Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “replicon”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 361 records · Page 20Linked to original sources
PubMed2000

Influenza virus (A/HK/156/97) hemagglutinin expressed by an alphavirus replicon system protects chickens against lethal infection with Hong Kong-origin H5N1 viruses.

Venezuelan equine encephalitis virus replicon particles (VRP) containing the gene expressing hemagglutinin (HA) from the human Hong Kong Influenza A isolate (A/HK/156/97) were evaluated as vaccines in chicken embryos and young chicks. Expressed HA was readily detected in bird-tissue staining with anti-H5 HA antibody and in chicken cells infected with the replicon preparations following immunoprecipitation with monoclonal antibody. Birds challenged with a dose of the lethal parent virus were protected to different extents depending on the age of the bird. In ovo and 1-day-old inoculated animals that received no boost with the VRP were partially protected; birds 2 weeks of age were completely protected with a single dose of VRP.

Age Factors↗
PubMed2002

Mutations in NS5B polymerase of hepatitis C virus: impacts on in vitro enzymatic activity and viral RNA replication in the subgenomic replicon cell culture.

Hepatitis C virus (HCV) nonstructural protein 5B (NS5B) is an RNA-dependent RNA polymerase (RdRp) essential for virus replication. Several consensus sequence motifs have been identified in NS5B, some of which have been shown to be critical for its enzymatic activity. A unique beta-hairpin structure located between amino acids 443 and 454 in the thumb subdomain has also been shown to play an important role in ensuring terminal initiation of RNA synthesis in vitro. However, the importance of these sequence and structural elements in viral RNA replication in infected cells has not been established, mainly due to the lack of a reliable cell culture system for HCV. In this study, we investigated the effect of several single amino acid substitutions and beta-hairpin truncations in NS5B on viral RNA replication by using the subgenomic replicon cell culture system. A strong correlation between in vitro polymerase activity and viral RNA replication was observed with most of the substitutions. Interestingly, truncations of the beta-hairpin (by four and eight amino acid residues, respectively), which did not reduce the in vitro enzymatic activity, completely abolished the ability of the replicon RNA to replicate in Huh-7 cells, demonstrating its essential role in viral RNA replication. Furthermore, a conservative substitution in motif D, from an arginine residue (AMTR(345)), which is conserved among all HCV isolates, to a lysine residue, resulted in significant improvements in both transient RNA replication and colony formation efficiencies. This result also correlates with a previous observation that the enzymatic activity of NS5B increased by about 50% when the same NS5B substitution was introduced (V. Lohmann, F. Korner, U. Herian, and R. Bartenschlager, J. Virol. 1997, 71, 8416-8428).

Amino Acid Motifs↗
PubMed1981

Isolation and characterization of lambda phages carrying the basic replicon of the resistance plasmid R1.

Specialized transducing lambda phages, lambda oriR1, harboring DNA from the resistance plasmid R1drd-19 and its copy mutant pKN103 were isolated. From measurements of CCC-DNA content it is concluded that upon infection the phages can establish themselves as self-replicating plasmids in recA hosts lysogenic for lambda. It is thought that this bypassing of lambda immunity is due to the presence of the R1 origin of replication. The plasmids are sensitive to the incompatibility expressed by plasmid R1. This has been shown mainly by transduction of lambda oriR1 into recipients containing R1 plasmids or plasmid pBR322 carrying the basic replicon. We were able to demonstrate that a copy mutant of plasmid R1 was insensitive to copA+, but sensitive to the concerted action of PstI fragments F1 and F2. This mutant was previously assumed to be of dominant type. Physical mapping of the lambda oriR1 derivatives verified that they carry the basic replicon of plasmid R1. The plasmids are not stably maintained, but are lost in a frequency of 1%-2% per cell generation, which is consistent with their lack of the R1par region.

Bacteriophage lambda↗
PubMed1996

Characterization of the replicon of plasmid pSW500 of Erwinia stewartii.

A 1.6-kb DNA region required for the replication of pSW500 from Erwinia stewartii SW2 has been identified. DNA sequencing analysis revealed that this DNA fragment consists of a DnaA box, seven 16-bp direct repeats, and a 1005-bp open reading frame. The seven direct repeats have been demonstrated to mediate the incompatibility function of the plasmid. Primer extension analysis showed that the 1005-bp ORF is transcribed in vivo and the +1 site of the transcript is located 113 bp upstream from the translation initiation codon of the ORF. Complementation studies showed that this ORF is required for the replication of the plasmid and may encode a replication protein, RepA. Gene fusion studies revealed that the expression of repA is autoregulated by RepA. We also found that the pSW500 replicon has a copy number of approximately two and that the plasmid is stably maintained in Escherichia coli, thus demonstrating that the replicon contains all the elements required for copy number control and plasmid stability in E. coli. Curing of pSW500 from E. stewartii SW2 revealed that loss of pSW500 did not have any obvious effect on morphology or physiology of the cells, suggesting that pSW500 does not encode a function that is indispensable for the survival of the organism.

