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PubMed2002

Modulation of the oligomeric structures of HIV-1 retroviral enzymes by synthetic peptides and small molecules.

The efficacy of antiretroviral agents approved for the treatment of HIV-1 infection is limited by the virus's ability to develop resistance. As such there is an urgent need for new ways of thinking about anti-HIV drug development, and accordingly novel viral and cellular targets critical to HIV-1 replication need to be explored and exploited. The retroviral RNA genome encodes for three enzymes essential for viral replication: HIV-1 protease (PR), HIV-1 reverse transcriptase (RT) and HIV-1 integrase (IN). The enzymatic functioning of each of these enzymes is entirely dependent on their oligomeric structures, suggesting that inhibition of subunit-subunit assembly or modulation of their quaternary structures provide alternative targets for HIV-1 inhibition. This review discusses the recent advances in the design and/or identification of synthetic peptides and small molecules that specifically target the subunit-subunit interfaces of these retroviral enzymes, resulting in the inactivation of their enzymatic functioning.

Anti-HIV Agents↗
PubMed2003

Evidence that the retroviral DNA integration process triggers an ATR-dependent DNA damage response.

Caffeine is an efficient inhibitor of cellular DNA repair, likely through its effects on ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related) kinases. Here, we show that caffeine treatment causes a dose-dependent reduction in the total amount of HIV-1 and avian sarcoma virus retroviral vector DNA that is joined to host DNA in the population of infected cells and also in the number of transduced cells. These changes were observed at caffeine concentrations that had little or no effect on overall cell growth, synthesis, and nuclear import of the viral DNA, or the activities of the viral integrase in vitro. Substantial reductions in the amount of host-viral-joined DNA in the infected population, and in the number of transductants, were also observed in the presence of a dominant-negative form of the ATR protein, ATRkd. After infection, a significant fraction of these cells undergoes cell death. In contrast, retroviral transduction is not impeded in ATM-deficient cells, and addition of caffeine leads to the same reduction that was observed in ATM-proficient cells. These results suggest that activity of the ATR kinase, but not the ATM kinase, is required for successful completion of the viral DNA integration process and/or survival of transduced cells. Components of the cellular DNA damage repair response may represent potential targets for antiretroviral drug development.

Active Transport, Cell Nucleus↗
PubMed1987

Integration host factor is required for the DNA inversion that controls phase variation in Escherichia coli.

The on-and-off expression (phase variation) of type 1 fimbriae, encoded by fimA, in Escherichia coli is controlled by the inversion of a promoter-containing 314-base-pair DNA element. This element is flanked on each side by a 9-base-pair inverted, repeat sequence and requires closely linked genes for inversion. Homology analysis of the products of these genes, fimB and fimE, reveals a strong similarity with the proposed DNA binding domain of lambda integrase, which mediates site-specific recombination in the presence of integration host factor. Integration host factor, encoded by himA and hip/himD, binds to the sequence 5' TNYAANNNRTTGAT 3', where Y = pyrimidine and R = purine, in mediating integration-excision. In analyzing the DNA flanking the fim 314-base-pair inversion sequence, we found the adjacent sequence 5' TTTAACTTATTGAT 3', which corresponds perfectly with the consensus integration host factor binding site. To characterize the role of himA in phase variation, we transduced either a deletion of himA or an insertionally inactivated hip/himD gene into an E. coli strain with a fimA-lacZ operon fusion. We found the rate of phase variation decreases sharply from 10(-3) to less than 10(-5) per cell per generation. Southern hybridization analysis demonstrates that the himA mutation results in a failure of the switch-generated genetic rearrangement. When the transductant was transformed with a himA+ plasmid, normal switching returned. Thus integration host factor is required for normal type 1 fimbriae phase variation in E. coli.

Bacterial Adhesion↗
PubMed1990

Finding sequence motifs in groups of functionally related proteins.

We have developed a method for rapidly finding patterns of conserved amino acid residues (motifs) in groups of functionally related proteins. All 3-amino acid patterns in a group of proteins of the type aa1 d1 aa2 d2 aa3, where d1 and d2 are distances that can be varied in a range up to 24 residues, are accumulated into an array. Segments of the proteins containing those patterns that occur most frequently are aligned on each other by a scoring method that obtains an average relatedness value for all the amino acids in each column of the aligned sequence block based on the Dayhoff relatedness odds matrix. The automated method successfully finds and displays nearly all of the sequence motifs that have been previously reported to occur in 33 reverse transcriptases, 18 DNA integrases, and 30 DNA methyltransferases.

