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At least 973 records · Page 54Linked to original sources
PubMed2026

Identification and characterization of anti-chikungunya virus compounds using a biosafe toolkit.

Chikungunya virus (CHIKV) is a re-emerging mosquito-borne alphavirus for which no specific antiviral therapy is currently available. During the large outbreak in Foshan, Guangdong Province, China, in July 2025, CHIKV rapidly spread to neighboring regions and caused more than 16,000 confirmed cases. In this study, the predominant outbreak strain of CHIKV was selected as the reference sequence to establish a panel of complementary biosafe tools for antiviral compound screening and mechanistic investigation. A virus replicon particle (VRP) system for CHIKV was first constructed and applied to compound library screening, resulting in the identification of three candidate antiviral compounds: MDL-12330A, bazedoxifene acetate, and anidulafungin. To further validate their antiviral activities and investigate their potential mechanisms, CHIKV functional evaluation systems were subsequently established, including vesicular stomatitis virus (VSV)- and murine leukemia virus (MLV)-based pseudovirus systems for viral entry, a replicon RNA system for post-entry replication-associated processes, a replication-defective nsP4 mutant replicon RNA system for primary translation, and a virus-like particle (VLP) system for viral particle assembly and budding assessment. Using these complementary systems, we systematically evaluated the antiviral profiles of the three candidate compounds across multiple stages of the CHIKV life cycle. This analysis revealed distinct stage-specific inhibitory patterns and provided insights into their potential antiviral mechanisms, which warrant validation using authentic CHIKV infection to assess their translational potential.

Chikungunya virus↗
PubMed1997

Mapping of replication origins and termination sites in the Duchenne muscular dystrophy gene.

The replication structure of the human dystrophin gene in cultured masculine erythroleukemia cells (line HEL 92.1.7) was studied using the replication direction assay. This gene is organized into at least six replicons ranging in size from 170 to more than 500 kb. One of the replicon junctions (sites of replication termination) was mapped to intron 44, i.e., roughly in the same area where the major recombination hot spot is located. A replicon junction was also found between the muscle and the brain promoters. The two replicons mapped in the present study are highly asymmetric, as the distances covered by the replication forks moving in opposite directions from the same origin differ by more than threefold.

Dystrophin↗
PubMed1994

Differential replication of plasmids during stringent and relaxed response of Escherichia coli.

Stringent control of DNA replication has been demonstrated for a few replicons like oriC, pBR322, and plasmids derived from coliphage lambda. In this study we investigated the replication of other plasmids harboring a well defined origin (orip15A, oripSC101, and oriRK2 = oriV) in amino-acid-starved stringent and relaxed strains of Escherichia coli. We found differential replication of plasmids during stringent and relaxed response. Inhibition of DNA synthesis or amplification of plasmid DNA in amino acid-starved relA+ and relA- cells depends on the kind of replicon and, surprisingly, on the nature of deprived amino acid. We conclude that there are no general rules for stringent control of DNA replication and each replicon must be considered separately. There are, however, possible explanations for the differences shown between replicons in their response to stringent and relaxed conditions.

Amino Acids↗
PubMed1989

Genetic structure, function and regulation of the transposable element IS21.

The IncP plasmid R68.45 and other plasmids carrying tandem repeats of the insertion sequence IS21 [= (IS21)2] produce replicon fusions via transposition at high frequencies in Escherichia coli and other gram-negative bacteria, whereas plasmids with a single IS21 copy, e.g. R68, give replicon fusions rarely. The 2131 bp nucleotide sequence of IS21 was determined; at the ends there were 11 bp inverted repeats with one mismatch. Two adjacent open reading frames, istA and istB, were located on one DNA strand of IS21. In E. coli maxicells, polypeptides of 46 kDa (the istA gene product) and 30 kDa (the istB gene product) were expressed by (IS21)2 plasmids, but not by IS21 plasmids. Genetic analysis of (IS21)2 plasmids indicates that the IS21-IS21 junctions form a promoter, which initiates transcription of the istAB operon in one of the two IS21 elements. A single IS21 element fused to an inducible external tac promoter expressed both proteins after induction, but did not promote effective replicon fusion, unless an IS21-IS21 junction (the preferred site for IS21 transposase action) was also present on the plasmid carrying the tac-IS21 construct. The sequences located between the IS21 elements in (IS21)2, 3 bp in R68.45 or 2 bp in pME28, were not recovered in the replicon fusion products. Homologous recombination between the directly oriented IS21 elements in the fusion products led to plasmids with a single IS21 insertion. Analysis of the latter showed that IS21 had a low, but not totally random specificity of insertion and created target duplications of 4 bp (occasionally 5 bp).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2002

