Functional Interactions of the HHCC Domain of Moloney Murine Leukemia Virus Integrase Revealed by Nonoverlapping Complementation and Zinc-Dependent Dimerization

The retroviral integrase (IN) is required for the integration of viral DNA into the host genome. The N terminus of IN contains an HHCC zinc finger-like motif, which is conserved among all retroviruses. To study the function of the HHCC domain of Moloney murine leukemia virus IN, the first N-terminal 105 residues were expressed independently. This HHCC domain protein is found to complement a completely nonoverlapping construct lacking the HHCC domain for strand transfer, 3′ processing and coordinated disintegration reactions, revealing trans interactions among IN domains. The HHCC domain protein binds zinc at a 1:1 ratio and changes its conformation upon binding to zinc. The presence of zinc within the HHCC domain stimulates selective integration processes. Zinc promotes the dimerization of the HHCC domain and protects it from N-ethylmaleimide modification. These studies dissect and define the requirement for the HHCC domain, the exact function of which remains unknown.     

Subcellular localization of feline immunodeficiency virus integrase and mapping of its karyophilic determinant

Feline immunodeficiency virus (FIV), like other members of the lentivirus subfamily, such as human immunodeficiency virus type 1 (HIV-1), can infect nondividing and terminally differentiated cells. The transport of the preintegration complex into the nucleus is cell cycle-independent, but the mechanism is not well understood. Integrase is a key component of the complex and has been suggested to play a role in nuclear import during HIV-1 replication. To determine its karyophilic property, FIV integrase fused with glutathione S-transferase and enhanced green fluorescent protein was expressed in various feline and human cells and the subcellular localization was visualized by fluorescence microscopy. Wild-type FIV integrase was karyophilic in all cell lines tested and capable of targeting the fusion protein to the nuclei of transfected cells. Analysis of deletion and point mutation variants of FIV integrase failed to reveal any canonical nuclear localization signal, and the karyophilic determinant was mapped to the highly conserved N-terminal zinc-binding HHCC motif. A region near the C-terminal domain enriched with basic amino acid residues also affected the nuclear import of integrase. However, the role of this region is only modulatory in comparison to that of the zinc-binding domain. The N-terminal zinc-binding domain does not bind DNA and instead is essential in integrase multimerization. We therefore postulate that the karyophilic property of FIV integrase requires subunit multimerization promoted by the HHCC motif. Alternatively, the HHCC motif may directly promote interaction between FIV integrase and cellular proteins involved in nuclear import.     

The core and carboxyl-terminal domains of the integrase protein of human immunodeficiency virus type 1 each contribute to nonspecific DNA binding.

The integrase protein of human immunodeficiency virus type 1 removes two nucleotides from the 3' ends of reverse-transcribed human immunodeficiency virus type 1 DNA (3' processing) and covalently inserts the processed ends into a target DNA (DNA strand transfer). Mutant integrase proteins that lack the amino-and/or carboxyl-terminal domains are incapable of catalyzing 3' processing and DNA strand transfer but are competent for an apparent reversal of the DNA strand transfer reaction (disintegration) in vitro. Here, we investigate the binding of integrase to DNA by UV cross-linking. Cross-linked complexes form with a variety of DNA substrates independent of the presence of divalent metal ion. Analysis with amino- and carboxyl-terminal deletion mutant proteins shows that residues 213 to 266 of the 288-residue protein are required for efficient cross-linking in the absence of divalent metal ion. Carboxyl-terminal deletion mutants that lack this region efficiently cross-link only to the branched disintegration DNA substrate, and this reaction is dependent on the presence of metal ion. Both the core and C-terminal domains of integrase therefore contribute to nonspecific DNA binding.     

Characterization of human immunodeficiency virus type 1 integrase expressed in Escherichia coli and analysis of variants with amino-terminal mutations.

