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PubMed2003

An in vitro Flaviviridae replicase system capable of authentic RNA replication.

We have established an in vitro replication system for bovine viral diarrhea virus (BVDV), a surrogate for the closely-related hepatitis C virus. In an in vitro reaction, BVDV replication complexes synthesize vRNA and replicative form (RF) and replicative intermediate (RI) RNAs. Kinetic and heparin trapping experiments demonstrate the recycling of RF and RI products and the initiation of vRNA synthesis in this system. Consistent with this, quantitative hybridization reveals the asymmetric synthesis of positive and negative strand RNA products. These findings support the notion that RF serves as a template and RI as a precursor in the synthesis of vRNA. Furthermore, the antiviral activity of an NS5B inhibitor was similar in BVDV replicase and infectivity assays. Together, these results indicate that the in vitro activity of BVDV replicase complexes recapitulates RNA replication that occurs in infected cells, providing a system in which to study both mechanisms and inhibitors of Flaviviridae replication.

Animals↗
PubMed1985

Analysis of the secondary structure of the poly(C) tract in foot-and-mouth disease virus RNAs.

Sodium bisulphite modification of foot-and-mouth disease virus (FMDV) RNA in solution indicates that the majority of the poly(C) tract in the RNA is single-stranded in concordance with previous results with encephalomyocarditis virus RNA. The reaction kinetics are biphasic; 60% of the cytidylic acid in the poly(C) tract reacts like synthetic poly(C), and the remainder with the kinetics of the cytidylic acid in the rest of the RNA. The reactivity of the poly(C) tract with poly(I) indicates that it is looped out and exposed in the RNA. The deamination reaction has also been used to investigate the structure of the replicative form (RF) and replicative intermediate (RI) isolated from infected cells. Analysis by gel electrophoresis of the long RNase A- and T1-resistant oligonucleotides of RI suggests that it has five single-stranded poly(C) tracts to every one which is base-paired. Bisulphite reactivity of the poly(C) tract and gel electrophoresis of the ribonuclease-resistant oligonucleotides of RF indicate that the poly(C) is base-paired to a poly(G) tract in this molecule. The presence of a poly(G) tract in RF and RI provides unequivocal evidence that the poly(C) is replicated via poly(G) in the negative strand.

Animals↗
PubMed1967

Replication of bacteriophage ribonucleic acid: some properties of native and denatured replicative intermediate.

Purified replicative form (RF) and replicative intermediate (RI) prepared from Escherichia coli cells infected with the ribonucleic acid (RNA) bacteriophage R17 were denatured with dimethyl sulfoxide at 37 C or in aqueous solvents of low ionic strength at 97 C. Denaturation was demonstrated for RF and RI by an increase in specific infectivity and a striking change in the hyperchromicity curves after treatment. RI denaturation was also demonstrated by a shift in the buoyant density in Cs(2)SO(4) from 1.619 to the buoyant density of single-stranded R17 RNA (1.627). Analysis of the denatured RI hyperchromicity curves and the equilibrium distributions of denatured RI in Cs(2)SO(4) gradients revealed, however, a residual double-stranded component. Velocity sedimentation of denatured RI was performed, and the weight distribution of S values was calculated. From the known relation between molecular weight and S values, it was possible to transform the weight distribution into a number distribution of chain lengths. This distribution was compared with that predicted from the steady-state hypothesis for RI. Deviations from the predicted distribution may be due to the residual double-stranded component.

Centrifugation, Density Gradient↗
PubMed1979

Effect of cordycepin triphosphate on in vitro RNA synthesis by plant viral replicases.

In vitro RNA synthesis by tobacco mosaic virus and cowpea chlorotic mottle virus replicase were inhibited by cordycepin triphosphate. Inhibition could be overcome with higher concentrations of ATP in assay mixtures but not with UTP. Products synthesized in vitro by tobacco mosaic virus RNA replicase in the presence of inhibitor revealed replicative form but not replicative intermediate RNAs. These results suggest that cordycepin triphosphate competes specifically with ATP and results in premature termination of viral RNA synthesis in vitro.

Journal Article↗
PubMed1970

Nucleic acid of rubella virus and its replication in hamster kidney cells.

Ribonucleic acid (RNA) has been isolated from partially purified rubella virus preparations and fractionated by rate zonal centrifugation in sucrose density gradients. The bulk of the RNA sedimented as a sharp band with a sedimentation coefficient of 38S. Rubella virus RNA appears to be single-stranded on the basis of its sensitivity to the degrading action of ribonuclease. Fractionation by precipitation with 1 m NaCl, followed by chromatography on cellulose columns, and by rate zonal centrifugation in sucrose density gradients of labeled RNA isolated from actinomycin D-treated and infected baby hamster kidney cells revealed the presence of the following virus-specific types of RNA: (i) single-stranded RNA with a heterogeneous sedimentation pattern, the 38S viral RNA becoming the predominant species only after long periods of labeling late after infection; (ii) double-stranded RNA with a sedimentation coefficient of 20S; (iii) RNA apparently composed of 20S double-stranded RNA and single-stranded branches. On the basis of their properties, the last two species were tentatively identified as the replicative form and the replicative intermediate of rubella virus RNA. Rubella virus RNA was infectious.