Dinucleotide compositional analysis of Sinorhizobium meliloti using the genome signature: distinguishing chromosomes and plasmids.

The symbiotic N(2)-fixing alpha-proteobacterium Sinorhizobium meliloti has three replicons: a circular chromosome (3.7 Mb) and two smaller replicons, pSymA (1.4 Mb) and pSymB (1.7 Mb). Sequence analysis has revealed that an essential gene is carried on pSymB, which brings into question whether pSymB should be considered a chromosome or a plasmid. Based on the criterion that essential genes define a chromosome, several species have been shown to have multiple chromosomes. Many of these species are part of the alpha subdivision of the Proteobacteria family. Here, additional justification is presented for designating the pSymB replicon as a chromosome. It is shown that chromosomes within a species share a more similar dinucleotide composition, or genome signature, than plasmids do with the host chromosome(s). Dinucleotide signatures were determined for each of the S. meliloti replicons, and, consistent with the suggestion that pSymB is a chromosome, it is shown that the pSymB signature more closely resembles that of the S. meliloti chromosome, while the pSymA signature is typical of other alpha-proteobacterial plasmids.

Genome, Bacterial↗
PubMed1988

DNA fiber replication during a morphogenetic switch in the shoot meristematic cells of a higher plant.

Chromosomal DNA fiber replication was investigated in the shoot meristem of mustard plants during the morphogenetic transition from the leaf-forming (vegetative) to the flower-forming (evoked) condition. The replicon size, determined using the modal class, was 15 micron in the vegetative meristem and shifted to 7.5 micron in the evoked meristem. The average fork rate was 1.05 micron.h-1 in the vegetative meristem and only slightly increased to 1.55 micron.h-1 during the morphogenetic switch. Replicon activation was asynchronous but the pattern of activation of replicons was the same in both kinds of meristems. Thus the shortening of the S phase at the floral transition in mustard was essentially achieved by an increase of the number of replicon origins per unit length of DNA. These results are similar to those obtained in amphibian and Drosophila embryogenesis.

DNA Replication↗
PubMed1983

Mode of inhibition of DNA replication in neocarzinostatin-treated HeLa cells.

The effect of antitumor antibiotic neocarzinostatin on DNA replication in HeLa cells was studied by pulse-labeling of DNA with [3H]thymidine and sedimentation analysis of the DNA with alkaline sucrose gradients. The drug, which produced DNA damage, primarily inhibited the replicon initiation in the cells at low doses (less than or equal to 0.1 microgram/ml), and at high doses (greater than or equal to 0.5 microgram/ml) inhibited the DNA chain elongation. An analysis of the number of single-strand breaks of parental DNA, induced by neocarzinostatin, indicated that inhibition of the initiation occurred with introduction of single-strand breaks of less than 1.5 . 10(4)/cell, while inhibition of the elongation occurred with introduction of single-strand breaks of more than 7.5 . 10(4)/cell. Assuming that the relative molecular mass of DNA/HeLa cell was about 10(13) Da, the target size of DNA for inhibition of replicon initiation was calculated to be about 10(9) Da, such being close to an average size of loop DNA in the cell and for inhibition of chain elongation, 1-2 . 10(8) Da which was of the same order of magnitude as the size of replicons. Recovery of inhibited DNA replication by neocarzinostatin occurred during post-incubation of the cells and seemed to correlate with the degree of rejoining of the single-strand breaks of parental DNA. Caffeine and theophylline enhanced the recovery of the inhibited replicon initiation, but did not aid in the repair of the breaks in parental DNA.

