Domain Of Integrase Research Articles

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).

602. Effectiveness of Gene Delivery Targeting HIV-1 Integrase in Inhibiting HIV Integration: Lack of HIV-1 Genomes in T-Lymphocytes during Repeated Challenges with HIV-1

HIV-1 integration into cell genomes is a key step in the HIV-1 replicative cycle. We applied a gene therapy strategy designed to block HIV replication at this early stage. To do this, we used a single chain antibody (SFv) against the DNA-binding domain of HIV-1 integrase (IN). SV40-derived vectors were used for gene transfer to human T-lymphocytes because of their high transduction efficiency and durability of transgene expression. HIV-1 replication was measured by supernatant p24 antigen levels. Protection by the test vector, SV(Aw), compared with mock- and SV(HBS) transduced cells used as negative controls, was assessed during 3 successive rounds of HIV-1 NL4-3 challenge. HIV-1 infectious dose escalated from 400 infectious units (IU) to 4000 IU.

Uracils as a Cellular Weapon Against Viruses and Mechanisms of Viral Escape

Uracil in DNA is a deleterious event that may arise either by cytosine deamination or misincorporation of dUTP. Consequently, cells from all free-living organisms have developed strategies to protect their genome against the presence of uracils, by using uracil DNA glycosylase (UNG) and deoxyuridine triphosphatase (dUTPase) enzymatic activities. In the viral kingdom, some (namely poxviruses and herpesviruses) but not all of the DNA viruses encode their own UNG and dUTPase to control uracilation of their genome. Some retroviruses, which are RNA viruses using DNA as an intermediate of replication, also encode dUTPase. Surprisingly, though most of nonprimate lentiviruses encode dUTPase, primate lentiviruses such as HIV-1, HIV-2 or SIV do not. Because these latter viruses also replicate in nondividing cells where the dUTP/dTTP ratio is high, it is probable that they have found other ways to fight against the emergence of uracilated-viral transcripts. Indeed, recent studies showed that HIV-1 efficiently controls both the cytosine deamination and the dUTP misincorporation. The viral Vif protein acts in preventing the packaging into viral particles of the host-derived cytosine deaminase APOBEC3G enzyme, while the viral integrase domain of the Gag-Pol precursor mediates the packaging of the host-derived uracil DNA glycosylase UNG2 enzyme. In the absence of Vif or UNG2, HIV-1 viral transcripts are heavily charged in uracil bases leading to inactivation of the virus.

Processing of Viral DNA Ends Channels the HIV-1 Integration Reaction to Concerted Integration*[boxs

Retroviral DNA made by reverse transcription is blunt-ended, and the viral integrase protein must remove two nucleotides from each 3′ end prior to integration into chromosomal DNA. Under most reaction conditions for integration in vitro, the majority of the reaction products are “half-site” products that result from integration of only one viral DNA end into one strand of the target DNA. Preprocessed DNA substrates are more efficient substrates for half-site reactions than are blunt-ended substrates, which require the removal of two nucleotides prior to integration. In contrast, we find that blunt-ended DNA is a better substrate for the biologically relevant reaction of concerted integration of pairs of viral DNA ends. The reaction pathway is channeled to concerted integration, and half-site integration products are reduced with blunt-ended DNA substrate that must first be processed by integrase. In addition, the terminal nucleotide requirements for concerted integration are more stringent than for the half-site reaction. Longer DNA is more efficient for the concerted reaction than is shorter DNA that is capable of efficient half-site integration. This suggests that nonspecific interactions of integrase with viral DNA distant from the termini contribute to the assembly of a complex that is competent for concerted integration. Finally, differential effects of mutation of a residue in the C-terminal domain of integrase on concerted versus half-site integration implicate protein-protein interactions involving this domain as important for concerted integration.

Molecular characterization of a gender-linked DNA marker and a related gene in Mercurialis annua L.

