Integrase Function Research Articles

Characterization and Expression Analysis of Staphylococcus aureus Pathogenicity Island 3: IMPLICATIONS FOR THE EVOLUTION OF STAPHYLOCOCCAL PATHOGENICITY ISLANDS

We describe the complete sequence of the 15.9-kb staphylococcal pathogenicity island 3 encoding staphylococcal enterotoxin serotypes B, K, and Q. The island, which meets the generally accepted definition of pathogenicity islands, contains 24 open reading frames potentially encoding proteins of more than 50 amino acids, including an apparently functional integrase. The element is bordered by two 17-bp direct repeats identical to those found flanking staphylococcal pathogenicity island 1. The island has extensive regions of homology to previously described pathogenicity islands, particularly staphylococcal pathogenicity islands 1 and bov. The expression of 22 of the 24 open reading frames contained on staphylococcal pathogenicity island 3 was detected either in vitro during growth in a laboratory medium or serum or in vivo in a rabbit model of toxic shock syndrome using DNA microarrays. The effect of oxygen tension on staphylococcal pathogenicity island 3 gene expression was also examined. By comparison with the known staphylococcal pathogenicity islands in the context of gene expression described here, we propose a model of pathogenicity island origin and evolution involving specialized transduction events and addition, deletion, or recombination of pathogenicity island "modules."

Bacterial Genomic Islands: Organization, Function, and Evolutionary Role

Data on the structural organization and evolutionary role of specific bacterial DNA regions known as genomic islands are reviewed. Emphasis is placed on the most extensively studied genomic islands, pathogenicity islands (PAIs), which are present in the chromosome of Gram-negative and Gram-positive pathogenic bacteria and absent from related nonpathogenic strains. PAIs are long DNA regions that harbor virulence genes and often differ in GC content from the remainder of the bacterial genome. Many PAI occur in the tRNA gene loci, which provide a convenient target for foreign gene insertion. Some PAI are highly homologous to each other and contain sequences similar to ISs, phage att sites, and plasmid ori sites, along with functional or defective integrase and transposase genes, suggesting horizontal transfer of PAI among bacteria.

Phage R4 integrase mediates site-specific integration in human cells

The R4 integrase is a site-specific, unidirectional recombinase derived from the genome of phage R4 of Streptomyces parvulus. Here we define compact attB and attP recognition sites for the R4 integrase and express the enzyme in mammalian cells. We demonstrate that R4 integrase functions in human cells, performing efficient and precise recombination between R4 attB and attP sites cloned on an extrachromosomal vector. We also provide evidence that the enzyme can mediate integration of an incoming plasmid bearing an attB or attP site into endogenous sequences in the human genome. Furthermore, when R4 attB and attP sites are placed into the human genome, either by random integration or at a specific sequence by using the φC31 integrase, they act as targets for integration of incoming plasmids bearing R4 att sites. The R4 integrase has immediate utility as a site-specific integration tool for genome engineering, as well as potential for further development.

Human immunodeficiency virus type 1 replication in the absence of integrase-mediated dna recombination: definition of permissive and nonpermissive T-cell lines.

Functional retroviral integrase protein is thought to be essential for productive viral replication. Yet, previous studies differed on the extent to which integrase mutant viruses expressed human immunodeficiency virus type 1 (HIV-1) genes from unintegrated DNA. Although one reason for this difference was that class II integrase mutations pleiotropically affected the viral life cycle, another reason apparently depended on the identity of the infected cell. Here, we analyzed integrase mutant viral infectivities in a variety of cell types. Single-round infectivity of class I integration-specific mutant HIV-1 ranged from <0.03 to 0.3% of that of the wild type (WT) across four different T-cell lines. Based on this approximately 10-fold influence of cell type on mutant gene expression, we examined class I and class II mutant replication kinetics in seven different cell lines and two primary cell types. Unexpectedly, some cell lines supported productive class I mutant viral replication under conditions that restricted class II mutant growth. Cells were defined as permissive, semipermissive, or nonpermissive based on their ability to support the continual passage of class I integration-defective HIV-1. Mutant infectivity in semipermissive and permissive cells as quantified by 50% tissue culture infectious doses, however, was only 0.0006 to 0.005% of that of WT. Since the frequencies of mutant DNA recombination in these lines ranged from 0.023 to <0.093% of the WT, we conclude that productive replication in the absence of integrase function most likely required the illegitimate integration of HIV-1 into host chromosomes by cellular DNA recombination enzymes.

