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At least 109 records · Page 6Linked to original sources
PubMed2006

Discovery of small molecule inhibitors of West Nile virus using a high-throughput sub-genomic replicon screen.

West Nile virus (WNV) is a positive-sense, single-stranded RNA virus of the family Flaviviridae. WNV persistently infects insect cells, but can causes acute cytopathic infection of mammalian cells and is an etiologic agent of viral encephalitis in humans. By using a cell line expressing a WNV subgenomic replicon [Rossi, S.L., Zhao, Q., O'Donnell, V.K., Mason, P.W., 2005. Adaptation of West Nile virus replicons to cells in culture and use of replicon-bearing cells to probe antiviral action. Virology 331 (2), 457-470], we developed a high-throughput assay and used it to screen a library of small molecule compounds for inhibitors of WNV replication in the absence of live virus. Here we report the identification of novel small molecule inhibitors for WNV replicon replication. We demonstrate that the compounds inhibited WNV replication-dependent luciferase expression in the replicon cells and reduced WNV viral protein accumulation and viral RNA copy number in the replicon cells. Two classes of compounds with multiple hits, parazolotrahydrothophenes and pyrozolopyrimidines, showed preliminary structure-activity relationships. In WNV infection assays, one pyrozolopyrimidine compound was confirmed to have antiviral activity. These compounds should be valuable for developing anti-WNV therapeutic drugs as well as research tools to study the mechanism of WNV replication.

Animals↗
PubMed2005

Packaging the replicon RNA of the Far-Eastern subtype of tick-borne encephalitis virus into single-round infectious particles: development of a heterologous gene delivery system.

The sub-genomic replicon of tick-borne encephalitis (TBE) virus (Far-Eastern subtype) was packaged into infectious particles by providing the viral structural proteins in trans. Sequential transfection of TBE replicon RNA and a plasmid that expressed the structural proteins led to the secretion of infectious particles that contained TBE replicon RNA. The secreted particles had single-round infectivity, which was inhibited by TBE virus-neutralizing antibody. The physical structure of the particles was almost identical to that of infectious virions, and the packaged replicon RNA showed no recombination with the mRNAs of the viral structural proteins. Furthermore, heterologous genes were successfully delivered and expressed by packaging TBE replicon RNA with inserted GFP and Neo genes. This replicon packaging system may be a useful tool for the molecular study of the TBE virus genome packaging mechanism, and for the development of vaccine delivery systems.

Animals↗
PubMed2005

Replication of a novel subgenomic HCV genotype 1a replicon expressing a puromycin resistance gene in Huh-7 cells.

Genotype 1a is a most prevalent genotype of hepatitis C virus in North America yet HCV replication has been studied predominantly with genotype 1b subgenomic replicons under neomycin selection in Huh-7 cells. Development of 1a-related dicistronic replicons under neo selection proved difficult and required either "conditioned" Huh-7 cells and/or chimeric genomes harboring pre-engineered adaptive mutations. We report the construction of a novel dicistronic genotype 1a(H77C) replicon expressing the puromycin N-acetyltransferase (PAC) gene as a selectable marker that, without prior introduction of adaptive mutations, allows establishment of puromycin-resistant Huh-7 colonies after transfection of naive Huh-7 cells. The large majority of HCV1a/PAC replicons did not reveal any adaptive mutations on short-term passage of Huh-7 cells. Continued passage led to mutations in the non-structural genes although these mutations did not significantly enhance replication of the original replicon. Transfection with total cellular RNA isolated from HCV1a/PAC replicon-containing cells led to a significant increase in colony-forming ability. The data identify PAC as an efficient selectable marker for studies of HCV replication, which may be useful with different genotypes in different host cell systems.

Antiviral Agents↗
PubMed2006

Derivation of a novel SARS-coronavirus replicon cell line and its application for anti-SARS drug screening.

The severe acute respiratory syndrome (SARS) outbreak in 2002, which had a high morbidity rate and caused worldwide alarm, remains untreated today even though SARS was eventually isolated and controlled. Development and high-throughput screening of efficacious drugs is therefore critical. However, currently there remains a lack of such a safe system. Here, the generation and characterization of the first selectable, SARS-coronavirus (SARS-CoV)-based replicon cell line which can be used for screening is described. Partial SARS-CoV cDNAs and antibiotic resistance/reporter gene DNA were generated and assembled in vitro to produce the replicon transcription template, which was then transcribed in vitro to generate the replicon RNA. The latter was introduced into a mammalian cell line and the transfected cells were selected for by antibiotic application. For the antibiotic-resistant cell lines thus generated, the expression of reporter gene was ensured by continued monitoring using fluorescent microscopy and flow cytometry. The suitability of this replicon cell line in drug screening was demonstrated by testing the inhibitory effect of several existing drugs and the results demonstrate that the SARS-CoV replicon cell lines provide a safe tool for the identification of SARS-CoV replicase inhibitors. The replicon cell lines thus developed can be applied to high-throughput screening for anti-SARS drugs without the need to grow infectious SARS-CoV.