Replication of a retroviral genome depends upon integration of the viral DNA into a chromosome of the host cell. The integration reaction is mediated by integrase, a viral enzyme. Human immunodeficiency virus type 1 integrase was expressed in Escherichia coli and purified to near homogeneity. Optimum conditions for the integration and 3'-end-processing activities of integrase were characterized by using an in vitro assay with short, double-stranded oligonucleotide substrates. Mutants containing amino acid substitutions within the HHCC region, defined by phylogenetically conserved pairs of histidine and cysteine residues near the N terminus, were constructed and characterized by using three assays: 3'-end processing, integration, and the reverse of the integration reaction (or disintegration). Mutations in the conserved histidine and cysteine residues abolished both integration and processing activities. Weak activity in both assays was retained by two other mutants containing substitutions for less highly conserved amino acids in this region. All mutants retained activity in the disintegration assay, implying that the active site for DNA cleavage-ligation is not located in this domain and that the HHCC region is not the sole DNA-binding domain in the protein. However, the preferential impairment of processing and integration rather than disintegration by mutations in the HHCC region is consistent with a role for this domain in recognizing features of the viral DNA. This hypothesis is supported by the results of disintegration assays performed with altered substrates. The results support a model involving separate viral and target DNA-binding sites on integrase.     

Identification of conserved amino acid residues critical for human immunodeficiency virus type 1 integrase function in vitro.

We have probed the structural organization of the human immunodeficiency virus type 1 integrase protein by limited proteolysis and the functional organization by site-directed mutagenesis of selected amino acid residues. A central region of the protein was relatively resistant to proteolysis. Proteins with altered amino acids in this region, or in the N-terminal part of the protein that includes a putative zinc-binding motif, were purified and assayed for 3' processing, DNA strand transfer, and disintegration activities in vitro. In general, these mutations had parallel effects on 3' processing and DNA strand transfer, suggesting that integrase may utilize a single active site for both reactions. The only proteins that were completely inactive in all three assays contained mutations at conserved amino acids in the central region, suggesting that this part of the protein may be involved in catalysis. In contrast, none of the mutations in the N-terminal region resulted in a protein that was inactive in all three assays, suggesting that this part of integrase may not be essential for catalysis. The disintegration reaction was particularly insensitive to these amino acid substitutions, indicating that some function that is important for 3' processing and DNA strand transfer may be dispensable for disintegration.     

Biochemical characteristics of functional domains using feline foamy virus integrase mutants

We constructed deletion mutants and seven point mutants by polymerase chain reaction to investigate the specificity of feline foamy virus integrase functional domains. Complementation reactions were performed for three enzymatic activities such as 3’-end processing, strand transfer, and disintegration. The complementation reactions with deletion mutants showed several activities for 3’-end processing and strand transfer. The conserved central domain and the combination of the N-terminal or C-terminal domains increased disintegration activity significantly. In the complementation reactions between deletion and point mutants, the combination between D107V and deletion mutants revealed 3’-end processing activities, but the combination with others did not have any activity, including strand transfer activities. Disintegration activity increased evenly, except the combination with glutamic acid 200. These results suggest that an intact central domain mediates enzymatic activities but fails to show these activities in the absence of the N-terminal or C-terminal domains. [BMB Reports 2013; 46(1):53-58]     

Characterization of a replication-defective human immunodeficiency virus type 1 att site mutant that is blocked after the 3' processing step of retroviral integration

Two activities of retroviral integrase, 3' processing and DNA strand transfer, are required to integrate viral cDNA into a host cell chromosome. Integrase activity has been analyzed in vitro using purified protein and recombinant DNA substrates that model the U3 and U5 ends of viral cDNA or by using viral preintegration complexes (PICs) that form during virus infection. Numerous studies have investigated changes in integrase or viral DNA for effects on both 3' processing and DNA strand transfer activities using purified protein, but similar analyses have not been carried out using PICs. Here, we analyzed PICs from human immunodeficiency virus type 1 (HIV-1) strain 604del, an integration-defective mutant lacking 26 bp of U5, and revE1, a revertant of 604del containing an additional 19-bp deletion, for levels of 3' processing activity that occurred in infected cells and for levels of in vitro DNA strand transfer activity. Whereas revE1 supported one-third to one-half of the level of wild-type DNA strand transfer activity, the level of 604del DNA strand transfer activity was undetectable. Surprisingly, integrase similarly processed the 3' ends of 604del and revE1 in vivo. We therefore conclude that 604del is blocked in its ability to replicate in cells after the 3' processing step of retroviral integration. Whereas Western blotting showed that wild-type, revE1, and 604del PICs contained similar levels of integrase protein, Mu-mediated PCR footprinting revealed only minimal protein-DNA complex formation at the ends of 604del cDNA. We propose that 604del is replication defective because proteins important for DNA strand transfer activity do not stably associate with this cDNA after in vivo 3' processing by integrase.     

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.     

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.     

Identification of the catalytic and DNA-binding region of the human immunodeficiency virus type I integrase protein.