Instability and site-specific excision of integration-proficient mycobacteriophage L5 plasmids: development of stably maintained integrative vectors

Integrative vectors expressing foreign genes are used as tools for the development of recombinant vaccines in mycobacteria since it is assumed that these vectors are stably maintained even without antibiotic selection. We here demonstrate that integration-proficient vectors are lost from the mycobacterial genome in high frequency. Loss of integrated vectors occurred in recA + and in recA - strains, indicating a RecA-independent mechanism. Loss of the integrated vector was prevented when integrase gene function was carried on a separate plasmid that is unable to replicate in mycobacteria, indicating that excision is a function of integrase. By providing attP in cis and integrase function in trans, vectors integrating at the attB site are stably maintained, even when carrying genes that deleteriously affect the host.

HIV-1 integrase inhibitors that compete with the target DNA substrate define a unique strand transfer conformation for integrase.

Diketo acids such as L-731,988 are potent inhibitors of HIV-1 integrase that inhibit integration and viral replication in cells. These compounds exhibit the unique ability to inhibit the strand transfer activity of integrase in the absence of an effect on 3' end processing. To understand the reasons for this distinct inhibitory profile, we developed a scintillation proximity assay that permits analysis of radiolabeled inhibitor binding and integrase function. High-affinity binding of L-731,988 is shown to require the assembly of a specific complex on the HIV-1 long terminal repeat. The interaction of L-731,988 with the complex and the efficacy of L-731, 988 in strand transfer can be abrogated by the interaction with target substrates, suggesting competition between the inhibitor and the target DNA. The L-731,988 binding site and that of the target substrate are thus distinct from that of the donor substrate and are defined by a conformation of integrase that is only adopted after assembly with the viral end. These results elucidate the basis for diketo acid inhibition of strand transfer and have implications for integrase-directed HIV-1 drug discovery efforts.

Efficient intracellular retrotransposition of an exogenous primate retrovirus genome

The foamy virus (FV) subgroup of Retroviridae reverse transcribe their RNA (pre-)genome late in the replication cycle before leaving an infected cell. We studied whether a marker gene-transducing FV vector is able to shuttle to the nucleus and integrate into host cell genomic DNA. While a potential intracellular retrotransposition of vectors derived from other retroviruses was below the detection limit of our assay, we found that up to 5% of cells transfected with the FV vector were stably transduced, harboring 1 to approximately 10 vector integrants. Generation of the integrants depended on expression of functional capsid, reverse transcriptase and integrase proteins, and did not involve an extracellular step. PCR analysis of the U3 region of the 5' long terminal repeat and determination of proviral integration sites showed that a reverse transcription step had taken place to generate the integrants. Co-expression of a mutated envelope allowing particle egress and avoiding extracellular infection resulted in a significantly increased rescue of cells harboring integrants, suggesting that accumulation of proviruses via intracellular retrotransposition represents an integral part of the FV replication strategy.

