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PubMed1998

Noncytopathic Sindbis virus RNA vectors for heterologous gene expression.

Infection of vertebrate cells with alphaviruses normally leads to prodigious expression of virus-encoded genes and a dramatic inhibition of host protein synthesis. Recombinant Sindbis viruses and replicons have been useful as vectors for high level foreign gene expression, but the cytopathic effects of viral replication have limited their use to transient studies. We recently selected Sindbis replicons capable of persistent, noncytopathic growth in BHK cells and describe here a new generation of Sindbis vectors useful for long-term foreign gene expression based on such replicons. Foreign genes of interest as well as the dominant selectable marker puromycin N-acteyltransferase, which confers resistance to the drug puromycin, were expressed as subgenomic transcripts of noncytopathic replicons or defective-interfering genomes complemented in trans by a replicon. Based on these strategies, we developed vectors that can be initiated via either RNA or DNA transfection and analyzed them for their level and stability of foreign gene expression. Noncytopathic Sindbis vectors express reasonably high levels of protein in nearly every cell. These vectors should prove to be flexible tools for the rapid expression of heterologous genes under conditions in which cellular metabolism is not perturbed, and we illustrate their utility with a number of foreign proteins.

Animals↗
PubMed1994

Evolution of the replication regions of IncI alpha and IncFII plasmids by exchanging their replication control systems.

The basic replicons of bacterial plasmids consist of two sets of genetic systems, the replication-structural system and the replication control system. Comparison of nucleotide sequences suggested that the basic replicons of plasmids P307 (IncFI) and pMU2200 (IncZ) were generated by reciprocal recombination between ancestors of R100 (IncFII) and ColIb-P9 (IncI alpha), or vice versa. The plasmids of each pair, P307/pMU2200 and R100/ColIb-P9, are structurally unrelated to each other. Based on this information, we constructed in vitro and analyzed P307-like chimeric replicons from ColIb-P9 and R100. When the replication-structural region of ColIb-P9 was combined with the whole replication control region of R100, the resultant replicon replicated stably as R100 did. These results revealed that the basic replicons of the plasmids diverged by exchanging their replication control systems. Thus, we propose that the replication control systems of plasmids, in some cases, evolved independently of their structural systems, although these two systems work together to maintain the replication functions. We also showed that the reciprocal recombination was specified by the unique secondary structures of RNA involved in the control of expression of the genes encoding the replication initiator proteins.

Amino Acid Sequence↗
PubMed2006

A link between translation of the hepatitis C virus polyprotein and polymerase function; possible consequences for hyperphosphorylation of NS5A.

Hyperphosphorylation of NS5A is thought to play a key role in controlling hepatitis C virus (HCV) RNA replication. Using a tetracycline-regulable baculovirus delivery system to introduce non-culture-adapted HCV replicons into HepG2 cells, we found that a point mutation in the active site of the viral polymerase, NS5B, led to an increase in NS5A hyperphosphorylation. Although replicon transcripts lacking elements downstream of NS5A also had altered NS5A hyperphosphorylation, this did not explain the changes resulting from polymerase inactivation. Instead, two additional findings may be related to the link between polymerase activity and NS5A hyperphosphorylation. Firstly, we found that disabling polymerase activity, either by targeted mutation of the polymerase active site or by use of a synthetic inhibitor, stimulated translation from the replicon transcript. Secondly, when the rate of translation of non-structural proteins from replicon transcripts was reduced by use of a defective encephalomyocarditis virus internal ribosome entry site, there was a substantial decrease in NS5A hyperphosphorylation, but this was not observed when non-structural protein expression was reduced by simply lowering replicon transcript levels using tetracycline. Therefore, one possibility is that the point mutation within the active site of NS5B causes an increase in NS5A hyperphosphorylation because of an increase in translation from each viral transcript. These findings represent the first demonstration that NS5A hyperphosphorylation can be modulated without use of kinase inhibitors or mutations within non-structural proteins and, as such, provide an insight into a possible means by which HCV replication is controlled during a natural infection.

Cell Line, Tumor↗
PubMed2003

Natural genomic design in Sinorhizobium meliloti: novel genomic architectures.