Amino Acid Sequence↗
PubMed2004

Sub-genomic replicons of Tick-borne encephalitis virus.

We constructed three sub-genomic replicons of Tick-borne encephalitis virus (TBEV) (Oshima REP, Oshima REP-GFP and Oshima REP-Neo) by deleting genes coding for structural proteins without or with insertion of green fluorescent protein (GFP) or Neo genes, respectively. BHK cells transfected with Oshima REP expressed the viral non-structural antigens in immunofluorescent and western blot analyses. GFP and viral antigens were co-expressed in the transfected cells with Oshima REP-GFP. G418-resistant cells harboring Oshima REP-Neo consistently expressed the antigens without showing any apparent CPE. These replicons constructed in this study will be useful in studies on the replication, assembly and packaging of TBEV, and to develop vaccines and gene-delivering systems.

Animals↗
PubMed1983

Decrease in the average size of replicons in a Werner syndrome cell line by Simian virus 40 infection.

We have measured the distance between replicon initiation sites as well as the rate of DNA chain elongation in Simian Virus 40 (SV40)-infected and uninfected Werner syndrome (WS) and normal cell lines by DNA fiber-autoradiography. There was no difference in the rate of chain elongation among these cell lines. On the other hand, the replicon center-to-center distance was clearly longer in WS fibroblasts than that in normal fibroblasts. SV40 infection changed the center-to-center distance in WS cells toward that in normal cells.

Autoradiography↗
PubMed1988

Synchronization of replicons in Ehrlich ascites cells.

Ehrlich ascites cells, in which replication units at the beginning of the S phase started and grew synchronously, were obtained by the following protocol: (1) selection of G1 cells by zonal centrifugation, (2) hypoxia for 12 h, (3) reaeration, (4) addition of cycloheximide (30 microM) within the first minute after reoxygenation. Studies on the effectiveness of the different steps revealed: (i) G1 cells reoxygenated after 12 h of hypoxia traverse two succeeding cell cycles highly synchronously. This was shown by monitoring the thymidine incorporation rate, the thymidine pulse-labeling index, and the mitotic index. (ii) Cycloheximide, like hypoxia, suppresses replicon initiation in Ehrlich ascites cells without interfering with DNA chain growth and DNA maturation. The reversibility of the suppression is less complete than in the case of hypoxia. This was shown by DNA fiber autoradiography and by analyzing the length distribution of pulse- or pulse/pulse-chase-labeled daughter DNA in alkaline sucrose gradients. The alkaline sedimentation patterns of daughter-strand DNA, pulse labeled immediately after the cycloheximide addition at the end of the elaborated protocol and 1 and 2 h later, indicated synchronous initiation and growth of a homogeneous population of DNA molecules to replicon-sized lengths.

Animals↗
PubMed1982

Replicon fusion in mouse cells is very sensitive to DNA damage induced by N-methyl-N'-nitro-N-nitrosoguanidine.

Incubation of mouse cells with N-methyl-N'-nitro-N-nitrosoguanidine causes a strong inhibition of DNA replication the extent of which varies with the cell line used. Analysis of the products synthesized in drug-treated cells indicates a particularly severe effect on the joining of replicons while other steps in DNA synthesis like initiation and chain elongation are much less affected. The data indicate that replicon fusion may be extremely sensitive to changes in the topology of DNA induced by the introduction of rare single-strand breaks during repair of N-methylated purines produced by incubation of cells with small amounts of the methylating agent.

Animals↗
PubMed1989

Genetic analysis of the lytic replicon of bacteriophage P1. I. Isolation and partial characterization.

Despite the extensive genetic analysis of bacteriophage P1, the region of the viral genome that is responsible for its lytic (vegetative) replication has not been identified. In this paper we describe the identification of various fragments of P1 DNA that can replicate an otherwise replication-defective lambda vector when they are cloned into that vector. The fragments share a 2800 base-pair segment of the P1 genome that is located adjacent to the immI region of the phage. Replication mediated by the cloned P1 fragments is abolished by the product of the P1 c1 gene, the repressor of phage lytic functions. Since these properties resemble those of the P1 lytic replicon, we suggest that the 2800 base-pair segment identified here contains that replicon.

Base Composition↗
PubMed1990

Isolation of the replication DNA region from a Rhizobium plasmid and examination of its potential as a replicon for Rhizobiaceae cloning vectors.