Two nuclear localization signals in the HIV-1 matrix protein regulate nuclear import of the HIV-1 pre-integration complex

Replication of HIV-1 in non-dividing and slowly proliferating cell populations depends on active import of the viral pre-integration complex (PIC) into the cell nucleus. While it is commonly accepted that this process is mediated by an interaction between the HIV-1 PIC and the cellular nuclear import machinery, controversial results have been reported concerning the mechanisms of this interaction. Here, we demonstrate that a recently identified nuclear localization signal within the HIV-1 matrix protein (MA), MA NLS-2, together with previously described MA NLS-1, mediates nuclear import of the HIV-1 PIC. Inactivation of both MA NLSs precluded nuclear translocation of MA and rendered the virus defective in nuclear import and replication in non-dividing macrophage cultures, even when functional Vpr and integrase (IN), two more viral proteins implicated in HIV-1 nuclear import, were present. Taken together, these results indicate that Vpr does not function as an independent nuclear import factor and demonstrate that HIV-1 MA, by virtue of its two nuclear localization signals, regulates HIV-1 nuclear import.

Inactivation of the SNF5 transcription factor gene abolishes the lethal phenotype induced by the expression of HIV-1 integrase in yeast.

The ubiquitous human transcription factor Ini1 has been shown to interact with HIV-1 integrase (IN) and to stimulate in vitro the reactions catalyzed by this enzyme. We have previously used a yeast model to study the effect of HIV-1 IN expression (Caumont, A.B., Jamieson, G.A., Pichuantes, S., Nguyen, A.T., Litvak, S., Dupont, C. -H., 1996. Expression of functional HIV-1 integrase in the yeast Saccharomyces cerevisiae leads to the emergence of a lethal phenotype: potential use for inhibitor screening. Curr. Genet. 29, 503-510). Here, we describe the effect of the inactivation of the gene encoding for SNF5, a yeast transcription factor homologous to Ini1, on the lethality induced by the expression of HIV-1 IN in yeast. We observed that the retroviral IN was unable to perform its lethal activity in cells where the SNF5 gene has been disrupted, suggesting that SNF5 may play a role in the lethal effect induced by IN in yeast. SNF5 inactivation affects neither yeast viability nor expression of HIV-1 IN. Given the homology between SNF5 and its human counterpart Ini1, our results suggest that this factor may be important for IN activity in infected cells. Moreover, given the important role proposed for this transcription factor in the integration step and the fact that it is dispensable for cell viability, the interaction between Ini1/ySNF5 and HIV-1 IN should become a potential target in the search for new antiretroviral agents.

Multiple integrations of human foamy virus in persistently infected human erythroleukemia cells.

Foamy viruses are complex retroviruses whose replication strategy resembles that of conventional retroviruses. However, foamy virus replication also resembles that of hepadnaviruses in many respects. Because hepadnaviruses replicate in an integrase-independent manner, we were interested in investigating the characteristics of human foamy virus (HFV) integration. We have shown that HFV requires a functional integrase protein for infectivity. Our analyses have revealed that in single-cell clones derived from HFV-infected erythroleukemia-derived cells (H92), there were up to 20 proviral copies per host cell genome as determined by Southern blot and fluorescent in situ hybridization analysis. Use of specific probes has also shown that a majority of the proviruses contain the complete tas gene, which encodes the viral transactivator, and are not derived from Deltatas cDNAs, which have been shown to arise rapidly in infected cells. To demonstrate that the multiple proviral sequences are due to integration instead of recombination, we have sequenced the junctions between the proviral sequences and the host genome and found that the proviruses have authentic long terminal repeat ends and that each integration is at a different chromosomal site. A virus lacking the Gag nuclear localization signal accumulates fewer proviruses, suggesting that nuclear translocation is important for high proviral load. Since persistently infected H92 clones are not resistant to superinfection, the relative importance of an intracellular versus extracellular mechanism in proviral acquisition has yet to be determined.

Inhibitors of strand transfer that prevent integration and inhibit HIV-1 replication in cells.

Integrase is essential for human immunodeficiency virus-type 1 (HIV-1) replication; however, potent inhibition of the isolated enzyme in biochemical assays has not readily translated into antiviral activity in a manner consistent with inhibition of integration. In this report, we describe diketo acid inhibitors of HIV-1 integrase that manifest antiviral activity as a consequence of their effect on integration. The antiviral activity of these compounds is due exclusively to inhibition of one of the two catalytic functions of integrase, strand transfer.