The dioecious Mercurialis annua L. was used as a model plant to study some aspects of the molecular basis of sex determination in plants. We report in this paper the characterization of a previously identified male specific DNA marker, OPB01-1562, from diploid dioecious M. annua. The marker co-segregated with male sex in the progeny of hormonally feminized males. Sequence analysis showed the presence of approximately 0.6 kb retrotransposon-like sequence at its 3' end. Homologous sequences were isolated from diploid female, hexaploid male and monoecious plants. These sequences contained RNaseH and integrase domains of reverse transcriptase and were most similar to pineapple retrotransposon dea1, hence were named M. annua retrotransposon-like sequences (MARL-1 to MARL-5). A 771 bp fragment isolated from a diploid female, named fem771, was homologous to the 5' end of OPB01-1562. Results from DNA blot hybridization suggested OPB01-1562 and fem771 to be from the same locus and MARL-1 from a different one. RNA blot hybridization with OPB01-1562 and MARL-1 detected an approximately 2.8 kb transcript which was expressed strongly in stems and flowers of females but not males. This transcript was named M. annua female expressed (Mafex). Sex linkage of OPB01-1562 and expression of Mafex detected by OPB01-1562 strongly suggested Mafex to be a candidate gene involved in sex determination in M. annua.

Development of broadly targeted human endogenous gammaretroviral pol-based real time PCRs : Quantitation of RNA expression in human tissues

Endogenous retroviral sequences (ERVs) are dynamic genomic components with profound influences on gene expression and genomic structure. Their extent of expression is not well known. Several broadly targeted real-time reverse transcription PCR (QPCRs) systems for surveillance of RNA expression of the major groups of human gammaretroviral ERVs were constructed. The highly conserved reverse transcriptase (RT) and integrase (IN) domains of the pol gene were used as targets for the PCRs, which were both probe-based (TaqMan ®) and probe-less (SYBR ® Green). Different levels of primer and probe degeneracy, with or without inosine, were tested. Several of the PCRs had sensitivities of a few HERV nucleic acid copies per PCR reaction. Specificities were approximately as expected from the fit of primers and probes. Gammaretroviral HERV RNA expression was studied in different human tissues. Each HERV group had a specific pattern of expression. HERV-E was highly expressed in testis, HERV-I/T in brain and testis, HERV-H in brain and testis, while HERV-W was highly expressed in placenta. Gammaretroviral RNA was not detected in plasma from 50 blood donors in saliva from 20 persons. In conclusion, a set of tools for investigation of gammaretroviral HERV RNA expression was created.

Role of Integrase in Reverse Transcription of the Saccharomyces cerevisiae Retrotransposon Ty1

Reverse transcriptase (RT) with its associated RNase H (RH) domain and integrase (IN) are key enzymes encoded by retroviruses and retrotransposons. Several studies have implied a functional role of the interaction between IN and RT during the replication of retroviral and retrotransposon genomes. In this study, IN deletion mutants were used to investigate the role of IN on the RT activity of the yeast Saccharomyces cerevisiae retrotransposon Ty1. We have identified two domains of Ty1 integrase which have effects on RT activity in vivo. The deletion of a domain spanning amino acid residues 233 to 520 of IN increases the exogenous specific activity of RT up to 20-fold, whereas the removal of a region rich in acidic amino acid residues between residues 521 and 607 decreases its activity. The last result complements our observation that an active recombinant RT protein can be obtained if a small acidic tail mimicking the acidic domain of IN is fused to the RT-RH domain. We suggest that interaction between these acidic amino acid residues of IN and a basic region of RT could be critical for the correct folding of RT and for the formation of an active conformation of the enzyme.

Synapsis in Phage Bxb1 Integration: Selection Mechanism for the Correct Pair of Recombination Sites

Recombination by site-specific recombinases is a highly concerted process that requires synapsis of the correct pair of DNA substrates. Phage-encoded serine-integrases are unusual among the serine-recombinase family, which includes transposon resolvases and DNA invertases, in that they utilize two simple but different DNA substrates (attB and attP) and do not require accessory sites, additional proteins, or DNA supercoiling. Synapsis must therefore be directed solely by integrase-DNA interactions. We show here that the Bxb1 serine-integrase binds as a dimer to its two DNA substrates (attB, attP) and recombinant products (attL, attR) with similar affinities. However, synapsis occurs only between attP and attB, and not between any of the other nine possible site combinations. The Bxb1 integrase domain structure, the unusual DNA-binding properties of the integrase, and the characterization of a mutant protein with altered site-discrimination, are consistent with synaptic selectivity being derived from DNA sequence-induced changes in the conformations of integrase-DNA complexes.

