X‐ray crystal structure of the N‐terminal region of Moloney murine leukemia virus integrase and its implications for viral DNA recognition

The retroviral integrase (IN) carries out the integration of a dsDNA copy of the viral genome into the host DNA, an essential step for viral replication. All IN proteins have three general domains, the N‐terminal domain (NTD), the catalytic core domain, and the C‐terminal domain. The NTD includes an HHCC zinc finger‐like motif, which is conserved in all retroviral IN proteins. Two crystal structures of Moloney murine leukemia virus (M‐MuLV) IN N‐terminal region (NTR) constructs that both include an N‐terminal extension domain (NED, residues 1–44) and an HHCC zinc‐finger NTD (residues 45–105), in two crystal forms are reported. The structures of IN NTR constructs encoding residues 1–105 (NTR_1–105) and 8–105 (NTR_8–105) were determined at 2.7 and 2.15 Å resolution, respectively and belong to different space groups. While both crystal forms have similar protomer structures, NTR_1–105 packs as a dimer and NTR_8–105 packs as a tetramer in the asymmetric unit. The structure of the NED consists of three anti‐parallel β‐strands and an α‐helix, similar to the NED of prototype foamy virus (PFV) IN. These three β‐strands form an extended β‐sheet with another β‐strand in the HHCC Zn²⁺ binding domain, which is a unique structural feature for the M‐MuLV IN. The HHCC Zn²⁺ binding domain structure is similar to that in HIV and PFV INs, with variations within the loop regions. Differences between the PFV and MLV IN NEDs localize at regions identified to interact with the PFV LTR and are compared with established biochemical and virological data for M‐MuLV. Proteins 2017; 85:647–656. © 2016 Wiley Periodicals, Inc.     

Assembly and catalysis of concerted two-end integration events by Moloney murine leukemia virus integrase

Retroviral integration results in the stable and coordinated insertion of the two termini of the linear viral DNA into the host genome. An in vitro concerted two-end integration reaction catalyzed by the Moloney murine leukemia virus (M-MuLV) integrase (IN) was used to investigate the binding and coordination of the two viral DNA ends. Comparison of the two-end integration and strand transfer assays indicates that zinc is required for efficient concerted integration utilizing plasmid DNA as target. Complementation assays using a pair of nonoverlapping integrase domains, consisting of the HHCC domain and the core/C-terminal region, yielded products containing the correct 4-base target site duplication. The efficiency of the coordinated two-end integration varied depending on the order of addition of the individual protein and DNA components in the complementation assay. Two-end integration was most efficient when the long terminal repeat (LTR) was premixed with either the target DNA or the HHCC domain. The preference for two-end integration through preincubation of the HHCC finger with the viral DNA supports the role of this domain in the recognition and/or positioning of the LTR.     

Coordinated disintegration reactions mediated by Moloney murine leukemia virus integrase.

The protein-DNA and protein-protein interactions important for function of the integrase (IN) protein of Moloney murine leukemia virus (M-MuLV) were investigated by using a coordinated-disintegration assay. A panel of M-MuLV IN mutants and substrate alterations highlighted distinctions between the intermolecular and intramolecular reactions of coordinated disintegration. Mispairing of the crossbone single-strand region and altered long terminal repeat (LTR) positioning affected the intermolecular, but not the intramolecular, reactions of coordinated disintegration. Partial components of the crossbone substrate were coordinated by M-MuLV IN, indicating a reliance on both LTR and target DNA determinants for substrate assembly. The intramolecular reaction was dependent on the presence of either the HHCC domain or a crossbone LTR 5' single-stranded tail. An M-MuLV IN mutant without the HHCC domain (Ndelta105) catalyzed reduced levels of double disintegration but not single disintegration. A separately purified HHCC domain protein (Cdelta232) stimulated double disintegration mediated by Ndelta105, suggesting a role of the N-terminal HHCC domain in stable IN-IN and IN-DNA interactions. Significantly, crossbone substrates lacking the LTR 5' tails were not recognized by the fingerless Ndelta105 protein. Collectively, these data suggest similar roles of the HHCC domain and 5' LTR tail in substrate recognition and modulation of IN activity.     

