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At least 379 records · Page 21Linked to original sources
PubMed2006

Epigenetic silencing of interferon-inducible genes is implicated in interferon resistance of hepatitis C virus replicon-harboring cells.

BACKGROUND/AIMS: We previously established hepatitis C virus (HCV) replicon-harboring cell lines possessing two interferon (IFN)-resistant phenotypes: a partially resistant phenotype (alphaR series) and a severely resistant phenotype (betaR series). We recently found that the severe IFN resistance of the betaR-series cells is caused by the functional disruption of type I IFN receptors. Here, we aimed to clarify the mechanism(s) underlying the partial IFN resistance of the alphaR-series cells. METHODS: alphaR-series cells were pre-treated with 5-azacytidine to evaluate the effects of DNA demethylation on IFN resistance. cDNA microarray analysis was carried out in order to compare 1alphaR cells, which belong to the alphaR series, treated with both 5-azacytidine and IFN-alpha with cells treated with 5-azacytidine or IFN-alpha alone. RESULTS: We found that the IFN-resistant phenotype of alphaR-series cells was impaired by treatment with 5-azacytidine. cDNA microarray analysis identified seven IFN-stimulated genes, which were up-regulated by 5-azacytidine treatment. We demonstrated here that the ectopic expression of each of these seven genes in 1alphaR cells frequently weakened the IFN resistance of these cells. CONCLUSIONS: The present results suggest that the epigenetic silencing of IFN-stimulated genes is implicated in the acquisition of a partially IFN-resistant phenotype of HCV replicon-harboring cells.

Antiviral Agents↗
PubMed2005

Identification of plasmids by PCR-based replicon typing.

The epidemiological importance of tracing plasmids conferring drug resistance prompted us to develop a PCR method based on replicons (inc/rep PCR) of the major plasmid incompatibility groups among Enterobacteriaceae. Eighteen pairs of primers were designed to perform 5 multiplex- and 3 simplex-PCRs, recognizing FIA, FIB, FIC, HI1, HI2, I1-Igamma, L/M, N, P, W, T, A/C, K, B/O, X, Y, F, and FIIA. The specificity of the method was tested on a collection of 61 reference plasmids and on 20 Salmonella enterica strains of different serotypes isolated in Italy. Results indicated that the inc/rep PCR method demonstrates high specificity and sensitivity in detecting replicons on reference plasmids and also revealed the presence of recurrent and common plasmids in epidemiologically unrelated Salmonella isolates of different serotypes. These results suggest that the method is potentially applicable to a large number of strains to trace the diffusion of specific multi-drug resistance plasmids in different environments.

Bacterial Typing Techniques↗
PubMed2004

Incompatibility and the partitioning site of the repABC basic replicon of the symbiotic plasmid from Rhizobium etli.

The basic replicon of the symbiotic plasmid (p42d) of Rhizobium etli CE3 is constituted by the repABC operon. Whereas RepC is essential for plasmid replication, RepA and RepB are involved in plasmid partitioning. Three incompatibility regions have been previously identified in this plasmid: the first one encodes RepA, a partitioning protein that also down-regulates the repABC transcription. The second region is situated within the repB-repC intergenic sequence (inc(alpha)), and the last one, inc(beta), is located in a 502 bp EcoRI fragment spanning the last 72-bp of the coding region of repC and the following downstream sequence. In this paper we show that: (1) The inc(beta) region is required for plasmid partitioning. (2) A 16-bp palindrome sequence, located 40 bp downstream of the repC gene of plasmid p42d, is necessary and sufficient to induce incompatibility towards the parental plasmid, and accounts for all the incompatibility properties of this region (inc(beta)). (3). The palindrome is the DNA target site for RepB binding. With these findings we propose that inc(beta) contains the partitioning site (par site) of the basic replicon of plasmid p42d, and that the 16-bp palindrome is the core sequence to nucleate the RepB binding.

Bacterial Proteins↗
PubMed2005

Genetic relationship among Bacillus licheniformis rolling-circle-replicating plasmids and complete nucleotide sequence of pBL63.1, an atypical replicon.