Animals↗
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↗
PubMed1987

Tobacco mosaic virus replicase and replicative structures.

The RNA-dependent RNA polymerase (replicase) mediating the replication of tobacco mosaic virus (TMV) has been investigated in a number of laboratories over a period of 20 years. Cell-free enzyme preparations have been prepared which can continue the synthesis of nascent complementary RNA, initiated in vivo; however, the enzyme does not require, nor does it respond to, exogenous viral RNA as a template. The presence in plants of a virus-stimulated, host-encoded RNA-dependent RNA polymerase (RdRp) has added confusion to this field; it is now generally conceded, however, that this enzyme is not the TMV replicase. Our recent studies have emphasized several aspects of TMV RNA replication. We have examined the nature of TMV replicative structures synthesized in vitro by utilizing a partially purified enzyme preparation isolated from TMV-infected tobacco tissue. Radiolabelled products of the reaction were analysed on agarose gels and fractions with the predicted electrophoretic migration and nuclease sensitivities of replicative form (RF) and replicative intermediate (RI) were isolated. These fractions were hybridized to a collection of bacteriophage M13 clones containing portions of the TMV genome of both plus and minus polarity. The nascent synthesis in the RI-like molecules was restricted to the plus viral strand, while the new synthesis in the RF-like molecules was of both plus and minus polarity. Solubilization of the membrane-bound replicase with the non-ionic detergent CHAPS has yielded complexes which remain in solution after high-speed centrifugation. The solubilized replication complexes have been utilized as starting material for enzyme purification by Sepharose 4B gel filtration chromatography. The intracellular site of synthesis of TMV RNA has been reinvestigated in the light of reports suggesting a nuclear site of replication. The conclusion for nuclear synthesis has been based on fractionation of subcellular homogenates of virus-infected leaves or mesophyll protoplasts and identification of virus-related proteins associated with these fractions. In our studies, however, we conclude that these procedures can be misleading in that the 126,000 Mr TMV protein (and replicase activity) were found in all fractions of the homogenate analysed. Double-stranded TMV RNA, on the other hand, was barely detectable in preparations of purified nuclei; instead it was concentrated in the post-nuclear supernatant, suggesting that the nucleus is not the site of TMV RNA synthesis.

Chromatography, Agarose↗
PubMed1980

The nature of the RNA products synthesized in vitro with a cell-free extract from TMV-infected tobacco leaves.

A cell-free extract containing TMV-RNA replicase was prepared from TMV-infected tobacco leaves. It could synthesize double-stranded RNAs in the presence of four nucleoside triphosphates (among them, UTP was tritium-labelled), magnesium ion and actinomycin D. It was confirmed by polyacrylamide-agarose gel electrophoresis, RNase treatment, thermal denaturation and self annealing that 3H-ds RNAs, obtained from phenol-SDS extraction and Serva cellulose column chromatography, consisted of replicative form (RF) and replicative intermediate (RI) of TMV-RNA, with molecular weights of 40 X 10(6) and 5.0 X 10(6), respectively. Molecular hybridization competition experiment showed that 60-70% of the nascent RNAs in the 3H-ds RNA were plus strand of tMV-RNA.

Cell-Free System↗
PubMed2003

SARS-coronavirus replicates in mononuclear cells of peripheral blood (PBMCs) from SARS patients.

BACKGROUND: The etiologic agent of severe acute respiratory syndrome (SARS) is a recently identified, positive single-stranded RNA (ssRNA) coronavirus (SARS-CoV). Little is known about the dynamic changes of the viral replicative form in SARS cases. OBJECTIVES: Evaluate whether SARS-CoV can infect and replicate in peripheral blood mononuclear cells (PBMCs) of infected persons and reveal any dynamic changes to the virus during the course of the disease. STUDY DESIGN: Peripheral blood mononuclear cells collected from SARS cases infected by the same infectious source were tested for both negative-stranded RNA (minus-RNA, "replicative intermediates") and positive-stranded RNA (genomic RNA) of SARS-CoV during the course of hospitalization by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: SARS-CoV minus-RNA was detected in PBMCs from SARS patients. The viral replicative forms in PBMCs were detectable during a period of 6 days post-onset of the disease, while the plus-RNA were detectable for a longer period (8-12 days post-onset). CONCLUSIONS: SARS-coronavirus can infect and replicate within PBMCs of SARS patients, but viral replication in PBMCs seems subject to self-limitation.

