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PubMedDate not supplied

Organization of HIV-1 pol is critical for Pol polyprotein processing.

The HIV pol sequentially encodes protease (PR), reverse transcriptase (RT), and integrase (IN) from the 5'-3' direction. We explored the significance of this gene arrangement. All six possible gene dispositions were examined. In two situations where PR was removed from the leading place and no two genes were in their original location, viral polyprotein processing was abolished. Processing of the polyprotein did not occur when IN was translocated to the front of PR-RT. However, in the following two arrangements, the polyprotein was processed but only at specific sites. First, PR remained in the leading position while the locations of RT and IN were exchanged; viral polyprotein was processed at a site between the upstream transframe peptide (TF) and PR. Second, PR was placed after RT-IN and located at the distal end of Pol. Processing occurred only at the created junction between TF and RT. These results indicated that cleavage after TF occurred autocatalytically but did not proceed to a second site, which needed an extraneous PR for trans-action. Therefore, arranging Pol in the order of PR-RT-IN warrants the streamline processing of the polyprotein once the autocleavage is initiated.

Amino Acid Sequence↗
PubMed2002

The clostridial mobilisable transposons.

Mobilisable transposons are transposable genetic elements that also encode mobilisation functions but are not in themselves conjugative. They rely on coresident conjugative elements to facilitate their transfer to recipient cells. Clostridial mobilisable transposons include Tn4451 and Tn4452 from Clostridium perfringens, and Tn4453a and Tn4453b from Clostridium difficile, all of which are closely related, and Tn5398 from C. difficile. The Tn4451 group of elements encodes resistance to chloramphenicol and is unusual in that transposition is dependent upon a large resolvase protein rather than a more conventional transposase or integrase. This group of elements also encodes the mobilisation protein TnpZ that, by acting at the RS(A) or oriT site located on the transposon, and in the presence of a coresident conjugative element, promotes the movement of the nonreplicating circular intermediate and of plasmids on which the transposon resides. The erythromycin resistance element Tn5398 is unique in that it encodes no readily identifiable transposition or mobilisation proteins. However, the element is still capable of intraspecific transfer between C. difficile isolates, by an unknown mechanism. The detailed analysis of these mobilisable clostridial elements provides evidence that the evolution and dissemination of antibiotic resistance genes is a complex process that may involve the interaction of genetic elements with very different properties.

Clostridium↗
PubMed2001

The structure and evolution of Penelope in the virilis species group of Drosophila: an ancient lineage of retroelements.

The Penelope element is the key element responsible for mobilization of other transposable elements in the course of hybrid dysgenesis in Drosophila virilis. Penelope has an unusually complex, highly variable organization in all studied species of the virlis group. Thc BRIDGE1 element from the fish Fugu rubripes is homologous to Penelope, and database searches detected additional homologous sequences among Expressed Sequence Tags from the flatworm Schistosoma mansonii and the nematode Ancylostoma caninum. Phylogenetic analysis shows that the reverse transcriptase of the Penelope group does not belong to any of the characterized major retroelement lineages, but apparently represents a novel branch of non-LTR retroelements. Sequence profile analysis results in the prediction that the C-terminal domain of the Penelope polyprotein is an active endonuclease related to intron-encoded endonucleases and the bacterial repair endonuclease UvrC, which could function as an integrase. No retroelements containing a predicted endonuclease of this family have been described previously. Phylogenetic analysis of Penelope copies isolated from several species of the virilis group reveals two subfamilies of Penelope elements, one of which includes full-length copies whose nucleotide sequences are almost identical, whereas the other one consists of highly diverged defective copies. Phylogenetic analysis of Penelope suggests both vertical transmission of the element and probable horizontal transfers. These findings support the notion that Penelope invasions occurred repeatedly in the evolution of the virilis group.

Amino Acid Sequence↗
PubMed1996

Molecular characterization of the xerC gene of Lactobacillus leichmannii encoding a site-specific recombinase and two adjacent heat shock genes.

Sequencing of four overlapping DNA fragments comprising 3.527 kb isolated from a L. leichmannii genomic library revealed three complete open reading frames (ORFs) and one that was truncated. The deduced amino acid sequences of the complete ORFs showed considerable similarities with the already known sequences of the xerC, hslV, and hslU gene products of Escherichia coli: the site-specific XerC recombinase, a member of the lambda integrase family, and the HtpI resp. HtpO heat shock proteins. The deduced amino acid sequence of the fourth, incomplete ORF upstream the xerC gene showed strong homology with the gid A gene product of B. subtilis.