The degree of biodiversity among Bacillus licheniformis plasmids and their relation to other Bacillus subtilis group plasmids has been evaluated. To attain this goal we surveyed the diversity and linkage of replication modules in a collection of 21 naturally occurring plasmids of B. licheniformis strains, isolated from different geographical areas. On the basis of rep gene sequence analysis it was possible to group the B. licheniformis plasmids rep genes in two main cluster. Comparison with known rep genes from Bacillus rolling-circle-replicating (RCR) plasmids revealed the presence in B. licheniformis plasmids of replication genes with a DNA sequence peculiar to B. licheniformis species together with rep genes with a very high sequence similarity to B. subtilis plasmids. Furthermore, the molecular organization of an atypical replicon, pBL63.1, was shown. This plasmid did not display any significant similarity with known Bacillus RCR plasmids. The complete nucleotide sequence evidenced a replication module with an unexpected similarity with Rep proteins from RCR plasmids of bacterial species phylogenetically distantly related to Bacillus. pBL63.1 represents an exception to the low-level diversity hypothesis among Bacillus RC replicons.

Bacillus↗
PubMed2006

Evaluation of human immunodeficiency virus type 1 subtype C gag, pol, and gagpol DNA and alphavirus replicon vaccines.

The worldwide HIV-1 vaccine research endeavor is focused increasingly on subtype C, which is now the predominant strain of the present HIV/AIDS epidemic. Expression cassettes of HIV-1 subtype C gag, pol and versions of gagpol fusion cassettes were constructed and evaluated for their relative abilities to induce cellular immune responses in mice. Animals were vaccinated with DNA or alphavirus replicon particle-based vaccines and cellular immune responses were measured by flow cytometry. Five new major histocompatibility complex (MHC) class I-restricted T cell epitopes in subtype C Gag and Pol were identified. Although two CD8(+) T cell epitopes within Gag were immunodominant in BALB/c and CB6F1 mice, the overall breadth of the T cell responses in mice immunized with plasmids or recombinant alphavirus replicon particles encoding gagpol fusion genes was improved over single antigen genes (i.e. gag or pol alone). The patterns of epitope dominance were consistent among mice although there were variations observed between different animals in the relative contributions of the various epitopes to the total response. These data are consistent with observations in non-human primates (Otten GR, Schaefer M, Doe B, Liu H, Magede JZ, Donnelly J, et al. Potent immunogenicity of an HIV-1 gag-pol fusion DNA vaccine delivered by in vivo electroporation. Vaccine 2005, in press) and support a subtype C in-frame gagpol fusion gene vaccine.

AIDS Vaccines↗
PubMed2005

Suppression of hepatitis C virus replicon by TGF-beta.

Hepatitis C virus (HCV) is one of the major causative agents of liver diseases, such as liver inflammation, fibrosis, cirrhosis, and hepatocellular carcinoma. Using an efficient HCV subgenomic replicon system, we demonstrate that transforming growth factor-beta (TGF-beta) suppresses viral RNA replication and protein expression from the HCV replicon. We further show that the anti-viral effect of this cytokine is associated with cellular growth arrest in a manner dependent on Smad signaling, not mitogen-activated protein kinase (MAPK) signaling. These results suggest a novel insight into the mechanisms of liver diseases caused by HCV.

Cell Line, Tumor↗
PubMed2006

Alphavirus replicon approach to promoterless analysis of IRES elements.

Here we describe a system for promoterless analysis of putative internal ribosome entry site (IRES) elements using an alphavirus (family Togaviridae) replicon vector. The system uses the alphavirus subgenomic promoter to produce transcripts that, when modified to contain a spacer region upstream of an IRES element, allow analysis of cap-independent translation of genes of interest (GOI). If the IRES element is removed, translation of the subgenomic transcript can be reduced >95% compared to the same transcript containing a functional IRES element. Alphavirus replicons, used in this manner, offer an alternative to standard dicistronic DNA vectors or in vitro translation systems currently used to analyze putative IRES elements. In addition, protein expression levels varied depending on the spacer element located upstream of each IRES. The ability to modulate the level of expression from alphavirus vectors should extend the utility of these vectors in vaccine development.

Alphavirus↗
PubMed2006

Mutations of the Hepatitis C virus protein NS4B on either side of the ER membrane affect the efficiency of subgenomic replicons.