Female↗
PubMed2000

Peripheral blood neutrophils from hepatitis C virus-infected patients are replication sites of the virus.

BACKGROUND AND OBJECTIVE: Hepatitis C virus (HCV) is able to cause not only acute and chronic liver disease, but also immunologic and hematologic disorders. In order to clarify the extra-hepatic tropism of HCV, and to understand the pathogenetic mechanisms of HCV infection, we evaluated viral replication in peripheral blood mononuclear cells. DESIGN AND METHODS: The presence of genomic and antigenomic (replicative) forms of HCV in B- and T-lymphocytes, monocytes, and polymorphonuclear leukocytes (PML) was determined by reverse transcriptase-polymerase chain reaction in 54 HCV-RNA positive patients and, as control groups, in 10 patients who had recovered from HCV infection without evidence of serum HCV-RNA, and in 10 HCV-negative subjects. RESULTS: In HCV-RNA positive patients, the genomic RNA was found in 94% of B-cells, in 14% of T-cells, in 40% of monocytes and in 77% of PML, while only 1 of the HCV-RNA negative subjects showed positivity in B-cells. The anti-genomic form of HCV-RNA was found in 52% of B-cells, in 3% of monocytes, and in 31% of PML. By contrast, it was never detected in T-cells and in HCV-RNA negative subjects. Neither genomic nor anti-genomic forms were found in HCV-negative cases. INTERPRETATION AND CONCLUSIONS: These data suggest that PML are replication sites of HCV. Whether the infection occurs at the level of the stem cells or subsequently during myeloid cell differentiation is, as yet, unknown. The absence of correlation between the presence of replicative forms and any clinical and/or laboratory data opens the question of the role of HCV replication in extra-hepatic sites.

Adult↗
PubMed1982

Intermediate stages in enzymatic replication of bacteriophage fd duplex DNA.

Using purified enzymes, double strand replication of phage fd DNA has been dissected into several intermediate steps. (i) Phage fd gene 2 protein cleaves supercoiled phage fd replicative form at a specific site in the viral strand (Meyer, T. F., Geider, K., Kurz, C., and Schaller, H. (1979) Nature 278, 365-367). (ii) Relaxed covalently closed circular replicative form DNA which is also formed by gene 2 protein as a side product in the initiation reaction preceding replication is converted into supercoils by DNA gyrase. (iii) The enzyme forms a noncovalent complex at the generated nick that is necessary for initiation of subsequent unwinding. (iv) The Escherichia coli rep helicase (rep protein) and E. coli DNA binding protein I unwind the double-stranded DNA. (v) Concomitant DNA replication by E. coli DNA polymerase III holoenzyme results in the formation of rolling circle intermediates. The double-stranded core of the rolling circle remains in an open form, thus allowing continued synthesis during several rounds of replication. (vi) Processing of replicated viral DNA can be subdivided into the cleavage and the circularization of viral single strands. Comparative studies of fd and phi X174 replication in vitro have revealed differences in the kinetics of individual steps besides an apparent contrast in the conformation of rolling circle intermediates in the electron microscopy where fd DNA features extended tails rather than looped-back structures observed for phi X174 DNA.

Adenosine Triphosphatases↗
PubMed1993

NS2 is required for efficient translation of viral mRNA in minute virus of mice-infected murine cells.

Detailed analysis of five NS2 mutants of the autonomous parvovirus minute virus of mice (MVMp) has revealed the following. At low multiplicities of infection, NS2 mutants killed NB324K cells as well as wild-type (wt) MVM did and grew to high titers, while in contrast they grew poorly and did not readily kill murine A9 cells. Following CaPO4 transfection of murine fibroblasts, NS2 mutant infectious clones generated approximately 10-fold less monomer replicative-form DNA than wt and no detectable progeny single-stranded DNA. On nonmurine semipermissive NB324K cells, however, these mutant plasmid clones generated near wt levels of all replicative DNA forms. After infection of highly synchronized murine fibroblasts by NS2 mutant virus at inputs equivalent to those of the wt, mutant monomer replicative-form DNA was decreased 5- to 10-fold compared with that of the wt, and progeny single-stranded DNA accumulation was decreased to an even greater extent. Both total and cytoplasmic NS2 mutant RNA was decreased, but the amount of total viral mRNA generated, relative to accumulated viral DNA in the same experiments, was similar to that seen in wt infection. The accumulation of virus-generated proteins was also decreased in NS2 mutant infection; however, the magnitude of this decrease, compared with that of wt infections, was significantly greater than the concomitant decrease in mutant-generated levels of accumulated cytoplasmic RNA, and this effect was most dramatic for VP2. There was no such disparity between the relative accumulation of mutant-generated RNA and protein in cells permissive for the growth of these mutants. These results suggest that translation of MVM viral RNA is specifically reduced in NS2 mutant infection of restrictive cells. Because the affected viral proteins are required for the efficient production of viral replicative DNA forms, these results reveal a fundamental, although perhaps not the only, role for NS2 in parvovirus infection.

