HIV-1 integrase can process a 3'-end crosslinked substrate.

Integrase of the human immunodeficiency virus type-1 (HIV-1) recognizes specific sequences located in the U3 and U5 regions at the ends of viral DNA. We synthesized DNA duplexes mimicking the U5 region and containing either 2'-aminonucleosides or non-nucleoside 1,3-propanediol insertions at the third and terminal positions and studied their interactions with HIV-1 integrase. Both modifications introduced a local structural distortion in the DNA double helix. Replacement of the terminal nucleosides by corresponding 2'-aminonucleosides had no significant effect on integrase activity. We used an integrase substrate bearing terminal 2'-aminonucleosides in both strands to synthesize a duplex with cross-linked strands. This duplex was then used to determine whether terminal base pair disruption is an obligatory step of retroviral DNA 3'-processing. Processing of the cross-linked analog of the integrase substrate yielded a product of the same length as 3'-processing of the wild-type substrate but the reaction efficiency was lower. Replacement of the third adenosine in the processed strand by a corresponding 2'-aminonucleoside did not affect integrase activity, whereas, its replacement by 1,3-propanediol completely inhibited 3'-processing. Both modifications of the complementary thymidine in the nonprocessed strand increased the initial rate of 3'-processing. The same effect was observed when both nucleosides, at the third position, were replaced by corresponding 2'-aminonucleosides. This indicates that the local duplex distortion facilitated the cleavage of the phosphodiester bond. Thus, a localized destabilization of the third A-T base pair is necessary for efficient 3'-processing, whereas 3'-end-fraying is important but not absolutely required.     

Peptide inhibitors of HIV-1 integrase dissociate the enzyme oligomers.

Integration of HIV-1 genome into host cell chromosome is mediated by viral integrase (IN). The IN catalytic core (CC, IN(50-212)) dimerizes through mutual interactions of its alpha1 and alpha5 helices. Peptides INH1 and INH5 reproducing these helix sequences strongly inhibited IN. For instance, an IC(50) of 80 nM was determined for INH5 in integration assays using wild-type IN (wtIN). In size exclusion chromatography, INH1 and INH5 perturbed the association-dissociation equilibrium of both dmIN (IN(1-288)/F185K/C280S) and CC, leading to monomers as surviving species, while in circular dichroism, binding of peptides to dmIN altered the protein conformation. Thus, enzyme deactivation, subunit dissociation, and protein unfolding are events which parallel one another. The target of INH5 in the enzyme was then identified. In fluorescence spectroscopy, C(0.5) values of 168 and 44 nM were determined for the binding affinity of INH5 to IN and CC, respectively, at 115 nM subunit concentration, while interaction of INH5 with INH1 was found stronger than interaction of INH5 with itself (23 times larger in term of dissociation constants). These results strongly suggested that the alpha1 helix is the privileged target of INH5. The latter could serve as a lead for the development of new chemotherapeutic agents against HIV-1.     

Inhibition of HIV-1 integrase dimerization and activity with crosslinked interfacial peptides

Alternative modes of inhibition for the design of anti-HIV therapies are sought due to the resistance of HIV to a number of the currently approved drugs. A non-active site strategy for generating potent inhibitors of HIV-1 integrase is described based on blocking protein association. Peptides α5 and α6 derived from the HIV-1 integrase dimeric interface have previously demonstrated efficacious dimerization inhibition of HIV-1 integrase. Due to the proximity of the termini of these peptides within the integrase structure, a focused library of tethered agents was designed based on crosslinking the peptides α5 and α6 to mimic a larger interfacial region. The best crosslinked inhibitors are approximately five-fold more potent against HIV-1 integrase than the individual peptides alone or in combination. The most active agents have an inhibitory constant in the mid-nM range and function via a dissociative mechanism of inhibition.     

A potent and orally active HIV-1 integrase inhibitor

A 1,6-naphthyridine inhibitor of HIV-1 integrase has been discovered with excellent inhibitory activity in cells, good pharmacokinetics, and an excellent ability to inhibit virus with mutant enzyme.     

6-oxocytidine containing oligonucleotides inhibit the HIV-1 integrase in vitro

Integration of the proviral DNA into the genome of infected cells is a key step of HIV-1 replication. Integration is catalyzed by the viral enzyme integrase (IN). 6-oxocytidine-containing oligonucleotides were found to be efficient inhibitors of integrase in vitro. The inhibitory effect is sequence-specific and strictly requires the presence of the 6-oxocytidine base. It is due to the impairment of the integrase binding to its substrate and does not involve an auto-structure of the oligonucleotide.     

Effect of acyclic nucleoside phosphonates on the HIV-1 integrase in vitro.