Animals↗
PubMed2003

Immunogenicity in pig-tailed macaques of poliovirus replicons expressing HIV-1 and SIV antigens and protection against SHIV-89.6P disease.

In the search for an effective vaccine against the human immunodeficiency virus (HIV), novel ways to deliver viral antigens are being evaluated. One such approach is the use of nonreplicating viral vectors encoding HIV and/or SIV genes that are expressed after infection of host cells. Nonreplicating poliovirus vectors, termed replicons, that expressed HIV-1/HXB2 and SIVmac239 gag and various HIV-1 env genes from different clades were tested for immunogenicity and protective efficacy against intravenous challenge of pig-tailed macaques with SHIV-89.6P. To maximize both cellular and humoral immune responses, a prime-boost regimen was used. Initially, macaques were immunized four times over 35 weeks by either the intranasal and intrarectal or the intramuscular (im) route with mixtures of poliovirus replicons expressing HIV-1 gag and multiple env genes. Immunization with replicons alone induced both serum antibodies and lymphocyte proliferative responses. After boosting with purified Env protein, neutralizing antibodies to SHIV-89.6P were induced in four of five immunized animals. In a second experiment, four macaques were immunized im three times over 27 weeks with replicons expressing the SIVmac239 gag and HIV-1/HXB2 env genes. All immunized animals were then boosted twice with purified HIV-1-89.6 rgp140-Env and SIVmac239 p55-Gag proteins. Four control animals received only the two protein inoculations. Immunized and control animals were then challenged intravenously with the pathogenic SHIV-89.6P. After challenge the animals were monitored for virus isolation from peripheral blood mononuclear cells and plasma viremia and for changes in virus-specific antibody titers. Naïve pig-tailed macaques experienced rapid loss of CD4(+) T cells and died between 38 and 62 weeks after infection. In contrast, macaques immunized with replicons and proteins rapidly cleared plasma virus and did not experience sustained loss of CD4(+) lymphocytes. Furthermore, two of the four macaques that were immunized only with purified proteins maintained high viral burdens and lost greater than 95% of their CD4(+) lymphocytes within 2 to 4 weeks after challenge. Thus, poliovirus replicons expressing HIV-1 and SIV antigens were immunogenic in pig-tailed macaques and appeared to enhance the protective effects observed after administration of purified proteins alone.

Animals↗
PubMed2002

Comparative characterization of repABC-type replicons of Paracoccus pantotrophus composite plasmids.

The repABC replicons have an unusual structure, since they carry genes coding for partitioning (repA, repB) and replication (repC) proteins, which are organized in an operon. So far, the presence of these compact bi-functional modules has been reported only in the megaplasmids of the Rhizobiaceae and within the plasmid pTAV1 (107kb) of Paracoccus versutus. We studied the distribution of repABC-type replicons within bacteria belonging to the genus Paracoccus. We found that repABC replicons occur only in the group of pTAV1-like plasmids: pKLW1, pHG16-a, pWKS2, and pPAN1, harbored by different strains of Paracoccus pantotrophus. A partial sequencing approach followed by phylogenetic analysis revealed that these replicons constitute a distinct evolutionary branch of repABC replicons. Incompatibility studies showed that they represent two incompatibility groups designated IncABC1 (pTAV1, pKLW1, and pHG16-a) and IncABC2 (pPAN1). Sequence comparison using available databases allowed the identification, within plasmid pRS241d of Rhodobacter sphaeroides 2.4.1, of an additional sequence highly homologous to the paracoccal repABC replicons, which has been included in comparative analyses.

Base Sequence↗
PubMed1997

Genetic map of the virulence plasmid of Salmonella enteritidis and nucleotide sequence of its replicons.