Moloney Murine Leukemia Virus Integrase Protein Augments Viral DNA Synthesis in Infected Cells

Mutations in the IN domain of retroviral DNA may affect multiple steps of the virus life cycle, suggesting that the IN protein may have other functions in addition to its integration function. We previously reported that the human immunodeficiency virus type 1 IN protein is required for efficient viral DNA synthesis and that this function requires specific interaction with other viral components but not enzyme (integration) activity. In this report, we characterized the structure and function of the Moloney murine leukemia virus (MLV) IN protein in viral DNA synthesis. Using an MLV vector containing green fluorescent protein as a sensitive reporter for virus infection, we found that mutations in either the catalytic triad (D184A) or the HHCC motif (H61A) reduced infectivity by approximately 1,000-fold. Mutations that deleted the entire IN (DeltaIN) or 34 C-terminal amino acid residues (Delta34) were more severely defective, with infectivity levels consistently reduced by 10,000-fold. Immunoblot analysis indicated that these mutants were similar to wild-type MLV with respect to virion production and proteolytic processing of the Gag and Pol precursor proteins. Using semiquantitative PCR to analyze viral cDNA synthesis in infected cells, we found the Delta34 and DeltaIN mutants to be markedly impaired while the D184A and H61A mutants synthesized cDNA at levels similar to the wild type. The DNA synthesis defect was rescued by complementing the Delta34 and DeltaIN mutants in trans with either wild-type IN or the D184A mutant IN, provided as a Gag-IN fusion protein. However, the DNA synthesis defect of DeltaIN mutant virions could not be complemented with the Delta34 IN mutant. Taken together, these analyses strongly suggested that the MLV IN protein itself is required for efficient viral DNA synthesis and that this function may be conserved among other retroviruses.     

N-Terminal domain of HTLV-I integrase. Complexation and conformational studies of the zinc finger.

The HTLV-I integrase N-terminal domain [50-residue peptide (IN50)], and a 35-residue truncated peptide formed by residues 9-43 (IN35) have been synthesized by solid-phase peptide synthesis. Formation of the 50-residue zinc finger type structure through a HHCC motif has been proved by UV-visible absorption spectroscopy. Its stability was demonstrated by an original method using RP-HPLC. Similar experiments performed on the 35-residue peptide showed that the truncation does not prevent zinc complex formation but rather that it significantly influences its stability. As evidenced by CD spectroscopy, the 50-residue zinc finger is unordered in aqueous solution but adopts a partially helical conformation when trifluoroethanol is added. These results are in agreement with our secondary structure predictions and demonstrate that the HTLV-I integrase N-terminal domain is likely to be composed of an helical region (residues 28-42) and a beta-strand (residues 20-23), associated with a HHCC zinc-binding motif. Size-exclusion chromatography showed that the structured zinc finger dimerizes through the helical region.     

Retroviral integrase domains: DNA binding and the recognition of LTR sequences.

Integration of retroviral DNA into the host chromosome requires a virus-encoded integrase (IN). IN recognizes, cuts and then joins specific viral DNA sequences (LTR ends) to essentially random sites in host DNA. We have used computer-assisted protein alignments and mutagenesis in an attempt to localize these functions within the avian retroviral IN protein. A comparison of the deduced amino acid sequences for 80 retroviral/retrotransposon IN proteins reveals strong conservation of an HHCC N-terminal 'Zn finger'-like domain, and a central D(35)E region which exhibits striking similarities with sequences deduced for bacterial IS elements. We demonstrate that the HHCC region is not required for DNA binding, but contributes to specific recognition of viral LTRs in the cutting and joining reactions. Deletions which extend into the D(35)E region destroy the ability of IN to bind DNA. Thus, we propose that the D(35)E region may specify a DNA-binding/cutting domain that is conserved throughout evolution in enzymes with similar functions.     

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.     

Implication of a Central Cysteine Residue and the HHCC Domain of Moloney Murine Leukemia Virus Integrase Protein in Functional Multimerization

Moloney murine leukemia virus (M-MuLV) IN-IN protein interactions important for catalysis of strand transfer and unimolecular and bimolecular disintegration reactions were investigated by using a panel of chemically modified M-MuLV IN proteins. Functional complementation of an HHCC-deleted protein (NΔ105) by an independent HHCC domain (CΔ232) was severely compromised by NEM modification of either subunit. Productive NΔ105 IN-DNA interactions with a disintegration substrate lacking a long terminal repeat 5′-single-stranded tail also required complementation by a functional HHCC domain. Virus encoding the C209A M-MuLV IN mutation exhibited delayed virion production and replication kinetics.     

