Identification of low molecular weight nuclear complexes containing integrase during the early stages of HIV-1 infection.

BACKGROUND: HIV-1 replication requires integration of its reverse transcribed viral cDNA into a host cell chromosome. The DNA cutting and joining reactions associated to this key step are catalyzed by the viral protein integrase (IN). In infected cells, IN binds the viral cDNA, together with viral and cellular proteins, to form large nucleoprotein complexes. However, the dynamics of IN complexes formation is still poorly understood. RESULTS: Here, we characterized IN complexes during the early stages of T-lymphocyte infection. We found that following viral entry into the host cell, IN was rapidly targeted to proteasome-mediated degradation. Interactions between IN and cellular cofactors LEDGF/p75 and TNPO3 were detected as early as 6 h post-infection. Size exclusion chromatography of infected cell extracts revealed distinct IN complexes in vivo. While at 2 h post-infection the majority of IN eluted within a high molecular weight complex competent for integration (IN complex I), IN was also detected in a low molecular weight complex devoid of full-length viral cDNA (IN complex II, ~440 KDa). At 6 h post-infection the relative proportion of IN complex II increased. Inhibition of reverse transcription or integration did not alter the elution profile of IN complex II in infected cells. However, in cells depleted for LEDGF/p75 IN complex II shifted to a lower molecular weight complex (IN complex III, ~150 KDa) containing multimers of IN. Notably, cell fractionation experiments indicated that both IN complex II and III were exclusively nuclear. Finally, IN complex II was not detected in cells infected with a virus harboring a mutated IN defective for LEDGF/p75 interaction and tetramerization. CONCLUSIONS: Our findings indicate that, shortly after viral entry, a significant portion of DNA-free IN that is distinct from active pre-integration complexes accumulates in the nucleus.

Ethyl malonate amides: a diketo acid offspring fragment for HIV integrase inhibition.

While searching for new HIV integrase inhibitors we discovered that some ethyl malonate amides (EMA) are active against this enzyme. Surprisingly, the main function can only very rarely be found among the reported drug candidates. We synthesised a series of compounds in order to establish and analyse the structure-activity relationship. The similarity to the important classes of HIV integrase inhibitors as well as the synthetic availability of the different targets including this pharmacophore makes EMA compounds an interesting object of investigations.

Dynamics of raltegravir resistance profile in an HIV type 2-infected patient.

The evolutionary dynamics of RAL resistance in the HIV-2 virus were examined through population and clonal sequence analysis of the IN from baseline, during treatment, and after stopping RAL therapy. The treatment failure of an RAL regimen in the HIV-2 patient studied was associated with the emergence of mutations via the N155H resistance pathway and subsequent switching to the Y143C mutational route. This study has also identified four novel secondary mutations, Q91R, S147G, A153G, and M183I, not previously reported in HIV-1 patients failing RAL therapy. Resistant variants involving the Y143C pathway were noted to have persisted beyond 4 weeks following the cessation of RAL therapy. All resistance-associated mutations were lost at 20 weeks after stopping RAL therapy. Our findings provide evidence supporting the supposition that substantial cross-resistance between strand transfer IN-Is is likely in HIV-2 as shown in HIV-1.

Scintillation proximity assays for mechanistic and pharmacological analyses of HIV-1 integration.

The early events of HIV-1 replication are highlighted by reverse transcription and integration, and the reverse transcriptase and integrase enzymes are important therapeutic targets. Integration proceeds through a series of steps including assembly of integrase on the viral donor DNA ends, 3'-processing, and DNA strand transfer. First generation integrase assays typically included all biochemical reagents in solution where excess donor substrate could serve as the target for DNA strand transfer. These conditions, though valuable for understanding mechanistic aspects of HIV-1 integration, fell short of critical pharmacological designs as most early inhibitors were found to block assembly instead of enzyme function. Second generation designs, which decoupled assembly from DNA strand transfer, afforded the specificity required to identify clinically relevant compounds. Here, we describe versatile scintillation proximity-based assays whereby integrase is assembled onto donor DNA that is immobilized onto the surface of beads. Immobilization and subsequent washing of excess donor DNA eliminates its potential to serve as target DNA, allowing investigation of the DNA strand transfer reaction in isolation. Assembled complexes can be used in high-throughput DNA strand transfer assays if radio labeled target DNA is employed or in integrase binding assays using a suitable radioligand.