Antibiotics, Antineoplastic↗
PubMedDate not supplied

Inhibition and recovery of DNA synthesis in human cells after exposure to ultraviolet light.

The inhibition of DNA synthesis in normal human cells by UV is a complex function of fluence because it has several causes. At low fluences, inhibition of replicon initiation is most important. This is made clear by the fact that it occurs to a lesser degree in cells from patients with ataxia telangiectasia (AT). Assuming that only leading strand synthesis is blocked by UV-induced lesions, single lesions between replicons in parental strands for leading strand synthesis inhibit DNA synthesis by acting as temporary blocks until they are replicated by extension of the lagging strand of the adjacent replicon. A more severe inhibition occurs when two lesions are induced between adjacent growing replicons, because one in four possible configurations may result in a long-lived unreplicated region (LLUR). In the absence of excision repair, these may eventually be replicated by activation of an otherwise unused origin within the LLUR. The frequency of LLURs increases steeply with fluence. Activation of normally unused origins to replicate LLURs may facilitate recovery from inhibition of DNA synthesis, but repair of lesions is probably more important. In excision-repair-defective cells, an LLUR without an origin to initiate its replication may be a lethal lesion.

Ataxia Telangiectasia↗
PubMed2005

Role of RNA structures present at the 3'UTR of dengue virus on translation, RNA synthesis, and viral replication.

We have developed a dengue virus replicon system that can be used to discriminate between translation and RNA replication. Using this system, we analyzed the functional role of well-defined RNA elements present at the 3'UTR of dengue virus in mammalian and mosquito cells. Our results show that deletion of individual domains of the 3'UTR did not significantly affect translation of the input RNA but seriously compromised or abolished RNA synthesis. We demonstrated that complementarity between sequences present at the 5' and 3' ends of the genome is essential for dengue virus RNA synthesis, while deletion of domains A2 or A3 within the 3'UTR resulted in replicons with decreased RNA amplification. We also characterized the vaccine candidate rDEN2Delta30 in the replicon system and found that viral attenuation is caused by inefficient RNA synthesis. Furthermore, using both the replicon system and recombinant viruses, we identified an RNA region of the 3'UTR that enhances dengue virus replication in BHK cells while is dispensable in mosquito cells.

3' Untranslated Regions↗
PubMed2006

C-E1 fusion protein synthesized by rubella virus DI RNAs maintained during serial passage.

Rubella virus (RUB) replicons are derivatives of the RUB infectious cDNA clone that retain the nonstructural open reading frame (NS-ORF) that encodes the replicase proteins but not the structural protein ORF (SP-ORF) that encodes the virion proteins. RUB defective interfering (DI) RNAs contain deletions within the SP-ORF and thus resemble replicons. DI RNAs often retain the 5' end of the capsid protein (C) gene that has been shown to modulate virus-specific RNA synthesis. However, when replicons either with or without the C gene were passaged serially in the presence of wt RUB as a source of the virion proteins, it was found that neither replicon was maintained and DI RNAs were generated. The majority DI RNA species contained in-frame deletions in the SP-ORF leading to a fusion between the 5' end of the C gene and the 3' end of the E1 glycoprotein gene. DI infectious cDNA clones were constructed and transcripts from these DI infectious cDNA clones were maintained during serial passage with wt RUB. The C-E1 fusion protein encoded by the DI RNAs was synthesized and was required for maintenance of the DI RNA during serial passage. This is the first report of a functional novel gene product resulting from deletion during DI RNA generation. Thus far, the role of the C-E1 fusion protein in maintenance of DI RNAs during serial passage remained elusive as it was found that the fusion protein diminished rather than enhanced DI RNA synthesis and was not incorporated into virus particles.

