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PubMed2004

Dominant negative effect of wild-type NS5A on NS5A-adapted subgenomic hepatitis C virus RNA replicon.

An efficient model is currently used to study hepatitis C virus (HCV) replication in cell culture. It involves transfection in Huh7, a hepatoma-derived cell line, of an antibiotic (neomycin) selectable HCV subgenomic replicon encoding the non-structural (NS) proteins from NS3 to NS5B. However, strong and sustained replication is achieved only on the appearance of adaptive mutations in viral proteins. The most effective of these adaptive mutations are concentrated mainly in NS5A, not only into the original Con1 but also in the recently established HCV-BK and HCV-H77 isolate-derived replicons. This suggests that the expression of wild-type (wt) NS5A may not allow efficient HCV RNA replication in cell culture. With the use of a beta-lactamase reporter gene as a marker for HCV replication and TaqMan RNA analysis, the replication of different HCV replicons in cotransfection experiments was investigated. Comparing wt with NS5A-adapted replicons, the strong evidence accumulated showed that the expression of wt NS5A was actually able to inhibit the replication of NS5A-adapted replicons. This feature was characterized as a dominant negative effect. Interestingly, an NS5B (R2884G)-adapted replicon, containing a wt NS5A, was dominant negative on an NS5A-adapted replicon but was not inhibited by the original Con1 replicon. In conclusion, these studies revealed that the original wt Con1 replicon is not only incompetent for replication in cell culture, but is also able to interfere with NS5A-adapted replicons.

Amino Acid Substitution↗
PubMed2004

Hepatitis C virus subgenomic replicons in the human embryonic kidney 293 cell line.

Hepatitis C virus (HCV) infects liver cells and its replication in other cells is incompletely defined. Human hepatoma Huh-7 cells harboring subgenomic HCV replicons were used in somatic cell fusion experiments with human embryonic kidney 293 cells as a means of examining the permissiveness of 293 cells for HCV subgenomic RNA replication. 293 cells were generally not permissive for replication of Huh-7 cell-adapted replicons. However, upon coculturing of the two cell lines, we selected rare replicon-containing cells, termed 293Rep cells, that resembled parental 293 cells. Direct metabolic labeling of cells with (33)P in the presence of actinomycin D and Northern blotting to detect the negative strand of the replicon demonstrated functional RNA replicons in 293Rep cells. Furthermore, Western blots revealed that 293Rep cells expressed the HCV nonstructural proteins as well as markers of the naïve 293 cells but not Huh-7 cells. Propidium iodide staining and fluorescence-activated cell sorting analysis of 293Rep cells revealed that clone 293Rep17 closely resembled naïve 293 cells. Transfection of total RNA from 293Rep17 into naïve 293 cells produced replicon-containing 293 cell lines with characteristics distinct from those of Huh-7-derived replicon cell lines. Relative to Huh-7 replicons, the 293 cell replicons were less sensitive to inhibition by alpha interferon and substantially more sensitive to inhibition by poly(I)-poly(C) double-stranded RNA. This study established HCV subgenomic replicons in nonhepatic 293 cells and demonstrated their utility in expanding the study of cellular HCV RNA replication.

Cell Fusion↗
PubMed2001

Repetitive intrathecal injections of poliovirus replicons result in gene expression in neurons of the central nervous system without pathogenesis.

Poliovirus-based vectors (replicons) can be used for gene delivery to motor neurons of the CNS. In the current study, a replicon encoding green fluorescent protein (GFP) was encapsidated into authentic poliovirions, using established procedures. Intrathecal delivery of encapsidated replicons encoding GFP to the CNS of mice transgenic for the human poliovirus receptor did not result in any functional deficits as judged by behavioral testing. Histological analysis of the CNS of mice given a single intrathecal injection of poliovirus replicons encoding GFP revealed no obvious pathogenesis in neurons (or other cell types) within the CNS. The expression of GFP was confined to motor neurons throughout the neuroaxis; a time course of expression of GFP revealed that expression was detectable 24 hr postinoculation and returned to background levels by 120 hr postinoculation. A procedure was devised to allow repetitive inoculation of replicons within the same animal. Behavioral testing of animals that had received 6 to 13 independent inoculations of replicons revealed no functional deficits. Histological analysis of the CNS from animals that had received 6 to 13 sequential inoculations of replicons revealed no obvious abnormalities in neurons or other cell types in the CNS; expression of GFP was demonstrated in neurons 24 to 72 hr after the final inoculation of the replicon. Furthermore, there was no obvious inflammatory response in the CNS after the multiple inoculations. These studies establish the safety and efficacy of replicons for gene delivery to the CNS and are discussed with respect to use of replicons as new therapeutic strategies for spinal cord injuries and/or neurological diseases.