Characterization of the Functional Domains of Human Foamy Virus Integrase Using Chimeric Integrases

Characterization of the Functional Domains of Human Foamy Virus Integrase Using Chimeric Integrases

Malazy, a degenerate, species-specific transposable element in Cercospora zeae-maydis

Two fungal pathogens, Cercospora zeae-maydis Groups I and II, cause gray leaf spot of maize. During the sequencing of a cosmid library from C. zeae-maydis Group I, we discovered a sequence with high similarity to Maggy, a transposable element from Magnaporthe grisea. The element from C. zeae-maydis, named Malazy, contained 194-base-pair terminal repeats and sequences with high similarity to reverse transcriptase and integrase, components of the POL gene in the gypsy-like retrotransposons in fungi. Sequences with similarity to other POL gene components, protease and ribonuclease, were not detected in Malazy. A single copy of the element was detected by PCR and Southern analyses in all six North American isolates of C. zeae-maydis Group I but was not detected in the four isolates of C. zeae-maydis Group II from three continents or in phylogenetically related species. Fragments of the core domains of reverse transcriptase and integrase contained a high frequency of stop codons that were conserved in all six isolates of Group I. Additional C:G to T:A transitions in occasional isolates usually were silent mutations, while two resulted in isolate-specific stop codons. The absence of Malazy from related species suggests that it was acquired after the divergence of C. zeae-maydis Groups I and II. The high frequency of stop codons and the presence of a single copy of the element suggest that it was inactivated soon after it was acquired. Because the element is inactive and because reading frames for other genes were not found in sequences flanking the element, Malazy does not appear to be the cause of differences leading to speciation or genetic diversity between C. zeae-maydis Groups I and II.

Malazy, a degenerate, species-specific transposable element in Cercospora zeae-maydis

Two fungal pathogens, Cercospora zeae-maydis Groups I and II, cause gray leaf spot of maize. During the sequencing of a cosmid library from C. zeae-maydis Group I, we discovered a sequence with high similarity to Maggy, a transposable element from Magnaporthe grisea. The element from C. zeae-maydis, named Malazy, contained 194-base-pair terminal repeats and sequences with high similarity to reverse transcriptase and integrase, components of the POL gene in the gypsy-like retrotransposons in fungi. Sequences with similarity to other POL gene components, protease and ribonuclease, were not detected in Malazy. A single copy of the element was detected by PCR and Southern analyses in all six North American isolates of C. zeae-maydis Group I but was not detected in the four isolates of C. zeae-maydis Group II from three continents or in phylo-genetically related species. Fragments of the core domains of reverse transcriptase and integrase contained a high frequency of stop codons that were conserved in all six isolates of Group I. Additional C:G to T:A transitions in occasional isolates usually were silent mutations, while two resulted in isolate-specific stop codons. The absence of Malazy from related species suggests that it was acquired after the divergence of C. zeae-maydis Groups I and II. The high frequency of stop codons and the presence of a single copy of the element suggest that it was inactivated soon after it was acquired. Because the element is inactive and because reading frames for other genes were not found in sequences flanking the element, Malazy does not appear to be the cause of differences leading to speciation or genetic diversity between C. zeae-maydis Groups I and II.