Animals↗
PubMed2003

Tissue culture and animal models for hepatitis C virus.

In recent years, significant advances have been achieved both in the development of animal- and tissue-culture models for HCV. Among all the new systems, the small animal model based on transgenic mice with chimeric mouse-human livers and the replicon system will presumably have the most profound impact on future HCV research. Yet, in spite of this progress, much more work will be required to optimizse both systems. In case of the mouse model, breeding homozygous Alb-uPa animals is difficult because of the toxicity of the transgene, and the transplantation of primary human hepatocytes into mice a few days after birth is technically challenging. These are immunodeficient, and, therefore, it will be desirable to furnish them with components of the human immune system in order to expand the applicability of this in vivo model to questions related to pathogenesis. Advances in cryopreservation techniques are urgently needed, moreover, as this would improve the availability of primary hepatocytes and in turn also the accessibility of this small animal model. As regards the replicon system, a number of open questions remain that will hopefully be answered by future research. Why, for instance, has replication in cell culture so far been achieved only with genotype 1b isolates, whereas an isolate with proven infectivity derived from genotype 1a failed to replicate in Huh-7 cells? And why can replicons so far be propagated only in this particular cell line? Is this attributable to the lack of certain inhibitory factors, or the presence of specific activators? What are the mechanisms underlying cell-culture adaptation. and what determines whether a certain Huh-7 cell replicates HCV RNA more efficiently? Finally, the replicon system may also lead the way to the development of systems for efficient virus production in cell culture, and ultimately also a permissive cell line. These developments would at last allow us to model the complete viral life cycle, something researchers have been struggling with ever since the first identification of HCV.

Animals↗
PubMed2004

Phosphorylation of hepatitis C virus nonstructural protein 5A modulates its protein interactions and viral RNA replication.

The study of the hepatitis C virus (HCV) has been hindered by the lack of in vitro model systems. The recent development of HCV subgenomic RNA replicons has permitted the study of viral RNA replication in cell culture; however, the requirements for efficient replication of replicons in this system are poorly understood. Many viral isolates do not function as replicons and most require conserved changes, termed adaptive mutations, to replicate efficiently. In this report, we focus on the HCV nonstructural protein 5A (NS5A), a frequent locus for adaptive mutation. We found the interaction between NS5A and human vesicle-associated membrane protein-associated protein A (hVAP-A), a cellular target N-ethylmaleimide-sensitive factor attachment protein receptor, to be required for efficient RNA replication: NS5A mutations that blocked interaction with hVAP-A strongly reduced HCV RNA replication. Further analyses revealed an inverse correlation between NS5A phosphorylation and hVAP-A interaction. A subset of the previously identified adaptive mutations suppressed NS5A hyperphosphorylation and promoted hVAP-A binding. Our results support a model in which NS5A hyperphosphorylation disrupts interaction with hVAP-A and negatively regulates viral RNA replication, suggesting that replicon-adaptive mutations act by preventing the phosphorylation-dependent dissociation of the RNA replication complex.

Genotype↗
PubMed1989

Inhibition and recovery of semiconservative DNA synthesis in normal and solar UV sensitive ICR 2A frog cell lines following the induction of non-dimer DNA damage by sunlamp UV greater than 315 nm.

Cultures of the solar UV-sensitive cell lines, DRP 36 and DRP 153, and of the parental ICR 2A cell line, were exposed to 150 kJ/m2 of sunlamp UV greater than 315 nm plus photoreactivating light. This treatment resulted in the induction primarily of non-dimer DNA damage. Following either a 0, 3, 6, 12 or 24 h incubation, the cultures were pulse-labelled with [3H]thymidine, and the synthesis of different size classes of replicon intermediates measured using the alkaline step elution assay. For all three cell lines tested, an immediate depression of low molecular weight DNA synthesis was observed. This was followed by an inhibition of all size classes of replicon intermediates. Within 12 h following irradiation, recovery of DNA synthesis was observed, which was generally most apparent for low molecular weight DNA. The ICR 2A cells exhibited a nearly full recovery in all size classes of DNA synthesized by 24 h. However, a much smaller recovery of DNA synthesis was detected for the DRP 36 and DRP 153 cultures. This continued inhibition was primarily in the synthesis of full replicon size DNA, and was most pronounced for the DRP 36 cells. Hence, it appears that replicon chain elongation continues to be inhibited in these solar UV-sensitive cell lines long after irradiation.