The non-structural protein NS4B of the Hepatitis C virus (HCV) is an integral membrane protein located in the endoplasmic reticulum (ER). Although the function of the NS4B in the viral life cycle is unknown a critical role in replication has been indicated. In order to investigate which components are involved we initially introduced restriction sites near the extremities of the NS4B in a subgenomic replicon that resulted in the alterations of six amino acid residues. This totally abolished replication. We subsequently introduced 14 single point mutations into different regions of NS4B based on the current topology model. One mutation abolished replication, while most conferred reduced replicon establishment and one mutation resulted in improved efficiency. Neither the protein processing nor the membrane altering capacity of NS4B was affected. Surprisingly, mutations situated in the ER lumen also conferred strong effects, despite the fact that replication occurs on the cytosolic side of the ER membrane. We conclude that the molecular integrity of NS4B is vital for HCV replication. Our results suggest that NS4B interacts with itself and with other viral and cellular factors, and may carry intrinsic capacities in order to allow replication.

Cell Line, Tumor↗
PubMed2000

Multiple replicons constitute the 6.5-megabase genome of Burkholderia pseudomallei.

Burkholderia pseudomallei is a causative agent of melioidosis, a fatal tropical infectious disease endemic in Southeast Asia and Northern Australia. In order to determine the size and characteristics of the bacterial genome, the B. pseudomallei genome and genes were analyzed by pulsed field gel electrophoresis of the undigested, intact megabase DNA, and by computational analysis of nucleotide sequences of B. pseudomallei genes which have been sequenced by several investigators and already deposited in a public database. The results showed that the B. pseudomallei genome consists of two large replicons, and that both contain ribosomal RNA gene sequences, indicating the presence of two chromosomes. The classical arabinose-negative B. pseudomallei isolate K96243 has chromosomes of approximately 3563 +/- 73 and 2974 +/- 40 kilobase-pairs in size, giving a total genome size of about 6.5 million base-pairs. The arabinose-positive nonvirulent biotype of B. pseudomallei also has two replicons which are smaller than those of the arabinose-negative biotype. Analysis of the publicly-available nucleotide sequences showed that the average B. pseudomallei gene is approximately 1031 base-pairs in size, with an average G + C content of 65.7%. The genome is gene-rich and about 89% of the coding capacity is used as coding sequences. It can therefore be estimated that the entire B. pseudomallei genome encodes about 5600 genes.

Burkholderia pseudomallei↗
PubMed2003

Morphine enhances hepatitis C virus (HCV) replicon expression.

Little information is available regarding whether substance abuse enhances hepatitis C virus (HCV) replication and promotes HCV disease progression. We investigated whether morphine alters HCV mRNA expression in HCV replicon-containing liver cells. Morphine significantly increased HCV mRNA expression, an effect which could be abolished by either of the opioid receptor antagonists, naltrexone or beta-funaltrexamine. Investigation of the mechanism responsible for this enhancement of HCV replicon expression demonstrated that morphine activated NF-kappaB promoter and that caffeic acid phenethyl ester, a specific inhibitor of the activation of NF-kappaB, blocked morphine-activated HCV RNA expression. In addition, morphine compromised the anti-HCV effect of interferon alpha (IFN-alpha). Our in vitro data indicate that morphine may play an important role as a positive regulator of HCV replication in human hepatic cells and may compromise IFN-alpha therapy.

Antiviral Agents↗
PubMed1986

Enhanced instability of IncFII basic replicon by the polA mutation.

IncFII plasmids consisting of a basic replicon and of an additional fragment(s) unrelated to plasmid maintenance were all less stable in polA1 than in its wild type. Reversion to UV-resistance recovered stability and vice versa. UV irradiation promoted instability in polA1 cells. Larger plasmids showed a greater instability and a fewer number of copies in a same host. Surprisingly, polA1 cells with Tn3 on the plasmid showed a higher ampicillin resistance than the wild type, apparently suggesting that the polA1 mutation increases the copy number. The size-dependency of instability was less marked in polA1 than in its parent. Comparison of the generation times has suggested a detrimental effect exerted by a basic replicon in polA1 hosts.

Ampicillin↗
PubMed2003

Trans-complementation of autonomously replicating Bovine viral diarrhea virus replicons with deletions in the E2 coding region.