Characterization and structural analysis of HIV-1 integrase conservation.

The HIV-1 integrase, responsible for the chromosomal integration of the newly synthesized double-stranded viral DNA into the host genomic DNA, represents a new and important target of potential clinical relevance. For instance, two integrase inhibitors, raltegravir and elvitegravir, have been shown to be promising in clinical trials, and the first has been recently made available for clinical practice. As is the case for other antiviral drugs, drug resistance to integrase inhibitors occurs both in vitro and/or in vivo through the selection of mutations within the HIV genome. Indeed, many integrase mutations have already been associated with resistance to all the different integrase inhibitors tested in in vitro and/or in vivo studies. Among them, about 40 substitutions have been specifically associated with the development of resistance to raltegravir and/or elvitegravir; some of them were also found in vivo in patients failing such integrase inhibitors. The relevance of integrase mutations in clinical practice has yet to be defined, in light of the lack of long-term follow-up of treated patients and the limited data about the prevalence of integrase inhibitor-associated mutations in integrase inhibitor-naive patients (either untreated, or treated with antiretrovirals not containing integrase inhibitors). Therefore, by structural analysis elaboration and literature discussion, the aim of this review is to characterize the conserved residues and regions of HIV-1 integrase and the prevalence of mutations associated with integrase inhibitor resistance, by matching data originated from a well-defined cohort of HIV-1 B subtype-infected individuals (untreated and antiretroviral-treated) and data originated from the public Los Alamos Database available in the literature (all patients integrase inhibitor-naive by definition). In integrase inhibitor-naive patients, 180 out of 288 HIV-1 integrase residues (62.5%) are conserved (< 1% variability). Residues involved in protein stability, multimerization, DNA binding, catalytic activity, and in the binding with the human cellular cofactor LEDGF/p75 are fully conserved. Some of these residues clustered into large defined regions of consecutive invariant amino acids, suggesting that consecutive residues in specific structural domains are required for the correct performance of HIV-1 integrase functions. All primary signature mutations emerging in patients failing raltegravir (Y143R, Q148H/K/R, N155H) or elvitegravir (T66I, E92Q, S147G, Q148H/K/R, N155H), as well as secondary mutations (H51Y, T66A/K, E138K, G140S/A/C, Y143C/H, K160N, R166S, E170A, S230R, D232N, R263K) were completely absent or highly infrequent (< 0.5%) in integrase inhibitor-naive patients, either infected with HIV-1 B subtype (drug-naive or antiretroviral-treated), or non-B subtypes/group N and O. Differently, other mutations (L74M, T97A, S119G/R, V151I, K156N, E157Q, G163K/R, V165I, I203M, T206S, S230N) occurred as natural polymorphisms with a different prevalence according to different HIV-1 subtype/circulating recombinant form/group. In conclusion, the HIV-1 integrase in vivo is an enzyme requiring the full preservation of almost two-thirds of its amino acids in the absence of specific integrase inhibitor pressure. Primary mutations associated with resistance to integrase inhibitors clinically relevant today are absent or highly infrequent in integrase inhibitor-naive patients. The characterization of the highly conserved residues (involved in protein stability, multimerization, DNA binding, catalytic activity, LEDGF binding, and some with still poorly understood function) could help in the rational design of new HIV-1 inhibitors with alternative mechanisms of action and more favorable resistance profiles.

Quantitative analysis of HIV-1 preintegration complexes.

Retroviral replication proceeds through the formation of a provirus, an integrated DNA copy of the viral RNA genome. The linear cDNA product of reverse transcription is the integration substrate and two different integrase activities, 3' processing and DNA strand transfer, are required for provirus formation. Integrase nicks the cDNA ends adjacent to phylogenetically-conserved CA dinucleotides during 3' processing. After nuclear entry and locating a suitable chromatin acceptor site, integrase joins the recessed 3'-OHs to the 5'-phosphates of a double-stranded staggered cut in the DNA target. Integrase functions in the context of a large nucleoprotein complex, called the preintegration complex (PIC), and PICs are analyzed to determine levels of integrase 3' processing and DNA strand transfer activities that occur during acute virus infection. Denatured cDNA end regions are monitored by indirect end-labeling to measure the extent of 3' processing. Native PICs can efficiently integrate their viral cDNA into exogenously added target DNA in vitro, and Southern blotting or nested PCR assays are used to quantify the resultant DNA strand transfer activity. This study details HIV-1 infection, PIC extraction, partial purification, and quantitative analyses of integrase 3' processing and DNA strand transfer activities.