HIV-1-Associated Uracil DNA Glycosylase Activity Controls dUTP Misincorporation in Viral DNA and Is Essential to the HIV-1 Life Cycle

Uracilation of DNA represents a constant threat to the survival of many organisms including viruses. Uracil may appear in DNA either by cytosine deamination or by misincorporation of dUTP. The HIV-1-encoded Vif protein controls cytosine deamination by preventing the incorporation of host-derived APOBEC3G cytidine deaminase into viral particles. Here, we show that the host-derived uracil DNA glycosylase UNG2 enzyme, which is recruited into viral particles by the HIV-1-encoded integrase domain, is essential to the viral life cycle. We demonstrate that virion-associated UNG2 catalytic activity can be replaced by the packaging of heterologous dUTPase into virion, indicating that UNG2 acts to counteract dUTP misincorporation in the viral genome. Therefore, HIV-1 prevents incorporation of dUTP in viral cDNA by UNG2-mediated uracil excision followed by a dNTP-dependent, reverse transcriptase-mediated endonucleolytic cleavage and finally by strand-displacement polymerization. Our findings indicate that pharmacologic strategies aimed toward blocking UNG2 packaging should be explored as potential HIV/AIDS therapeutics.

The Sinbad retrotransposon from the genome of the human blood fluke, Schistosoma mansoni, and the distribution of related Pao-like elements

BackgroundOf the major families of long terminal repeat (LTR) retrotransposons, the Pao/BEL family is probably the least well studied. It is becoming apparent that numerous LTR retrotransposons and other mobile genetic elements have colonized the genome of the human blood fluke, Schistosoma mansoni.ResultsA proviral form of Sinbad, a new LTR retrotransposon, was identified in the genome of S. mansoni. Phylogenetic analysis indicated that Sinbad belongs to one of five discreet subfamilies of Pao/BEL like elements. BLAST searches of whole genomes and EST databases indicated that members of this clade occurred in species of the Insecta, Nematoda, Echinodermata and Chordata, as well as Platyhelminthes, but were absent from all plants, fungi and lower eukaryotes examined. Among the deuterostomes examined, only aquatic species harbored these types of elements. All four species of nematode examined were positive for Sinbad sequences, although among insect and vertebrate genomes, some were positive and some negative. The full length, consensus Sinbad retrotransposon was 6,287 bp long and was flanked at its 5'- and 3'-ends by identical LTRs of 386 bp. Sinbad displayed a triple Cys-His RNA binding motif characteristic of Gag of Pao/BEL-like elements, followed by the enzymatic domains of protease, reverse transcriptase (RT), RNAseH, and integrase, in that order. A phylogenetic tree of deduced RT sequences from 26 elements revealed that Sinbad was most closely related to an unnamed element from the zebrafish Danio rerio and to Saci-1, also from S. mansoni. It was also closely related to Pao from Bombyx mori and to Ninja of Drosophila simulans. Sinbad was only distantly related to the other schistosome LTR retrotransposons Boudicca, Gulliver, Saci-2, Saci-3, and Fugitive, which are gypsy-like. Southern hybridization and bioinformatics analyses indicated that there were about 50 copies of Sinbad in the S. mansoni genome. The presence of ESTs representing transcripts of Sinbad in numerous developmental stages of S. mansoni along with the identical 5'- and 3'-LTR sequences suggests that Sinbad is an active retrotransposon.ConclusionSinbad is a Pao/BEL type retrotransposon from the genome of S. mansoni. The Pao/BEL group appears to be comprised of at least five discrete subfamilies, which tend to cluster with host species phylogeny. Pao/BEL type elements appear to have colonized only the genomes of the Animalia. The distribution of these elements in the Ecdysozoa, Deuterostomia, and Lophotrochozoa is discontinuous, suggesting horizontal transmission and/or efficient elimination of Pao-like mobile genetic elements from some genomes.

The C-terminal chromodomain-like module in the integrase domain is crucial for high transposition efficiency of the retrotransposon MAGGY

MAGGY is a Ty3/Gypsy retrotransposon, which was identified in the rice blast fungus Magnaporthe oryzae. Some Ty3/Gypsy retrotransposons, including MAGGY, contain a chromodomain-like module (CLM) in the C-terminus of the integrase domain. We have made a series of MAGGY mutants to examine the role of the CLM in the transposition activity of the element. Introduction of a mutation at different positions in the MAGGY integrase revealed that a loss or alteration of the CLM resulted in a drastic decrease in the transposition activity of the element. Our results indicate that the CLM may confer high transposition activity to the element.

