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PubMed2002

Systemic, mucosal, and heterotypic immune induction in mice inoculated with Venezuelan equine encephalitis replicons expressing Norwalk virus-like particles.

Norwalk-like viruses (NLVs) are a diverse group of single-stranded, nonenveloped, positive-polarity RNA viruses and are the leading cause of epidemic acute gastroenteritis in the United States. In this study, the major capsid gene of Norwalk virus, the prototype NLV, has been cloned and expressed in mammalian cells using a Venezuelan equine encephalitis (VEE) replicon expression system. Upon infection of baby hamster kidney (BHK) cells with VEE replicon particles (VRPs), the Norwalk virus capsid proteins self-assemble to generate high titers of Norwalk virus-like particles (VLPs) that are morphologically and antigenically analogous to wild-type Norwalk virus. Mice inoculated subcutaneously with VRPs expressing the Norwalk virus capsid protein (VRP-NV1) developed systemic and mucosal immune responses to Norwalk VLPs, as well as heterotypic antibody responses to the major capsid protein from another genogroup I NLV strain (NCFL) isolated from a recent outbreak. A second Norwalk virus capsid clone (NV2) containing three amino acid codon mutations from the NV1 clone was also expressed using VEE replicons (VRP-NV2), but upon infection of BHK cells failed to confer VLP self-assembly. Mice inoculated with VRP-NV2 elicited reduced systemic and mucosal immune responses to Norwalk VLPs, demonstrating the importance and potential utility of endogenous VLP presentation for maximum immune induction. Inoculation with either VRP-NV1 or VRP-NV2 resulted in serum antibody responses far superior to the induction in mice dosed orally with VLPs that were prepared using the VEE-NV1 replicon construct, a regimen similar to current models for NLV vaccination. Expression of NLV VLPs in mammalian cells offers a powerful approach for the design of novel NLV vaccines, either alone or in combination with current vaccination models.

Amino Acid Sequence↗

PubMed2002

Cell-free replication of the hepatitis C virus subgenomic replicon.

The hepatitis C virus (HCV) contains a plus-strand RNA genome. The 5' noncoding region (NCR) of the viral genome functions as an internal ribosome entry site, and its unique 3' NCR is required for the assembly of the replication complex during initiation of HCV RNA replication. Lohmann et al. (V. Lohmann, F. Korner, J.-O. Koch, U. Herian, L. Theilman, and R. Batenschlager, Science 285:110-113, 1999) developed a subgenomic HCV replicon system, which represents an important tool in studying HCV replication in cultured cells. In this study, we describe a cell-free replication system that utilizes cytoplasmic lysates prepared from Huh-7 cells harboring the HCV subgenomic replicons. These lysates, which contain ribonucleoprotein complexes associated with cellular membranes, were capable of incorporating [alpha(32)P]CTP into newly synthesized RNA from subgenomic replicons in vitro. Replicative forms (RFs) and replicative intermediates (RIs) were synthesized from the endogenous HCV RNA templates. Consistent with previous observations, RFs were found to be resistant to RNase A digestion, whereas RIs were sensitive to RNase treatment. The radiolabeled HCV RF-RI complexes contained both minus and plus strands and were specific to the lysates derived from replicon-expressing cells. The availability of a cell-free replication system offers opportunities to probe the mechanism(s) of HCV replication. It also provides a novel assay for potential therapeutic agents.

Cell Line↗

PubMed2004

Genetic interactions between hepatitis C virus replicons.

To investigate interactions between hepatitis C virus (HCV) RNA replication complexes, a system was developed to simultaneously select different HCV subgenomic replicons within the same cell. Transcomplementation of defective replicons was not observed, suggesting an isolated and independent nature of the HCV RNA replication complex. In contrast, a high level of competition between replicons was observed, such that the presence and increased fitness of one replicon reduced the capacity of a second one to stably replicate. These results suggest that at least one factor in Huh7 cells required for HCV RNA replication is limiting and saturable.

Genetic Complementation Test↗

PubMed2005

Hepatitis C virus replicons escape RNA interference induced by a short interfering RNA directed against the NS5b coding region.