Quantitative analysis of the interactions between HIV-1 integrase and retroviral reverse transcriptases.

The RT (reverse transcriptase) of HIV-1 interacts with HIV-1 IN (integrase) and inhibits its enzymatic activities. However, the molecular mechanisms underling these interactions are not well understood. In order to study these mechanisms, we have analysed the interactions of HIV-1 IN with HIV-1 RT and with two other related RTs: those of HIV-2 and MLV (murine-leukaemia virus). All three RTs inhibited HIV-1 IN, albeit to a different extent, suggesting a common site of binding that could be slightly modified for each one of the studied RTs. Using surface plasmon resonance technology, which monitors direct protein-protein interactions, we performed kinetic analyses of the binding of HIV-1 IN to these three RTs and observed interesting binding patterns. The interaction of HIV-1 RT with HIV-1 IN was unique and followed a two-state reaction model. According to this model, the initial IN-RT complex formation was followed by a conformational change in the complex that led to an elevation of the total affinity between these two proteins. In contrast, HIV-2 and MLV RTs interacted with IN in a simple bi-molecular manner, without any apparent secondary conformational changes. Interestingly, HIV-1 and HIV-2 RTs were the most efficient inhibitors of HIV-1 IN activity, whereas HIV-1 and MLV RTs showed the highest affinity towards HIV-1 IN. These modes of direct protein interactions, along with the apparent rate constants calculated and the correlations of the interaction kinetics with the capacity of the RTs to inhibit IN activities, are all discussed.

Genetic analyses of DNA-binding mutants in the catalytic core domain of human immunodeficiency virus type 1 integrase.

The catalytic core domain (CCD) of human immunodeficiency virus type 1 (HIV-1) integrase (IN) harbors the enzyme active site and binds viral and chromosomal DNA during integration. Thirty-five CCD mutant viruses were constructed, paying particular attention to conserved residues in the Phe(139)-Gln(146) flexible loop and abutting Ser(147)-Val(165) amphipathic alpha helix that were implicated from previous in vitro work as important for DNA binding. Defective viruses were typed as class I mutants (specifically blocked at integration) or pleiotropic class II mutants (additional particle assembly and/or reverse transcription defects). Whereas HIV-1(P145A) and HIV-1(Q146K) grew like the wild type, HIV-1(N144K) and HIV-1(Q148L) were class I mutants, reinforcing previous results that Gln-148 is important for DNA binding and uncovering for the first time an important role for Asn-144 in integration. HIV-1(Q62K), HIV-1(H67E), HIV-1(N120K), and HIV-1(N155K) were also class I mutants, supporting findings that Gln-62 and Asn-120 interact with viral and target DNA, respectively, and suggesting similar integration-specific roles for His-67 and Asn-155. Although results from complementation analyses established that IN functions as a multimer, the interplay between active-site and CCD DNA binding functions was unknown. By using Vpr-IN complementation, we determined that the CCD protomer that catalyzes integration also preferentially binds to viral and target DNA. We additionally characterized E138K as an intramolecular suppressor of Gln-62 mutant virus and IN. The results of these analyses highlight conserved CCD residues that are important for HIV-1 replication and integration and define the relationship between DNA binding and catalysis that occurs during integration in vivo.

Class II integrase mutants with changes in putative nuclear localization signals are primarily blocked at a postnuclear entry step of human immunodeficiency virus type 1 replication.