Partial sequencing of a genomic library of the virulence plasmid of Salmonella enteritidis has been used to localize in the restriction map of this plasmid the genetic loci already described in other Salmonella plasmids. The comparison of the vestigial tra region with the corresponding genes in the F plasmid allowed us to define the extent of the deletions that the S. enteritidis plasmid should have suffered. The putative replicons of the plasmid, repB and repC, were isolated and both proved to be functional in Escherichia coli, but repC was segregationally unstable. The nucleotide sequence of repB showed the typical organization of RepFIIA replicons, although the similarity was lower than usual in this group of replicons. The highest homology was found with the replicon of the virulence plasmid pYVe439-80 from Yersinia enterocolitica (72.5%). Replicon repC also showed a maximum identity of 72.6% with known replicons, namely the RepFIB of pColV-K30 and P307, both virulence plasmids isolated from E. coli. We conclude that the S. enteritidis plasmid could arise from the S. typhimurium plasmid through deletions, and that they are evolutionary distant from other IncFI and IncFII plasmids.

Amino Acid Sequence↗
PubMed1982

In the higher plant Pisum sativum maturation of nascent DNA is blocked by cycloheximide, but only after 4-8 replicons are joined.

Velocity sedimentation in alkaline sucrose gradients, single cell autoradiography and cytophotometry were used to determine if protein synthesis is required for the maturation of nascent replicons to chromosomal-sized molecules in cultured pea-root cells. The results obtained showed that cycloheximide at 5 and 10 microgram/ml, added either before or during labeling with tritiated thymidine, blocked maturation of nascent DNA at an intermediate size of 72-140 X 10(6) daltons single-stranded DNA. To reach this size, nascent replicons - which are 18 X 10(6) daltons single-stranded DNA each - were replicated and groups of 4-8 replicons were joined even though protein synthesis was reduced to 15% of the control. Further maturation of the nascent molecules to chromosomal size, however, was prevented and this resulted in the accumulation of nascent molecules in the 72-140 X 10(6) daltons range. The experiments also showed that the joining of nascent replicons is not an absolute function of late S or G2 phase of the cell cycle, since cells treated with cycloheximide and blocked in mid-S phase had nascent DNA of a size corresponding to 4-8 joined replicons. Finally, the results support the hypothesis that at least one step in the process of nascent DNA maturation may require replication, during late-S phase, of DNA segments that are interspersed within replicon-clusters that replicate early in the S phase.

Cell Nucleus↗
PubMed2002

Replication of a hepatitis A virus replicon detected by genetic recombination in vivo.

Unlike other picornaviruses, hepatitis A virus (HAV) replicates so inefficiently in cell culture that the study of its RNA biosynthesis presents a major experimental challenge. To assess viral RNA replication independent of particle formation, a subgenomic replicon representing a self-replicating RNA was constructed by replacing the P1 domain encoding the capsid proteins with the firefly luciferase sequence. Although translation of the HAV replicon was as efficient as a similar poliovirus replicon, the luciferase activity derived from replication of the HAV construct was more than 100-fold lower than that of poliovirus. The replication capacity of the HAV replicon was clearly demonstrated by its ability to recombine genetically with a non-viable, full-length HAV genome that served as capsid donor and thus to rescue a fully infectious virus. In contrast to a replication-deficient replicon, co-expression of the genetically marked and replication-competent HAV replicon with several lethally mutated HAV genomes resulted in the successful rescue of infectious HAV with a unique genetic marker. Our data suggest: (i) that autonomous HAV RNA replication does not require sequences for the HAV structural proteins; and (ii) that low-level genome replication can unequivocally be demonstrated by the rescue of infectious virus after co-expression with non-viable genomes.

Animals↗
PubMed2006

Multiple gene genealogical analyses reveal both common and distinct population genetic patterns among replicons in the nitrogen-fixing bacterium Sinorhizobium meliloti.

Sinorhizobium meliloti is a Gram-negative alpha-proteobacterium that can form symbiotic relationships with alfalfa and fix atmospheric nitrogen. The complete genome of a laboratory strain, Rm1021, was published in 2001, and the genome of this strain is arranged in three replicons: a chromosome of 3.65 million base pairs (Mb), and two megaplasmids, pSymA (1.35 Mb) and pSymB (1.68 Mb). However, the potential difference in genetic variation among the three replicons in natural strains remains poorly understood. In this study, a total of 16 gene fragments were sequenced, four from pSymA and six each from the chromosome and pSymB, for 49 natural S. meliloti strains. The analyses identified significant differences in divergence among genes, with the mean Hasegawa-Kishino-Yano-1985 (HKY85) distance ranging from 0.00157 to 0.04109 between pairs of strains. Overall, genes on pSymA showed the highest mean HKY85 distance, followed by those on pSymB and the chromosome. Although evidence for recombination was found, the authors' population genetic analyses revealed overall significant linkage disequilibria among genes within both pSymA and the chromosome. However, genes on pSymB were in overall linkage equilibrium, consistent with frequent recombination among genes on this replicon. Furthermore, the genealogical comparisons among the three replicons identified significant incongruence, indicating reassortment among the three replicons in natural populations. The results suggest both shared and distinct patterns of molecular evolution among the three replicons in the genomes of natural strains of S. meliloti.