The complete nucleotide sequence of the genome of Sinorhizobium meliloti, the symbiont of alfalfa, was reported in 2001 by an international consortium of laboratories. The genome comprises a chromosome of 3.65 megabases (Mb) and two megaplasmids, pSymA and pSymB, of 1.35 Mb and 1.68 Mb, respectively. Based on the nucleotide sequence of the whole genome, we designed a pathway of consecutive rearrangements leading to novel genomic architectures. In a first step we obtained derivative strains containing two replicons; in a second step we obtained a strain containing the genetic information in one single replicon of 6.68 MB. From this last architecture we isolated revertants containing two replicons, and from these we could return to the original architecture showing the three replicons. We found that the relative frequency of excision of cointegrated replicons is higher at the site used for the cointegration than at other sites. This might conciliate two apparently opposed facts: the highly dynamic state of genomic architecture in S. meliloti and the common observation that different isolates and derived cellular clones of S. meliloti usually present the architecture of one chromosome and two distinct megaplasmids. Different aspects that must be considered to obtain full advantage of the strategy of natural genomic design are discussed.

DNA Transposable Elements↗
PubMed1995

A physical genome map of the Burkholderia cepacia type strain.

Burkholderia cepacia (basonym Pseudomonas cepacia), the type species of the new genus Burkholderia, is of interest, not only because of its broad catabolic capacity and its ability to antagonize soil-borne plant pathogens, but also because of its causative role in infections in man, which are particularly evident in patients with cystic fibrosis. A physical map of the 8.1 Mb genome of the B. cepacia type-strain ATCC 25416 was constructed by applying two-dimensional pulsed-field gel electrophoresis techniques. Placed onto the macrorestriction map were 38 SpeI, 11 SwaI, 11 PacI, 11 PmeI and six I-CeuI sites, resulting in an average resolution of 105 kbp. Random single-hit linearization by irradiation and restriction mapping uncovered the presence of four circular replicons of 3.65 Mb, 3.17 Mb, 1.07 Mb and 200 kbp in size. The largest replicon harbours four rrn operons while the other two Megabase-size replicons each contain a single rrn operon, suggesting that the genome has three chromosomes and a large plasmid. Within the beta subdivision of proteobacteria, the existence of multiple replicons is not confined to B. cepacia. The phylogenetically related species Burkholderia glumae, Burkholderia pickettii, Burkholderia solanacearum, Alcaligenes eutrophus and the so far unassigned Pseudomonas glathei were also found to harbour more than one Megabase-size replicon.

Base Sequence↗
PubMed2005

Mutations conferring resistance to a hepatitis C virus (HCV) RNA-dependent RNA polymerase inhibitor alone or in combination with an HCV serine protease inhibitor in vitro.

Compounds A-782759 (an N-1-aza-4-hydroxyquinolone benzothiadiazine) and BILN-2061 are specific anti-hepatitis C virus (HCV) agents that inhibit the RNA-dependent RNA polymerase and the NS3 serine protease, respectively. Both compounds display potent activity against HCV replicons in tissue culture. In order to characterize the development of resistance to these anti-HCV agents, HCV subgenomic 1b-N replicon cells were cultured with A-782759 alone or in combination with BILN-2061 at concentrations 10 times above their corresponding 50% inhibitory concentrations in the presence of neomycin. Single substitutions in the NS5B polymerase gene (H95Q, N411S, M414L, M414T, or Y448H) resulted in substantial decreases in susceptibility to A-782759. Similarly, replicons containing mutations in the NS5B polymerase gene (M414L or M414T), together with single mutations in the NS3 protease gene (A156V or D168V), conferred high levels of resistance to both A-782759 and BILN-2061. However, the A-782759-resistant mutants remained susceptible to nucleoside and two other classes of nonnucleoside NS5B polymerase inhibitors, as well as interferon. In addition, we found that the frequency of replicons resistant to both compounds was significantly lower than the frequency of resistance to the single compound. Furthermore, the dually resistant mutants displayed significantly reduced replication capacities compared to the wild-type replicon. These findings provide strategic guidance for the future treatment of HCV infection.

Binding Sites↗
PubMed2005

Replication fitness and NS5B drug sensitivity of diverse hepatitis C virus isolates characterized by using a transient replication assay.