Animals↗
PubMed1989

Evidence for chromosomal replicons as units of sister chromatid exchanges.

Chromosomal replicons have been described as the cytological counterpart of DNA replicon clusters and have previously been studied in vitro using premature chromosome condensation-sister chromatid differentiation (PCC-SCD) techniques. Chromosomal replicons are visualized as small SCD segments in S-phase cells, and measurement of these segments can provide estimates of relative chromosomal replicon size corresponding to DNA replicon clusters functioning coordinately in S-phase. Current hypotheses of sister chromatid exchange (SCE) formation postulate that sites of SCE induction are associated with active replicons or replicon clusters. We have applied the PCC-SCD technique to in vivo studies of mouse bone marrow cells that have been treated with cyclophosphamide (CP) for two cell cycles. We have been able to visualize chromosomal replicons, as well as SCEs which have been induced in vivo by CP treatment, simultaneously in the same cells. Chromosomal replicons visualized as small SCD segments were measured in PCC cells classified at early or late S-phase based on SCD segment size prevalence. Early S-phase (E/S) PCC cells contained 90% of the SCD segments measured clustered in a segment size range of 0.1 to 0.8 micron with a peak value around 0.3 to 0.6 micron regardless of CP treatment. As the cells progressed through S-phase, late S-phase (L/S) PCC cells were characterized by the appearance of larger SCD segments and even whole SCD chromosomes in addition to small SCD segments.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Characterization of the basic replicons of the chimeric R/Ent plasmid pCG86 and the related Ent plasmid P307.

Restriction-enzyme fragments that can replicate autonomously after circularization were isolated from the chimeric R/Ent plasmid pCG86 and the Ent plasmid P307. Two such regions containing a basic replicon were located in each plasmid. One of the basic replicons of P307, RepFIB, is almost identical with one of the basic replicons of pCG86. The other basic replicon in P307, RepFIC, is partly homologous with the second basic replicon in pCG86, RepFIIA/RepFIC. The latter is a hybrid basic replicon and is in addition partly homologous with RepFIIA, a basic replicon present in IncFII R plasmids. By restriction-enzyme mapping and nucleotide-sequence analysis we have determined a site in the hybrid replicon where it ceases to be homologous with the RepFIIA basic replicon contained in the IncFII miniplasmid pSM1. The 2410-bp region of homology with pSM1 corresponds with a segment containing the origin of replication and all the genes responsible for replication control of pSM1.

Base Sequence↗
PubMed1996

The bacteriophage P1 lytic replicon: directionality of replication and cis-acting elements.

We have identified the direction of replication of a bacteriophage P1 lytic replicon. This was accomplished by constructing lambda P1 lysogens that contain a functional P1 lytic replicon and analysing which of two nearby bacterial DNA markers flanking the lambda prophage were amplified when that replicon was activated. We demonstrate that both DNA markers are coordinately amplified, a result consistent with lytic replication proceeding in a bidirectional fashion. To analyze the role of various elements comprising the lytic replicon, we assessed the ability of a wild type replicon to complement a defective replicon that contains a transposon inserted between an essential lytic replication gene (repL) and the promoter (P53) at which transcription of that gene is initiated. We show that the wild type replicon cannot complement the mutant replicon. The simplest hypothesis to explain this result is that either P53 or repL protein functions primarily in cis for the replicon to operate.

Bacteriophage P1↗
PubMed2003

The effect of ribavirin and IMPDH inhibitors on hepatitis C virus subgenomic replicon RNA.