RNA interference represents an exciting new technology that could have therapeutic applications for the treatment of viral infections. Hepatitis C virus (HCV) is a major cause of chronic liver disease and affects over 270 million individuals worldwide. The HCV genome is a single-stranded RNA that functions as both an mRNA and a replication template, making it an attractive target for therapeutic approaches using short interfering RNA (siRNA). We have shown previously that double-stranded siRNA molecules designed to target the HCV genome block gene expression and RNA synthesis from hepatitis C replicons propagated in human liver cells. However, we now show that this block is not complete. After several treatments with a highly effective siRNA, we have shown growth of replicon RNAs that are resistant to subsequent treatment with the same siRNA. However, these replicon RNAs were not resistant to siRNA targeting another part of the genome. Sequence analysis of the siRNA-resistant replicons showed the generation of point mutations within the siRNA target sequence. In addition, the use of a combination of two siRNAs together severely limited escape mutant evolution. This suggests that RNA interference activity could be used as a treatment to reduce the devastating effects of HCV replication on the liver and the use of multiple siRNAs could prevent the emergence of resistant viruses.

Amino Acid Sequence↗

PubMed2005

Proteome analysis of liver cells expressing a full-length hepatitis C virus (HCV) replicon and biopsy specimens of posttransplantation liver from HCV-infected patients.

The development of a reproducible model system for the study of hepatitis C virus (HCV) infection has the potential to significantly enhance the study of virus-host interactions and provide future direction for modeling the pathogenesis of HCV. While there are studies describing global gene expression changes associated with HCV infection, changes in the proteome have not been characterized. We report the first large-scale proteome analysis of the highly permissive Huh-7.5 cell line containing a full-length HCV replicon. We detected >4,200 proteins in this cell line, including HCV replicon proteins, using multidimensional liquid chromatographic (LC) separations coupled to mass spectrometry. Consistent with the literature, a comparison of HCV replicon-positive and -negative Huh-7.5 cells identified expression changes of proteins involved in lipid metabolism. We extended these analyses to liver biopsy material from HCV-infected patients where a total of >1,500 proteins were detected from only 2 mug of liver biopsy protein digest using the Huh-7.5 protein database and the accurate mass and time tag strategy. These findings demonstrate the utility of multidimensional proteome analysis of the HCV replicon model system for assisting in the determination of proteins/pathways affected by HCV infection. Our ability to extend these analyses to the highly complex proteome of small liver biopsies with limiting protein yields offers the unique opportunity to begin evaluating the clinical significance of protein expression changes associated with HCV infection.

Amino Acid Sequence↗

PubMed2005

In vitro and in vivo mutational analysis of the 3'-terminal regions of hepatitis e virus genomes and replicons.

Hepatitis E virus (HEV) replication is not well understood, mainly because the virus does not infect cultured cells efficiently. However, Huh-7 cells transfected with full-length genomes produce open reading frame 2 protein, indicative of genome replication (6). To investigate the role of 3'-terminal sequences in RNA replication, we constructed chimeric full-length genomes with divergent 3'-terminal sequences of genotypes 2 and 3 replacing that of genotype 1 and transfected them into Huh-7 cells. The production of viral proteins by these full-length chimeras was indistinguishable from that of the wild type, suggesting that replication was not impaired. In order to better quantify HEV replication in cell culture, we constructed an HEV replicon with a reporter (luciferase). Luciferase production was cap dependent and RNA-dependent RNA polymerase dependent and increased following transfection of Huh-7 cells. Replicons harboring the 3'-terminal intergenotypic chimera sequences were also assayed for luciferase production. In spite of the large sequence differences among the 3' termini of the viruses, replication of the chimeric replicons was surprisingly similar to that of the parental replicon. However, a single unique nucleotide change within a predicted stem structure at the 3' terminus substantially reduced the efficiency of replication: RNA replication was partially restored by a covariant mutation. Similar patterns of replication were obtained when full-length genomes were inoculated into rhesus macaques, suggesting that the in vitro system could be used to predict the effect of 3'-terminal mutations in vivo. Incorporation of the 3'-terminal sequences of the swine strain of HEV into the genotype 1 human strain did not enable the human strain to infect swine.

Animals↗

PubMed2005

Replicon cell culture system as a valuable tool in antiviral drug discovery against hepatitis C virus.