Amino Acid Sequence↗
PubMed1992

Active nuclear import of human immunodeficiency virus type 1 preintegration complexes.

After cell infection by the human immunodeficiency virus type 1 (HIV-1), nascent viral DNA in the form of a high molecular weight nucleoprotein preintegration complex must be transported to the nucleus of the host cell prior to integration of viral DNA with the host genome. The mechanism used by retroviruses for nuclear targeting of preintegration complexes and dependence on cell division has not been established. Our studies show that, after infection, the preintegration complex of HIV-1 was rapidly transported to the nucleus of the host cell by a process that required ATP but was independent of cell division. Functional HIV-1 integrase, an essential component of the preintegration complex, was not required for nuclear import of these complexes. The ability of HIV-1 to use host cell active transport processes for nuclear import of the viral preintegration complex obviates the requirement for host cell division in establishment of the integrated provirus. These findings are pertinent to our understanding of early events in the life cycle of HIV-1 and to the mode of HIV-1 replication in terminally differentiated nondividing cells such as macrophages (monocytes, tissue macrophages, follicular dendritic cells, and microglial cells).

Adenosine Triphosphate↗
PubMed1993

The site of antiviral action of 3-nitrosobenzamide on the infectivity process of human immunodeficiency virus in human lymphocytes.

The C-nitroso compound 3-nitrosobenzamide, which has been shown to remove zinc from the retroviral-type zinc finger of p7NC nucleocapsid proteins, inhibits acute infection of human immunodeficiency virus type 1 in cultured human lymphocytes. The attachment of the virus to lymphocytes and the activities of critical viral enzymes, such as reverse transcriptase, protease, and integrase, are not affected by 3-nitrosobenzamide. However, the process of reverse transcription to form proviral DNA is effectively abolished by the drug, identifying the mode of action of 3-nitrosobenzamide as interrupting the role of p7NC in accurate proviral DNA synthesis during the infectious phase of the virus life cycle.

Antiviral Agents↗
PubMed1994

Identification of residues in the Mu transposase essential for catalysis.

A tetramer of Mu transposase (MuA) cleaves the phage Mu DNA and joins these ends to a target DNA to catalyze transposition. Substitution mutations at Asp-269 or Glu-392 within MuA destroy both the DNA cleavage and joining activities without blocking tetramer assembly, indicating that the mutations specifically affect catalysis. Although inactive under standard reaction conditions (10 mM Mg2+), the mutant proteins are partially resuscitated by 10-20 mM Mn2+, concentrations 5- to 10-fold higher than optimal for wild-type MuA. Amino acid sequence alignment and the similar effects of mutations suggests that Asp-269 and Glu-392 of MuA may be analogs of the first Asp and final Glu of a conserved triad of acidic amino acids present in many transposases and the retroviral integrases (the D-D-35-E motif). The higher Mn2+ optima observed with MuA derivatives altered at these positions supports a role for the conserved acidic amino acids in coordinating divalent metal ions in the active sites of transposases.

Amino Acid Sequence↗
PubMed1997

Penelope, a new family of transposable elements and its possible role in hybrid dysgenesis in Drosophila virilis.

A hybrid dysgenesis syndrome occurs in Drosophila virilis when males from an established laboratory strain are crossed to females obtained from the wild, causing the simultaneous mobilization of several different transposable elements. The insertion sequence responsible for the mutant phenotype of a dysgenic yellow allele has been characterized and named Penelope. In situ hybridization and Southern analyses reveal the presence of more than 30 copies of this element in the P-like parental strain, whereas Penelope is absent in all M-like strains tested. Penelope contains one 2.5-kb-long ORF that could encode products with homology to integrase and reverse transcriptase. Northern analysis and whole-mount in situ hybridization show strong induction of a 2.6-kb RNA in the ovaries of dysgenic females that is expressed at very low levels in the parental strains or in the progeny from the reciprocal cross. Injection of Penelope-containing plasmids into preblastoderm embryos of an M-like strain results in mutant progeny caused by insertion of Ulysses and perhaps other transposons, suggesting that Penelope expression might be responsible for the observed dysgenesis syndrome and the simultaneous mobilization of other transposable elements.