Integrase (IN) is an essential enzyme in the human immunodeficiency virus type-1 (HIV-1) replication cycle and, thus, a potential target for chemotherapeutic agents. Because various nucleotide analogues have been reported to inhibit IN in vitro, we investigated the effect of acyclic nucleoside phosphonates. Both unphosphorylated and diphosphorylated derivatives were inhibitory to IN at concentrations ranging between 60 and 800 microM, with diphosphorylated derivatives being 5- to 8-fold more potent than unphosphorylated counterparts.     

High-level expression of active HIV-1 integrase from a synthetic gene in human cells.

A synthetic gene encoding for HIV-1 integrase was designed to circumvent the intrinsic instability and the repressor elements present in the wild-type gene. High-level expression of HIV-1 integrase was obtained in various human cell lines independently of viral accessory proteins. A human 293T cell line was selected that stably expresses HIV-1 integrase and has growth kinetics comparable to the parental cell line. The enzyme was localized in the nucleus and remained stably associated with the chromosomes during mitosis. Lentiviral vector particles carrying the inactivating D64V mutation in the integrase gene were capable of stably transducing 293T cells when complemented in the producer cells with integrase expressed from the synthetic gene. When the cell line that stably expresses integrase was infected with the defective viral particles, complementation of integrase activity was detected as well. Expression of active HIV-1 integrase in human cells will facilitate the study of the interplay between host and viral factors during integration.     

Disulfide-linked integrase oligomers involving C280 residues are formed in vitro and in vivo but are not essential for human immunodeficiency virus replication

The human immunodeficiency virus type 1 integrase (IN) forms an oligomer that integrates both ends of the viral DNA. The nature of the active oligomer is unclear. Recombinant IN obtained under reducing conditions is always in the form of noncovalent oligomers. However, disulfide-linked oligomers of IN were recently observed within viral particles. We show that IN produced from a baculovirus expression system can form disulfide-linked oligomers. We investigated which residues are responsible for the disulfide bridges and the relationship between the ability to form covalent dimers and IN activity. Only the mutation of residue C280 was sufficient to prevent the formation of intermolecular disulfide bridges in oligomers of recombinant IN. IN activity was studied under and versus nonreducing conditions: the formation of disulfide bridges was not required for the in vitro activities of the enzyme. Moreover, the covalent dimer does not dissociate into individual protomers on disulfide bridge reduction. Instead, IN undergoes a spontaneous multimerization process that yields a homogenous noncovalent tetramer. The C280S mutation also completely abolished the formation of disulfide bonds in the context of the viral particle. Finally, the replication of the mutant virus was investigated in replicating and arrested cells. The infectivity of the virus was not affected by the C280S IN mutation in either dividing or nondividing cells. The disulfide-linked form of the IN oligomers observed in the viral particles is thus not required for viral replication.     

Dihydroxypyridopyrazine-1,6-dione HIV-1 integrase inhibitors

A series of potent novel dihydroxypyridopyrazine-1,6-dione HIV-1 integrase inhibitors was identified. These compounds inhibited the strand transfer process of HIV-1 integrase and viral replication in cells. Compound 6 is active against replication of HIV with a CIC(95) of 0.31 microM and exhibits no shift in potency in the presence of 50% normal human serum. It displays a good pharmacokinetic profile when dosed in rats and no covalent binding with microsomal proteins in both in vitro and in vivo models.     

Peptides derived from HIV-1 Rev inhibit HIV-1 integrase in a shiftide mechanism

The HIV-1 Integrase protein (IN) mediates the integration of the viral cDNA into the host genome. IN is an emerging target for anti-HIV drug design, and the first IN-inhibitor was recently approved by the FDA. We have developed a new approach for inhibiting IN by "shiftides": peptides derived from its cellular binding protein LEDGF/p75 that inhibit IN by shifting its oligomerization equilibrium from the active dimer to an inactive tetramer. In addition, we described two peptides derived from the HIV-1 Rev protein that interact with IN and inhibit its activity in vitro and in cells. In the current study, we show that the Rev-derived peptides also act as shiftides. Analytical gel filtration and cross-linking experiments showed that IN was dimeric when bound to the viral DNA, but tetrameric in the presence of the Rev-derived peptides. Fluorescence anisotropy studies revealed that the Rev-derived peptides inhibited the DNA binding of IN. The Rev-derived peptides inhibited IN catalytic activity in vitro in a concentration-dependent manner. Inhibition was much more significant when the peptides were added to free IN before it bound the viral DNA than when the peptides were added to a preformed IN-DNA complex. This confirms that the inhibition is due to the ability of the peptides to shift the oligomerization equilibrium of the free IN toward a tetramer that binds much weaker to the viral DNA. We conclude that protein-protein interactions of IN may serve as a general valuable source for shiftide design.     

Complementation of integrase function in HIV-1 virions.