Discovery of potential therapeutics against hepatitis C virus (HCV) infection has been hampered in the past decade by the inability to grow this virus in tissue culture and by the lack of robust small animal models. This situation has been improved by the recent development of a selectable HCV replicon cell culture system. For the first time, drug discovery scientists are able to screen large compound collections using the replicon cell culture system to identify small molecules with the potential to inhibit HCV RNA replication. The replicon system has also been used to elucidate inhibitors' antiviral mechanism of action and to optimize antiviral potency. In this review, we will summarize the recent development of HCV replicon cell culture system and its use in anti-HCV drug discovery. The antiviral activities of promising lead compounds are also reviewed.

Antiviral Agents↗

PubMed2002

[The cloning of plasmid replicon ori165 from Bacillus thuringiensis subsp. thenebrionis].

An EcoRI DNA fragment containing the plasmid replicon, named ori165, was cloned from the plasmids of B. thuringiensis subsp. tenebrionis strains YBT-1765. The replicon ori165 was the first replicon cloned from this subspecie. Its size is approximately 11 kb. The replicon was minimized to an 8 kb fragment by deletion analysis. Southern hybridization exhibited that ori165 was coming from the plasmid detected with the highest molecular weight. A shuttle vector pBMB6071 based on ori165 was constructed. The stabilities of pBMB6071 in different B. thuringiensis host had great discrepancy. In strain 4Q7, a crystal-negative derivative of B. thuringiensis subsp. isrealensis, the stability was 100%. ori165 was compatible with ori1030 and ori2062 in strain BMB171, a crystal-negative derivative of B. thuringiensis subsp. kurstaki.

Bacillus thuringiensis↗

PubMed1991

The same mammalian replicon yields distinct recombination products in different cell lines.

We have observed previously that some chimeric replicons inclusive of a partly duplicated polyomavirus (Py) genome yield unit-length Py DNA (P155) at high frequency when transfected into normal or Py-transformed mouse cells. We demonstrate here that one such replicon generates either P155 or illegitimate recombination products in other mouse cells, transformed by simian virus 40. Use of the polymerase chain reaction indicates that each of the illegitimate products carried a different deletion, but that all deletions mapped within a rather well defined portion of the precursor replicon. Thus, these products were organized as if two hotspots for recombination existed in the Py late-coding region, one being located within or near one of the duplicated sequences characteristic of the chimeric replicon. Since this particular hotspot has already been shown to be involved in the generation of P155, the data reported here could indicate that a single recombination mechanism can yield either homologous (P155) or illegitimate products. How the DNA interacts with certain proteins, such as papovavirus large tumor antigen, could explain why one or the other type of product is formed.

Animals↗

PubMed1980

[Organization of mammalian genome replication: data on the high rate of DNA replication in replicons of structural heterochromatin].

By the autoradiography, the increase of the mean rate of replication fork movement in DNA was shown for human skin fibroblasts during S-period induced by reseeding the stationary culture; the portion of high-rate replicons growing parallel to the increase in numbers of cells, in which the constitutive heterochromatin was replicating. Furthermore, the heterogeneity of replicons was investigated in regard to the rate of DNA chain growth in asynchronous cell populations of human and three rodent species (Microtus agrestis, Tscherskia triton, Cricetulus griseus). A positive correlation was found between the portion of replicons, with a relatively high rate of replication (more than 60 micron/h), and the relative amounts of C-heterochromatin in genomes of these species. On the basis of these studies it has been assumed, that the replicons with the high rate of DNA chain growth belong to the constitutive heterochromatin.

Animals↗

PubMedDate not supplied

[Pseudomonas mallei and Pseudomonas pseudomallei: introduction and maintenance of natural and recombinant plasmid replicons].

Comparative analysis of recipient activity of Pseudomonas mallei, Pseudomonas pseudomallei, and Pseudomonas cepacia strains towards naturally occurring and recombinant plasmid replicons was carried out. Autonomic broad host range vector plasmids based on RSF1010(IncQ) and pSa(IncW) replicons as well as integrative vectors based on pSUP202(Co1E1) replicon have been constructed. The study has shown that naturally occurring plasmids RSF1010(IncQ), pSa(IncW), R15(IncN), and RP4(IncP) are being efficiently transferred and stably maintained in investigated Pseudomonas strains. However, recombinant plasmids with the mini-replicon pSa which are stable in Escherichia coli have shown segregative instability in Pseudomonas strains, whereas derivatives of plasmid RSF1010 demonstrated different stability depending on the type of insertion. Plasmid pSUP202 derivative integrative vector pSM525 is efficiently introduced and stably maintained in P. mallei C-5 strain. Two vector systems for genetic manipulations in P.mallei and P.pseudomallei cells have been developed.