Animals↗
PubMed1978

Enrichment and visualization of small replication units from cultured mammalian cells.

DNA from cultured Chinese hamster cells has been fractionated to yield a population of DNA enriched for replicating molecules. Molecules containing replication structures were analyzed by electron microscopy, and replicon size was estimated. The enrichment procedure takes advantage of single-stranded regions characteristic of replicating molecules, and the greater affinity of mercuric ion for single-stranded rather than native DNA. After interaction with low concentrations of HgCl2, DNA with bound mercury is separated from the bulk of the DNA by virtue of its increased buoyant density in an isopycnic Cs2SO4 gradient. When DNA from cells labeled with [3H]thymidine for 45 s is interacted with HgCl2 and banded in Cs2SO4, the DNA with the highest specific activity is found in a dense region of the gradient. The high specific activity DNA behaves kinetically like nascent DNA since the radioactivity can be chased into main band if the cells are incubated for a further 2 h in excess unlabeled thymidine. Electron microscope analysis of the DNA in the enriched fraction confirmed that it contains a substantial fraction of molecules with replication structures. The level of enrichment is about 25-fold compared to unfractionated DNA or DNA taken from the main band of the Hg++/Cs2SO4 gradient. Of the replicating molecules visualized, 85% possessed a single replication structure. All molecules with multiple replication forms contained replicon sizes less than 5 micron, ranging from 0.2 to 4.5 micron. Replicon size was determined by measuring the distance from the center of one replication structure to the center of the adjacent replication structure on the same molecule. The replicons observed in this study are far smaller than can be detected by DNA fiber autoradiography and are in the same size range as the very small replication units reported in embryonic systems.

Animals↗
PubMed1986

Effect of benzo[a]pyrene-diol-epoxide-I on growth of nascent DNA in synchronized human fibroblasts.

In logarithmically growing cultures of human fibroblasts, benzo[a]pyrene-diol-epoxide-I (BPDE-I) treatment inhibits both replicon initiation and DNA chain elongation. In this study we have focused on the effect of BPDE-I treatment on the growth of nascent DNA strands in synchronized human fibroblasts. Cells were collected at the beginning of the S phase by replating confluent cultures in the presence of aphidicolin for 24 h. At 1 h after release from aphidicolin, cells were incubated with [3H]thymidine for 10-15 min and treated with BPDE-I for 10 min. Following different periods of incubation with unlabeled thymidine, cells were lysed on top of alkaline sucrose gradients for analysis of the size distribution of nascent DNA and its average molecular weight. After a brief pulse with the radiolabeled precursor, a unimodal distribution of nascent DNA strands was observed in control cells. Upon chase this distribution was displaced to higher average molecular weights at an approximate rate of 1 X 10(6) daltons/min. Treatment with low doses of BPDE-I (less than 0.2 microM) did not decrease this rate. Indeed the nascent DNA in BPDE-I-treated cells appeared to grow faster than in control cells. This higher rate of DNA strand growth may be related to the inhibition of replicon initiation. Treatment with 0.3 microM or 0.7 microM BPDE-I, which are doses that interfere with DNA synthesis in operating replicons in asynchronous cells, also inhibited the growth of nascent DNA strands in synchronized cells by 22 and 64%, respectively. Because of the simultaneous inhibition of replicon initiation, these values are probably underestimations of the total inhibition of DNA strand growth. The use of synchronized cells in this type of study should aid in the elucidation of mechanisms of replication of carcinogen-damaged DNA.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed2006

Site-specific mutation of the interferon sensitivity-determining region (ISDR) modulates hepatitis C virus replication.