Animals↗
PubMed1974

A cleavage map of bacteriophage phiX174 genome.

Restriction endonucleases isolated from Hemophilus influenzae, Hemophilus parainfluenzae, and Hemophilus aegyptius were used to cleave varphiX174 replicative form DNA into three sets of specific DNA fragments. The order of these fragments in the varphiX replicative form molecule was determined by (1) analysis of partial digest products, (2) analysis of overlapping sets of fragments produced by two different restrictive enzymes. On the basis of these results, a detailed physical map of the varphiX174 genome has been constructed with respect to the cleavage sites of all three enzymes.

Bacteriophages↗
PubMed1986

Spontaneous curing of a minute virus of mice carrier state by selection of cells with an intracellular block of viral replication.

We previously described a persistent infection established by the lymphotropic minute virus of mice in mouse L cells at the level of the cell population (D. Ron, P. Tattersall, and J. Tal, J. Virol. 52:63-69, 1984). This carrier state is maintained by a series of consecutive phenotypic changes which take place in both the cells and the virus and is cured spontaneously after 150 to 200 cell generations (D. Ron and J. Tal, J. Virol. 55:424-430, 1985). We show here that the cure was caused by the selection of virus-resistant cells in the culture. The resistance of these survivor cells to virus replication was due to an intracellular block. Infection of a spontaneously cured culture with the fibrotropic parental minute virus of mice resulted in a restrictive infection in which the viral replicative-form DNA was formed and amplified, but the synthesis of single-stranded progeny DNA was markedly reduced. The lymphotropic strain was blocked in these cells at an earlier stage, with little or no amplification of viral replicative-form DNA observed. These data indicate that the replication of minute virus of mice requires host-coded helper functions in at least two stages of its growth cycle.

Animals↗
PubMed1982

Clonal inheritance of the pattern of DNA methylation in mouse cells.

DNA-mediated gene transfer was used to investigate the mode of inheritance of 5-methylcytosine in mouse L cells. Unmethylated phi X174 replicative form DNA remains unmethylated after its introduction and integration into these cells. On the other hand, phi X174 replicative form DNA that was methylated in vitro at its C-C-G-G residues retains these methylations as shown by restriction enzyme analysis with Hpa II and Msp I to detect methylation at this specific site. Although these unselected methylated vectors are prone to lose 30-40% of their methyl moieties upon transfection, this demethylation appears to be random. Once established, the resulting methylation pattern is stable for at least 100 cell generations. In order to examine the specificity of methylation inheritance, fully hemimethylated duplex phi X174 DNA was synthesized in vitro from primed single-strand phi X174 DNA by using 5-methyl deoxycytidine 5'-triphosphate. This molecule was inserted into mouse L cells by cotransformation and subsequently was analyzed by a series of restriction enzymes. Only methylations located at C-G residues were conserved after many generations of cell growth. The results suggest that the inheritance of the cellular DNA methylation pattern is based on a C-G-specific methylase that operates on newly replicated hemimethylated DNA.

5-Methylcytosine↗
PubMed1996

[Combined therapy with recombinant alfa-2 and gamma interferons in patients with chronic hepatitis delta virus infection].

5 patients with chronic HD-viral infection received combined therapy with alpha 2 and gamma-interferons in daily doses 4000000 U for 30 days. In 1 patient with low-replicative form of hepatitis an AlAT activity decline and disappearance of serum IgM anti-D observed in the course of the treatment returned to pretreatment values one month after the end of therapy. In the other patient with high-replicative form of hepatitis a long suppression of viral replication was achieved, AlAT activity returned to normal. Low titers of IgM anti-D persisted for 5 months after therapy. The treatment failed in delta cirrhosis as activity of AlAT and IgM anti-D titers remained unchanged.

Alanine Transaminase↗
PubMed1976

Novel replicative properties of a capsid mutant of bacteriophage phi chi 174.

A capsid mutant of bacteriophage phi chi 174 demonstrates altered requirements for the conversion of viral single-stranded DNA to double-stranded replicative form DNA. In the presence of puromycin at 42 C, wild-type phi chi 174 is unable to complete this replicative event, whereas phi chi ahb is able to do so. Furthermore, in contrast to wild-type phi chi 174, formation of phi chi ahb parental replicative form DNA is sensitive to rifampin under certain experimental conditions. These data suggest that the mutant capsid proteins of phi chi ahb influence the biosynthesis of phi chi ahb complementary strand DNA.

Capsid↗