Burkholderia pseudomallei↗

PubMed1993

RepFIB: a basic replicon of large plasmids.

The distribution of the RepFIB replicon among a total of 20 plasmid incompatibility groups was determined using Southern blot and polymerase chain reaction analysis. The presence of the replicon was confirmed in 25 of 55 plasmids tested. The majority of plasmids carrying RepFIB are from the IncF incompatibility groups, but one plasmid from IncI and one plasmid from IncP also possess the replicon. Seven different examples of RepFIB were sequenced in the minimal replicon region to obtain 1525 bp of sequence information covering the repA gene and flanking repeat regions for comparison. An analysis of these sequences plus three sequences previously reported showed almost perfect conservation of the predicted protein sequence of RepA and of the flanking DNA repeats. DNA sequence data were analyzed using maximum parsimony techniques to describe the possible evolutionary relationships of the 10 examples of RepFIB.

Amino Acid Sequence↗

PubMed1983

The cdc9 ligase joins completed replicons in baker's yeast.

The drug hydroxyurea has been found to affect the conditional DNA ligase mutant cdc9 in the same way as a wild type. Specific concentrations inhibit the joining of completed replicons leading to a substantial accumulation of these molecules. Upon removal of hydroxyurea and further incubation of cdc9 cells at the permissive temperature the replicons joined together, while in sharp contrast at the restrictive temperature no such joining occurred. However, a revertant of cdc9 able to grow at the restrictive temperature was also able to join replicons under these conditions, so the cdc9 ligase must be responsible for the assembly of completed replicons.

DNA Ligases↗

PubMed1987

In vivo DNA cloning with a mini-Mu replicon cosmid and a helper lambda phage.

A mini-Mu bacteriophage, containing the cohesive-end packaging site (cos) from a lambda-phi 80 hybrid phage, a high-copy-number plasmid replicon, and a kanamycin-resistance gene for independent selection, was constructed to clone genes in vivo. This mini-Mu element can be derepressed to transpose at a high frequency. DNA segments that become flanked by copies of this mini-Mu element in the same orientation can be packaged by a helper lambda phage. The resulting lambda lysate can be used to infect recipient cells where the injected DNA can circularize by annealing at the cos termini. Drug-resistant transductants obtained carry the mini-Mu-replicon cosmid element with inserts of different nucleotide sequences. These are analogous to recombinant DNA clones generated in vitro with restriction endonuclease cutting and ligase joining reactions replaced by the Mu transposition process. Clones of particular genes were isolated by their ability to complement specific mutations. Both recA+ and recA- recipient cells can be used with equal efficiency. Clones obtained with a helper lambda phage require the presence of the cos site in the mini-Mu replicon. They carry larger inserts than those isolated with the same mini-Mu element and Mu as a helper phage. The mini-Mu replicon-cosmid bacteriophage contains a lac-gene fusing segment for isolating fusions of lac operon DNA to gene control regions in the cloned sequences. Independent clones of a particular gene can be used to prepare a restriction map of the gene and its flanking regions.

Bacteriophage lambda↗

PubMed2006

Evaluation of replicative capacity and genetic stability of West Nile virus replicons using highly efficient packaging cell lines.

A stable cell system for high-efficiency packaging of West Nile virus (WNV) subgenomic replicons into virus-like particles (VLPs) was developed. VLPs could be propagated on these packaging cells and produced infectious foci similar to foci produced by WNV. Focus size correlated with the replicative capacity of WNV replicons, indicating that genome copy number, rather than amount of trans-complementing structural proteins, was rate-limiting in packaging of VLPs. Comparison of VLP production from replicon genomes encoding partial or complete C genes indicated that portions of C downstream of the cyclization sequence could improve genome replication or that cis expression of C could enhance packaging. Interestingly, a rapid loss of replicon-encoded reporter gene activity was detected within two serial passages of reporter gene-containing VLPs. The loss of reporter activity correlated with gene deletion and better VLP growth, indicating a powerful selection pressure for WNV genomes lacking reporter genes.

