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At least 685 records · Page 38Linked to original sources
PubMed2000

Differential effects of C-terminal molecular tagged integrase on replication competent moloney murine leukemia virus.

Moloney murine leukemia virus (M-MuLV) proviruses carrying integrase (IN) protein tagged either with a simian virus 40 (SV40) nuclear localization signal (NLS) or various antigenic epitopes were generated. Hexahistidine (His(6)), hemagluttinin (HA), or two consecutive HA sequences (2XHA) were fused to the C-terminus of IN as antigenic markers. These epitope-tagged IN proteins were stably expressed through multiple rounds of infection. The IN-His(6), IN-HA, and IN-2XHA proteins, purified from virus, could be immunoprecipitated with antibodies against His(6) and HA, respectively. An M-MuLV provirus encoding the SV40 large T antigen NLS fused to IN at the same position as the epitope tags was also passaged through cells. In contrast to the stability of the epitope tags, the SV40 NLS sequence was rapidly mutated by a frameshift mutation that introduced negatively charged amino acids into the basic NLS. The instability of the NLS suggests that the strong nuclear localization of the IN-SV40 NLS may have detrimental effects on virus assembly. These observations have implications for studying nuclear transport properties of M-MuLV and for engineering a murine-based retroviral vector for gene therapy.

Amino Acid Sequence↗
PubMed2001

Mutational analysis of the N-terminus of Moloney murine leukemia virus integrase.

The retroviral integrase (IN) carries out the integration of viral DNA into the host genome. The IN protein consists of three domains: the N-terminal HHCC motif, the catalytic core region, and the C-terminus. The Moloney murine leukemia virus (M-MuLV) IN encodes a unique 45-amino-acid domain N-terminal to the HHCC motif. The function of the N-terminus of M-MuLV IN was studied through deletional and mutational analyses. The IN 1-105 domain was dissected into two halves expressing either the unique N-terminus or the HHCC domain. Although the parental IN 1-105 could functionally complement the core-C-terminus for integration reactions, neither half of the N-terminus was sufficient. Partial complementation of strand transfer, but not 3prime prime or minute processing, could be obtained through mixing the two halves. The dimerization of the M-MuLV N-terminus was dependent on the expression of the intact 1-105. Critical basic amino acids within the HHCC domain which are required for 3' processing and strand transfer reactions were identified through alanine mutagenesis. Loss of in vitro strand transfer activity correlated with loss of viral titer in vivo for this cluster of basic amino acids within the HHCC domain.

Alanine↗
PubMed1995

Site-specific integration of the phage phi CTX genome into the Pseudomonas aeruginosa chromosome: characterization of the functional integrase gene located close to and upstream of attP.

The site-specific integration of the phage phi CTX genome, which carries the gene for a pore-forming cytotoxin, into the Pseudomonas aeruginosa chromosome was analysed. The 1,167 bp integrase gene, int, located immediately upstream of the attachment site, attP, was characterized using plasmid constructs, harbouring the integration functions, and serving as an integration probe in both P. aeruginosa and Escherichia coli. The attP plasmids p1000/p400 in the presence of the int plasmid pIBH and attP-int plasmids pINT/pINTS can be stably integrated into the P. aeruginosa chromosome. Successful recombination between the attP plasmid p1000 and the attB plasmid p5.1, in the presence of the int plasmid pIBH in E. coli HB101 showed that the int gene is active in trans in E. coli. The int gene product was detected as a 43 kDa protein in E. coli maxicells harbouring pINT. Proposed integration arm regions downstream of attP are not necessary for the integration process. pINT and phage phi CTX could be integrated together into P. aeruginosa chromosomal DNA, yielding double integrates.

Attachment Sites, Microbiological↗
PubMed1993

Expression of a processed and a non-processed form of the integrase protein of HIV-1 in the baculovirus system.

The integrase (IN) protein of HIV-1 was expressed as a processed and a non-processed protein in the eucaryotic baculovirus expression system. In immunoblots we could demonstrate that recombinant baculoviruses containing the complete gag and pol reading frames of HIV-1 expressed a gag/pol precursor polyprotein. The specific proteolytic activity of the recombinant protease on the gag and pol precursor proteins was used for the generation of processed gag (p 17, p 24, p 16) and pol (RT/RNaseH, IN) proteins. The non-processed IN protein, expressed as a polyhedrin fusion protein, was produced at much higher level than the processed protein.

Animals↗
PubMed1998

Generation of HIV-1/HIV-2 cross-reactive peptide antisera by small sequence changes in HIV-1 reverse transcriptase and integrase immunizing peptides.