The innate genetic variability characteristic of chronic hepatitis C virus (HCV) infection makes drug resistance a concern in the clinical development of HCV inhibitors. To address this, a transient replication assay was developed to evaluate the replication fitness and the drug sensitivity of NS5B sequences isolated from the sera of patients with chronic HCV infection. This novel assay directly compares replication between NS5B isolates, thus bypassing the potential sequence and metabolic differences which may arise with independent replicon cell lines. Patient-derived NS5B sequences were similar to those of the established HCV genotypes, but isolates from each patient shared genetic variability specific to that patient, with additional genetic variability observed across the individual isolates. Every sample provided functional NS5B isolates which supported subgenomic replication, frequently to levels comparable to that of laboratory-optimized replicons. All isolates were equivalently sensitive to an active-site nucleoside inhibitor, but the sensitivities to a panel of nonnucleoside inhibitors which targeted three distinct sites on NS5B varied among the isolates. In con1, the original laboratory-optimized replicon, the NS5B S282T substitution confers resistance to the nucleoside inhibitor but impairs replication. This substitution was engineered into both genotype 1a and genotype 1b isolates. Replication was severely debilitated, demonstrating that no compensatory residues were encoded within these genetically diverse sequences to increase the replication fitness of the mutated replicons. This work describes a transient replicon-based assay that can support the clinical development of compounds which target NS5B and demonstrates its utility by examining several patient-derived NS5B isolates for replication fitness and differential sensitivity to NS5B inhibitors.

Animals↗
PubMed2001

Candidate vaccine against botulinum neurotoxin serotype A derived from a Venezuelan equine encephalitis virus vector system.

A candidate vaccine against botulinum neurotoxin serotype A (BoNT/A) was developed by using a Venezuelan equine encephalitis (VEE) virus replicon vector. This vaccine vector is composed of a self-replicating RNA containing all of the VEE nonstructural genes and cis-acting elements and also a heterologous immunogen gene placed downstream of the subgenomic 26S promoter in place of the viral structural genes. In this study, the nontoxic 50-kDa carboxy-terminal fragment (H(C)) of the BoNT/A heavy chain was cloned into the replicon vector (H(C)-replicon). Cotransfection of BHK cells in vitro with the H(C)-replicon and two helper RNA molecules, the latter encoding all of the VEE structural proteins, resulted in the assembly and release of propagation-deficient, H(C) VEE replicon particles (H(C)-VRP). Cells infected with H(C)-VRP efficiently expressed this protein when analyzed by either immunofluorescence or by Western blot. To evaluate the immunogenicity of H(C)-VRP, mice were vaccinated with various doses of H(C)-VRP at different intervals. Mice inoculated subcutaneously with H(C)-VRP were protected from an intraperitoneal challenge of up to 100,000 50% lethal dose units of BoNT/A. Protection correlated directly with serum enzyme-linked immunosorbent assay titers to BoNT/A. The duration of the immunity achieved was tested at 6 months and at 1 year postvaccination, and mice challenged at these times remained refractory to challenge with BoNT/A.

Animals↗
PubMed1993

Presence of one linear and one circular chromosome in the Agrobacterium tumefaciens C58 genome.

Analysis of the entire Agrobacterium tumefaciens C58 genome by pulsed-field gel electrophoresis (PFGE) reveals four replicons: two large molecules of 3,000 and 2,100 kb, the 450-kb cryptic plasmid, and the 200-kb Ti plasmid. Digestion by PacI or SwaI generated 12 or 14 fragments, respectively. The two megabase-sized replicons, used as probes, hybridize with different restriction fragments, showing that these replicons are two independent genetic entities. A 16S rRNA probe and genes encoding functions essential to the metabolism of the organism were found to hybridize with both replicons, suggesting their chromosomal nature. In PFGE, megabase-sized circular DNA does not enter the gel. The 2.1-Mb chromosome always generated an intense band, while the 3-Mb band was barely visible. After linearization of the DNA by X-irradiation, the intensity of the 3-Mb band increased while that of the 2.1-Mb remained constant. This suggests that the 3-Mb chromosome is circular and that the 2.1-Mb chromosome is linear. To confirm this hypothesis, genomic DNA, trapped in an agarose plug, was first submitted to PFGE to remove any linear DNA present. The plug was then recovered, and the remaining DNA was digested with either PacI or SwaI and then separated by PFGE. The fragments corresponding to the small chromosome were found to be absent, while those corresponding to the circular replicon remained, further proof of the linear nature of the 2.1-Mb chromosome.

