFWW was found to inhibit this reaction, suggesting that the hexapeptide acts at or near the catalytic site of IN. Identification of an IN hexapeptide inhibitor provides proof of concept for the approach, and, moreover, this peptide may be useful for structure-function analysis of IN.     

Genetic analysis of homomeric interactions of human immunodeficiency virus type 1 integrase using the yeast two-hybrid system.

The retroviral integrase protein (IN) is responsible for catalyzing a concerted integration reaction in which the two termini of linear viral DNA are joined to host DNA. To probe the potential for IN to form protein multimers, we used the yeast two-hybrid system. The coexpression of a GAL4 DNA binding domain-IN fusion and a GAL4 activation domain-IN fusion together resulted in the successful activation of a GAL4-responsive LacZ reporter gene. The system was used to examine a variety of IN deletion mutants. The results suggest that the central region of the protein is necessary for multimerization and that the N-terminal zinc finger region is not important.     

Integration of human immunodeficiency virus DNA: adduct interference analysis of required DNA sites.

The integration (IN) protein encoded by human immunodeficiency virus directs the integration of viral DNA into host DNA. We have probed the DNA sites required for the function of IN protein by attaching adducts to model DNA substrates and assaying their effects on integration in vitro. These experiments reveal that modifications in a short region on both DNA strands at the ends of the viral DNA block IN protein function. Modification of the target DNA near the point of DNA strand transfer also blocks IN protein function. Further experiments suggest that distinct subsets of the identified interactions are important for separate steps in the integration process.     

Requirement for a conserved serine in both processing and joining activities of retroviral integrase.

Retroviruses encode a protein, the integrase (IN), that is required for insertion of the viral DNA into the host cell chromosome. IN alone can carry out the integration reaction in vitro. The reaction involves endonucleolytic cleavage near the 3' ends of both viral DNA strands (the processing step), followed by joining of these new viral DNA ends to host DNA (the joining step). Based on their evolutionary conservation, we have previously identified at least 11 amino acid residues of IN that may be essential for the reaction. Here we report that even conservative replacements of one of these residues, an invariant serine, produce severe reductions in both the processing and joining activities of Rous sarcoma virus IN in vitro. Replacement of the analogous serine of the type 1 human immunodeficiency virus IN had similar effects on processing activity. These results suggest that this single conserved serine is a component of the active site and that one active site is used for both processing and joining. Replacement of this serine with certain amino acids resulted in a loss or reduction in DNA binding activities, while other replacements at this position appeared to affect later steps in catalysis. All of the defective Rous sarcoma virus INs were able to compete with the wild-type protein, which supports a model in which IN functions in a multimeric complex.     

Diketo acid inhibitor mechanism and HIV-1 integrase: implications for metal binding in the active site of phosphotransferase enzymes

The process of integrating the reverse-transcribed HIV-1 DNA into the host chromosomal DNA is catalyzed by the virally encoded enzyme integrase (IN). Integration requires two metal-dependent reactions, 3' end processing and strand transfer. Compounds that contain a diketo acid moiety have been shown to selectively inhibit the strand transfer reaction of IN in vitro and in infected cells and are effective as inhibitors of HIV-1 replication. To characterize the molecular basis of inhibition, we used functional assays and binding assays to evaluate a series of structurally related analogs. These studies focused on investigating the role of the conserved carboxylate and metal binding. We demonstrate that an acidic moiety such as a carboxylate or isosteric heterocycle is not required for binding to the enzyme complex but is essential for inhibition and confers distinct metal-dependent properties on the inhibitor. Binding requires divalent metal and resistance is metal dependent with active site mutants displaying resistance only when the enzymes are evaluated in the context of Mg(2+). The mechanism of action of these inhibitors is therefore likely a consequence of the interaction between the acid moiety and metal ion(s) in the IN active site, resulting in a functional sequestration of the critical metal cofactor(s). These studies thus have implications for modeling active site inhibitors of IN, designing and evaluating analogs with improved efficacy, and identifying inhibitors of other metal-dependent phosphotransferases.     

Retroviral integration: structure of the initial covalent product and its precursor, and a role for the viral IN protein.

An essential step in the life cycle of a retrovirus is the integration of a DNA copy of the viral genome into a host cell chromosome. We have analyzed the structure of the initial covalent product of an in vitro retroviral integration reaction and determined the structure of the ends of the unintegrated linear viral DNA molecules present in vivo in cells infected with murine leukemia virus (MLV). Our results lead to the following conclusions: (i) Circularization of viral DNA plays no role in integration. The direct precursor to the integrated MLV provirus is a linear molecule. (ii) The initial step in the integration reaction is probably a cleavage that removes the terminal 2 bases from each 3' end of the viral DNA. This cleavage depends on a virally encoded protein, IN, that has previously been shown genetically to be required for integration. (iii) The resulting viral 3' ends are joined to target DNA to form the initial recombination intermediate.     

