Lys-34, dispensable for integrase catalysis, is required for preintegration complex function and human immunodeficiency virus type 1 replication

Retroviral integrases (INs) function in the context of preintegration complexes (PICs). Two conserved Lys residues in the N-terminal domain of human immunodeficiency virus type 1 (HIV-1) IN were analyzed here for their roles in integration and virus replication. Whereas HIV-1(K46A) grew like the wild type, HIV-1(K34A) was dead. Yet recombinant IN(K34A) protein functioned in in vitro integration assays, and Vpr-IN(K34A) efficiently transcomplemented the infectivity defect of an IN active site mutant virus in cells. HIV-1(K34A) was therefore similar to a number of previously characterized mutant viruses that failed to replicate despite encoding catalytically competent IN. To directly analyze mutant PIC function, a sensitive PCR-based integration assay was developed. HIV-1(K34A) and related mutants failed to support detectable levels (<1% of wild type) of integration. We therefore concluded that mutations like K34A disrupted higher-order interactions important for PIC function/maturation compared to the innate catalytic activity of IN enzyme.     

Functional Interactions of Human Immunodeficiency Virus Type 1 Integrase with Human and Yeast HSP60

Integration of human immunodeficiency virus type 1 (HIV-1) proviral DNA in the nuclear genome is catalyzed by the retroviral integrase (IN). In addition to IN, viral and cellular proteins associated in the high-molecular-weight preintegration complex have been suggested to be involved in this process. In an attempt to define host factors interacting with IN, we used an in vitro system to identify cellular proteins in interaction with HIV-1 IN. The yeast Saccharomyces cerevisiae was chosen since (i) its complete sequence has been established and the primary structure of all the putative proteins from this eucaryote has been deduced, (ii) there is a significant degree of homology between human and yeast proteins, and (iii) we have previously shown that the expression of HIV-1 IN in yeast induces a lethal phenotype. Strong evidences suggest that this lethality is linked to IN activity in infected human cells where integration requires the cleavage of genomic DNA. Using IN-affinity chromatography we identified four yeast proteins interacting with HIV-1 IN, including the yeast chaperonin yHSP60, which is the counterpart of human hHSP60. Yeast lethality induced by HIV-1 IN was abolished when a mutated HSP60 was coexpressed, therefore suggesting that both proteins interact in vivo. Besides interacting with HIV-1 IN, the hHSP60 was able to stimulate the in vitro processing and joining activities of IN and protected this enzyme from thermal denaturation. In addition, the functional human HSP60-HSP10 complex in the presence of ATP was able to recognize the HIV-1 IN as a substrate.     

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.     

Biochemical and random mutagenesis analysis of the region carrying the catalytic E152 amino acid of HIV-1 integrase

HIV-1 integrase (IN) catalyzes the integration of the proviral DNA into the cellular genome. The catalytic triad D₆₄, D₁₁₆ and E₁₅₂ of HIV-1 IN is involved in the reaction mechanism and the DNA binding. Since the integration and substrate binding processes are not yet exactly known, we studied the role of amino acids localized in the catalytic site. We focused our interest on the V₁₅₁E₁₅₂S₁₅₃ region. We generated random mutations inside this domain and selected mutated active INs by using the IN-induced yeast lethality assay. In vitro analysis of the selected enzymes showed that the IN nuclease activities (specific 3′-processing and non-sequence-specific endonuclease), the integration and disintegration reactions and the binding of the various DNA substrates were affected differently. Our results support the hypothesis that the three reactions may involve different DNA binding sites, enzyme conformations or mechanisms. We also show that the V₁₅₁E₁₅₂S₁₅₃ region involvement in the integration reaction is more important than for the 3′-processing activity and can be involved in the recognition of DNA. The IN mutants may lead to the development of new tools for studying the integration reaction, and could serve as the basis for the discovery of integration-specific inhibitors.     

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.     

Characterization of Mutant HIV-1 Integrase Carrying Amino Acid Changes in the Catalytic Domain

To gain insight into the importance of conserved residues in the core domain of HIV-1 IN, we performed site-directed mutagenesis of the full-length enzyme, overexpressed the mutant proteins in E. coli, purified and analyzed their 3-processing, integration and disintegration activities in vitro. Change of E152V in the DD(35)E motif abolished all detectable activities of IN. Alteration of two highly conserved residues, P145 and K156, by isoleucine, resulted in a substantial loss or completely abolished the three activities of the enzyme. Mutant P90D weakly reduced the 3-processing but severely affected the two other IN activities. Results obtained from double and triple mutations, P90D/P145I and P145I/F185K/C280S, clearly suggest a crucial role of P145 in the catalytic function of IN, whereas the mutants V150E, L158F and L172M had no detectable effect on any of the IN activities. Taken together, these results allowed us to conclude that all the conserved amino acids in the core domain of IN are not equally important for catalytic functions: like D64, D116 and E152, our data suggest that P90, P145 and K156 are also essential for all three enzymatic activities of HIV-1 IN in vitro, whereas V150, L158 and L172 appear to be less critical.     