Amino Acid Sequence↗
PubMed1997

The catalytic domain of lambda site-specific recombinase.

The Escherichia coli phage lambda integrase protein (Int) belongs to the large Int family of site-specific recombinases. It is a heterobivalent DNA binding protein that makes use of a high energy covalent phosphotyrosine intermediate to catalyze integrative and excisive recombination at specific chromosomal sites (att sites). A 293-amino acid carboxy-terminal fragment of Int (C65) has been cloned, characterized, and used to further dissect the protein. From this we have cloned and characterized a 188-amino acid, protease-resistant, carboxy-terminal fragment (C170) that we believe is the minimal catalytically competent domain of Int. C170 has topoisomerase activity and converts att suicide substrates to the covalent phosphotyrosine complexes characteristic of recombination intermediates. However, it does not show efficient binding to att site DNA in a native gel shift assay. We propose that lambda Int consists of three functional and structural domains: residues 1-64 specify recognition of "arm-type" DNA sequences distant from the region of strand exchange; residues 65-169 contribute to specific recognition of "core-type" sequences at the sites of strand exchange and possibly to protein-protein interactions; and residues 170-356 carry out the chemistry of DNA cleavage and ligation. The finding that the active site nucleophile Tyr-342 is in a uniquely protease-sensitive region complements and reinforces the recently solved C170 crystal structure, which places Tyr-342 at the center of a 17-amino acid flexible loop. It is proposed that C170 is likely to represent a generic Int family domain that thus affords a specific route to studying the chemistry of DNA cleavage and ligation in these recombinases.

Amino Acid Sequence↗
PubMed1997

High-resolution functional mapping of a cloned gene by genetic footprinting.

We describe an efficient method for introducing and analyzing a comprehensive set of mutations in a cloned gene to map its functional organization. The technique, genetic footprinting, uses a retroviral integrase to generate a comprehensive library of mutants, each of which bears a single insertion of a defined oligonucleotide at a random position in the gene of interest. This mutant library is selected for gene function en masse. DNA samples are isolated from the library both before and after selection, and the mutations represented in each sample are then analyzed. The analysis is designed so that a mutation at a particular location gives rise to an electrophoretic band of discrete mobility. For the whole library, this results in a ladder of bands, each band representing a specific mutation. Mutants in which the inserted sequence disrupts a feature that is required for the selected function, ipso facto, fail the selection. The corresponding bands are therefore absent from the ladder of bands obtained from the library after selection, giving rise to a footprint representing features of the gene that are essential for the selected function. Because the sequence of the inserted oligonucleotide is known, and its position can be inferred precisely from the electrophoretic mobility of the corresponding band, the precise location and sequence of mutations that disrupt gene function can be determined without isolating or sequencing individual mutants. This method should be generally applicable for saturation mutagenesis and high-resolution functional mapping of cloned DNA sequences.

Chromosome Mapping↗
PubMed1998

A site-specific recombinase is required for competitive root colonization by Pseudomonas fluorescens WCS365.

A colonization mutant of the efficient root-colonizing biocontrol strain Pseudomonas fluorescens WCS365 is described that is impaired in competitive root-tip colonization of gnotobiotically grown potato, radish, wheat, and tomato, indicating a broad host range mutation. The colonization of the mutant is also impaired when studied in potting soil, suggesting that the defective gene also plays a role under more natural conditions. A DNA fragment that is able to complement the mutation for colonization revealed a multicistronic transcription unit composed of at least six ORFs with similarity to lppL, lysA, dapF, orf235/233, xerC/sss, and the largely incomplete orf238. The transposon insertion in PCL1233 appeared to be present in the orf235/233 homologue, designated orf240. Introduction of a mutation in the xerC/sss homologue revealed that the xerC/sss gene homologue rather than orf240 is crucial for colonization. xerC in Escherichia coli and sss in Pseudomonas aeruginosa encode proteins that belong to the lambda integrase family of site-specific recombinases, which play a role in phase variation caused by DNA rearrangements. The function of the xerC/sss homologue in colonization is discussed in terms of genetic rearrangements involved in the generation of different phenotypes, thereby allowing a bacterial population to occupy various habitats. Mutant PCL1233 is assumed to be locked in a phenotype that is not well suited to compete for colonization in the rhizosphere. Thus we show the importance of phase variation in microbe-plant interactions.