A cooperative and specific DNA-binding mode of HIV-1 integrase depends on the nature of the metallic cofactor and involves the zinc-containing N-terminal domain

HIV-1 integrase catalyzes the insertion of the viral genome into chromosomal DNA. We characterized the structural determinants of the 3′-processing reaction specificity—the first reaction of the integration process—at the DNA-binding level. We found that the integrase N-terminal domain, containing a pseudo zinc-finger motif, plays a key role, at least indirectly, in the formation of specific integrase–DNA contacts. This motif mediates a cooperative DNA binding of integrase that occurs only with the cognate/viral DNA sequence and the physiologically relevant Mg²⁺ cofactor. The DNA-binding was essentially non-cooperative with Mn²⁺ or using non-specific/random sequences, regardless of the metallic cofactor. 2,2′-Dithiobisbenzamide-1 induced zinc ejection from integrase by covalently targeting the zinc-finger motif, and significantly decreased the Hill coefficient of the Mg²⁺-mediated integrase–DNA interaction, without affecting the overall affinity. Concomitantly, 2,2′-dithiobisbenzamide-1 severely impaired 3′-processing (IC₅₀ = 11–15 nM), suggesting that zinc ejection primarily perturbs the nature of the active integrase oligomer. A less specific and weaker catalytic effect of 2,2′-dithiobisbenzamide-1 is mediated by Cys 56 in the catalytic core and, notably, accounts for the weaker inhibition of the non-cooperative Mn²⁺-dependent 3′-processing. Our data show that the cooperative DNA-binding mode is strongly related to the sequence-specific DNA-binding, and depends on the simultaneous presence of the Mg²⁺ cofactor and the zinc effector.Integration of HIV-1 DNA into the host genome ensures stable maintenance of the viral genome in the host organism and, therefore, is a key process in the virus life cycle. Integrase (IN) is responsible for two distinct, consecutive catalytic steps in the integration process (1). The first of these two reactions is 3′-processing, which corresponds to the specific cleavage of two nucleotides from the 3′-ends of the linear viral DNA. The hydroxyl groups of newly recessed 3′-ends are then used in the second reaction— strand transfer—for the covalent joining of viral and cellular (or target) DNAs, resulting in full-site integration. For both reactions, IN functions as a multimer, most likely a dimer for 3′-processing and a tetramer (dimer of a dimer) for concerted integration (2–7). Two other reactions occur in vitro, a disintegration reaction that represents, in first approximation, the reversal of the half-site integration process (8) and a specific internal cleavage occurring on a symmetrical DNA site (9). All reactions require a metallic cofactor, Mg²⁺ or Mn²⁺, and, except for disintegration (10,11), all reactions require the full-length IN. There are several experimental evidences to suggest that Mg²⁺ is more physiologically relevant as a cofactor, particularly because Mg²⁺-dependent catalysis exhibits weaker non-specific endonucleolytic cleavage and the tolerance of sequence variation at the ends of the viral DNA is much greater in the presence of Mn²⁺ than in the presence of Mg²⁺ (12–15).The emergence of viral strains resistant against available drugs and the dynamic nature of the HIV-1 genome support a continued effort towards the discovery and characterization of novel targets and anti-viral drugs. Due to its central role in the HIV-1 life cycle, IN represents a promising therapeutic target. In the past, in vitro IN assays were extensively used to find IN inhibitors (16). Current inhibitors can be separated into two main classes, depending on their mechanisms of action: (i) Compounds that competitively prevent the DNA binding of IN to the viral DNA. These compounds are mainly directed against the 3′-processing reaction as they bind to the donor site within the catalytic site—i.e. the ‘specific’ DNA-binding site for the viral DNA -. This group is referred to as ‘integrase DNA-binding inhibitors’ (INBI) and includes styrylquinoline compounds (17,18). (ii) The second class includes compounds that cannot bind to the DNA-free IN. They bind to the pre-formed IN–viral DNA complex. These compounds preferentially inhibit strand transfer over the 3′-processing reaction [this family of compounds is referred to as ‘integrase strand transfer inhibitors’ (INSTI)], probably by displacing the viral DNA end from the active site (7,19–21). It is not clear whether this mechanism alone accounts for the inhibitory properties of INSTIs or whether these compounds also prevent the binding of the target DNA to the acceptor site—i.e. the ‘non-specific’ DNA binding site. INSTI compounds have generally good ex vivo activity against HIV replication, probably due to their ability to inhibit pre-assembled viral DNA/IN complexes. Raltegravir which is currently used in clinical treatment of HIV-1 belongs to this class. For both anti-IN classes, resistance mutations were identified (17,20,22,23). Difficulties in deeply understanding their mechanisms of action are closely related to the absence of structural data that clearly delineate the donor and the acceptor DNA binding sites in the active site. Although structural information is now available