Genes, Bacterial↗
PubMed2005

Development and characterization of a transient-replication assay for the genotype 2a hepatitis C virus subgenomic replicon.

Dicistronic, subgenomic hepatitis C virus (HCV) replicons were constructed containing sequences from JFH1, a genotype 2a strain, that also incorporated the firefly luciferase gene under the control of the HCV internal ribosome entry site element. Luciferase activity in Huh-7 cell extracts containing in vitro-transcribed subgenomic JFH1 RNA was monitored over a 72 h period to examine early stages of HCV replication in the absence of any selective pressure. Enzyme activities produced by the replicon were almost 200-fold greater than those generated from corresponding genotype 1b replicons and correlated with an accumulation of NS5A protein and replicon RNA. Transient replication was sensitive to IFN treatment in a dose-dependent manner and, in addition to Huh-7 cells, the U2OS human osteosarcoma cell line supported efficient replication of the JFH1 replicon. Thus, this system based on JFH1 sequences offers improvements over prior genotype 1b replicons for quantitative measurement of viral RNA replication.

Cell Line↗
PubMed2006

Tagging of NS5A expressed from a functional hepatitis C virus replicon.

Knowledge of how hepatitis C virus (HCV) proteins associate with components of the host cell to form a functional replication complex is still limited. To address this issue, HCV replicon constructs were generated where either green fluorescent protein (GFP) or the Propionibacterium shermanii transcarboxylase domain (PSTCD) was introduced into the NS5A coding region. Insertion of both GFP and PSTCD was tolerated well, allowing formation of stable replicon-containing cell lines that contained viral protein and transcript levels that were comparable to those of an unmodified parental replicon. Cell lines generated from the GFP-tagged NS5A replicon allowed live-cell visualization of the location of NS5A. Cell lines generated from the PSTCD-tagged replicons allowed rapid and efficient precipitation of the PSTCD-tagged NS5A, as well as other HCV non-structural proteins, using streptavidin-coated magnetic beads. Both replicons represent useful tools that offer different but complementary ways of examining replication-complex formation in cells.

Carboxyl and Carbamoyl Transferases↗
PubMed2004

Introduction of NS5A mutations enables subgenomic HCV replicon derived from chimpanzee-infectious HC-J4 isolate to replicate efficiently in Huh-7 cells.

Hepatitis C virus (HCV) subgenomic replicon has been reported to replicate efficiently and continuously in human hepatoma Huh-7 cells. To extend the previous results to other isolated HCV clones, we constructed another HCV replicon from HC-J4, one of chimpanzee-infectious HCV clones. An HCV replicon derived from HC-J4 (RpJ4) consists of HCV-5' untranslated region, neomycin phosphotransferase gene, the encephalomyocarditis virus internal ribosomal entry site, HCV nonstructural region, NS3 to NS5B, and HCV-3' untranslated region. The adaptive mutations known to be required for HCV-Con1 replicon were introduced in RpJ4 replicon, aa.(amino acids number according to HC-J4) 2197 serine to proline, deletion of serine at aa.2201, and aa.2204 serine to isoleucine (RpJ4-S2197P, RpJ4-S22001del, and RpJ4-S2204I). RpJ4/ISDR mutant and RpJ4-S2201del/ISDR mutant were also constructed by introducing six amino acid mutations into the interferon sensitivity determining region (ISDR). After transfection into Huh-7 cells and G418 selection, RpJ4 and RpJ4/ISDR mutants did not produce any colony. In contrast, G418-resistant cells were transduced efficiently by RpJ4-S2197P, RpJ4-S2204I, RpJ4-S2201del and RpJ4-S2201del/ISDR mutant, with the RpJ4-S2201del/ISDR mutant being most efficient. Hence the HCV replicon derived from HC-J4 can replicate efficiently following the introduction of adaptive mutations into the upstream region of ISDR. Moreover, additional introduction of mutations into ISDR further enhanced its replication. These findings demonstrate that the genetic structure of the NS5A domain is critical in HCV replications.