The recent development of in vitro hepatitis C virus (HCV) RNA replication systems has provided useful tools for studying the intracellular anti-HCV activity of ribavirin. Ribavirin has been shown to: (1) induce "error catastrophe" in poliovirus, Proc. Natl. Acad. Sci. USA 98, 6895-6900), (2) be a pseudo-substrate of the HCV RNA-dependent RNA polymerase (RdRp) in vitro, J. Biol. Chem. 276, 46094-46098), and (3) increase mutations in HCV RNA in the binary T7 polymerase/HCV cDNA replication system, J. Virol. 76, 8505-8517). These findings have led to the hypothesis that ribavirin may also induce error catastrophe in HCV. However, the functional relevance of ribavirin-induced HCV RNA mutagenesis is unclear. By use of a colony formation assay, in which RNA is isolated from the HCV subgenomic replicon system following treatment, the impact of ribavirin, inosine-5'-monophosphate dehydrogenase (IMPDH) inhibitors, and the combination was assessed. Ribavirin reduced HCV replicon colony-forming efficiency (CFE) in a dose-dependent fashion, suggesting that ribavirin may be misincorporated into replicon RNA and result in an anti-replicon effect analogous to error catastrophe. This effect was markedly suppressed by addition of exogenous guanosine. Combination treatment with ribavirin and mycophenolic acid (MPA) or VX-497, both potent, nonnucleoside IMPDH inhibitors, led to a greatly enhanced anti-replicon effect. This enhancement was reversed by inclusion of guanosine with the treatment. In contrast, MPA or VX-497 alone had only marginal effects on both the quantity and quality (CFE) of replicon RNA, suggesting that although IMPDH inhibition is an important contributing factor to the overall ribavirin anti-HCV replicon activity, IMPDH inhibition by itself is not sufficient to exert an anti-HCV effect. Sequencing data targeting the neo gene segment of the HCV replicon indicated that ribavirin together with MPA or VX-497 increased the replicon error rate by about two-fold. Taken together these results further suggest that lethal mutagenesis may be an effective anti-HCV strategy. The colony formation assay provides a useful tool for evaluating mutagenic nucleoside analogs for HCV therapy. Finally, the data from combination treatment indicate potential therapeutic value for an enhanced anti-HCV effect when using ribavirin in combination with IMPDH inhibition.

Antimetabolites↗
PubMed1997

Immunization of mice with poliovirus replicons expressing the C-fragment of tetanus toxin protects against lethal challenge with tetanus toxin.

In this study, we describe the construction of poliovirus genomes or "replicons" which contain the C fragment gene of tetanus toxin substituted for the poliovirus P1 capsid. Upon transfection of replicon RNA into cells, we immunoprecipitated a protein corresponding to the C-fragment of tetanus toxin using tetanus-specific antibodies. Using a recombinant vaccinia virus expressing poliovirus P1 capsid protein (VV-P1) to provide P1 protein, the replicon RNA was encapsidated; stocks of the replicons were generated by passage with VV-P1. The immunogenicity of the replicons was determined by immunization of transgenic mice which are susceptible to poliovirus. A serum antibody response to poliovirus and tetanus toxoid was detected in all of the immunized mice. Protection against a lethal dose of tetanus toxin generally correlated with the levels of serum anti-tetanus antibodies. To address whether pre-existing antibodies to poliovirus limit the effectiveness of the replicon as a vaccine vector, mice were first immunized with the inactivated poliovirus vaccine followed by immunization with the replicons expressing C-fragment protein. Anti-tetanus antibodies were detected in these mice after a single administration of the replicon; these antibodies conferred protection upon challenge with tetanus toxin. These results demonstrate the potential use of poliovirus replicons encoding foreign proteins to induce a protective antibody response, even in the presence of pre-existing antibodies to poliovirus.

Animals↗
PubMed2006

Sindbis virus replicon particles encoding calreticulin linked to a tumor antigen generate long-term tumor-specific immunity.