Amino Acid Sequence↗
PubMed2003

CEN plasmid segregation is destabilized by tethered determinants of Ty 5 integration specificity: a role for double-strand breaks in CEN antagonism.

The yeast retrotransposon Ty 5 integrates preferentially into heterochromatin at the telomeres and HM loci. Target specificity is mediated by a six amino acid sequence motif (the targeting domain, TD) of integrase that interacts with Sir4p, a structural component of heterochromatin. When tethered to CEN plasmids as part of a Gal4p DNA binding domain (GBD) fusion protein, TD destabilizes plasmid segregation in a manner similar to that observed for CEN + HM or CEN +TEL antagonism. This instability is caused by the ability of TD to nucleate components of heterochromatin on the CEN plasmid, because CEN +TD antagonism is abrogated by sir2, sir3 and sir4 mutations and by TD mutations that prevent interaction with Sir4p. In strains that acquire resistance to CEN +TD antagonism, the CEN plasmid has either recombined with a 2 mu plasmid or sustained deletions in sequences required to bind GBD-TD. CEN +TD and CEN + HM antagonism is exacerbated by mutations in components of the Ku-mediated non-homologous end-joining pathway. These observations suggest that CEN antagonism is caused by DNA breaks that result from competition between CEN - and Sir-specific segregation pathways.

Amino Acid Motifs↗
PubMed1997

Control of Escherichia coli type 1 fimbrial gene expression in stationary phase: a negative role for RpoS.

Expression of type 1 fimbriae in Escherichia coli is subject to phase-dependent control, with many regulatory inputs from proteins which organise or rearrange the structure of DNA. Inversion of a DNA segment carrying the promoter for expression of fimA, the gene encoding the fimbrial subunit protein, makes a key contribution to the switching of cells between fimbriate and afimbriate states. We have discovered that transcription of fimA is repressed as cells enter stationary phase. This repression is not seen in isogenic strains deficient in rpoS, the gene coding for the stationary phase-specific sigma factor RpoS. RpoS-deficient strains are also altered in the frequency of inversion of the fimA promoter segment. In the strains used in this study, inversion is catalysed by an integrase-like site-specific recombinase encoded by the fimB gene. We report that fimB transcription is repressed strongly as wild-type cells enter stationary phase but that this repression is alleviated in cells deficient in RpoS. These data suggest that RpoS has a negative regulatory role which may be indirect, at both the fimA and the fimB promoters.

Bacterial Proteins↗
PubMed1997

Zebedee: a novel copia-Ty1 family of transposable elements in the genome of the medically important mosquito Aedes aegypti.

We have utilised PCR to directly identify a novel family of copia-Ty1 retrotransposable elements (RTPs) in the genome of the mosquito Aedes aegypti. Two members of the family have been sequenced in their entirety and their structural characteristics determined. ZebedeeI is 3505 bp long and appears to be flanked by 21bp direct repeat sequences. A single open reading frame (ORF) of 972 amino acids has the coding potential for a polyprotein with homology corresponding to the conserved amino acid motifs of Long Terminal Repeat (LTR) retrotransposon protease, integrase and reverse transcriptase. ZebedeeII likewise shares significant homology with these regions and also appears to be flanked by short direct terminal repeat sequences of 22 bp. Fifty copies of the 22 bp repeat sequence are present abutting the 5' end of ZebedeeII, with two (partial) representatives of this repeat sequence being present at the 3' end. The Zebedee family appears to have a low middle repetitive copy number in different strains of Ae. aegypti; and transcripts of the elements have been detected in cultured mosquito cells by RT-PCR. Despite the lack of a gag homologue or the LTR hallmarks of previously characterised copia-Ty1 RTPs, phylogenetic analyses place Zebedee within this group, showing considerable homology to copia from Drosophila melanogaster.

Aedes↗
PubMed1998

Molecular characterization of a retrotransposon in Drosophila melanogaster, nomad, and its relationship to other retrovirus-like mobile elements.