Unusual structure of the attB site of the site-specific recombination system of Lactobacillus delbrueckii bacteriophage mv4.

The temperate phage mv4 integrates its genome into the chromosome of Lactobacillus delbrueckii subsp. bulgaricus by site-specific recombination within the 3' end of a tRNA(Ser) gene. Recombination is catalyzed by the phage-encoded integrase and occurs between the phage attP site and the bacterial attB site. In this study, we show that the mv4 integrase functions in vivo in Escherichia coli and we characterize the bacterial attB site with a site-specific recombination test involving compatible plasmids carrying the recombination sites. The importance of particular nucleotides within the attB sequence was determined by site-directed mutagenesis. The structure of the attB site was found to be simple but rather unusual. A 16-bp DNA fragment was sufficient for function. Unlike most genetic elements that integrate their DNA into tRNA genes, none of the dyad symmetry elements of the tRNA(Ser) gene were present within the minimal attB site. No inverted repeats were detected within this site either, in contrast to the lambda site-specific recombination model.

Integrated pararetroviral sequences define a unique class of dispersed repetitive DNA in plants.

Although integration of viral DNA into host chromosomes occurs regularly in bacteria and animals, there are few reported cases in plants, and these involve insertion at only one or a few sites. Here, we report that pararetrovirus-like sequences have integrated repeatedly into tobacco chromosomes, attaining a copy number of approximately 10(3). Insertion apparently occurred by illegitimate recombination. From the sequences of 22 independent insertions recovered from a healthy plant, an 8-kilobase genome encoding a previously uncharacterized pararetrovirus that does not contain an integrase function could be assembled. Preferred boundaries of the viral inserts may correspond to recombinogenic gaps in open circular viral DNA. An unusual feature of the integrated viral sequences is a variable tandem repeat cluster, which might reflect defective genomes that preferentially recombine into plant DNA. The recurrent invasion of pararetroviral DNA into tobacco chromosomes demonstrates that viral sequences can contribute significantly to plant genome evolution.

Multiple Integrase Functions Are Required to Form the Native Structure of the Human Immunodeficiency Virus Type I Intasome

Mu-mediated polymerase chain reaction footprinting was used to investigate the protein-DNA structure of human immunodeficiency virus type I (HIV-I) preintegration complexes. Preintegration complexes were partially purified from cells after using an established coculture infection technique as well as a novel technique using cell-free supernatant from transfected cells as the source of virus. Footprinting revealed that bound proteins protected the terminal 200-250 base pairs of each viral end from nuclease attack. Bound proteins also caused strong transpositional enhancements near each end of HIV-I. In contrast, regions of viral DNA internal to the ends did not show evidence of strong protein binding. The end regions of preintegrative HIV-I apparently form a unique nucleoprotein structure, which we term the intasome to distinguish it from the greater preintegration complex. Our novel system also allowed us to analyze the structure and function of preintegration complexes isolated from cells infected with integrase mutant viruses. Complexes were derived from viruses defective for either integrase catalysis, integrase binding to the viral DNA substrate, or an unknown function in the carboxyl-terminal domain of the integrase protein. None of these mutant complexes supported detectable integration activity. Despite the presence of the mutant integrase proteins in purified samples, none of these nucleoprotein complexes displayed the native intasome structure detected in wild-type preintegration complexes. We conclude that multiple integrase functions are required to form the native structure of the HIV-I intasome in infected cells.