We have generated peptide antisera against selected regions in HIV-1 and HIV-2 reverse transcriptase (RT) and integrase (IN) to investigate the specificity of determinants governing the immune response. Peptides representing homologous regions (>50%) in the N- and C-termini and central portions of these proteins were synthesized and injected into rabbits. HIV-1 and HIV-2 IN peptide antisera inhibited IN-mediated cleavage of an HIV-1 DNA oligonucleotide substrate in a 3' processing assay, while anti-RT or normal sera had no effect. None of the RT sera inhibited RT activity. In Western blots, HIV-2 antisera directed against RT or IN peptides recognized HIV-2 RT and IN proteins, respectively, as expected, but also cross-reacted with the corresponding HIV-1 proteins. By contrast, corresponding HIV-1 antisera were type-specific. In some cases, HIV-1 cross-reactive antisera could be generated by immunization with HIV-1 chimeric peptides with as few as two residues in the HIV-1 sequence changed to the corresponding HIV-2 amino acids. The finding that a type-specific response can be converted to a cross-reactive response suggests alternate strategies for developing new diagnostic reagents which detect HIV-1 and HIV-2. In addition, our results provide a general model for generating HIV peptide vaccines with dual specificity against HIV-1 and HIV-2.

Amino Acid Sequence↗
PubMedDate not supplied

Target integration by a chimeric Sp1 zinc finger domain-Moloney murine leukemia virus integrase in vivo.

A specificity protein 1 (Sp1) zinc finger domain containing two tandem zinc fingers was fused to the C terminus of the integrase (IN) protein of the Moloney murine leukemia virus (MuLV). The integrity of the MuLV IN was completely preserved, since the fusion was conducted at the last amino acid residue of the protein. The vector pMIN-Sp1, which carried the fused MuLV IN-Sp1 zinc finger domain gene, was cotransfected with a wild-type MuLV vector pMLV-K to NIH/3T3 cells. A nonradioactive reverse transcriptase assay was performed on culture supernatants collected from the cotransfected cells to confirm the production of recombinant viruses. The expression of the fusion protein and the integration of the MuLV genome by the fusion protein were confirmed by a Northern and then a Southern hybridization analysis on the total RNA or genomic DNA extracted from cells infected by viruses collected from the supernatants of the cotransfected cells. Regions of the host chromosome that were selected by the fusion protein as the integration targets were sequenced using the TOPO(TM) cloning method on a series of PCR products generated with a nested set of primers. The percentage of positive clones screened that contained the DNA-binding sequence of the fused Sp1 zinc finger domain was around 13% (5 out of 39 clones). It was found that the Sp1 DNA-binding sequence was only present in regions that were proximal to one of the long terminal repeats of the integrated viral genome, suggesting that the fusion protein could select a target sequence for integration. The host flanking sequences determined for all the positive clones were also used as queries to perform a BLAST search on the GenBank mouse EST entries. Although matching scores for sequences of some of the clones computed were more significant than others, it was difficult to judge whether or not the integration in these clones had been targeted to some gene sequences. Most of the integration sites might exist in the introns, since we found that the probability of the gene sequences containing an Sp1 DNA-binding site was low.

3' Flanking Region↗
PubMed2001

HIV-1 integrase inhibitory phenylpropanoid glycosides from Clerodendron trichotomum.

Seven phenylpropanoid glycosides named acteoside (1), acteoside isomer (2), leucosceptoside A (3), plantainoside C (4), jionoside D (5), martynoside (6), and isomartynoside (7) were isolated from Clerodendron trichotomum. Compounds 1 and 2 showed potent inhibitory activities against HIV-1 integrase with IC50 values of 7.8 +/- 3.6 and 13.7 +/- 6.0 microM, respectively.

Disaccharides↗
PubMed2001

The clc element of Pseudomonas sp. strain B13 and other mobile degradative elements employing phage-like integrases.

Genes for metabolic pathways in bacteria that degrade aromatic or aliphatic pollutants have mostly been confined to either plasmid DNAs or to the chromosome. For a few pathways, including classical pathways for chlorocatechol and biphenyl degradation, recent evidence has been obtained for location of the pathway genes on mobile DNA elements which employ phage-like integrases. This enables the DNA elements to integrate into specific sites on the chromosome and yet to excise and transfer to other host bacteria. This mini-review gives an overview of those elements and their relationship to an increasing number of phage-like elements associated with bacterial virulence.