Alphavirus vectors have emerged as a promising strategy for the development of cancer vaccines and gene therapy applications. In this study, we used the replication-defective vaccine vector SIN replicon particles from a new packaging cell line (PCL) to develop SIN replicon particles encoding calreticulin (CRT) linked to a model tumor antigen, human papillomavirus type 16 (HPV16) E7 protein. The linkage of CRT to E7 in SIN replicon particles resulted in a significant increase in E7-specific CD8(+) T-cell precursors and a strong antitumor effect against E7-expressing tumors in vaccinated mice. SINrep5-CRT/E7 replicon particles enhanced presentation of E7 through the major histocompatibility complex (MHC) class I pathway by infecting dendritic cells (DCs) directly and pulsing DCs with lysates of cells infected by SINrep5-CRT/E7 replicons. Vaccination of immunocompromised (BALB/c nu/nu) mice with SINrep5-CRT/E7 replicon particles also generated significant reduction of lung tumor nodules, suggesting that antiangiogenesis may contribute to the antitumor effect of SINrep5-CRT/E7 replicon particles. Furthermore, SINrep5-CRT/E7 replicon particles generated long-term in vivo tumor protection effects and antigen-specific memory immunities. We concluded that the CRT strategy used in the context of SIN replicon particles facilitated the generation of a highly effective vaccine for cancer prophylaxis and immunotherapy.

Analysis of Variance↗
PubMed2005

Development of a cell-based high-throughput specificity screen using a hepatitis C virus-bovine viral diarrhea virus dual replicon assay.

The hepatitis C virus (HCV) replicon is a unique system for the development of a high-throughput screen (HTS), since the analysis of inhibitors requires the quantification of a decrease in a steady-state level of HCV RNA. HCV replicon replication is dependent on host cell factors, and any toxic effects may have a significant impact on HCV replicon replication. Therefore, determining the antiviral specificity of compounds presents a challenge for the identification of specific HCV inhibitors. Here we report the development of an HCV/bovine viral diarrhea virus (BVDV) dual replicon assay suitable for HTS to address these issues. The HCV reporter enzyme is the endogenous NS3 protease contained within the HCV genome, while the BVDV reporter enzyme is a luciferase enzyme engineered into the BVDV genome. The HTS uses a mixture of HCV and BVDV replicon cell lines placed in the same well of a 96-well plate and isolated in the same cell backgrounds (Huh-7). The format consists of three separate but compatible assays: the first quantitates the amount of cytotoxicity based upon the conversion of Alamar blue dye via cellular enzymes, while the second indirectly quantitates HCV replicon replication through measurement of the amount of NS3 protease activity present. The final assay measures the amount of luciferase activity present from the BVDV replicon cells, as an indicator of the specificity of the test compounds. This HCV/BVDV dual replicon assay provides a reliable format to determine the potency and specificity of HCV replicon inhibitors.

Animals↗
PubMed1989

Participation of the lytic replicon in bacteriophage P1 plasmid maintenance.

P1 bacteriophage carries at least two replicons: a plasmid replicon and a viral lytic replicon. Since the isolated plasmid replicon can maintain itself stably at the low copy number characteristic of intact P1 prophage, it has been assumed that this replicon is responsible for driving prophage replication. We provide evidence that when replication from the plasmid replicon is prevented, prophage replication continues, albeit at a reduced rate. The residual plasmid replication is due to incomplete repression of the lytic replicon by the c1 immunity repressor. Incomplete repression was particularly evident in lysogens of the thermoinducible P1 c1.100 prophage, whose replication at 32 degrees C remained almost unaffected when use of the plasmid replicon was prevented. Moreover, the average plasmid copy number of P1 in a P1 c1.100 lysogen was elevated with respect to the copy number of P1 c1+. The capacity of the lytic replicon to act as an auxiliary in plasmid maintenance may contribute to the extraordinary stability of P1 plasmid prophage.

Alleles↗
PubMed1998

Encapsidation of the flavivirus kunjin replicon RNA by using a complementation system providing Kunjin virus structural proteins in trans.