The integrase (IN) protein of the human immunodeficiency virus (HIV) is required for specific cleavage of the viral DNA termini, and subsequent integration of the viral DNA into target DNA. To identify the various domains of the IN protein we generated a series of IN deletion mutants as fusions to maltose-binding protein (MBP). The deletion mutants were tested for their ability to bind DNA, to mediate site-specific cleavage of the viral DNA ends, and to carry out integration and disintegration reactions. We found that the DNA-binding region resides between amino acids 200 and 270 of the 288-residues HIV-1 IN protein. The catalytic domain of the protein was mapped between amino acids 50 and 194. For the specific activities of IN, cleavage of the viral DNA and integration, both the DNA-binding domain and the conserved amino-terminal region of IN are required. These regions are dispensable however, for disintegration activity.     

Functional domains of Moloney murine leukemia virus integrase defined by mutation and complementation analysis.

Retroviral integrases perform two catalytic steps, 3' processing and strand transfer, that result in the stable insertion of the retroviral DNA into the host genome. Mutant M-MuLV integrases were constructed to define the functional domains important for 3' processing, strand transfer, and disintegration by in vitro assays. N-terminal mutants had no detectable 3' processing activity, and only one mutant which lacks the HHCC domain, Ndelta105, had strand transfer activity. Strand transfer mediated by Ndelta105 showed preference for one site in the target DNA. Disintegration activity of N-terminal mutants decreased only minimally. In contrast, all C-terminal mutants truncated by more than 28 amino acids had no integration or disintegration activity. Activity on a single-strand disintegration substrate did not require a functional HHCC domain but did require most of the C-terminal region. Complementation analysis found that the HHCC region alone was able to function in trans to a promoter containing only the DD(35)E and C-terminal regions and to enhance integration site selection. Increasing the reducing conditions or adding the HHCC domain to Ndelta105 reaction mixtures restored the wild-type strand transfer activity and range of target sites. The reducing agent affected Cys-209 in the DD(35)E region. The presence of C-209 was required for complementation of Ndelta105 by the HHCC region.     

Infectivity of Moloney murine leukemia virus defective in late assembly events is restored by late assembly domains of other retroviruses

The p12 region of the Moloney murine leukemia virus (M-MuLV) Gag protein contains a PPPY motif important for efficient virion assembly and release. To probe the function of the PPPY motif, a series of insertions of homologous and heterologous motifs from other retroviruses were introduced at various positions in a mutant gag gene lacking the PPPY motif. The assembly defects of the PPPY deletion mutant could be rescued by insertion of a wild-type PPPY motif and flanking sequences at several ectopic positions in the Gag protein. The late assembly domain (L-domain) of Rous sarcoma virus (RSV) or human immunodeficiency virus type 1 (HIV-1) could also fully or partially restore M-MuLV assembly when introduced into matrix, p12, or nucleocapsid domains of the mutant M-MuLV Gag protein lacking the PPPY motif. Strikingly, mutant viruses carrying the RSV or the HIV-1 L-domain at the original location of the deleted PPPY motif were replication competent in rodent cells. These data suggest that the PPPY motif of M-MuLV acts in a partially position-independent manner and is functionally interchangeable with L-domains of other retroviruses. Electron microscopy studies revealed that deletion of the entire p12 region resulted in the formation of tube-like rather than spherical particles. Remarkably, the PPPY deletion mutant formed chain structures composed of multiple viral particles linked on the cell surface. Many of the mutants with heterologous L-domains released virions with wild-type morphology.     

Single residue mutation in integrase catalytic core domain affects feline foamy viral DNA integration

DD(35)E motif in catalytic core domain (CCD) of integrase (IN) is extremely involved in retroviral integration step. Here, nine single residue mutants of feline foamy virus (FFV) IN were generated to study their effects on IN activities and on viral replication. As expected, mutations in the highly conserved D107, D164, and E200 residues abolished all IN catalytic activities (3′-end processing, strand transfer, and disintegration) as well as viral infectivity by blocking viral DNA integration into cellular DNA. However, Q165, Y191, and S195 mutants, which are located closely to DDE motif were observed to have diverse levels of enzymatic activities, compared to those of the wild type IN. Their mutant viruses produced by one-cycle transfection showed different infectivity on their natural host cells. Therefore, it is likely that effects of single residue mutation at DDE motif is critical on viral replication depending on the position of the residues.     

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.     

Reversion of a human immunodeficiency virus type 1 integrase mutant at a second site restores enzyme function and virus infectivity.