Dissecting the role of the N-terminal domain of human immunodeficiency virus integrase by trans-complementation analysis

The human immunodeficiency virus (HIV) integrase protein (IN) catalyzes two reactions required to integrate HIV DNA into the human genome: 3' processing of the viral DNA ends and integration. IN has three domains, the N-terminal zinc-binding domain, the catalytic core, and the C-terminal SH3 domain. Previously, it was shown that IN proteins mutated in different domains could complement each other. We now report that this does not require any overlap between the two complementing proteins; an N-terminal domain, provided in trans, can restore IN activity of a mutant lacking this domain. Only the zinc-coordinating form of the N-terminal domain can efficiently restore IN activity of an N-terminal deletion mutant. This suggests that interaction between different domains of IN is needed for functional multimerization. We find that the N-terminal domain of feline immunodeficiency virus IN can support IN activity of an N-terminal deletion mutant of HIV type 2 IN. These cross-complementation experiments indicate that the N-terminal domain contributes to the recognition of specific viral DNA ends.     

The human polycomb group EED protein interacts with the integrase of human immunodeficiency virus type 1

Human EED, a member of the superfamily of WD-40 repeat proteins and of the Polycomb group proteins, has been identified as a cellular partner of the human immunodeficiency virus type 1 (HIV-1) matrix (MA) protein (R. Peytavi et al., J. Biol. Chem. 274:1635-1645, 1999). In the present study, EED was found to interact with HIV-1 integrase (IN) both in vitro and in vivo in yeast. In vitro, data from mutagenesis studies, pull-down assays, and phage biopanning suggested that EED-binding site(s) are located in the C-terminal domain of IN, between residues 212 and 264. In EED, two putative discrete IN-binding sites were mapped to its N-terminal moiety, at a distance from the MA-binding site, but EED-IN interaction also required the integrity of the EED last two WD repeats. EED showed an apparent positive effect on IN-mediated DNA integration reaction in vitro, in a dose-dependent manner. In situ analysis by immunoelectron microscopy (IEM) of cellular distribution of IN and EED in HIV-1-infected cells (HeLa CD4(+) cells or MT4 lymphoid cells) showed that IN and EED colocalized in the nucleus and near nuclear pores, with maximum colocalization events occurring at 6 h postinfection (p.i.). Triple colocalizations of IN, EED, and MA were also observed in the nucleoplasm of infected cells at 6 h p.i., suggesting the ocurrence of multiprotein complexes involving these three proteins at early steps of the HIV-1 virus life cycle. Such IEM patterns were not observed with a noninfectious, envelope deletion mutant of HIV-1.     

Inhibition of early steps of HIV-1 replication by SNF5/Ini1

To replicate, human immunodeficiency virus, type 1 (HIV-1) needs to integrate a cDNA copy of its RNA genome into a chromosome of the host cell, a step controlled by the viral integrase (IN) protein. Viral integration involves the participation of several cellular proteins. SNF5/Ini1, a subunit of the SWI/SNF chromatin remodeling complex, was the first cofactor identified to interact with IN. We report here that SNF5/Ini1 interferes with early steps of HIV-1 replication. Inhibition of SNF5/Ini1 expression by RNA interference increases HIV-1 replication. Using quantitative PCR, we show that both the 2-long terminal repeat circle and integrated DNA forms accumulate upon SNF5/Ini1 knock down. By yeast two-hybrid assay, we screened a library of HIV-1 IN random mutants obtained by PCR random mutagenesis using SNF5/Ini1 as prey. Two different mutants of interaction, IN E69G and IN K71R, were impaired for SNF5/Ini1 interaction. The E69G substitution completely abolished integrase catalytic activity, leading to a replication-defective virus. On the contrary, IN K71R retained in vitro integrase activity. K71R substitution stimulates viral replication and results in higher infectious titers. Taken together, these results suggest that, by interacting with IN, SNF5/Ini1 interferes with early steps of HIV-1 infection.     