Amino Acid Sequence↗
PubMed1998

Evolution of rhizobia by acquisition of a 500-kb symbiosis island that integrates into a phe-tRNA gene.

Nodulation and nitrogen fixation genes of Mesorhizobium loti are encoded on the chromosome of the bacterium. Nevertheless, there is strong evidence that these genes can be transferred from an inoculant strain to nonsymbiotic mesorhizobia in the field environment. Here we report that the chromosomal symbiotic element of M. loti strain ICMP3153 is transmissible in laboratory matings to at least three genomic species of nonsymbiotic mesorhizobia. The element is 500 kb in size, integrates into a phe-tRNA gene, and encodes an integrase of the phage P4 family just within its left end. The entire phe-tRNA gene is reconstructed at the left end of the element upon integration, whereas the 3' 17 nucleotides of the tRNA gene are present as a direct repeat at the right end. We termed the element a symbiosis island on the basis of its many similarities to pathogenicity islands. It may represent a class of genetic element that contributes to microbial evolution by acquisition.

Amino Acid Sequence↗
PubMed1997

Purification and characterization of HP1 Cox and definition of its role in controlling the direction of site-specific recombination.

The protein that activates site-specific excision of the HP1 genome from the Hemophilus influenzae chromosome, HP1 Cox, was purified. Native Cox consists of four 8.9-kDa protomers. Tetrameric Cox self-associates to octamers; the apparent dissociation constant was 8 microM protomer, suggesting that under reaction conditions Cox is largely tetrameric. Cox binding sites consist of two direct repeats of the consensus motif 5'-GGTMAWWWWA; one Cox tetramer binds to each motif. Cox binding interfered with the interaction of HP1 integrase with one of its binding sites, IBS5. This competition is central to directional control, as shown by studies on mutated sites. Both Cox binding sites were necessary for Cox to fully inhibit integration and activate excision, although Cox continued to affect recombination when the single binding site proximal to IBS5 remained intact. Eliminating the IBS5 site completely prevented integration but greatly enhanced excision. Excisive recombination continued to require Cox even when IBS5 was inactivated. Cox must therefore play a positive role in assembling the nucleoprotein complexes producing excisive recombination, by inducing the formation of a critical conformation in those complexes.

Amino Acid Sequence↗
PubMed1997

Xer recombination in Escherichia coli. Site-specific DNA topoisomerase activity of the XerC and XerD recombinases.

Xer site-specific recombination functions in maintaining circular replicons in the monomeric state in Escherichia coli. Two recombinases of the bacteriophage lambda integrase family, XerC and XerD, are required for recombination at the chromosomal site, dif, and at a range of plasmid-borne sites. Xer recombination core sites contain the 11-base pair binding sites for each recombinase separated by a 6 to 8-base pair central region. We report that both XerC and XerD act as site-specific type I topoisomerases by relaxing supercoiled plasmids containing a dif site. Relaxation by either XerC or XerD occurs in the absence of the partner recombinase and requires only a single recombination core site. XerC or XerD relaxation activities are completely inhibited by the addition of the partner recombinase, providing that the DNA recognition sequence for the inhibiting partner is present.

Binding Sites↗
PubMed2000

Trans complementation of variant Cre proteins for defects in cleavage and synapsis.

The Cre recombinase is a member of the integrase family of conservative site-specific recombinases. These proteins share five conserved catalytic residues, one of which is a tyrosine that acts as the nucleophile to attack the scissile phosphodiester bond in the DNA target. Recombination by the Cre recombinase takes place in a supramolecular structure called a synapse that consists of four molecules of Cre bound to two DNA target sequences called lox sites. The synapse is held together by an intricate network of protein-protein interactions. They bend the two sites into square planar structure that resembles a Holliday intermediate. We have studied three mutant Cre proteins that appear to have defects in synapsis (Cre A36V, Cre T41F, and Cre G314R). We found that they were unable to carry out strand cleavage but that cleavage occurred if they were mixed with a cleavage-defective Cre protein that lacks the catalytic nucleophilic tyrosine residue. The three variant proteins could also be complemented for the formation of a novel structure ("complexV"), which may be a cleaved synaptic intermediate. We suggest that these three mutant proteins have a defect in DNA bending and discuss the relationship between bending, synapsis, and cleavage.