Agrobacterium tumefaciens↗
PubMed2002

Dynamics of genome architecture in Rhizobium sp. strain NGR234.

Bacterial genomes are usually partitioned in several replicons, which are dynamic structures prone to mutation and genomic rearrangements, thus contributing to genome evolution. Nevertheless, much remains to be learned about the origins and dynamics of the formation of bacterial alternative genomic states and their possible biological consequences. To address these issues, we have studied the dynamics of the genome architecture in Rhizobium sp. strain NGR234 and analyzed its biological significance. NGR234 genome consists of three replicons: the symbiotic plasmid pNGR234a (536,165 bp), the megaplasmid pNGR234b (>2,000 kb), and the chromosome (>3,700 kb). Here we report that genome analyses of cell siblings showed the occurrence of large-scale DNA rearrangements consisting of cointegrations and excisions between the three replicons. As a result, four new genomic architectures have emerged. Three consisted of the cointegrates between two replicons: chromosome-pNGR234a, chromosome-pNGR234b, and pNGR234a-pNGR234b. The other consisted of a cointegrate of the three replicons (chromosome-pNGR234a-pNGR234b). Cointegration and excision of pNGR234a with either the chromosome or pNGR234b were studied and found to proceed via a Campbell-type mechanism, mediated by insertion sequence elements. We provide evidence showing that changes in the genome architecture did not alter the growth and symbiotic proficiency of Rhizobium derivatives.

DNA Transposable Elements↗
PubMed1991

Cloning, sequencing, and functional analysis of a Marek's disease virus origin of DNA replication.

Previously, we isolated a replicon from a defective Marek's disease virus (MDV), analogous to defective herpes simplex viruses (amplicons). Defective viruses contain cis-acting elements required for DNA synthesis and virus propagation such as an origin of DNA replication and a packaging-cleavage signal site. In this report, the MDV replicon was utilized to locate an origin of MDV DNA replication. A comparison of MDV replicon sequences with other herpesvirus replication origin sequences revealed a 90-bp sequence containing 72% identity to the lytic origin (oris) of herpes simplex virus type 1. This 90-bp sequence displayed no similarity to betaherpesvirus or gammaherpesvirus replication origins. The 90-bp sequence is arranged as an imperfect palindrome centered around an A+T-rich region. This sequence also contains a 9-bp motif (5'CGTTCGCAC3') highly conserved in alphaherpesvirus replication origins. To test functionality of the 90-bp putative MDV replication origin, we conducted DpnI replication assays with subclones generated from the 4-kbp MDV replicon. A 700-bp MDV replicon subfragment containing the 90-bp putative MDV replication origin sequence is capable of replicating in chicken embryo fibroblast cells cotransfected with helper virus DNA. In conclusion, we identified a functional origin of DNA replication in MDV. Similarity of MDV origin sequences to those of alphaherpesviruses supports the current contention that MDV is more closely related to alphaherpesviruses than to gammaherpesviruses.

Animals↗
PubMed2001

Enhancement of hepatitis C virus RNA replication by cell culture-adaptive mutations.

Studies of the Hepatitis C virus (HCV) replication cycle have been made possible with the development of subgenomic selectable RNAs that replicate autonomously in cultured cells. In these replicons the region encoding the HCV structural proteins was replaced by the neomycin phosphotransferase gene, allowing the selection of transfected cells that support high-level replication of these RNAs. Subsequent analyses revealed that, within selected cells, HCV RNAs had acquired adaptive mutations that increased the efficiency of colony formation by an unknown mechanism. Using a panel of replicons that differed in their degrees of cell culture adaptation, in this study we show that adaptive mutations enhance RNA replication. Transient-transfection assays that did not require selection of transfected cells demonstrated a clear correlation between the level of adaptation and RNA replication. The highest replication level was found with an adapted replicon carrying two amino acid substitutions located in NS3 and one in NS5A that acted synergistically. In contrast, the nonadapted RNA replicated only transiently and at a low level. The correlation between the efficiency of colony formation and RNA replication was corroborated with replicons in which the selectable marker gene was replaced by the gene encoding firefly luciferase. Upon transfection of naive Huh-7 cells, the levels of luciferase activity directly reflected the replication efficiencies of the various replicon RNAs. These results show that cell culture-adaptive mutations enhance HCV RNA replication.