Animals↗

PubMed1977

The organization of repeated nucleotide sequences in the replicons of mammalian DNA.

Chinese hamster ovary cells were irradiated with 100-5,000 rads of X-rays and inhibition of the initiation of replicons after irradiation was demonstrated by analyzing nascent DNA sedimented in alkaline sucrose gradients. The renaturation kinetics of DNA synthesized during 60 min of incubation after irradiation was compared with that of DNA synthesized during the 60 min after sham irradiation and with that of parental DNA. Nascent DNA from cells whose replicon initiation was inhibited renatured faster than nascent DNA from control cells in the COt range of repeated nucleotide sequences, suggesting that regions of the replicon not close to origins are enriched in repeated sequences and that regions close to origins are enriched in unique sequences. A class of repeated nucleotide sequences may be involved in the regulation of replicon initiation.

Base Sequence↗

PubMed2003

Boosting with recombinant vaccinia increases HPV-16 E7-Specific T cell precursor frequencies and antitumor effects of HPV-16 E7-expressing Sindbis virus replicon particles.

Immunotherapy using the heterologous prime-boost regimen has emerged as an attractive approach for generating antigen-specific T-cell-mediated immune responses against tumors and infectious diseases. We have previously linked the Mycobacterium tuberculosis heat-shock protein 70 (HSP70) to the HPV-16 E7 antigen creating a chimera, E7/HSP70. We found that nucleic acid vaccines encoding E7/HSP70 can generate strong antitumor immunity. Recently, replication-defective Sindbis virus replicon particle vaccines have been considered as an important vector system for vaccine development. In this study, we assessed whether the combination of E7/HSP70 Sindbis virus replicon particles (SINrep5-E7/HSP70) and E7/HSP70 vaccinia (Vac-E7/HSP70) can further enhance E7-specific immune responses using sequential vaccination. We found that priming with SINrep5-E7/HSP70 and boosting with Vac-E7/HSP70 generated the highest number of E7-specific CD8(+) T cells and best antitumor effect compared to other combinations. Moreover, our data showed that at the dosage and route of immunization used in this study, mice treated with the Sindbis virus replicon particle prime-vaccinia boost regimen generated stronger antitumor responses compared to mice treated with the DNA prime-vaccinia boost vaccine regimen. Our results encourage the use of the Sindbis virus replicon particle prime-vaccinia boost regimen in future clinical trials.

Animals↗

PubMed1992

Conserved sequence motifs in the initiator proteins for rolling circle DNA replication encoded by diverse replicons from eubacteria, eucaryotes and archaebacteria.

An amino acid motif was identified that consists of the sequence HisHydrHisHydrHydrHydr (Hydr--bulky hydrophobic residue) and is conserved in two vast classes of proteins, one of which is involved in initiation and termination of rolling circle DNA replication, or RCR (Rep proteins), and the other in mobilization (conjugal transfer) of plasmid DNA (Mob proteins). Based on analogies with metalloenzymes, it is hypothesized that the two conserved His residues in this motif may be involved in metal ion coordination required for the activity of the Rep and Mob proteins. Rep proteins contained two additional conserved motifs, one of which was located upstream, and the other downstream from the 'two His' motif. The C-terminal motif encompassed the Tyr residue(s) forming the covalent link with nicked DNA. Mob proteins were characterized by the opposite orientation of the conserved motifs, with the (putative) DNA-linking Tyr being located near their N-termini. Both Rep and Mob protein classes further split into several distinct families. Although it was not possible to find a motif or pattern that would be unique for the entire Rep or Mob class, unique patterns were derived for large subsets of the proteins of each class. These observations allowed the prediction of the amino acid residues involved in DNA nicking, which is required for the initiation of RCR or conjugal transfer of single-stranded (ss) DNA, in Rep and Mob proteins encoded by a number of replicons of highly diverse size, structure and origin. It is conjectured that recombination has played a major part in the dissemination of genes encoding related Rep or Mob proteins among the replicons exploiting RCR. It is speculated that the eucaryotic small ssDNA replicons encoding proteins with the conserved RCR motifs and replicating via RCR-related mechanisms, such as geminiviruses and parvoviruses, may have evolved from eubacterial replicons.

Amino Acid Sequence↗