Docking dinucleotides to HIV-1 integrase carboxyl-terminal domain to find possible DNA binding sites

HIV-1 integrase mediates a processed viral DNA into the host genome DNA. The binding modes between HIV-1 integrase and viral/host DNA have not been clarified until now. The C-terminal domain of integrase has been found to be a DNA-binding domain. In this work, we have explored the possible DNA binding sites of dimeric C-terminal domain by docking dinucleotides to HIV-1 integrase. The docking results suggest that two symmetrical DNA-binding sites are likely located on the outside surface of the dimeric C-terminal domain and not located in the groove. Those sites are in agreement with the experimental data.

Active site binding modes of the β-diketoacids: a multi-active site approach in HIV-1 integrase inhibitor design

Predicting a bioactive conformation of a ligand is of paramount importance in rational drug design. The task becomes very difficult when the receptor site possesses a region with unusual conformational flexibility. Significant conformational differences are present in the active site regions in the available crystal structures of the core domains of HIV-1 integrase (IN). Among all reported IN inhibitors, the β-diketoacid class of compounds has proved to be of most promise and indeed S-1360 was the first IN inhibitor to enter clinical studies. With an aim to predict the bioactive (active site bound) conformation of S-1360, we performed extensive docking studies using three different reported crystal structures where the active site or partial active site region was resolved. For comparison we extended our studies to include 5CITEP (the first compound cocrystallized with IN core domain) and a bis-diketoacid (BDKA). We found that the conformation of S-1360 when bound in one of the active sites matches that of the experimentally observed results of IN escape mutants resistant to S-1360. Therefore, we propose that this active site conformation is the biologically relevant conformation and can be used for the future structure-based drug design studies selectively targeting IN.

Multiple mutations in human immunodeficiency virus-1 integrase confer resistance to the clinical trial drug S-1360

Study of HIV-1 resistance development to the diketo analogue S-1360, the first HIV-1 integrase strand transfer inhibitor that has entered clinical development. HIV-1(IIIB) was passaged in cell culture in the presence of increasing concentrations of S-1360 (IIIB/S-1360(res)). The IIIB/S-1360(res) strains selected for 30, 50 and 70 passages in the presence of S-1360 were evaluated genotypically by sequencing analysis and phenotypically using the MT-4/MTT assay. Multiple mutations, nine in total, emerged progressively in the catalytic domain of integrase as a result of the selection process. They included T66I and L74M that have both been associated with resistance against the diketo acid L-708,906. After 30, 50 and 70 passages in the presence of S-1360, IIIB/S-1360(res) displayed a four-, eight- and more than 62-fold reduced susceptibility for S-1360, respectively. Phenotypic cross-resistance to L-708,906 was modest for the IIIB/S-1360(res) strain selected during 50 passages, but pronounced for the strain selected during 70 passages. Interesting, all IIIB/S-1360(res) strains remained fully susceptible to the pyranodipyrimidine V-165, an integrase DNA binding inhibitor. Recombination of the mutant integrase genes into wild-type background by integrase-chimeric virus technology entirely reproduced the resistance profile of the IIIB/S-1360(res) strains. As for the replication kinetics of the selected and recombined strains, reduced replication fitness was measured for all strains when compared with their respective wild-type strains. The accumulation of integrase mutations coincided with an increasing level of (cross-)resistance of IIIB/S-1360(res). Integrase-chimeric virus technology confirmed that the integrase mutations are indeed fully responsible for the resistance phenotype of IIIB/S-1360.