The number of amino acid substitutions in the interferon sensitivity-determining region (ISDR) in the nonstructural 5A (NS5A) gene of hepatitis C virus (HCV) is closely associated with the interferon (IFN) response and viral load. Several HCV replicon-based studies have reported that ISDR sequences had an influence on viral replication in vitro. However, it is unclear as to how different ISDR sequences affect HCV replication. Various clinically observed ISDR sequences were introduced into HCV replicons and their contribution to viral replication was investigated using a colony formation assay and/or a transient replication assay. A mapping study of the ISDR was performed to identify the amino acid positions that critically affect replication. While no colonies were formed in the colony formation assay using HCV replicons with few mutations (0, 1 and 3) in the ISDR, numerous colonies (>200) appeared when using constructs with six mutations. Introduction of various distinct ISDR sequences with multiple mutations resulted in replication enhancement in transient assays. A mapping study identified several specific sites in the ISDR that critically affected replication, including codon 2209 which, in patients, was closely associated with a strong response to IFN. ISDR sequences associated with a clinical IFN response and viral load modulated the replication of HCV replicons, suggesting the importance of the ISDR sequence in HCV infection.

Amino Acid Sequence↗
PubMed2006

Triaryl pyrazoline compound inhibits flavivirus RNA replication.

Triaryl pyrazoline {[5-(4-chloro-phenyl)-3-thiophen-2-yl-4,5-dihydro-pyrazol-1-yl]-phenyl-methanone} inhibits flavivirus infection in cell culture. The inhibitor was identified through high-throughput screening of a compound library using a luciferase-expressing West Nile (WN) virus infection assay. The compound inhibited an epidemic strain of WN virus without detectable cytotoxicity (a 50% effective concentration of 28 microM and a compound concentration of >or=300 microM required to reduce 50% cell viability). Besides WN virus, the compound also inhibited other flaviviruses (dengue, yellow fever, and St. Louis encephalitis viruses), an alphavirus (Western equine encephalitis virus), a coronavirus (mouse hepatitis virus), and a rhabdovirus (vesicular stomatitis virus). However, the compound did not suppress an orthomyxovirus (influenza virus) or a retrovirus (human immunodeficiency virus type 1). Mode-of-action analyses in WN virus showed that the compound did not inhibit viral entry or virion assembly but specifically suppressed viral RNA synthesis. To examine the mechanism of inhibition of dengue virus, we developed two replicon systems for dengue type 1 virus: (i) a stable cell line that harbored replicons containing a luciferase reporter and a neomycin phosphotransferase selection marker and (ii) a luciferase-expressing replicon that could differentiate between viral translation and RNA replication. Analyses of the compound in the dengue type 1 virus replicon systems showed that it weakly suppressed viral translation but significantly inhibited viral RNA synthesis. Overall, the results demonstrate that triaryl pyrazoline exerts a broad spectrum of antiflavivirus activity through potent inhibition of viral RNA replication. This novel inhibitor could be developed for potential treatment of flavivirus infection.

Animals↗
PubMed1996

Characterization and sequence analysis of a stable cryptic plasmid from Enterococcus faecium 226 and development of a stable cloning vector.

A small cryptic plasmid, pMBB1, isolated from Enterococcus faecium 226 was characterized. The plasmid contained an extremely stable replicon which has limited homology to the lactococcal plasmid pCI305. Sequence analysis of the replicon detected one open reading frame of 822 bp capable of encoding a 32-kDa protein. No detectable single-stranded intermediates were found for the replicon, suggesting that pMBB1 may be included in the same family as pCI305, although pCI305 exhibits a more narrow host range. A small stably maintained vector able to replicate in a variety of lactic acid bacteria, containing a large multiple cloning region, was constructed by using the pMBB1 replicon.

Amino Acid Sequence↗