The nomad element was identified as a retrovirus-like transposon in Drosophila melanogaster. DNA sequence analysis showed that the nomad element contains three long ORFs that are similar to the gag, pol and env genes of retrovirus- and the copia-like elements of D. melanogaster. The nomad element terminates with 519-bp terminal repeats, each of which contains eukaryotic consensus transcription initiation and termination signals. nomad elements are located at approximately 10-15 sites within the euchromatic arms of the genome and at the chromocenter, as shown by in situ hybridization. The host DNA sequence TANA was found to be duplicated on each side of the nomad element and appears to be a preferential target site for insertion of nomad elements. Analysis of the zinc finger motif in the pol gene product of retrotransposons known to have target site preference suggests involvement of the integrase subunit in target site selection for those retrotransposons that display insert site specificity. A comparison of the predicted amino acid sequence of the pol-like genes of several known retrotransposons was made and the phylogenetic relationship between nomad and other retrovirus-like mobile elements was determined. It was clear from this conceptual protein analysis and from analysis of their structural characteristics that retrotransposons of the gypsy class can be generally classified as members of one of two distinct groups. The phylogenetic relationships of these groups are also discussed.

Amino Acid Sequence↗
PubMed2000

REAL, an LTR retrotransposon from the plant pathogenic fungus Alternaria alternata.

A retrotransposon was isolated and characterized from strain 15A of the Japanese pear pathotype of Alternaria alternata, which causes black spot disease in certain cultivars of Japanese pear by producing a host-specific toxin known as AK-toxin. The element, which we have named REAL (Retrotransposon of Alternaria alternata), is 6046 bp in size and contains direct long terminal repeats (LTRs) of 218 bp. Target-site duplication of 5 bp was found. REAL contains two long overlapping ORFs. The first ORF shows homology to retroviral gag genes. The second ORF has homology to protease, reverse transcriptase, RNase H and integrase domains of the retroelement pol genes, in that order. Phylogenetic comparison of reverse transcriptase domains from retrotransposons placed REAL in the Ty3/gypsy group of LTR retrotransposons, most closely related to grasshopper from Magnaporthe grisea. Northern analysis detected REAL transcripts of about 2.0 and 6.0 kb. The 6.0-kb species corresponds to a full-length transcript of the element. The element was found by Southern analysis in 12 out of 13 strains of the Japanese pear pathotype, and the banding patterns, copy numbers and signal intensities in these strains were variable. REAL-related elements were also found in some, but not all, of the other strains tested, including nonpathogenic A. alternata and other pathotypes, which cause diseases on different plant species by producing distinct hostspecific toxins. These results suggest that the distribution of REAL in A. alternata is not pathotype specific.

Alternaria↗
PubMed1999

Genetic diversity of HIV-1 group M from Cameroon and Republic of Congo.

We analyzed 57 HIV-1 isolates from Cameroon and the Republic of Congo, with respect to the env C2V3 and/or the pol integrase regions. The results indicated that the topology of the pol tree correlated well with that of the env tree for four clusters of subtype D, F G and H, suggesting that these trees reflect the true evolution of the overall genome structures of these subtypes. However, of 22 Cameroonian isolates that were classified as subtype A based on env, 20 of them diverged in their pol sequence into two lineages that were completely different from the prototypical subtype A, tentatively designated as subtypes A1 and A2. The subtype A1 isolates (6 out of 22) were related in their env C2V3 regions with prototypical subtype A strain, but in their pol regions, they formed an independent cluster that diverged from known HIV-1 subtypes so far reported (except for subtypes I and J). The subtype A2 isolates (14 out of 22), which represent the major epidemic type of HIV-1 in Cameroon, clustered distinctly in both the env and pol trees with the recently described A/G mosaic strains from Nigeria and Djibouti. These two lineages were not spreading in the neighboring Republic of Congo.

Amino Acid Sequence↗
PubMed2006

Characterization of the Sulfolobus host-SSV2 virus interaction.

The Sulfolobus spindle virus, SSV2, encodes a tyrosine integrase which furthers provirus formation in host chromosomes. Consistently with the prediction made during sequence analysis, integration was found to occur in the downstream half of the tRNA(Gly) (CCC) gene. In this paper we report the findings of a comparative study of SSV2 physiology in the natural host, Sulfolobus islandicus REY15/4, versus the foreign host, Sulfolobus solfataricus, and provide evidence of differently regulated SSV2 life cycles in the two hosts. In fact, whereas a significant induction of SSV2 replication takes place during the growth of the natural host REY15/4, the cellular content of SSV2 DNA remains fairly low throughout the incubation of the foreign host. The accumulation of episomal DNA in the former case cannot be traced to decreased packaging activity because of a simultaneous increase in the virus titre in the medium. In addition, the interaction between SSV2 and its natural host is characterized by the concurrence of host growth inhibition and the induction of viral DNA replication. When this virus-host interaction was investigated using S. islandicus REY15A, a strain which is closely related to the natural host, it was found that the SSV2 replication process was induced in the same way as in the natural host REY15/4.