Targeting E2F1–DNA complexes with microgonotropen DNA binding agents

Microgonotropen (MGT) DNA binding drugs, which consist of an A+T-selective DNA minor groove binding tripyrrole peptide and polyamine chains attached to a central pyrrole that extend drug contact into the DNA major groove, were found to be extraordinarily effective inhibitors of E2 factor 1 (E2F1) association with its DNA promoter element (5′-TTTCGCGCCAAA). The most active of these drugs, MGT-6a, was three orders of magnitude more effective than distamycin and inhibited complexes between E2F1 and the dihydrofolate reductase promoter by 50% at 0.00085 μM. A relationship was found between the measured equilibrium constants for binding of MGTs to the A+T region of d(GGCGA₃T₃GGC)/d(CCGCT₃A₃CCG) and their inhibition of complex formation between E2F1 and the DNA promoter element. A representative of the potent MGT inhibitors was significantly more active on inhibition of E2F1–DNA complex formation compared with disruption of a preexisting complex.     

A covalent complex between retroviral integrase and nicked substrate DNA.

Purified retroviral integrase (IN) from avian sarcoma-leukosis viruses can appropriately process the termini of linear viral DNA, cleave host DNA in a sequence-independent manner, and catalyze integrative recombination; an exogenous source of energy is not required for these reactions. Using DNA substrates containing radioactive phosphate groups, we demonstrate that IN becomes covalently joined to the new 5' phosphate ends of DNA produced at sites of cleavage. Most of the phosphodiester linkages between IN and DNA involve serine, but some involve threonine. Computer-assisted alignment of 80 retroviral and retrotransposon IN sequences identified one serine that is conserved in all of these proteins and three less-conserved threonine residues. These results identify candidate active-site residues and provide support for the participation of a covalent IN-DNA intermediate in retroviral integration.     

Mutational analysis of the integrase protein of human immunodeficiency virus type 2.

Purified integrase protein (IN) can nick linear viral DNA at a specific site near the ends and integrate nicked viral DNA into target DNA. We have made a series of 43 site-directed point mutants of human immunodeficiency virus type 2 IN and assayed purified mutant proteins for the following activities: site-specific cleavage of viral DNA (donor cut), integration (strand transfer), and disintegration. In general, the different activities were similarly affected by the mutations. We found three mutations that (almost) totally abolished IN function: Asp-64-->Val, Asp-116-->Ile, and Glu-152-->Leu, whereas 25 mutations did not affect IN function. A few mutations affected the different activities differentially. Near the amino terminus a zinc finger-like sequence motif His-Xaa3-His-Xaa20-30-Cys-Xaa2-Cys is present in all retroviral IN proteins. Two mutations in this region (His-12-->Leu and Cys-40-->Ser) strongly inhibited donor cut but had less effect on strand transfer. The central region of IN is most highly conserved between retroviral INs. Three mutants in this region (Asn-117-->Ile, Asn-120-->Leu, and Lys-159-->Val) were inhibited in strand transfer but were inhibited less strongly in donor cut. Mutation of Asn-120 (to glycine, leucine, or glutamate) resulted in changes in integration-site preference, suggesting that Asn-120 is involved in interactions with target DNA. We did not find a mutant in which one activity was lost and the others were unaffected, supporting the notion that IN has only one active site for the catalysis of donor cut and strand transfer.     

Structure of the catalytic domain of avian sarcoma virus integrase with a bound HIV-1 integrase-targeted inhibitor

The x-ray structures of an inhibitor complex of the catalytic core domain of avian sarcoma virus integrase (ASV IN) were solved at 1.9- to 2.0-Å resolution at two pH values, with and without Mn²⁺ cations. This inhibitor (Y-3), originally identified in a screen for inhibitors of the catalytic activity of HIV type 1 integrase (HIV-1 IN), was found in the present study to be active against ASV IN as well as HIV-1 IN. The Y-3 molecule is located in close proximity to the enzyme active site, interacts with the flexible loop, alters loop conformation, and affects the conformations of active site residues. As crystallized, a Y-3 molecule stacks against its symmetry-related mate. Preincubation of IN with metal cations does not prevent inhibition, and Y-3 binding does not prevent binding of divalent cations to IN. Three compounds chemically related to Y-3 also were investigated, but no binding was observed in the crystals. Our results identify the structural elements of the inhibitor that likely determine its binding properties.     

Mechanism of protein remodeling by ClpA chaperone

ClpA, a newly discovered ATP-dependent molecular chaperone, remodels bacteriophage P1 RepA dimers into monomers, thereby activating the latent specific DNA binding activity of RepA. We investigated the mechanism of the chaperone activity of ClpA by dissociating the reaction into several steps and determining the role of nucleotide in each step. In the presence of ATP or a nonhydrolyzable ATP analog, the initial step is the self-assembly of ClpA and its association with inactive RepA dimers. ClpA-RepA complexes form rapidly and at 0°C but are relatively unstable. The next step is the conversion of unstable ClpA-RepA complexes into stable complexes in a time- and temperature-dependent reaction. The transition to stable ClpA-RepA complexes requires binding of ATP, but not ATP hydrolysis, because nonhydrolyzable ATP analogs satisfy the nucleotide requirement. The stable complexes contain approximately 1 mol of RepA dimer per mol of ClpA hexamer and are committed to activating RepA. In the last step of the reaction, active RepA is released upon exchange of ATP with the nonhydrolyzable ATP analog and ATP hydrolysis. Importantly, we discovered that one cycle of RepA binding to ClpA followed by ATP-dependent release is sufficient to convert inactive RepA to its active form.