The DNA region essential for replication and stability of a native plasmid (pTM5) from Rhizobium sp. (Hedysarum) has been identified and isolated within a 5.4-kb PstI restriction fragment. The isolation of this region was accomplished by cloning endonuclease-restricted pTM5 DNA into a ColE1-type replicon and selecting the recombinant plasmids containing the pTM5 replicator (pTM5 derivative plasmids) by their ability to replicate in Rhizobium. DNA homology studies revealed that pTM5-like replicons are present in cryptic plasmids from some Rhizobium sp. (Hedysarum) strains but not in plasmids from strains of other Rhizobium species or Agrobacterium tumefaciens. The pTM5 derivative plasmids were able to replicate in Escherichia coli and A. tumefaciens and in a wide range of Rhizobium species. On the basis of stability assays in the absence of antibiotic selective pressure, the pTM5 derivative plasmids were shown to be highly stable in both free-living and symbiotic cells of Rhizobium sp. (Hedysarum). The stability of these plasmids in other species of Rhizobium and in A. tumefaciens varied depending on the host and on the plasmid. Most pTM5 derivative plasmids tested showed significantly higher symbiotic stability than RK2 derivative plasmids pRK290 and pAL618 in Rhizobium sp. (Hedysarum), R. meliloti, and R. leguminosarum by. phaseoli. Consequently, we consider that the constructed pTM5 derivative plasmids are potentially useful as cloning vectors for Rhizobiaceae.

Cloning, Molecular↗
PubMed1992

Molecular, genetic, and functional analysis of the basic replicon of pVA380-1, a plasmid of oral streptococcal origin.

The 4.2-kb cryptic plasmid pVA380-1 has been used as a vector for the cloning of antibiotic resistance genes directly in streptococci, and in the construction of Escherichia coli/Streptococcus shuttle vectors. The results of subcloning experiments located the basic replicon of pVA380-1 within a 2.5-kb region. The nucleotide base sequence of this region was determined and contained a single complete open reading frame (ORF) encoding a 237-amino-acid peptide with a predicted size of 29 kDa. This peptide and a region of the DNA molecule 5' to the ORF encoding it shared homology with the replication protein and plus origin, respectively, of the Staphylococcus aureus plasmid pUB110. Data from Tn5 mutagenesis and complementation studies indicated that the protein product of the ORF was required for pVA380-1 replication in streptococci. Deletion of a region of the basic replicon distal to the plus origin and ORF produced an unstable derivative, and resulted in the accumulation of single-stranded replicative intermediates, consistent with the loss of a minus origin. All of these results suggest that pVA380-1 replicates by a rolling circle mode, and is most closely related to the pC194 family of single-stranded DNA plasmids.

Amino Acid Sequence↗
PubMed1988

Kinetics of replicon initiation during S phase of Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells were synchronized by a thymidine-hydroxyurea block. At different times after release from the block, cells were treated with trioxsalen and long-wavelength ultraviolet light to crosslink DNA in vivo and were labelled with [3H]thymidine for 30 min. This technique permits labelling of only the short nescent DNA fragments initiated between crosslinks. The amount of radioactivity incorporated in these fragments during the labelling period reflects the number of replicon initiation events and allows us to follow the replicon initiation pattern after removing the inhibitor. It was shown that the rate of initiation was high at the beginning of S phase and then steadily decreased.

Animals↗
PubMed1988

Replicon initiation in normal human cells and in ataxia telangiectasia cells: its differential inhibition by cycloheximide and bleomycin.

Treatment of normal human fibroblasts (NHFs) with cycloheximide, which inhibits protein synthesis, resulted in partial inhibition of their DNA synthesis, as determined by incorporation of radioactive thymidine and resistance of the cells to subsequent treatment with bleomycin. The effects of treatments of ataxia telangiectasia fibroblasts (ATFs) with cycloheximide and then bleomycin on their DNA synthesis were very similar to those on DNA synthesis of NHFs. The fact that treatment with bleomycin only caused transient inhibition of DNA synthesis within an hour in NHFs but not ATFs was confirmed. Studies by alkali-density gradient centrifugation showed that the cycloheximide mainly inhibited formation of short fragments of DNA in both NHFs and ATFs, as bleomycin does in NHFs. These findings suggest that these two chemicals both inhibit replicon initiation, and thus provide evidence that the genetic defect in ATFs is related to replicon initiation.

Ataxia Telangiectasia↗
PubMed1991

Effect of UV light on sister-chromatid exchanges, activation of alternative sites of replicon initiation and thymidine incorporation in CHO AA8, UV61 and UV5 cells.

The relative importance of the UV-induced pyrimidine(5-6)pyrimidine and the pyrimidine(6-4)-pyrimidone lesions in sister-chromatid exchanges (SCEs), activation of alternative sites of replicon initiation and thymidine incorporation were examined using wild-type Chinese hamster ovary (CHO) AA8 cells which remove both lesions, mutant CHO UV61 cells which remove only the (6-4) lesion and mutant CHO UV5 cells which remove neither lesion. Our data suggest that both lesions play a role in each end point examined. The relative importance of these lesions is dependent on the end point studied as well as the fluence used. For SCE induction and the activation of alternative sites of replicon initiation, the (6-4) lesion appears to play a predominant role, while for the thymidine incorporation studies the (6-4) lesion appears to play the predominant role at low fluences while the role of the (5-6) lesion increases at higher fluences.