DNA↗
PubMed2002

Changes in the mechanism of DNA integration in vitro induced by base substitutions in the HIV-1 U5 and U3 terminal sequences.

We have reconstituted concerted human immunodeficiency virus type 1 (HIV-1) integration with specially designed mini-donor DNA, a supercoiled plasmid acceptor, purified bacterial-derived HIV-1 integrase (IN), and host HMG-I(Y) protein (Hindmarsh, P., Ridky, T., Reeves, R., Andrake, M., Skalka, A. M., and Leis, J. (1999) J. Virol. 73, 2994-3003). Integration in this system is dependent upon the mini donor DNA having IN recognition sequences at both ends and the reaction products have all of the features associated with integration of viral DNA in vivo. Using this system, we explored the relationship between the HIV-1 U3 and U5 IN recognition sequences by analyzing substrates that contain either two U3 or two U5 terminal sequences. Both substrates caused severe defects to integration but with different effects on the mechanism indicating that the U3 and the U5 sequences are both required for concerted DNA integration. We have also used the reconstituted system to compare the mechanism of integration catalyzed by HIV-1 to that of avian sarcoma virus by analyzing the effect of defined mutations introduced into U3 or U5 ends of the respective wild type DNA substrates. Despite sequence differences between avian sarcoma virus and HIV-1 IN and their recognition sequences, the consequences of analogous base pair substitutions at the same relative positions of the respective IN recognition sequences were very similar. This highlights the common mechanism of integration shared by these two different viruses.

Base Pairing↗
PubMed2004

Functional mapping of Cre recombinase by pentapeptide insertional mutagenesis.

Cre is a site-specific recombinase from bacteriophage P1. It is a member of the tyrosine integrase family and catalyzes reciprocal recombination between specific 34-bp sites called loxP. To analyze the structure-function relationships of this enzyme, we performed large scale pentapeptide insertional mutagenesis to generate insertions of five amino acids at random positions in the protein. The high density of insertion mutations into Cre allowed us to identify an unexpected degree of functional tolerance to insertions into the 4-5 beta-hairpin and into the loop between helices J and K (both of which contact the DNA in the minor groove) and also into helix A. The phenotypes of the majority of inserts allowed us to confirm a variety of predictions made on the basis of sequence conservation, known three-dimensional structure, and proposed catalytic mechanism. In particular, most insertions into conserved regions or secondary structure elements inactivated Cre, and most insertions located in nonconserved, unstructured regions preserved Cre activity. Less expectedly, the non-conserved and poorly structured loops and linkers between helices A-B, E-F, and M-N did not tolerate insertions, thus identifying these as critical regions for recombinase activity. We purified and characterized in vitro several representatives of these "unexpected" Cre insertion mutants. The role of those regions in the recombination process is discussed.

Amino Acid Sequence↗
PubMed2005

Synthesis, processing, and composition of the virion-associated HTLV-1 reverse transcriptase.

It is not known whether the low infectivity and low virion-associated polymerase activity of human T-cell lymphotropic virus type-1 (HTLV-1) are due to the quantity or quality of the reverse transcriptase (RT), because the protein has not yet been fully characterized. We have developed anti-RT antibodies and constructed HTLV-1 expression plasmids that express truncated or hemagglutinin-tagged Pol polyproteins to examine the maturation and composition of HTLV-1 RT. We detected virion-associated proteins corresponding to RT-integrase (IN) (pr98) and RT (p62) as well as smaller proteins containing the polymerase (p49) or RNase H domains. We have identified the amino acid sequences in the Pol polyprotein that are cleaved by HTLV-1 protease to yield RT and IN. We have also identified the cleavage sites within RT that give rise to the p49 polymerase fragment. Immunoprecipitation of an epitope-tagged p62 subunit coprecipitated p49, indicating that the HTLV-1 RT complex can exist as a p62/p49 heterodimer analogous to the RT of HIV-1 (p66/p51).

Amino Acid Sequence↗