Amino Acid Sequence↗
PubMed2006

VX-950, a novel hepatitis C virus (HCV) NS3-4A protease inhibitor, exhibits potent antiviral activities in HCv replicon cells.

The NS3-4A serine protease of hepatitis C virus (HCV) is essential for viral replication and therefore has been one of the most attractive targets for developing specific antiviral agents against HCV. VX-950, a highly selective, reversible, and potent peptidomimetic inhibitor of the HCV NS3-4A protease, is currently in clinical development for the treatment of hepatitis C. In this report, we describe the in vitro characterization of anti-HCV activities of VX-950 in subgenomic HCV replicon cells. Incubation with VX-950 resulted in a time- and dose-dependent reduction of HCV RNA and proteins in replicon cells. Moreover, following a 2-week incubation with VX-950, a reduction in HCV RNA levels of 4.7 log(10) was observed, and this reduction resulted in elimination of HCV RNA from replicon cells, since there was no rebound in replicon RNA after withdrawal of the inhibitor. The combination of VX-950 and alpha interferon was additive to moderately synergistic in reducing HCV RNA in replicon cells with no significant increase in cytotoxicity. The benefit of the combination was sustained over time: a 4-log(10) reduction in HCV RNA level was achieved following a 9-day incubation with VX-950 and alpha interferon at lower concentrations than when either VX-950 or alpha interferon was used alone. The combination of VX-950 and alpha interferon also suppressed the emergence of in vitro resistance mutations against VX-950 in replicon cells.

Animals↗
PubMed1998

Genes for 2,4,5-trichlorophenoxyacetic acid metabolism in Burkholderia cepacia AC1100: characterization of the tftC and tftD genes and locations of the tft operons on multiple replicons.

Burkholderia cepacia AC1100 uses the chlorinated aromatic compound 2, 4,5-trichlorophenoxyacetic acid (2,4,5-T) as a sole source of carbon and energy. The enzyme which converts the first intermediate in the pathway, 2,4,5-trichlorophenol, to 5-chlorohydroquinone has been purified and consists of two subunits of 58 and 22 kDa, encoded by the tftC and tftD genes (48). A degenerate primer was designed from the N terminus of the 58-kDa polypeptide and used to isolate a clone containing the tftC and tftD genes from a genomic library of AC1100. The derived amino acid sequences of tftC and tftD show significant homology to the two-component monooxygenases HadA of Burkholderia pickettii, HpaBC of Escherichia coli, and HpaAH of Klebsiella pneumonia. Expression of the tftC and tftD genes appeared to be induced when they were grown in the presence of 2,4,5-T, as shown by RNA slot blot and primer extension analyses. Three sets of cloned tft genes were used as probes to explore the genomic organization of the pathway. Pulsed-field gel electrophoresis analyses of whole chromosomes of B. cepacia AC1100 demonstrated that the genome is comprised of five replicons of 4.0, 2.7, 0.53, 0.34, and 0.15 Mbp, designated I to V, respectively. The tft genes are located on the smaller replicons: the tftAB cluster is on replicon IV, tftEFGH is on replicon III, and copies of the tftC and the tftCD operons are found on both replicons III and IV. When cells were grown in the absence of 2,4,5-T, the genes were lost at high frequency by chromosomal deletions and rearrangements to produce 2,4,5-T-negative mutants. In one mutant, the tftA and tftB genes translocated from one replicon to another, with the concomitant loss of tftEFGH and one copy of tftCD.

2,4,5-Trichlorophenoxyacetic Acid↗
PubMed2001

Complete DNA sequence of Yersinia enterocolitica serotype 0:8 low-calcium-response plasmid reveals a new virulence plasmid-associated replicon.