Adaptation, Physiological↗
PubMed2003

Viral and cellular determinants of hepatitis C virus RNA replication in cell culture.

Studies on the replication of hepatitis C virus (HCV) have been facilitated by the development of selectable subgenomic replicons replicating in the human hepatoma cell line Huh-7 at a surprisingly high level. Analysis of the replicon population in selected cells revealed the occurrence of cell culture-adaptive mutations that enhance RNA replication substantially. To gain a better understanding of HCV cell culture adaptation, we characterized conserved mutations identified by sequence analysis of 26 independent replicon cell clones for their effect on RNA replication. Mutations enhancing replication were found in nearly every nonstructural (NS) protein, and they could be subdivided into at least two groups by their effect on replication efficiency and cooperativity: (i). mutations in NS3 with a low impact on replication but that enhanced replication cooperatively when combined with highly adaptive mutations and (ii). mutations in NS4B, -5A, and -5B, causing a strong increase in replication but being incompatible with each other. In addition to adaptive mutations, we found that the host cell plays an equally important role for efficient RNA replication. We tested several passages of the same Huh-7 cell line and found up to 100-fold differences in their ability to support replicon amplification. These differences were not due to variations in internal ribosome entry site-dependent translation or RNA degradation. In a search for cellular factor(s) that might be responsible for the different levels of permissiveness of Huh-7 cells, we found that replication efficiency decreased with increasing amounts of transfected replicon RNA, indicating that viral RNA or proteins are cytopathic or that host cell factors in Huh-7 cells limit RNA amplification. In summary, these data show that the efficiency of HCV replication in cell culture is determined both by adaptation of the viral sequence and by the host cell itself.

Amino Acid Substitution↗
PubMed2003

In vitro selection and characterization of hepatitis C virus serine protease variants resistant to an active-site peptide inhibitor.

The hepatitis C virus (HCV) serine protease is necessary for viral replication and represents a valid target for developing new therapies for HCV infection. Potent and selective inhibitors of this enzyme have been identified and shown to inhibit HCV replication in tissue culture. The optimization of these inhibitors for clinical development would greatly benefit from in vitro systems for the identification and the study of resistant variants. We report the use HCV subgenomic replicons to isolate and characterize mutants resistant to a protease inhibitor. Taking advantage of the replicons' ability to transduce resistance to neomycin, we selected replicons with decreased sensitivity to the inhibitor by culturing the host cells in the presence of the inhibitor and neomycin. The selected replicons replicated to the same extent as those in parental cells. Sequence analysis followed by transfection of replicons containing isolated mutations revealed that resistance was mediated by amino acid substitutions in the protease. These results were confirmed by in vitro experiments with mutant enzymes and by modeling the inhibitor in the three-dimensional structure of the protease.

Binding Sites↗
PubMed2004

Rubella virus capsid protein modulates viral genome replication and virus infectivity.

The structural proteins (SP) of the Togaviridae can be deleted in defective interfering RNAs. The dispensability of viral SP has allowed construction of noninfectious viral expression vectors and replicons from viruses of the Alphavirus and Rubivirus genera. Nevertheless, in this study, we found that the SP of rubella virus (RUB) could enhance expression of reporter genes from RUB replicons in trans. SP enhancement required capsid protein (CP) expression and was not due to RNA-RNA recombination. Accumulation of minus- and plus-strand RNAs from replicons was observed in the presence of SP, suggesting that SP specifically affects RNA synthesis. By using replicons containing an antibiotic resistance gene, we found 2- to 50-fold increases in the number of cells surviving selection in the presence of SP. The increases depended significantly on the amount of transfected RNA. Small amounts of RNA or templates that replicated inefficiently showed more enhancement. The infectivity of infectious RNA was increased by at least 10-fold in cells expressing CP. Moreover, virus infectivity was greatly enhanced in such cells. In other cells that expressed higher levels of CP, RNA replication of replicons was inhibited. Thus, depending on conditions, CP can markedly enhance or inhibit RUB RNA replication.