Autonomously replicating Bovine viral diarrhea virus (BVDV) genomes (replicons) were constructed from the full-length BVDV cDNA clone pA/BVDV/Ins- (G. Meyers et al., J. Virol. 70, 8606-8613, 1996). The sequences coding for envelope protein E2, for E2 without the C-terminal transmembrane region, or for E2 and nonstructural protein p7 were deleted, and the resulting mutants were tested for their ability to replicate after transfection. All deletion mutants were able to replicate and to express the inserted green fluorescent protein but did not produce infectious progeny virus in bovine kidney PT cells. The replicons were also tested for their ability to be trans-complemented in the bovine cell line PT_805, which constitutively expresses BVDV structural proteins. E2-negative BVDV mutants were complemented and >10(6) infectious units were obtained at 24 h after transfection. Complementing PT_805 cells could only inefficiently be infected using trans-complemented virions, however, and low levels of virus production were observed when complemented BVDV was passaged using PT_805 cells. Similarly, infection of PT_805 cells with BVDV was highly inefficient, but transfection of full-length BVDV NCP7 RNA into PT_805 resulted in 10,000-fold higher virus titers when compared to those obtained 24 h after transfection of parental PT cells. We concluded that self-replicating E2-deleted BVDV RNAs can be efficiently trans-complemented by constitutively expressed E2, and that expression of BVDV structural proteins markedly influences susceptibility of cells to BVDV infection as well as BVDV titers after transfection of full-length BVDV RNA.

Animals↗
PubMed2003

Kunjin RNA replication and applications of Kunjin replicons.

The Kunjin virus (KUNV) has provided a useful laboratory model for Flavivirus RNA replication. The synthesis of progeny RNA(+) strands occurs via asymmetric and semiconservative replication on a template of recycling double-stranded RNA (dsRna) or replicative form (RF). Kinetics of viral RNA synthesis indicated a cycle period of about 15 min during which, on average, a single nascent RNA (+) strand displaces the pre-existing RNA(+) strand in the replicative intermediate. Data on the composition of the replication complex (RC) in KUNV-infected cells were obtained from several sources, including analyses of the partially-purified still active RC, immunogold labeling of cryosections using monospecific antibodies to the nonstructural proteins and to the dsRNA, radioimmunoprecipitations of cell lysates using antibodies to dsRNA and to an RC-associated cell marker, and pull-down assays of cell lysates using fusion proteins GST-NS2A and GST-NS4A. These results yeilded a consensus composition of NS1, NS2A, NS3, NS4A, and NS5 strongly associated with the dsRNA template. The RC was located in induced membranes described as vesicle packets. The RNA-dependent RNA polymerase activity late in infection did not require continuing protein synthesis. Replication of genomic RNA was completely dependent on the presence of conserved complementary or cyclization sequences near the 5' and 3' ends. Assembly of the RC during translation in cis and the relationships, particularly those of NS1 and NS5 among the components, were deduced from an extensive set of complementation experiments in trans involving mutations/deletions in all the nonstructural proteins and use of KUN or alphahavirus replicons as helpers. The KUN replicon has found useful applications also as a noncytopathic vector for the continuing expression of foreign genes, delivered either as packaged RNA or as plasmid DNA.

Amino Acid Sequence↗
PubMed1998

[Is the replicon model applicable to higher eukaryotes?].

Thirty-five years ago, the Replicon model was proposed by Jacob, Brenner and Cuzin to explain the regulation of the Escherichia coli DNA replication. In this model, a genetic element, the replicator, would function as a target for a positive-acting initiator protein to drive the initiation of replication. This simple idea has been extremely useful in providing a framework to explain how the initiation of DNA replication occurs in all organisms. The identification of autonomously replicating sequences (ARSs) in budding yeast was the first extension of the Replicon model to eukaryotic chromosomes. In the higher eukaryotes, many biochemically defined replication start sites have been identified; nevertheless there is little genetic data indicating that these sites contain DNA sequences that are essential for replication. Moreover, in early Xenopus or Drosophila embryos, specific DNA sequences are not required either for initiating DNA replication or for preventing rereplication within a single cell cycle. This apparently fundamental difference between replicators in yeast and metazoan embryos may be more superficial than initially thought. In fact, during the past several years, an eukaryotic initiator conserved from yeast to man and also present in embryonic cells, the origin recognition complex (ORC), has been characterized, suggesting that the initiation mechanism should be essentially the same in prokaryotes and eukaryotes. In addition, the efficient once-per-cell-cycle replication of DNA is ensured in eukaryotes by a simple two-step mechanism in which the assembly of stable prereplicative complexes (PreRCs) at origins precedes and is temporally separated from the firing of these origins. Regulation of this process by cyclin-dependent kinases ensures that when origins fire, the cell is no longer competent to form new PreRCs. Now, it is important to understand how these complexes are remodeled or disassembled during replication initiation to trigger the transition from a stable origin-bound complex to a mobile replication machine.