HIV Integrase Structure and Function

HIV integrase consists of three domains, the structures of which have been individually determined by X-ray crystallography or NMR spectroscopy. The core domain, spanning residues 50-212, is responsible for the catalytic activity of the enzyme. The crystal structure of a dimer of this domain shows similarity to other proteins that carry out polynucleotidyl transfer, including MuA transposase and RNase H. The small N-terminal domain folds into a dimeric helix-turn-helix structure, which is stabilized by the coordination of zinc with conserved His and Cys residues. The function of this domain is unclear; however, it is required for integration activity and enhances tetramerization in the context of the full-length integrase. The C-terminal domain, which has an SH3-like fold, is involved in DNA binding. The structure of this domain reveals a large saddle-shaped cleft that is formed by dimerization. This cleft contains a number of positively charged residues, and its dimensions are appropriate for accommodating a double-stranded DNA helix. Although the C-terminal domain was originally believed to be involved in target DNA binding, more recent evidence suggests that it may bind to both the ends of the viral DNA and to the target DNA. Although the individual domain structures provide some insights into the function of the protein, a more detailed understanding of the complete mechanism by which integrase binds, cleaves, and transfers DNA requires a greater knowledge of how these domains are arranged in the active multimer.

In Vivo Analysis of Retroviral Integrase Structure and Function

There are two retroviral integration loci. One encodes the transacting IN protein, which is cleaved from the carboxyl terminus of the Gag-Pol polyprotein precursor during virus assembly. The second locus is the cis-acting attachment (att) site, comprising the terminal sequences at the U3 and U5 ends of linear viral cDNA. Integrase and att site mutant viruses can be blocked at different steps of the viral replication cycle. Class I IN mutants are blocked specifically at the integration step. Class II IN mutants, on the other hand, display pleiotropic defects, most notably in virion morphogenesis and/or reverse transcription. Mutations in the U5 end att site can also disrupt reverse transcription in addition to integration. It is prudent to use caution when interpreting results of in vivo mutagenesis experiments that target retroviral IN and the att site.

Schizosaccharomyces pombe retrotransposon Tf2 mobilizes primarily through homologous cDNA recombination.

The Tf2 retrotransposon, found in the fission yeast Schizosaccharomyces pombe, is nearly identical to its sister element, Tf1, in its reverse transcriptase-RNase H and integrase domains but is very divergent in the gag domain, the protease, the 5' untranslated region, and the U3 domain of the long terminal repeats. It has now been demonstrated that a neo-marked copy of Tf2 overexpressed from a heterologous promoter can mobilize into the S. pombe genome and produce true transposition events. However, the Tf2-neo mobilization frequency is 10- to 20-fold lower than that of Tf1-neo, and 70% of the Tf2-neo events are homologous recombination events generated independently of a functional Tf2 integrase. Thus, the Tf2 element is primarily dependent on homologous recombination with preexisting copies of Tf2 for its propagation. Finally, production of Tf2-neo proteins and cDNA was also analyzed; surprisingly, Tf2 was found to produce its reverse transcriptase as a single species in which it is fused to protease, unlike all other retroviruses and retrotransposons.

Biochemical characterization of the HIV-1 integrase 3'-processing activity and its inhibition by phosphorothioate oligonucleotides.

To better understand HIV-1 integrase (IN) functions, we determined the kinetic parameters of the 3'-processing reaction. Steady-state kinetic analysis performed using Dixon plots indicated that the concentration of active enzyme was 10-fold lower than that calculated by protein determination. The turnover number was low, suggesting that IN remained bound to DNA after cleavage. The catalytic efficiency increased 10-fold from 30 to 37 degrees C and 2-fold from 37 to 42 degrees C. In enzyme assays carried out at 37 degrees C, both single- and double-stranded phosphorothioate oligos bound to IN with an efficiency comparable to that of the phosphodiester duplex substrate. The competition efficiency of single-stranded oligos was directly related to the sequence length. On the other hand, phosphorothioate duplex U5 LTRs modified in the plus strand were capable of both competing with the substrate and directly inhibiting the 3'-processing activity. These results suggest that, in addition to other modes of action (inhibition of gp120-CD4 interaction and reverse transcriptase), phosphorothioate hetero- and homopolimeric oligos also potently inhibit the IN activity.

Tdd-3, a tRNA gene-associated poly(A) retrotransposon from Dictyostelium discoideum.