The integration of a DNA copy of the retroviral RNA genome into the host cell genome is essential for viral replication. The virion-associated integrase protein, encoded by the 3' end of the viral pol gene, is required for integration. Stable virus-producing T-cell lines were established for replication-defective human immunodeficiency virus type 1 carrying single amino acid substitutions at conserved residues in the catalytic domain of integrase. Phenotypically reverted virus was detected 12 weeks after transfection with the integrase mutant carrying the P-109-->S mutation (P109S). Unlike the defective P109S virus, the revertant virus (designated P109SR) grew in CD4+ SupT1 cells. In addition to the Ser substitution at Pro-109, P109SR had a second substitution of Ala for Thr at position 125 in integrase. Site-directed mutagenesis was used to show that the P109S T125A genotype was responsible for the P109SR replication phenotype. The T125A substitution also rescued the in vitro enzyme activities of recombinant P109S integrase protein. P109S integrase did not display detectable 3' processing or DNA strand transfer activity, although 5 to 10% of wild-type disintegration activity was detected. P109S T125A integrase displayed nearly wild-type levels of 3' processing, DNA strand transfer, and disintegration activities, confirming that T125A is a second-site intragenic suppressor of P109S. P109S integrase ran as a large aggregate on a size exclusion column, whereas wild-type integrase ran as a monomer and P109S T125A integrase ran as a mixed population. Pro-109 and Thr-125 are not immediately adjacent in the crystal structure of the integrase catalytic domain. We suggest that the T125A substitution restores integrase function by stabilizing a structural alteration(s) induced by the P109S mutation.     

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.     

Molecular and genetic determinants of rous sarcoma virus integrase for concerted DNA integration

Site-directed mutagenesis of recombinant Rous sarcoma virus (RSV) integrase (IN) allowed us to gain insights into the protein-protein and protein-DNA interactions involved in reconstituted IN-viral DNA complexes capable of efficient concerted DNA integration (termed full-site). At 4 nM IN, wild-type (wt) RSV IN incorporates approximately 30% of the input donor into full-site integration products after 10 min of incubation at 37 degrees C, which is equivalent to isolated retrovirus preintegration complexes for full-site integration activity. DNase I protection analysis demonstrated that wt IN was able to protect the viral DNA ends, mapping approximately 20 bp from the end. We had previously mapped the replication capabilities of several RSV IN mutants (A48P and P115S) which appeared to affect viral DNA integration in vivo. Surprisingly, recombinant RSV A48P IN retained wt IN properties even though the virus carrying this mutation had significantly reduced integrated viral DNA in comparison to wt viral DNA in virus-infected cells. Recombinant RSV P115S IN also displayed all of the properties of wt RSV IN. Upon heating of dimeric P115S IN in solution at 57 degrees C, it became apparent that the mutation in the catalytic core of RSV IN exhibited the same thermolabile properties for 3' OH processing and strand transfer (half-site and full-site integration) activities consistent with the observed temperature-sensitive defect for integration in vivo. The average half-life for inactivation of the three activities were similar, ranging from 1.6 to 1.9 min independent of the IN concentrations in the assay mixtures. Wt IN was stable under the same heat treatment. DNase I protection analysis of several conservative and nonconservative substitutions at W233 (a highly conserved residue of the retrovirus C-terminal domain) suggests that this region is involved in protein-DNA interactions at the viral DNA attachment site. Our data suggest that the use of recombinant RSV IN to investigate efficient full-site integration in vitro with reference to integration in vivo is promising.     

Characterization of the functional domains of human foamy virus integrase using chimeric integrases

Retroviral integrases insert viral DNA into target DNA. In this process they recognize their own DNA specifically via functional domains. In order to analyze these functional domains, we constructed six chimeric integrases by swapping domains between HIV-1 and HFV integrases, and two point mutants of HFV integrase. Chimeric integrases with the central domain of HIV-1 integrase had strand transfer and disintegration activities, in agreement with the idea that the central domain determines viral DNA specificity and has catalytic activity. On the other hand, chimeric integrases with the central domain of HFV integrase did not have any enzymatic activity apart from FFH that had weak disintegration activity, suggesting that the central domain of HFV integrase was defective catalytically or structurally. However, these inactive chimeras were efficiently complemented by the point mutants (D164A and E200A) of HFV integrase, indicating that the central domain of HFV integrase possesses potential enzymatic activity but is not able to recognize viral or target DNA without the help of its homologous N-terminal and C-terminal domains.