Structural Basis for Functional Tetramerization of Lentiviral Integrase

Experimental evidence suggests that a tetramer of integrase (IN) is the protagonist of the concerted strand transfer reaction, whereby both ends of retroviral DNA are inserted into a host cell chromosome. Herein we present two crystal structures containing the N-terminal and the catalytic core domains of maedi-visna virus IN in complex with the IN binding domain of the common lentiviral integration co-factor LEDGF. The structures reveal that the dimer-of-dimers architecture of the IN tetramer is stabilized by swapping N-terminal domains between the inner pair of monomers poised to execute catalytic function. Comparison of four independent IN tetramers in our crystal structures elucidate the basis for the closure of the highly flexible dimer-dimer interface, allowing us to model how a pair of active sites become situated for concerted integration. Using a range of complementary approaches, we demonstrate that the dimer-dimer interface is essential for HIV-1 IN tetramerization, concerted integration in vitro, and virus infectivity. Our structures moreover highlight adaptable changes at the interfaces of individual IN dimers that allow divergent lentiviruses to utilize a highly-conserved, common integration co-factor.     

Structural analysis of a mutant of the HIV-1 integrase zinc finger domain that forms a single conformation

HIV-1 integrase consists of three functional domains, an N-terminal zinc finger domain, a catalytic core domain and a C-terminal DNA binding domain. NMR analysis of an isolated N-terminal domain (IN(1-55)) has shown that IN(1-55) exists in two conformational states [E and D forms; Cai et al. (1997) Nat. Struct. Biol. 4, 567-577]. The two forms differ in the coordination of the zinc ion by two histidine residues. In the present study, structural analysis of a mutant of IN(1-55), Y15A, by NMR spectroscopy indicated that the mutant protein folds correctly but takes only the E form. Since the Y15A mutation abrogates the HIV-1 infectivity, Y15 might have some important role in the full-length integrase activity during the virus infection cycle. Our results suggest a possible role of Y15 in structural transition between the E and D forms of HIV-1 integrase to allow the optimal tetramerization.     

Nuclear localization of human immunodeficiency virus type 1 preintegration complexes (PICs): V165A and R166A are pleiotropic integrase mutants primarily defective for integration,not PIC nuclear import

Retroviral replication requires the integration of reverse-transcribed viral cDNA into a cell chromosome. A key barrier to forming the integrated provirus is the nuclear envelope, and numerous regions in human immunodeficiency virus type 1 (HIV-1) have been shown to aid the nuclear localization of viral preintegration complexes (PICs) in infected cells. One region in integrase (IN), composed of Val-165 and Arg-166, was reportedly essential for HIV-1 replication and nuclear localization in all cell types. In this study we confirmed that HIV-1(V165A) and HIV-1(R166A) were replication defective and that less mutant viral cDNA localized to infected cell nuclei. However, we present three lines of evidence that argue against a specific role for Val-165 and Arg-166 in PIC nuclear import. First, results of transient transfections revealed that V165A FLAG-tagged IN and green fluorescent protein-IN fusions carrying either V165A or R166A predominantly localized to cell nuclei. Second, two different strains of previously described class II IN mutant viruses displayed similar nuclear entry profiles to those observed for HIV-1(V165A) and HIV-1(R166A), suggesting that defective nuclear import may be a common phenotype of replication-defective IN mutant viruses. Third, V165A and R166A mutants were defective for in vitro integration activity, when assayed both as PICs isolated from infected T-cells and as recombinant IN proteins purified from Escherichia coli. Based on these results, we conclude that HIV-1(V165A) and HIV-1(R166A) are pleiotropic mutants primarily defective for IN catalysis and that Val-165 and Arg-166 do not play a specific role in the nuclear localization of HIV-1 PICs in infected cells.     

Minimal Core Domain of HIV-1 Integrase for Biological Activity

The human immunodeficiency virus type-1 (HIV-1) integrase (IN) mediates insertion of viral DNA into human DNA, which is an essential step in the viral life cycle. In order to study minimal core domain in HIV-1 IN protein, we constructed nine deletion mutants by using PCR amplification. The constructs were expressed in Escherichia coli, and the proteins were subsequently purified and analyzed in terms of biological activity such as enzymatic and DNA-binding activities. The mutant INs with an N-terminal or C-terminal deletion showed strong disintegration activity though they failed to show endonucleolytic and strand transfer activities, indicating that the disintegration reaction does not require the fine structure of the HIV-1 IN protein. In the DNA-binding analysis using gel mobility shift assay and UV cross-linking method, it was found that both the central and C-terminal domains are essential for proper DNA-IN protein interaction although the central or C-terminal domain alone was able to be in close contact with DNA substrate. Therefore, our results suggest that the C-terminal domain act as a DNA-holding motive, which leads to proper interaction for enzymatic reaction between the IN protein and DNA.