Biodegradation, Environmental↗
PubMed1997

Nucleotide sequence of the murine leukaemia virus amphotropic strain 4070A integrase (IN) coding region and comparative structural analysis of the inferred polypeptide.

The complete nucleotide sequence of the integrase (IN) protein coding region of the murine leukaemia virus (MLV) amphotropic strain 4070A is presented. The sequence comprises 1,224 nucleotides, encoding a 408-residue polypeptide of M(r) 46,312. Alignment of the inferred 4070A IN amino acid sequence with the IN proteins of other MLV showed that substitutions are confined largely to segments within the N- and C-terminal domains. In the N-terminal domain the majority of substitutions occur as contiguous 2- to 6-residue blocks, whereas in the C-terminal domain they occur as isolated entities except within a short segment characterized by deletions/insertions. Selection appears to act on the C-terminal 19 residues of IN rather than on the N-terminal residues of ENV (encoded by overlapping reading frames), suggesting a functional role for this segment. Phylogenetic analyses grouped the sequences into two clusters, one comprising IN from the amphotropic strain 4070A and three ecotropic MLV (CAS-BR-E, Moloney and Friend), the other consisting of IN from three ecotropic MLV (two radiation-induced viruses and AKV) and a mink cell focus-forming (MCF) MLV virus. The same dichotomy and cluster composition was obtained from analysis of the long terminal repeat (LTR) regions from these viruses (consistent with the functional interrelationship of IN and LTR) but not from analysis of envelope protein sequences (consistent with the functional independence of ENV proteins from both IN and LTR). Secondary structure predictions supported features determined from the catalytic domain of human immunodeficiency virus and avian sarcoma virus IN, and identified probable structures within the relatively long N- and C-terminal domains of MLV IN proteins.

Amino Acid Sequence↗
PubMed2000

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.

Binding Sites↗
PubMed2005

Model of full-length HIV-1 integrase complexed with viral DNA as template for anti-HIV drug design.

We report structural models of the full-length integrase enzyme (IN) of the human immunodeficiency virus type 1 (HIV-1) and its complex with viral and human DNA. These were developed by means of molecular modeling techniques using all available experimental evidence, including X-ray crystallographic and NMR structures of portions of the full-length protein. Special emphasis was placed on obtaining a model of the enzyme's active site with the viral DNA apposed to it, based on the hypothesis that such a model would allow structure-based design of inhibitors that retain activity in vivo. This was because bound DNA might be present in vivo after 3'-processing but before strand transfer. These structural models were used to study the potential binding modes of various diketo-acid HIV-1 IN inhibitors (many of them preferentially inhibiting strand transfer) for which no experimentally derived complexed structures are available. The results indicate that the diketo-acid IN inhibitors probably chelate the metal ion in the catalytic site and also prevent the exposure of the 3'-processed end of the viral DNA to human DNA.

Amino Acid Sequence↗
PubMed2005

Site-specific recombination in Arabidopsis plants promoted by the Integrase protein of coliphage HK022.

The gene encoding the wild type Integrase protein of coliphage HK022 was integrated chromosomally and expressed in Arabidopsis thaliana plants. Double-transgenic plants cloned with the int gene as well as with a T-DNA fragment carrying the proper att sites in a tandem orientation showed that Int catalyzed a site-specific integration reaction (attP x attB) as well as a site-specific excision reaction (attL x attR). The reactions took place without the need to provide any of the accessory proteins that are required by Int in the bacterial host. When expressed in tobacco plants a GFP-Int fusion exhibits a predominant nuclear localization.

Arabidopsis↗
PubMed1991

Development of an acid-soluble assay for measuring retrovirus integrase 3'-OH terminal nuclease activity.

A quantitative and efficient assay was developed to measure the 3'-OH terminal DNA endonuclease activity of the avian myeloblastosis virus (AMV) integrase protein. A retroviral-like linearized plasmid containing long terminal repeat (LTR) sequences at its recessed 3'-OH termini was filled in and labeled with the Escherichia coli Klenow DNA polymerase fragment. The 32P-labeled nucleotide was located at the penultimate position. The labeled linearized plasmid or restriction fragments derived from it were incubated with AMV IN and release of the label was quantitated by conversion to acid-soluble counts. The structure of the released product was characterized on 23% sequencing gels. Results indicate that AMV integration protein is functioning as an endonuclease releasing a dinucleotide and that the activity is stoichiometric with a preference for the cleavage of the U3 LTR terminus over that of the U5 LTR terminus.