Integrase has been implicated in human immunodeficiency virus type 1 (HIV-1) nuclear import. Integrase analyses, however, can be complicated by the pleiotropic nature of mutations: whereas class I mutants are integration defective, class II mutants display additional assembly and/or reverse transcription defects. We previously determined that HIV-1(V165A), originally reported as defective for nuclear import, was a class II mutant. Here we analyzed mutants containing changes in other putative nuclear localization signals, including (186)KRK(188)/(211)KELQKQITK(219) and Cys-130. Previous work established HIV-1(K186Q), HIV-1(Q214L/Q216L), and HIV-1(C130G) as replication defective, but phenotypic classification was unclear and nuclear import in nondividing cells was not addressed. Consistent with previous reports, most of the bipartite mutants studied here were replication defective. These mutants as well as HIV-1(V165A) synthesized reduced cDNA levels, but a normal fraction of mutant cDNA localized to dividing and nondividing cell nuclei. Somewhat surprisingly, recombinant class II mutant proteins were catalytically active, and class II Vpr-integrase fusion proteins efficiently complemented class I mutant virus. Since a class I Vpr-integrase mutant efficiently complemented class II mutant viruses under conditions in which class II Vpr-integrases failed to function, we conclude that classes I and II define two distinct complementation groups and suggest that class II mutants are primarily defective at a postnuclear entry step of HIV-1 replication. HIV-1(C130G) was also defective for reverse transcription, but Vpr-integrase(C130G) did not efficiently complement class I mutant HIV-1. Since HIV-1(C130A) grew like the wild type, we conclude that Cys-130 is not essential for replication and speculate that perturbation of integrase structure contributed to the pleiotropic HIV-1(C130G) phenotype.

The use of a new in vitro reaction substrate reproducing both U3 and U5 regions of the HIV-1 3'-ends increases the correlation between the in vitro and in vivo effects of the HIV-1 integrase inhibitors.

Human Immunodeficiency Virus type 1 (HIV-1) integrase (IN) is an attractive target for the development of new antiviral therapies. Recently, several HIV-1 recombinant IN (rIN) in vitro inhibitors have been described. However, the great majority of them failed to block the virus replication in cell-based assays, suggesting the inadequacy of the in vitro assay systems used for inhibitor screening. To improve these systems, we designed a 40(mer) duplex DNA reaction substrate consisting of recognition sequences from both U3 and U5 HIV-1 long terminal repeat (LTR) termini. The HIV-1 rIN was able to catalyze its enzyme activities recognizing both ends of the 40(mer) dsDNA. Using this substrate we assayed the effects on rIN catalysis of different classes of compounds which inhibit the HIV-1 rIN in vitro when the reaction substrate is the standard 21(mer) U5 dsDNA, and that are either active or inactive on the HIV-1 replication. We also compared the efficacy of these compounds when added to the reaction before or after the formation of the rIN-dsDNA complex. In this system, the enzyme preincubation with the two-ended 40(mer) dsDNA before the addition of the compounds allowed a strong correlation between the effects of hydroxylated aromatics derivatives on rIN activity in cell-free assays and their effects on viral replication in cell-culture assays. This increase in drug selectivity of the rIN in vitro assay was explored by investigating whether it was due to the length of the 40(mer), longer than the standard 21(mer), or to presence of both viral ends, versus only one viral end. To this purpose we designed four 40(mer) oligonucleotides containing either only one viral end or two-repetitive ends, finding that the architecture of the rIN-dsDNA complex and its compound susceptibility is significantly influenced by the sequence of the dsDNA substrate.

Use of patient-derived human immunodeficiency virus type 1 integrases to identify a protein residue that affects target site selection.

To identify parts of retroviral integrase that interact with cellular DNA, we tested patient-derived human immunodeficiency virus type 1 (HIV-1) integrases for alterations in the choice of nonviral target DNA sites. This strategy took advantage of the genetic diversity of HIV-1, which provided 75 integrase variants that differed by a small number of amino acids. Moreover, our hypothesis that biological pressures on the choice of nonviral sites would be minimal was validated when most of the proteins that catalyzed DNA joining exhibited altered target site preferences. Comparison of the sequences of proteins with the same preferences then guided mutagenesis of a laboratory integrase. The results showed that single amino acid substitutions at one particular residue yielded the same target site patterns as naturally occurring integrases that included these substitutions. Similar results were found with DNA joining reactions conducted with Mn(2+) or with Mg(2+) and were confirmed with a nonspecific alcoholysis assay. Other amino acid changes at this position also affected target site preferences. Thus, this novel approach has identified a residue in the central domain of HIV-1 integrase that interacts with or influences interactions with cellular DNA. The data also support a model in which integrase has distinct sites for viral and cellular DNA.