Animals↗
PubMed1999

Evolution of the linear DNA replicons of the Borrelia spirochetes.

Members of the spirochete genus Borrelia carry numerous linear DNA replicons with covalently closed hairpin telomeres. The genome of one member of this genus, B. burgdorferi B31, has now been completely characterized and contains a linear chromosome, twelve linear plasmids and nine circular extra-chromosomal elements. The phylogenetic position of the Borrelia spirochetes strongly suggests that a progenitor with circular replicons acquired the ability to replicate linear DNA molecules.

Base Sequence↗
PubMedDate not supplied

Cloning of the initiation region of a mammalian chromosomal replicon.

In eukaryotic cells, each chromosome is divided into several thousand tandemly arranged replicons, each synthesized at a characteristic time during the S period. Although as yet no eukaryotic DNA sequence known for certain to be a eukaryotic chromosomal replication origin has been isolated, electron microscopic and biochemical evidence suggests that eukaryotic DNA synthesis is initiated at specific sites. We have attempted to establish a system in which functional chromosomal origins could be identified in vivo before their isolation by molecular cloning. For this purpose, we have developed a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400) that contains 1,000 copies of a 135-kilobase (kb) early-replicating sequence, and which includes the gene for dihydrofolate reductase (DHFR). We have shown that initiation of DNA synthesis within each repeated unit (amplicon) is restricted to a small subset of restriction fragments at the beginning of the S period, suggesting that these fragments contain or flank origins of DNA synthesis. Here we report molecular cloning and restriction mapping experiments showing that all of these early-labelled fragments (ELFs) are derived from a single locus within each repeated unit. This result implies that synthesis of each amplicon initiates from a single origin of replication at the onset of S, and that an amplicon is formally equivalent to a replicon.

Animals↗
PubMed2003

Transfection-independent production of alphavirus replicon particles based on poxvirus expression vectors.

This report describes a transfection-independent system for packaging alphavirus replicon vectors using modified vaccinia virus Ankara (MVA) vectors to express all of the RNA components necessary for the production of Venezuelan equine encephalitis (VEE) virus replicon particles (VRP). Infection of mammalian cells with these recombinant MVA vectors resulted in robust expression of VEE structural genes, replication of the alphavirus vector and high titers of VRP. In addition, VRP packaging was achieved in a cell type (fetal rhesus lung) that has been approved for the manufacturing of vaccines destined for human use.

Alphavirus↗
PubMed2006

Mucosal and systemic adjuvant activity of alphavirus replicon particles.

Vaccination represents the most effective control measure in the fight against infectious diseases. Local mucosal immune responses are critical for protection from, and resolution of, infection by numerous mucosal pathogens. Antigen processing across mucosal surfaces is the natural route by which mucosal immunity is generated, as peripheral antigen delivery typically fails to induce mucosal immune responses. However, we demonstrate in this article that mucosal immune responses are evident at multiple mucosal surfaces after parenteral delivery of Venezuelan equine encephalitis virus replicon particles (VRP). Moreover, coinoculation of null VRP (not expressing any transgene) with inactivated influenza virions, or ovalbumin, resulted in a significant increase in antigen-specific systemic IgG and fecal IgA antibodies, compared with antigen alone. Pretreatment of VRP with UV light largely abrogated this adjuvant effect. These results demonstrate that alphavirus replicon particles possess intrinsic systemic and mucosal adjuvant activity and suggest that VRP RNA replication is the trigger for this activity. We feel that these observations and the continued experimentation they stimulate will ultimately define the specific components of an alternative pathway for the induction of mucosal immunity, and if the activity is evident in humans, will enable new possibilities for safe and inexpensive subunit and inactivated vaccines.

Adjuvants, Immunologic↗