Animals↗
PubMed2005

Screening for hepatitis C virus antiviral activity with a cell-based secreted alkaline phosphatase reporter replicon system.

We describe a phased screening system for discovery of compounds with antiviral activity against hepatitis C virus (HCV). The primary assay utilizes dicistronic subgenomic HCV replicons in which the upstream cistron was modified to express the human immunodeficiency virus (HIV) tat protein. When these replicons are stably transfected into Huh-7-derived cells that express secreted alkaline phosphatase (SEAP) under transcriptional control of the HIV long terminal repeat promoter, there is a strong correlation between intracellular HCV RNA abundance and the activity of SEAP secreted into the culture medium. Thus, active compounds are easily identified by direct enzymatic quantification of SEAP in the medium without post-assay processing. Compounds that reduce SEAP activity without causing cellular toxicity are next evaluated in a second Huh-7-derived cell line constitutively expressing SEAP under control of the tat-HIV promoter axis, independent of HCV RNA replication. This specificity control identifies compounds that cause reductions in SEAP that are unrelated to suppression of HCV RNA replication. Compounds showing HCV-specific activity in primary assays are next evaluated by real-time RT-PCR to directly quantify reductions in HCV RNA. We have found excellent agreement between the SEAP and RT-PCR assays. This phased system provides an efficient and cost-effective screen for compounds with antiviral activity against HCV.

Alkaline Phosphatase↗
PubMed2006

Antiviral activity of CHO-SS cell-derived human omega interferon and other human interferons against HCV RNA replicons and related viruses.

The fully glycosylated human omega interferon produced from CHO-SS cells (glycosylated IFN-omega) has been shown to be well-tolerated in man and to induce a sustained virologic response in patients infected with hepatitis C virus (HCV). We examined the antiviral activity of glycosylated IFN-omega and various human IFNs (IFN-alpha, -beta, -gamma and non-glycosylated bacterial (Escherichia coli) recombinant IFN-omega (non-glycosylated IFN-omega)) against HCV RNA replicons and several viruses related to HCV. Since none of the IFNs displayed cytotoxicity we compared their activities based on the effective concentration of the IFN that inhibited virus growth by 50% (EC50). Glycosylated IFN-omega was found to be the most potent antiviral agent of all the IFNs tested against bovine viral diarrhea virus (BVDV), yellow fever virus and West Nile virus. With HCV RNA replicons, non-glycosylated IFN-omega was comparable in activity to IFN-alpha while glycosylated IFN-omega was markedly more potent, indicating that glycosylation has an important effect on its activity. Drug combination analysis of glycosylated IFN-omega+ribavirin (RBV) in BVDV showed a synergy of antiviral effects similar to IFN-alpha+RBV, as well as a unique antagonism of RBV cytotoxic effects by glycosylated IFN-omega. Transcription factor (TF) profiling indicated that IFN-alpha or glycosylated IFN-omega treatment upregulated the same 17 TFs. IFN-alpha and glycosylated IFN-omega also upregulated 9 and 40 additional unique TFs, respectively. The differences in the expression of these TFs were modest, but statistically significantly different for eight of the TFs that were upregulated exclusively by glycosylated IFN-omega. The activation of these additional TFs by glycosylated IFN-omega might contribute to its high potency.

Animals↗
PubMed2006

Inhibition of hepatitis C virus RNA replication by short hairpin RNA synthesized by T7 RNA polymerase in hepatitis C virus subgenomic replicons.

RNA interference (RNAi) is a cellular process that induces gene silencing by which small duplexes of RNA specifically target a homologous sequence for cleavage by cellular ribonucleases. Here, to test the RNAi method for blocking hepatitis C virus (HCV) RNA replication, we created four short hairpin RNAs (shRNAs) targeting the HCV internal ribosome entry site/Core gene transcript using T7 RNA polymerase. shRNA suppressed the replication of HCV RNA in the HCV replicon. On the other hand, short interfering RNAs synthesized using the T7 RNA polymerase system trigger a potent induction of interferon-alpha and -beta in a variety of cells. We examined whether the shRNAs synthesized using the T7 RNA polymerase system activated double-stranded RNA-dependent protein kinase, 2'-5' oligoadenylate synthetase, or interferon-regulatory factor-3. Our results demonstrated that the T7-transcribed shRNA did not activate these proteins in Huh-7 cells and the HCV replicon. These shRNAs are a promising new strategy for anti-HCV gene therapeutics.

Base Sequence↗