Sequences within Pr160gag-pol affecting the selective packaging of primer tRNA(Lys3) into HIV-1.

The selective packaging of the primer tRNA(Lys3) into HIV-1 particles is dependent upon the viral incorporation of the Pr160gag-pol precursor protein. In order to map a tRNA(Lys3) binding site within this precursor, we have studied the effects of mutations in Pr160gag-pol upon the selective incorporation of tRNA(Lys3). Many of these mutations were placed in a protease-negative HIV-1 proviral DNA to prevent viral protease degradation of the mutant Gag-Pol protein. C-terminal deletions of protease-negative Gag-Pol that removed the entire integrase sequence and the RNase H and connection subdomains of reverse transcriptase did not inhibit the incorporation of either the truncated Gag-Pol or the tRNA(Lys3), indicating that these regions are not required for tRNA(Lys3) binding. On the other hand, larger C-terminal deletions, which also remove the thumb subdomain sequence, did prevent tRNA(Lys3) packaging, without inhibiting viral incorporation of the truncated Gag-Pol, indicating a possible interaction between thumb subdomain sequences and tRNA(Lys3). While point mutations K249E, K249Q, and R307E in the primer grip region of the thumb subdomain have been reported to inhibit the in vitro interaction of mature reverse transcriptase with the anticodon loop of tRNA(Lys3), we find that these mutations do not inhibit tRNA(Lys3) packaging into the virus, which supports other work indicating that the anticodon loop of tRNA(Lys3) is not involved in interactions with Pr160gag-pol during tRNA(Lys3) packaging.

Inhibition of HIV-1 by an anti-integrase single-chain variable fragment (SFv): delivery by SV40 provides durable protection against HIV-1 and does not require selection.

Human immunodeficiency virus type 1 (HIV-1) encodes several proteins that are packaged into virus particles. Integrase (IN) is an essential retroviral enzyme, which has been a target for developing agents to inhibit virus replication. In previous studies, we showed that intracellular expression of single-chain variable antibody fragments (SFvs) that bind IN, delivered via retroviral expression vectors, provided resistance to productive HIV-1 infection in T-lymphocytic cells. In the current studies, we evaluated simian-virus 40 (SV40) as a delivery vehicle for anti-IN therapy of HIV-1 infection. Prior work suggested that delivery using SV40 might provide a high enough level of transduction that selection of transduced cells might be unnecessary. In these studies, an SV40 expression vector was developed to deliver SFv-IN (SV(Aw)). Expression of the SFv-IN was confirmed by Western blotting and immunofluorescence staining, which showed that > 90% of SupT1 T-lymphocytic cells treated with SV(Aw) expressed the SFv-IN protein without selection. When challenged, HIV-1 replication, as measured by HIV-1 p24 antigen expression and syncytium formation, was potently inhibited in cells expressing SV40-delivered SFv-IN. Levels of inhibition of HIV-1 infection achieved using this approach were comparable to those achieved using murine leukemia virus (MLV) as a transduction vector, the major difference being that transduction using SV40 did not require selection in culture whereas transduction with MLV did require selection. Therefore, the SV40 vector as gene delivery system represents a novel therapeutic strategy for gene therapy to target HIV-1 proteins and interfere with HIV-1 replication.

Assaying the activity of HIV-1 integrase with DNA-coated plates.

Integration of reverse transcribed viral DNA of HIV into host chromosomes is mediated by the viral enzyme, integrase. This enzymatic activity can be monitored in vitro by integration of a small labeled DNA (donor) into a second unlabeled DNA (target). The methodology usually involves isotope labeling and gel electrophoresis. To simplify the measurement, a method mimicking enzyme-linked immunosorbent assay (ELISA) procedures was developed. Fragments of DNA were adsorbed directly on 96-well plates and used as the target DNA. The donor was a synthetic 21-bp DNA duplex of HIV-1 U5 LTR; biotin was incorporated into the 5' end of one strand whose two nucleotides at the 3' end were specifically removed during the integration. As a result of integration, the biotin-labeled donor DNA was joined with the target DNA and became immobilized on plates. These integration products were then measured by binding of avidin-alkaline phosphatase on plates. The method is simple and straightforward and can easily be adapted for high throughput screening of integrase inhibitors.