Avian Myeloblastosis Virus↗
PubMed1983

Retroregulation: control of integrase expression by the b2 region of bacteriophages lambda and 434.

Genetic fusions constructed by a combination of in vivo and in vitro techniques have been used to investigate the cis-regulatory role of a 250-base-pair segment of b2 DNA in the expression of int transcripts originating from three different promoters PI, PL, and PTRP. It has been established that (a) the PI and PTRP transcripts, which produce functional int protein, are efficiently terminated by the b2 segment, (b) in the same test system, the PL transcript, which does not result in functional integrase, is not terminated by the b2 segment, (c) this b2 (sib) effect does not depend on genes lying between int and N on the phage genome, (d) the sib effect is also observed with the b2 DNA of phage 434, which resembles that of lambda between -197 and att but diverges radically from it to the left of base -197.

Bacteriophage lambda↗
PubMed1995

HIV nuclear import is governed by the phosphotyrosine-mediated binding of matrix to the core domain of integrase.

The karyophilic properties of the viral matrix (MA) protein govern HIV nuclear import in nondividing cells such as macrophages. A critical regulator of this process is the C-terminal tyrosine phosphorylation of MA during virus maturation. Here, we reveal the mechanism of this phenomenon, by demonstrating that tyrosine phosphorylation induces the binding of MA to integrase (IN). This leads to the incorporation of MA molecules into virus cores, and subsequently into uncoated viral nucleoprotein complexes. A direct interaction between tyrosine-phosphorylated MA and the central domain of IN can be demonstrated in vitro. It is blocked by phosphotyrosine, indicating that IN recognizes the phosphorylated C-terminal residue of MA. These results explain how the karyophilic potential of MA is conferred to the HIV nucleoprotein complex.

Base Sequence↗
PubMed1993

The human immunodeficiency virus integrase protein.

The DNA integration step in the replication cycle of the human immunodeficiency virus (HIV) has been recognized as an important target in antiviral strategies. There are two main reasons for this. First, integration of HIV DNA into the human genome is required for replication of this retrovirus. Second, since the integration reaction does not have an obvious cellular counterpart, drugs that specifically inhibit integration may not be toxic for the cell. Here, we focus on the only protein known to be required for retroviral integration, the integrase (IN) protein.

DNA Nucleotidyltransferases↗
PubMed1987

Functional elements of DNA upstream from the integrase operon that are conserved in bacteriophages 434 and lambda.

A 1488-bp restriction fragment of bacteriophage 434 DNA contains the integrase promoter and an adjacent nucleotide sequence (t'I) resembling a Rho-independent terminator. To identify and quantitate transcription termination, DNA segments were cloned into a plasmid between the galactose promoter and assayable galactokinase gene and tested for termination. Whereas the entire fragment effectively terminated transcription, a 331-bp restriction fragment containing the t'I terminator had only weak terminator activity. Random sequential deletions of the 434 DNA segment defined a strong terminator 650-bp upstream from t'I. This proposed Rho-independent terminator called tL4 consists of a 7-bp stem and 6-nt loop followed by a uridine-rich region in the RNA. Phage lambda contains an even stronger tL4 terminator that differs in 4 nt from 434 tL4. Thus, despite some sequence divergence, terminator activity has been conserved in these phages. The 434 DNA segment was also tested for promoter activity. Rightward promoter activity (opposite to pL in the phage) was located about 200 bp to the right of tL4 and was followed by an open reading frame (ORF) capable of encoding a 91 amino acid protein. Promoter activity in the same approximate location was also found in phage lambda. Thus the rightward promoter, the tL4 and t'I terminators, and ORF-55 all are elements in this segment of the genome that are conserved for function despite sequence divergence.

Amino Acid Sequence↗
PubMed2005

Identification of heavy metal complexes of a hexapeptide inhibitor of the human immunodeficiency virus integrase protein by using a voltammetric approach.

Complexation of the hexapeptide Hys-Cys-Lys-Phe-Trp-Trp, inhibitor of the human immunodeficiency virus integrase protein, with the heavy metal ions Cd2+, Pb2+, and Zn2+ has been investigated using differential pulse polarography. In the case of Pb2+, no significant complexation is detected, whereas in the cases of Cd2+ and Zn2+, strong and electrochemically inert ML2 complexes predominate. In contrast, ML complexes are present in a low proportion or are absent. When possible, the corresponding conditional stability constants have been determined at both pH 7.0 and pH 7.5, showing that Zn2+ complexes are slightly more stable than Cd2+ complexes.

Cadmium↗