regarding the IN–viral DNA interaction, based on the recent crystal structure of the full-length primate foamy virus (PFV-1) IN in complex with its cognate processed viral DNA, the target DNA binding mode and the precise location of the acceptor site remains open to debate (7). Moreover, it is a difficult task to experimentally discriminate between the two DNA binding sites and no significant or only modest difference can be evidenced in vitro (depending on the method used for monitoring IN–DNA interactions) between the HIV-1 IN binding to the cognate viral DNA sequence and a non-specific random sequence in terms of overall affinity, suggesting that the specific and the non-specific DNA-binding modes display similar binding free energies (5,24). The basis of DNA binding specificity remains essentially unknown.HIV-1 IN (288 amino acids) contains three functional domains. The central domain or catalytic core domain (IN^50–213 or CC) contains the catalytic triad (DDE) that coordinates one or two metallic cofactors [probably a pair coordinated by three carboxylate groups of the triad, based on the X-ray structure of the PFV-1 IN (7)] and is essential for enzymatic activity; this domain alone can perform the disintegration reaction (10,11). This domain is flanked by the N-terminal (IN^1-49) and the C-terminal (IN^214–288) domains. The C-terminal domain is involved in IN–DNA contacts, together with the CC domain (25,26). The N-terminal domain contains a conserved non-conventional HHCC motif that binds zinc to ensure proper domain folding and promotes IN multimerization (27–29). It is worth noting that the integrity of the HHCC motif is crucial in the stringent Mg²⁺-context but appears dispensable under the less stringent Mn²⁺ condition (30), suggesting, at least, an indirect role of the zinc-binding domain in the establishment of specific and physiologically relevant IN–DNA complexes. In the structure of the PFV-1 IN–viral DNA complex, the N-terminal domain is also involved in the interaction with DNA (7).In this article, we found that IN binds cooperatively to the cognate viral DNA sequence only in the presence of Mg²⁺. The presence of Mn²⁺ or, most importantly, the use of non-specific random sequences, regardless of the metallic cofactor, dramatically reduced the Hill coefficient. This finding suggests that the cooperative DNA-binding mode of IN is strongly related to the formation of specific IN–DNA contacts. To gain deeper insight into the role of the zinc-binding domain in the cooperative/multimerization process, in relationship with the establishment of specific protein–DNA contacts, we studied the effect of DIBA-1 (2,2′-dithiobisbenzamide-1) (Figure 1A) on IN activity. This compound is a zinc ejector affecting many proteins containing zinc fingers, including HIV-1 nucleocapsid or estrogen receptor (31–34). Here, we found that DIBA-1 induced zinc ejection from the IN N-terminal domain by covalently targeting the HHCC motif. In the presence of Mg²⁺, DIBA-1 did not affect significantly the overall affinity of IN for the DNA substrate but dramatically reduced the Hill coefficient. Concomitantly, DIBA-1 strongly inhibited the catalytic step, with IC₅₀ values against the 3′-processing reaction of 11–15 nM. Interestingly, we found a secondary DIBA-1 binding site in the catalytic core (involving residue Cys 56), suggesting a second mechanism of action of DIBA-1, independent of zinc ejection. The prevalence of the two distinct mechanisms was dependent on the cofactor context, with the second one accounting for the weaker DIBA-1 inhibitory effect under Mn²⁺ conditions (IC₅₀ = 115–126 nM). DIBA-1 behaves as a non-competitive/catalytic inhibitor that did not disturb the fractional saturation of DNA sites, regardless of the mechanism considered. Open in a separate windowFigure 1.DIBA-1 induces the ejection of zinc from IN. (A) Structure of 2,2′-dithiobisbenzamide-1 (DIBA-1) (MW, 614 Da). (B) Ejection of zinc was measured by optical absorbance at 495 nm using a sample containing full-length IN (1 µM) and PAR (10⁻⁴ M) in a Tris buffer (20 mM pH 7.0) containing 15% DMSO (v/v) in the absence of reducing agent (black squares) or in the presence of 4 mM β-mercaptoethanol (white circles). The concentration of DIBA-1 for complete zinc release—[DIBA-1]_eq—was estimated graphically. This value was determined as a function of the initial concentration of IN (C). The slope of the straight line indicates that the zinc ejection coincides with the reaction of two DIBA-1 molecules per IN protomer.Altogether, our results show that, although it is a difficult task to discriminate between the specific viral sequence and a non-specific random sequence in terms of overall affinity, these sequences lead to distinct DNA-binding properties in terms of cooperativity. Moreover, our results highlight that the Mg²⁺-dependent catalytic activity of IN is strongly sensitive to the loss of cooperative DNA binding. Such a cooperative DNA-binding mode accounts for specific activity and requires: (i) the cognate viral DNA sequence, (ii) Mg²⁺ as a catalytic cofactor and (iii) zinc which can be considered as a positive allosteric effector. Development of non-competitive compounds acting on the N-terminal domain may be of interest for anti-IN pharmacology.     