Characterization of human immunodeficiency virus type 1 Pr160 gag-pol mutants with truncations downstream of the protease domain

We have constructed a series of HIV-1 Gag-pol mutants by progressive deletion of the pol sequence downstream of the viral protease (PR) domain. Effects of the truncation mutations on virus particle production and Gag particle processing were analyzed. Analysis indicated that removal of the integrase (IN) domain had no major effect on the efficiency of particle processing, but resulted in a marked reduction in virus particle budding. Deletion of both the IN and RNase H domains, however, restored the production of virus particles to wild-type level. The proteolytic processing of virus particle was significantly impaired when the p51RT domain was truncated. All of the truncated Gag-pol proteins could be incorporated into virus particles and demonstrated an immunofluorescence staining pattern similar to that of the wild type (wt). Our data are consistent with the proposal that signals for directing the Gag-pol transport and particle incorporation are determined by its N-terminal Gag domain. Truncated Gag-pol retaining an intact p51RT was able to complement a PR-defective mutant to produce infectious pseudotyped virions, with a virus titer 20–70% of that of wt. Pseudotyped virions produced by the Gag-pol lacking an intact p51RT were noninfectious or poorly infectious. This suggests that an intact p51RT domain is required for the Gag-pol to mediate production of mature infectious virus particles in trans.

Requirement for integrase during reverse transcription of human immunodeficiency virus type 1 and the effect of cysteine mutations of integrase on its interactions with reverse transcriptase.

Retroviral integrase catalyzes the essential step of integrating a double-stranded DNA copy of the viral genome into a host cell chromosome. Mutational studies have revealed that integrase is involved in additional steps of viral replication, but the mechanism for the pleiotropic effect is not well characterized. Since Cys residues generally play crucial roles in protein structure and function, we introduced Cys-to-Ser substitutions at positions 56, 65, and 130 of human immunodeficiency virus type 1 (HIV-1) integrase to determine their effects on integration activity and viral replication. None of the substitutions significantly affected the enzymatic activities in vitro. When introduced into the NL4-3 molecular clone of HIV-1, mutant viruses encoding Cys mutations at positions 56 and 65 of integrase replicated similarly to the wild-type virus in CD4(+)-T-cell lines, whereas the C130S-containing virus was noninfectious. The entry and postintegration steps of the viral life cycle for all mutant viruses were normal, and all had particle-associated reverse transcriptase (RT) activity. However, early reverse-transcribed DNA products were absent in the lysate of cells infected with the C130S mutant virus, indicating that the mutation abolished the ability of the virus to initiate endogenous reverse transcription. Coimmunoprecipitation using purified integrase and RT showed that the C-terminal domain of wild-type HIV-1 integrase interacted with RT. The interaction between integrase and RT was not affected in the presence of a reducing or alkylating agent, suggesting that the interaction did not involve a disulfide linkage. The C130S substitution within the core region may disrupt the protein recognition interface of the C-terminal domain and abolish its ability to interact with RT. Our results indicate that integrase plays an important role during the reverse-transcription step of the viral life cycle, possibly through physical interactions with RT.

Structural and evolutionary analysis of the transcribed sequence of Boudicca, a Schistosoma mansoni retrotransposon.

Boudicca is a gypsy-like, long terminal repeat (LTR) retrotransposon that has colonized the genome of the human blood fluke, Schistosoma mansoni. Previous studies have indicated that more than 1000 copies of Boudicca reside within the S. mansoni genome, although many of them may be degenerate and inactive. Messenger RNAs transcribed from genomic copies of Boudicca were investigated by reverse transcription PCR. Overlapping RT-PCR products corresponding to the gag and pol polyproteins of Boudicca, along with relevant sequences of genomic fragments of Boudicca, were assembled into contigs. Consensus sequences from these contigs were used to predict the sequence and structure of transpositionally active copies of the Boudicca retrotransposon. They verified that Boudicca has a kabuki-like Cys-His box motif at the active site of its gag protein, a classic DTG motif as the active site of the protease domain of the pol ORF2, and indicated a contiguous integrase domain at the C-terminus of pol with strong identity to integrase from the LTR retrotransposons CsRn1 and kabuki, as well as to the conserved integrase core domain, GenBank rve (). Models of the secondary structure of the Boudicca transcript suggested that the first AUG was occluded by a stem loop structure, which in turn suggested a method of regulation of expression, at the level of translation, of Boudicca proteins. In addition, phylogenetic analysis targeting discrete domains of Boudicca revealed a generalized radiation in sequences among the multiple copies of Boudicca resident in the schistosome genome.