Kunjin virus (KUN) replicon RNA was encapsidated by a procedure involving two consecutive electroporations of BHK-21 cells, first with KUN replicon RNA C20DXrep (with prME and most of C deleted) and about 24 h later with a recombinant Semliki Forest virus (SFV) replicon RNA(s) expressing KUN structural proteins. The presence of KUN replicon RNA in encapsidated particles was demonstrated by its amplification and expression in newly infected BHK-21 cells, detected by Northern blotting with a KUN-specific probe and by immunofluorescence analysis with anti-NS3 antibodies. No infectious particles were produced when C20DXrep RNA and recombinant SFV RNAs were electroporated simultaneously. When the second electroporation was performed with a single SFV replicon RNA expressing the KUN contiguous prME genes and the KUN C gene together but under control of two separate 26S subgenomic promoters (SFV-prME-C107), a 10-fold-higher titer of infectious particles was achieved than when two different SFV replicon RNAs expressing the KUN C gene (SFV-C107) and prME genes (SFV-prME) separately were used. No SFV replicon RNAs expressing KUN structural proteins were encapsidated in secreted particles. Infectious particles pelleted by ultracentrifugation of the culture fluid from cells sequentially transfected with C20DXrep and SFV-prME-C107 RNAs were neutralized by preincubation with monoclonal antibodies to KUN E protein. Radioimmunoprecipitation analysis with anti-E antibodies of the culture fluid of the doubly transfected cells showed the presence of C, prM/M, and E proteins in the immunoprecipitated particles. Reverse transcription-PCR analysis showed that the immunoprecipitated particles also contained KUN-specific RNA. The encapsidated replicon particles sedimented more slowly than KUN virions in a 5 to 25% sucrose density gradient and were uniformly spherical, with an approximately 35-nm diameter, compared with approximately 50 nm for KUN virions. The results of this study demonstrate for the first time packaging of flavivirus RNA in trans, and they exclude a role in packaging for virtually all of the structural region. Possible applications of the developed packaging system include the definition of the packaging signal(s) in flavivirus RNA as well as the amino acid motif(s) in the structural proteins involved in RNA encapsidation, virion assembly, and secretion. Furthermore, it could facilitate the development of a noninfectious vaccine delivery system based on encapsidation of a noncytopathic flavivirus replicon expressing heterologous genes.

Animals↗
PubMed2003

An alphavirus replicon particle chimera derived from venezuelan equine encephalitis and sindbis viruses is a potent gene-based vaccine delivery vector.

Alphavirus replicon particle-based vaccine vectors derived from Sindbis virus (SIN), Semliki Forest virus, and Venezuelan equine encephalitis virus (VEE) have been shown to induce robust antigen-specific cellular, humoral, and mucosal immune responses in many animal models of infectious disease and cancer. However, since little is known about the relative potencies among these different vectors, we compared the immunogenicity of replicon particle vectors derived from two very different parental alphaviruses, VEE and SIN, expressing a human immunodeficiency virus type 1 p55(Gag) antigen. Moreover, to explore the potential benefits of combining elements from different alphaviruses, we generated replicon particle chimeras of SIN and VEE. Two distinct strategies were used to produce particles with VEE-p55(gag) replicon RNA packaged within SIN envelope glycoproteins and SIN-p55(gag) replicon RNA within VEE envelope glycoproteins. Each replicon particle configuration induced Gag-specific CD8(+) T-cell responses in murine models when administered alone or after priming with DNA. However, Gag-specific responses varied dramatically, with the strongest responses to this particular antigen correlating with the VEE replicon RNA, irrespective of the source of envelope glycoproteins. Comparing the replicons with respect to heterologous gene expression levels and sensitivity to alpha/beta interferon in cultured cells indicated that each might contribute to potency differences. This work shows that combining desirable elements from VEE and SIN into a replicon particle chimera may be a valuable approach toward the goal of developing vaccine vectors with optimal in vivo potency, ease of production, and safety.

AIDS Vaccines↗
PubMed2005

Establishment of a subgenomic replicon for bovine viral diarrhea virus in Huh-7 cells and modulation of interferon-regulated factor 3-mediated antiviral response.