Proviral integration is essential for HIV-1 replication and represents an important potential target for antiviral drug design. Although much is known about the integration process from studies of purified integrase (IN) protein and synthetic target DNA, provirus formation in virally infected cells remains incompletely understood since reconstituted in vitro assays do not fully reproduce in vivo integration events. We have developed a novel experimental system in which IN-mutant HIV-1 molecular clones are complemented in trans by Vpr-IN fusion proteins, thereby enabling the study of IN function in replicating viruses. Using this approach we found that (i) Vpr-linked IN is efficiently packaged into virions independent of the Gag-Pol polyprotein, (ii) fusion proteins containing a natural RT/IN processing site are cleaved by the viral protease and (iii) only the cleaved IN protein complements IN-defective HIV-1 efficiently. Vpr-mediated packaging restored IN function to a wide variety of IN-deficient HIV-1 strains including zinc finger, catalytic core and C-terminal domain mutants as well as viruses from which IN was completely deleted. Furthermore, trans complemented IN protein mediated a bona fide integration reaction, as demonstrated by the precise processing of proviral ends (5'-TG...CA-3') and the generation of an HIV-1-specific (5 bp) duplication of adjoining host sequences. Intragenic complementation between IN mutants defective in different protein domains was also observed, thereby providing the first evidence for IN multimerization in vivo.     

Stereospecificity of reactions catalyzed by HIV-1 integrase

The retroviral integrase catalyzes two successive chemical reactions essential for integration of the retroviral genome into a host chromosome: 3' end processing, in which a dinucleotide is cleaved from each 3' end of the viral DNA; and the integration reaction itself, in which the resulting recessed 3' ends of the viral DNA are joined to the host DNA. We have examined the stereospecificity of human immunodeficiency virus type 1 integrase for phosphorothioate substrates in these reactions and in a third reaction, disintegration, which is macroscopically the reverse of integration. Integrase preferentially catalyzed end processing and integration of a substrate with the (R(p))-phosphorothioate stereoisomer at the reaction center and disintegration of a substrate with an (S(p))-phosphorothiate at the reaction center. These results suggest a model for the architecture of the active site of integrase, and its interactions with key features of the viral and target DNA.     

HIV-1 integrase interacts with yeast microtubule-associated proteins

The human immunodeficiency virus type 1 (HIV-1) integrase (IN) mediates the insertion of viral DNA into the human genome. In addition to IN, cellular and viral proteins are associated to proviral DNA in the so-called preintegration complex (PIC). We previously reported that the expression of HIV-1 IN in yeast leads to the emergence of a lethal phenotype. This effect may be linked to the IN activity on infected human cells where integration requires the cleavage of genomic DNA. To isolate and characterize potential cellular partners of HIV-1 IN, we used it as a bait in a two-hybrid system with a yeast genomic library. IN interacted with proteins belonging to the microtubule network, or involved in the protein synthesis apparatus. We focused our interest on one of the selected inserts, L2, which corresponds to the C-end half of the yeast STU2p, a microtubule-associated protein (MAP). STU2p is an essential component of the yeast spindle pole body (SPB), which is able to bind microtubules in vitro. After expressing and purifying L2 as a recombinant protein, we showed its binding to IN by ELISA immunodetection. L2 was also able to inhibit IN activity in vitro. In addition, the effect of L2 was tested using the "lethal yeast phenotype". The coexpression of IN and the L2 peptide abolished the lethal phenotype, thus showing important in vivo interactions between IN and L2. The identification of components of the microtubule network associated with IN suggest a role of this complex in the transport of HIV-1 IN present in the PIC to the nucleus, as already described for other human viruses.     

Azido-containing aryl beta-diketo acid HIV-1 integrase inhibitors

Aryl beta-diketo acids (ADK) comprise a general class of potent HIV-1 integrase (IN) inhibitors, which can exhibit selective inhibition of strand transfer reactions in extracellular recombinant IN assays and provide potent antiviral effects in HIV-infected cells. Recent studies have shown that polycyclic aryl or aryl rings bearing aryl-containing substituents are components of potent members of this class. Reported herein is the first use of azido functionality as an aryl replacement in beta-diketo acid IN inhibitors. The ability of azido-containing inhibitors to exhibit potent inhibition of IN and antiviral protection in HIV-infected cells, renders the azide group of potential value in the further development of ADK-based IN inhibitors.     

Binding aspects of baicalein to HIV-1 integrase

Human immunodeficiency virus type 1 (HIV-1) integrase is an essential enzyme in the life cycle of the virus. It is responsible for catalyzing the insertion of the viral genome into the host cell chromosome. This integrase is an attractive target for the design of a HIV antiviral drug, because integrase has no human counterpart. In order to know the interaction mode of HIV-1 integrase with its inhibitor, we investigated the effect of the inhibitor, baicalein, on the conformation of the HIV-1 integrase catalytic domain [IN-(50-212/F185K)] using fluorescence and circular dichroism (CD) spectroscopy. We found that baicalein binds to the hydrophobic region of the HIV-1 integrase catalytic core domain. This binding of baicalein induces the conformational change of the enzyme. We also found that the binding ratio of baicalein to the HIV-1 integrase catalytic domain is 2:1.