Archaeal Viruses↗
PubMed2006

New antiretroviral drugs.

Despite the availability of 21 antiretroviral drugs approved for the treatment of HIV infection, current combination regimens remain hampered by issues of toxicity, convenience, cost, incomplete viral suppression, and drug resistance. Expansion of the currently available therapeutic options through the reformulation of available agents, discovery of new compounds with antiretroviral activity, and the exploitation of novel drug targets are critical. This review describes the status of new nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, protease inhibitors, and fusion inhibitors. We also summarize new classes of antiretroviral therapy in clinical development including the attachment inhibitors, chemokine receptor antagonists, integrase inhibitors, and maturation inhibitors.

Anti-HIV Agents↗
PubMed1988

Micropia: a retrotransposon of Drosophila combining structural features of DNA viruses, retroviruses and non-viral transposable elements.

The retrotransposon micropia was first described from Y-chromosomal fertility genes of Drosophila hydei. Screening a Drosophila melanogaster genomic library yielded several clones representing micropia elements in D. melanogaster. The DNA sequences of two elements from D. hydei (micropia-DhMiF2 and micropia-DhMiF8) and two elements from D. melanogaster (micropia-Dm2 and micropia-Dm11) permitted a detailed analysis of the spatial organization of micropia constituents. Micropia represents the typical gene organization represented by "core"-protein domains followed by a protease, reverse transcriptase, RNase and integrase domain. New features of the micropia family compared with other retrotransposons are: (1) a region of similarity to class I major histocompatibility complex antigens of mammals; (2) only one main open reading frame of about 4000 bases length; (3) a non-protein-coding region of about 500 base-pairs length between the 3' end of the open reading frame and the 5' start of the 3' long terminal repeat. This region includes 32 base-pair tandem repeats; (4) within the long terminal repeats, 82 base-pair tandem repeats with four potential ecdysteroid receptor binding sites. Because micropia combines many evolutionary features of different viruses, non-viral transposable elements, chromosomal genes and repetitive sequence organizations, this retrotransposon may be seen as a "minigenome" reflecting evolutionary principles of the construction of genomic components.

Amino Acid Sequence↗
PubMed1992

Functional analysis of Box II mutations in yeast site-specific recombinases Flp and R. Significance of amino acid conservation within the Int family and the yeast sub-family.

The site-specific recombinases Flp and R from Saccharomyces cerevisiae and Zygosaccharomyces rouxii, respectively, are related proteins that share approximately 30% amino acid matches. They exhibit a common reaction mechanism that appears to be conserved within the larger Integrase family of site-specific recombinases. Two regions of the proteins, designated as Box I and Box II, harbor, in addition to amino acid conservation, a significantly high degree of nucleotide sequence homology within their coding segments. Box II also contains two amino acids, a histidine and an arginine, that are invariant throughout the Int family. We have performed functional analysis of Flp and R variants carrying point mutations within the Box II segment. Several positions within Box II can tolerate substitutions with no effect, or only modest effects on recombination. Alterations of the Int family residues, His305 and Arg308, in the R protein lead to the arrest of recombination at the strand cleavage or the strand exchange step. This is very similar to previously observed "step-arrest" phenotypes in Flp variants altered at these positions and has strong implications for the catalytic mechanism of recombination. Flp and R variants at His305 and His309 can be complemented in half-site strand transfer by a corresponding Tyr343 to phenylalanine variant. In contrast to Arg308 Flp variants, which are efficiently complemented in half-site strand transfer by Flp(Y343F), no strong complementation has been observed between Arg308 variants of R and R (Y343F).

DNA Mutational Analysis↗
PubMed1992

Recognition of distantly related protein sequences using conserved motifs and neural networks.