We describe the development of a selectable, bi-cistronic subgenomic replicon for bovine viral diarrhea virus (BVDV) in Huh-7 cells, similar to that established for hepatitis C virus (HCV). The selection marker and reporter (Luc-Ubi-Neo) in the BVDV replicon was fused with the amino-terminal protease N(pro), and expression of the nonstructural proteins (NS3 to NS5B) was driven by an encephalomyocarditis virus internal ribosome entry site. This BVDV replicon allows us to compare RNA replication of these two related viruses in a similar cellular background and to identify antiviral molecules specific for HCV RNA replication. The BVDV replicon showed similar sensitivity as the HCV replicon to interferons (alpha, beta, and gamma) and 2'-beta-C-methyl ribonucleoside inhibitors. Known nonnucleoside inhibitor molecules specific for either HCV or BVDV can be easily distinguished by using the parallel replicon systems. The HCV replicon has been shown to block, via the NS3/4A serine protease, Sendai virus-induced activation of interferon regulatory factor 3 (IRF-3), a key antiviral signaling molecule. Similar suppression of IRF-3-mediated responses was also observed with the Huh-7-BVDV replicon but was independent of NS3/4A protease activity. Instead, the amino-terminal cysteine protease N(pro) of BVDV appears to be, at least partly, responsible for suppressing IRF-3 activation induced by Sendai virus infection. This result suggests that different viruses, including those closely related, may have developed unique mechanisms for evading host antiviral responses. The parallel BVDV and HCV replicon systems provide robust counterscreens to distinguish viral specificity of small-molecule inhibitors of viral replication and to study the interactions of the viral replication machinery with the host cell innate immune system.

Animals↗
PubMed2005

Replication-competent chimeric hepatitis C virus subgenomic replicons.

OBJECTIVE: To utilize chimeric hepatitis C virus (HCV) replicons to select adaptive mutation(s) that allow replication of a genotype 1a replicon. METHODS: We used a genetic approach to gradually apply selective pressure by generating chimeric replicons through sequential replacement of nonstructural genes of a 1b replicon with genotype 1a sequences. RESULTS: A chimeric replicon containing a genotype 1a NS5A protein did not replicate in a transient assay, but could be used to establish stable cell lines using G418 selection. The cell lines contained a K1846T mutation in NS4B which functioned as an adaptive mutation that now allowed the chimera to replicate at levels similar to wild-type replicons. Similarly, replication of a 1a NS5A5B chimera was only observed after establishment of stable cell lines, even in the presence of the K1846T mutation. Sequence analysis of this cell line revealed an additional adaptive mutation of M1496L in NS3. Lastly, by including the K1846T mutation in a replicon that was entirely genotype 1a sequence, stable 1a cell lines could be established. CONCLUSION: These studies identify an NS4B adaptive mutation, K1846T, which allows establishment of a replication-competent 1a replicon and demonstrate the utility of this chimeric approach for establishing replicons for various HCV genotypes.

Amino Acid Substitution↗
PubMed2003

Antiviral effect and virus-host interactions in response to alpha interferon, gamma interferon, poly(i)-poly(c), tumor necrosis factor alpha, and ribavirin in hepatitis C virus subgenomic replicons.

The recently developed hepatitis C virus (HCV) subgenomic replicon system was utilized to evaluate the efficacy of several known antiviral agents. Cell lines that persistently maintained a genotype 1b replicon were selected. The replicon resident in each cell line had acquired adaptive mutations in the NS5A region that increased colony-forming efficiency, and some replicons had acquired NS3 mutations that alone did not enhance colony-forming efficiency but were synergistic with NS5A mutations. A replicon constructed from the infectious clone of the HCV-1 strain (genotype 1a) was not capable of inducing colony formation even after the introduction of adaptive mutations identified in the genotype 1b replicon. Alpha interferon (IFN-alpha), IFN-gamma, and ribavirin exhibited antiviral activity, while double-stranded RNA (dsRNA) and tumor necrosis factor alpha did not. Analysis of transcript levels for a series of genes stimulated by IFN (ISGs) or dsRNA following treatment with IFN-alpha, IFN-gamma, and dsRNA revealed that both IFNs increased ISG transcript levels, but that some aspect of the dsRNA response pathway was defective in Huh7 cells and replicon cell lines in comparison to primary chimpanzee and tamarin hepatocytes. The colony-forming efficiency of the replicon was reduced or eliminated following replication in the presence of ribavirin, implicating the induction of error-prone replication. The potential role of error-prone replication in the synergy observed between IFN-alpha and ribavirin in attaining sustained viral clearance is discussed. These studies reveal characteristics of Huh7 cells that may contribute to their unique capacity to support HCV RNA synthesis and demonstrate the utility of the replicon system for mechanistic studies on antiviral agents.