Animals↗
PubMed1981

Initiation points for cellular deoxyribonucleic acid replication in human lymphoid cells converted by Epstein-Barr virus.

Replicon size was estimated in two Epstein-Barr virus (EBV)-negative human lymphoma lines, BJAB and Ramos, and four EBV-positive lines derived from the former ones by infection (conversion) with two viral strains, B95-8 and P3HR-1. Logarithmic cultures were pulse-labeled with [3H]thymidine, and the deoxyribonucleic acid was spread on microscopic slides and autoradiographed by the method of Huberman and Riggs. After developing, replication forks were visualized as silver grain tracks on the autoradiograms. Average replicon size was estimated by scoring the number of replication forks per constant length of deoxyribonucleic acid and by measuring distances between centers of adjacent tracks, followed by detailed statistical analyses. Three of the four EBV-converted cell lines, BJAB/B95-8, Ra/B95-8, and Ra/HRIK, were found to have significantly shorter replicons (41, 21, 54% shorter, respectively), i.e., more initiation points, than their EBV-negative parents. BJAB/HRIK had replicons which were only slightly shorter (11%) than those of BJAB. However, analysis of track length demonstrated that extensive track fusion occurred during the labeling of BJAB/ HRIK, implying that its true average replicon size is shorter than the observed value. The results indicate that in analogy to simian virus 40, EBV activates new initiation points for cellular DNA replication in EBV-transformed cells.

Autoradiography↗
PubMed1991

Relationship of eukaryotic DNA replication to committed gene expression: general theory for gene control.

The historic arguments for the participation of eukaryotic DNA replication in the control of gene expression are reconsidered along with more recent evidence. An earlier view in which gene commitment was achieved with stable chromatin structures which required DNA replication to reset expression potential (D. D. Brown, Cell 37:359-365, 1984) is further considered. The participation of nonspecific stable repressor of gene activity (histones and other chromatin proteins), as previously proposed, is reexamined. The possible function of positive trans-acting factors is now further developed by considering evidence from DNA virus models. It is proposed that these positive factors act to control the initiation of replicon-specific DNA synthesis in the S phase (early or late replication timing). Stable chromatin assembles during replication into potentially active (early S) or inactive (late S) states with prevailing trans-acting factors (early) or repressing factors (late) and may asymmetrically commit daughter templates. This suggests logical schemes for programming differentiation based on replicons and trans-acting initiators. This proposal requires that DNA replication precede major changes in gene commitment. Prior evidence against a role for DNA replication during terminal differentiation is reexamined along with other results from terminal differentiation of lower eukaryotes. This leads to a proposal that DNA replication may yet underlie terminal gene commitment, but that for it to do so there must exist two distinct modes of replication control. In one mode (mitotic replication) replicon initiation is tightly linked to the cell cycle, whereas the other mode (terminal replication) initiation is not cell cycle restricted, is replicon specific, and can lead to a terminally differentiated state. Aberrant control of mitotic and terminal modes of DNA replication may underlie the transformed state. Implications of a replicon basis for chromatin structure-function and the evolution of metazoan organisms are considered.

Animals↗
PubMed1986

Replication from one of the three origins of the plasmid R6K requires coupled expression of two plasmid-encoded proteins.

The minimal beta-replicon of plasmid R6K contains an open reading frame for a 151-amino acid protein in addition to the seven 22-base pair direct nucleotide sequence repeats, the structural gene (pir) for the pi initiation protein, and the beta-origin sequence that have been shown to be required for replication activity. In this work, a site-specific mutation by linker insertion in this putative coding sequence, designated bis, resulted in a nonfunctional beta-replicon. The nonfunctional beta-replicon was complementable in trans and the protein coded by the bis sequence was detected in an immunoblot assay as a hybrid product from a bis-lac z fused gene. The bis gene is not required for a functional alpha or gamma origin replication origin of R6K. A site-specific mutation in the upstream pir gene was shown to lead to a loss of synthesis of the bis product and inactivation of the beta-replicon. Trans-complementation of this mutation for beta-replicon activity required the wild-type sequence of the pir gene joined to the intact bis sequence. These results indicate that the bis product is required for activity specifically of the beta-origin, and its synthesis is coupled in cis to the expression of pi protein from an unaltered pir gene.

Bacterial Proteins↗