A sensitive technique for protein sequence motif recognition based on neural networks has been developed. It involves three major steps. (1) At each appropriate alignment position of a set of N matched sequences, a set of N aligned oligopeptides is specified with preselected window length. N neural nets are subsequently and successively trained on N-1 amino acid spans after eliminating each ith oligopeptide. A test for recognition of each of the ith spans is performed. The average neural net recognition over N such trials is used as a measure of conservation for the particular windowed region of the multiple alignment. This process is repeated for all possible spans of given length in the multiple alignment. (2) The M most conserved regions are regarded as motifs and the oligopeptides within each are used to train intensively M individual neural networks. (3) The M networks are then applied in a search for related primary structures in a databank of known protein sequences. The oligopeptide spans in the database sequence with strongest neural net output for each of the M networks are saved and then scored according to the output signals and the proper combination that follows the expected N- to C-terminal sequence order. The motifs from the database with highest similarity scores can then be used to retrain the M neural nets, which can be subsequently utilized for further searches in the databank, thus providing even greater sensitivity to recognize distant familial proteins. This technique was successfully applied to the integrase, DNA-polymerase and immunoglobulin families.

Aldehyde Dehydrogenase↗
PubMed1990

Two bases are deleted from the termini of HIV-1 linear DNA during integrative recombination.

Proviral integration is a required step in the retrovirus life cycle. The mechanism of integration involves specific modification of the ends of linear viral DNA and subsequent recombination with host sequences. Integration results in the limited loss of sequence information at the termini of the viral genome. The composition of the intact linear DNA termini were inferred by sequencing the 2-long terminal repeat (2-LTR) circle junction that is formed when the linear molecule undergoes intramolecular, blunt-end ligation. The junction sequence contained the nucleotides GTAC that were not present at the ends of the integrated provirus. Comparison with the sequence of the LAV-1 strain of HIV-1 demonstrated that the GT dinucleotide derived from the right-hand terminus (U5) of the linear viral DNA and the AC dinucleotide came from the left-hand terminus (U3). Therefore, the corrected size of the LAV-1 LTR is 637 bp. This conclusion was confirmed independently by assessing the structure of linear viral DNA in acutely infected T cells. A portion of the population of linear HIV-1 DNA molecules were specifically deleted at their 3' ends; the extent of this deletion was 2 bases. This result is consistent with the activity of viral integrase protein on linear viral DNA and it accounts for the structure of integrated HIV-1 proviruses.

Base Sequence↗
PubMed1991

Moloney murine leukemia virus IN protein from disrupted virions binds and specifically cleaves its target sequence in vitro.

The integration of retroviral DNA plays an essential role in the viral life cycle. Previous studies of the Moloney murine leukemia virus (M-MuLV) have shown that viral integration is mediated by the integrase (IN) protein acting on the 13-bp inverted repeats that flank the linear viral DNA produced during reverse transcription. Prior studies have also shown that when the M-MuLV IN protein is produced in Escherichia coli it retains an ability to specifically associate with the viral inverted repeats (Krogstad and Champoux, 1990). In this study we present evidence that the IN protein present in detergent-disrupted virions is capable of specifically interacting with double-stranded oligonucleotides that correspond to the viral inverted repeats, and that this interaction may change after integration-related processing of the viral att sites. We further present evidence that, in vitro, detergent-disrupted virions are capable of specifically cleaving ds-IR oligonucleotides in an IN-dependent reaction that mimics the trimming step that precedes integration.

Animals↗
PubMed1992

Identification of amino acid residues critical for endonuclease and integration activities of HIV-1 IN protein in vitro.

HIV-IN protein, tagged with a hexahistidine tail was expressed in Escherichia coli and purified by a one-step nickel chelate affinity chromatography procedure. The purified IN protein was characterized in terms of its endonuclease and integrase properties in vitro. Specific cleavage and integration of HIV U5 LTR ends were observed in the presence of 2-5 mM Mg2+ or Ca2+. In the presence of 2 mM Mn2+, cleavage and integration occurred at additional sites indicating a decreased specificity. The properties of mutant IN proteins were examined in vitro. Deletion of 39 amino acids from the N-terminus and a minimum of 25 residues from the C-terminus impaired IN-mediated cleavage and integration activities. The results of site-directed mutagenesis experiments showed that residues critical for IN function are highly conserved. Mutations of conserved residues Asp64, Pro109, Asp116, and Glu152 adversely affected IN function in vitro. Mutations of nonconserved residues Gly189 and Thr112 had no effect. Mutation of a conserved Thr115 to Ala caused a near complete loss of Mg(2+)-dependent integration activity, but only partially effected endonucleolytic cleavage activity of IN. These results suggest that not all conserved residues are involved in both endonucleolytic cleavage and integration activities of HIV-IN.

Amino Acid Sequence↗