The full-length 5218-bp sequence of the mobile genetic element Tdd-3 from Dictyostelium discoideum is described. Tdd-3 encodes two overlapping open reading frames (ORFs) flanked by non-redundant, untranslated regions. The deduced amino acid sequence of ORF2 is homologous to reverse transcriptases (RTs) encoded by the class of poly(A) retrotransposons. ORF2 also encodes a putative protein domain related to the family of apurinic/apyrimidinic (AP) endonucleases, whose retroelement-encoded homologs have recently been proposed to represent the integrase function of poly(A) retrotransposons. Comparison of several genomic Tdd-3 copies revealed that element insertion is orientation specific and occurs about 100 bp downstream of tRNA genes in the D. discoideum genome. These properties of Tdd-3 suggest that the element is a tRNA gene-associated poly(A) retroelement present in the D. discoideum genome. Analysis of several cloned cDNAs derived from Tdd-3-specific plus strand RNAs indicate that the element is transcribed and polyadenylated during the growth of D. discoideum cells.

Efficient gap repair catalyzed in vitro by an intrinsic DNA polymerase activity of human immunodeficiency virus type 1 integrase.

Cleavage and DNA joining reactions, carried out by human immunodeficiency virus type 1 (HIV-1) integrase, are necessary to effect the covalent insertion of HIV-1 DNA into the host genome. For the integration of HIV-1 DNA into the cellular genome to be completed, short gaps flanking the integrated proviral DNA must be repaired. It has been widely assumed that host cell DNA repair enzymes are involved. Here we report that HIV-1 integrase multimers possess an intrinsic DNA-dependent DNA polymerase activity. The activity was characterized by its dependence on Mg2+, resistance to N-ethylmaleimide, and inhibition by 3'-azido-2',3'-dideoxythymidine-5'-triphosphate, coumermycin A1, and pyridoxal 5'-phosphate. The enzyme efficiently utilized poly(dA)-oligo(dT) or self-annealing oligonucleotides as a template primer but displayed relatively low activity with gapped calf thymus DNA and no activity with poly(dA) or poly(rA)-oligo(dT). A monoclonal antibody binding specifically to an epitope comprised of amino acids 264 to 273 near the C terminus of HIV-1 integrase severely inhibited the DNA polymerase activity. A deletion of 50 amino acids at the C terminus of integrase drastically altered the gel filtration properties of the DNA polymerase, although the level of activity was unaffected by this mutation. The DNA polymerase efficiently extended a hairpin DNA primer up to 19 nucleotides on a T20 DNA template, although addition of the last nucleotide occurred infrequently or not at all. The ability of integrase to repair gaps in DNA was also investigated. We designed a series of gapped molecules containing a single-stranded region flanked by a duplex U5 viral arm on one side and by a duplex nonviral arm on the other side. Molecules varied structurally depending on the size of the gap (one, two, five, or seven nucleotides), their content of T's or C's in the single-stranded region, whether the CA dinucleotide in the viral arm had been replaced with a nonviral sequence, or whether they contained 5' AC dinucleotides as unpaired tails. The results indicated that the integrase DNA polymerase is specifically designed to repair gaps efficiently and completely, regardless of gap size, base composition, or structural features such as the internal CA dinucleotide or unpaired 5'-terminal AC dinucleotides. When the U5 arm of the gapped DNA substrate was removed, leaving a nongapped DNA template-primer, the integrase DNA polymerase failed to repair the last nucleotide in the DNA template effectively. A post-gap repair reaction did depend on the CA dinucleotide. This secondary reaction was highly regulated. Only two nucleotides beyond the gap were synthesized, and these were complementary to and dependent for their synthesis on the CA dinucleotide. We were also able to identify a specific requirement for the C terminus of integrase in the post-gap repair reaction. The results are consistent with a direct role for a heretofore unsuspected DNA polymerase function of HIV-1 integrase in the repair of short gaps flanking proviral DNA integration intermediates that arise during virus infection.