Amino Acid Sequence↗
PubMed1998

Characterization of a theta plasmid replicon with homology to all four large plasmids of Bacillus megaterium QM B1551.

A replicon from one of an array of seven indigenous compatible plasmids of Bacillus megaterium QM B1551 has been cloned and sequenced. The replicon hybridized with all four of the large plasmids (165, 108, 71, and 47 kb) of strain QM B1551. The cloned 2374-bp HindIII fragment was sequenced and contained two upstream palindromes and a large (>419-amino-acid) open reading frame (ORF) truncated at the 3' end. Unlike most plasmid origins, a region of four tandem 12-bp direct repeats was located within the ORF. The direct repeats alone were incompatible with the replicon, suggesting that they are iterons and that the plasmid probably replicates by theta replication. The ORF product was shown to act in trans. A small region with similarity to the B. subtilis chromosomal origin membrane binding region was detected as were possible binding sites for DnaA and IHF proteins. Deletion analysis showed the minimal replicon to be a 1675-bp fragment containing the incomplete ORF plus 536 bp upstream. The predicted ORF protein of >48 kDa was basic and rich in glutamate + glutamine (16%). There was no significant amino acid similarity to any gene, nor were there any obvious motifs present in the ORF. The data suggest that this is a theta replicon with an expressed rep gene required for replication. The replicon contains its iterons within the gene and has no homology to reported replicons. It is the first characterization of a B. megaterium replicon.

Amino Acid Sequence↗
PubMed1997

Replicon-helper systems from attenuated Venezuelan equine encephalitis virus: expression of heterologous genes in vitro and immunization against heterologous pathogens in vivo.

A replicon vaccine vector system was developed from an attenuated strain of Venezuelan equine encephalitis virus (VEE). The replicon RNA consists of the cis-acting 5' and 3' ends of the VEE genome, the complete nonstructural protein gene region, and the subgenomic 26S promoter. The genes encoding the VEE structural proteins were replaced with the influenza virus hemagglutinin (HA) or the Lassa virus nucleocapsid (N) gene, and upon transfection into eukaryotic cells by electroporation, these replicon RNAs directed the efficient, high-level synthesis of the HA or N proteins. For packaging of replicon RNAs into VEE replicon particles (VRP), the VEE capsid and glycoproteins were supplied in trans by expression from helper RNA(s) coelectroporated with the replicon. A number of different helper constructs, expressing the VEE structural proteins from a single or two separate helper RNAs, were derived from attenuated VEE strains Regeneration of infectious virus was not detected when replicons were packaged using a bipartite helper system encoding the VEE capsid protein and glycoproteins on two separate RNAs. Subcutaneous immunization of BALB/c mice with VRP expressing the influenza HA or Lassa virus N gene (HA-VRP or N-VRP, respectively) induced antibody responses to the expressed protein. After two inoculations of HA-VRP, complete protection against intranasal challenge with influenza was observed. Furthermore, sequential immunization of mice with two inoculations of N-VRP prior to two inoculations of HA-VRP induced an immune response to both HA and N equivalent to immunization with either VRP construct alone. Protection against influenza challenge was unaffected by previous N-VRP immunization. Therefore, the VEE replicon system was characterized by high-level expression of heterologous genes in cultured cells, little or no regeneration of plaque-forming virus particles, the capability for sequential immunization to multiple pathogens in the same host, and induction of protective immunity against a mucosal pathogen.

Animals↗