Mechanism of action of the HIV-1 integrase inhibitory peptide LEDGF 361-370

The HIV-1 integrase protein (IN) mediates integration of the viral cDNA into the host genome and is a target for anti-HIV drugs. We have recently described a peptide derived from residues 361-370 of the IN cellular partner protein LEDGF/p75, which inhibited IN catalytic activity in vitro and HIV-1 replication in cells. Here we performed a comprehensive study of the LEDGF 361-370 mechanism of action in vitro, in cells and in vivo. Alanine scan, fluorescence anisotropy binding studies, homology modeling and NMR studies demonstrated that all residues in LEDGF 361-370 contribute to IN binding and inhibition. Kinetic studies in cells showed that LEDGF 361-370 specifically inhibited integration of viral cDNA. Thus, the full peptide was chosen for in vivo studies, in which it inhibited the production of HIV-1 RNA in mouse model. We conclude that the full LEDGF 361-370 peptide is a potent HIV-1 inhibitor and may be used for further development as an anti-HIV lead compound.     

Chromosomal integration of LTR-flanked DNA in yeast expressing HIV-1 integrase: down regulation by RAD51

HIV-1 integrase (IN) is the key enzyme catalyzing the proviral DNA integration step. Although the enzyme catalyzes the integration step accurately in vitro, whether IN is sufficient for in vivo integration and how it interacts with the cellular machinery remains unclear. We set up a yeast cellular integration system where integrase was expressed as the sole HIV-1 protein and targeted the chromosomes. In this simple eukaryotic model, integrase is necessary and sufficient for the insertion of a DNA containing viral LTRs into the genome, thereby allowing the study of the isolated integration step independently of other viral mechanisms. Furthermore, the yeast system was used to identify cellular mechanisms involved in the integration step and allowed us to show the role of homologous recombination systems. We demonstrated physical interactions between HIV-1 IN and RAD51 protein and showed that HIV-1 integrase activity could be inhibited both in the cell and in vitro by RAD51 protein. Our data allowed the identification of RAD51 as a novel in vitro IN cofactor able to down regulate the activity of this retroviral enzyme, thereby acting as a potential cellular restriction factor to HIV infection.     

HRP2 determines the efficiency and specificity of HIV-1 integration in LEDGF/p75 knockout cells but does not contribute to the antiviral activity of a potent LEDGF/p75-binding site integrase inhibitor

The binding of integrase (IN) to lens epithelium-derived growth factor (LEDGF)/p75 in large part determines the efficiency and specificity of HIV-1 integration. However, a significant residual preference for integration into active genes persists in Psip1 (the gene that encodes for LEDGF/p75) knockout (KO) cells. One other cellular protein, HRP2, harbors both the PWWP and IN-binding domains that are important for LEDGF/p75 co-factor function. To assess the role of HRP2 in HIV-1 integration, cells generated from Hdgfrp2 (the gene that encodes for HRP2) and Psip1/Hdgfrp2 KO mice were infected alongside matched control cells. HRP2 depleted cells supported normal infection, while disruption of Hdgfrp2 in Psip1 KO cells yielded additional defects in the efficiency and specificity of integration. These deficits were largely restored by ectopic expression of either LEDGF/p75 or HRP2. The double-KO cells nevertheless supported residual integration into genes, indicating that IN and/or other host factors contribute to integration specificity in the absence of LEDGF/p75 and HRP2. Psip1 KO significantly increased the potency of an allosteric inhibitor that binds the LEDGF/p75 binding site on IN, a result that was not significantly altered by Hdgfrp2 disruption. These findings help to rule out the host factor-IN interactions as the primary antiviral targets of LEDGF/p75-binding site IN inhibitors.