The complete nucleotide sequence and organization of the Yersinia enterocolitica serotype 0:8 low-calcium-response (LCR) plasmid, pYVe8081, were determined. The 67,720-bp plasmid encoded all the genes known to be part of the LCR stimulon except for ylpA. Eight of 13 intact open reading frames of unknown function identified in pYVe8081 had homologues in Yersinia pestis plasmid pCD1 or in Y. enterocolitica serotype 0:9 plasmid pYVe227. A region of approximately 17 kbp showed no DNA identity to pCD1 or pYVe227 and contained six potential new genes, a possible new replicon, and two intact insertion sequence (IS) elements. One intact IS element, ISYen1, was a new IS belonging to the IS256 family. Several vestigial IS elements appeared different from the IS distribution seen in the other LCR plasmids. The RepA proteins encoded by Y. enterocolitica serotype 0:8 pYVeWA and pYVe8081 were identical. The putative pYVe8081 replicon showed significant homology to the IncL/M replicon of pMU407.1 but was only distantly related to the replicons of pCD1 and pYVe227. In contrast, the putative partitioning genes of pYVe8081 showed 97% DNA identity to the spy/sopABC loci of pCD1 and pYVe227. Sequence analysis suggests that Yersinia LCR plasmids are from a common ancestor but that Y. enterocolitica serotype 0:8 plasmid replicons may have evolved independently via cointegrate formation following a transposition event. The change in replicon structure is predicted to change the incompatibility properties of Y. enterocolitica serotype 0:8 plasmids from those of Y. enterocolitica serotype 0:9 and Y. pestis LCR plasmids.

Amino Acid Sequence↗
PubMed1993

Isolation and location on the R27 map of two replicons and an incompatibility determinant specific for IncHI1 plasmids.

Two replicons were isolated independently from different IncHI1 plasmids. One was isolated from R27, and a second was isolated from pIP522. We demonstrate, by DNA-DNA hybridization experiments, that these maintenance regions are different and that they are specific to, and carried by, all IncHI1 plasmids tested. In view of this specificity we decided to designate the replicon isolated from R27 as RepHI1A and the replicon isolated from pIP522 as RepHI1B. These two autoreplicative regions are not related to a third replicon present in all IncHI1 plasmids that bears homology with RepFIA and that expresses the characteristic incompatibility of IncHI1 subgroup plasmids toward F factor (D. Saul, D. Lane, and P. L. Bergquist, Mol. Microbiol. 2:219-225, 1988; D. E. Taylor, R. W. Hedges, and P. L. Bergquist, J. Gen. Microbiol. 131:1523-1530, 1985). These results demonstrate that all IncHI1 plasmids tested contain at least three replicons. An incompatibility (Inc) region that hybridizes specifically to all the IncHI1 plasmids was previously isolated (M. Couturier, F. Bex, P. L. Bergquist, and W. K. Maas, Microbiol. Rev. 52:375-395, 1988). Although this Inc locus is not located in an autoreplicative region of IncHI1 plasmids, we observed that this locus stabilizes a low-copy-number replicon. This Inc locus is probably a component of an active partition locus involved in the maintenance of IncHI1 plasmids. The nucleotide sequence of the Inc region contains direct repeats of 31 bp. In addition, this incompatibility determinant hybridizes specifically with IncHI1 plasmids but expresses incompatibility toward plasmids of both IncHI subgroups (IncHI1 and IncHI2). In this communication, we present the mapping of these maintenance elements on the R27 genome.

Base Sequence↗
PubMed2001

Identification of the partitioning site within the repABC-type replicon of the composite Paracoccus versutus plasmid pTAV1.

The replicator region of composite plasmid pTAV1 of Paracoccus versutus (included in mini-replicon pTAV320) belongs to the family of repABC replicons commonly found in plasmids harbored by Agrobacterium and Rhizobium spp. The repABC replicons encode three genes clustered in an operon, which are involved in partitioning (repA and repB) and replication (repC). In order to localize the partitioning site of pTAV320, the two identified incompatibility determinants of this mini-replicon (inc1, located in the intergenic sequence between repB and repC; and inc2, situated downstream of the repC gene) were PCR amplified and used together with purified RepB fusion protein (homologous to the type B partitioning proteins binding to the partitioning sites) in an electrophoretic mobility shift assay. The protein bound only inc2, forming two complexes in a protein concentration-dependent manner. The inc2 region contains two long (14-bp) repeated sequences (R1 and R2). Disruption of these sequences completely eliminates RepB binding ability. R1 and R2 have sequence similarities with analogous repeats of another repABC replicon of plasmid pPAN1 of Paracoccus pantotrophus DSM 82.5 and with centromeric sequences of the Bacillus subtilis chromosome. Excess RepB protein resulted in destabilization of the inc2-containing plasmid in Escherichia coli. On the other hand, the inc2 region could stabilize another unstable replicon in P. versutus when RepA and RepB were delivered in trans, proving that this region has centromere-like activity. Thus, it was demonstrated that repA, repB, and inc2 constitute a functional system for active partitioning of pTAV320.

Bacterial Proteins↗