WO1993000442A1 - De novo cell-free synthesis of picornavirus - Google Patents
De novo cell-free synthesis of picornavirus Download PDFInfo
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- WO1993000442A1 WO1993000442A1 PCT/US1992/005177 US9205177W WO9300442A1 WO 1993000442 A1 WO1993000442 A1 WO 1993000442A1 US 9205177 W US9205177 W US 9205177W WO 9300442 A1 WO9300442 A1 WO 9300442A1
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- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/32011—Picornaviridae
- C12N2770/32051—Methods of production or purification of viral material
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- This invention is directed to an in vitro process for the de novo synthesis of infectious picornaviruses and a cell-free medium useful in the process. More particularly, the invention is directed to an in vitro process using a cell-free medium to synthesize 0 mature, infectious picornaviruses de novo. Most particularly, the invention is directed to using a cell- free medium from which the nuclei and mitochondria of the cells have been removed, to synthesize infectious picornavirus and rhinovirus. -.
- the invention was made with Government support under No. AI15122-17 awarded by National Institute of Health. The Government has certain rights in this invention.
- the Picornaviridae among the smallest ribonucleic acid-containing viruses known, comprise one of the largest and most important families of human and agricultural pathogens. Poliovirus, human rhinovirus, human hepatitis A virus, encephaloniyocarditis virus and food-and-mouth disease virus are all members of the picornavirus family.
- picornaviruses Because of the economic and medical importance of picornaviruses, they have been the subject of much study. Studies on the molecular biology of these viruses became possible when it was discovered that poliovirus could be propagated in cultured cells, such as HeLa cells, and other picornaviruses can be propagated in L cells or BHK cells. Crucial advances, such as the development of - plaque assay for inf ctivity and of methods of purification of viruses, proteins and polypeptides opened the way for structural analysis of these viruses.
- the family of picornavirus is currently divided into four genera: enteroviruses, cardioviruses, 5 rhinoviruses and aphthoviruses.
- the poliovirus the prototype of picornaviruses, is an enterovirus.
- the etiologic agents of the common cold are rhinoviruses.
- the foot-and-mouth disease viruses are aphthoviruses.
- the encephalomyocartditis virus is a cardiovirus.
- Present •.A knowledge indicates that revision of this classification is probably necessary. However, for the present purposes, this classification scheme suffices.
- Members of each genus is then subclassified into serotypes, and then to strains. Morphologically, the members of the four genera - j ⁇ c; are not distinguishable by electronmicroscopy.
- HRVs Human rhinoviruses
- a better understanding of the biochemical mechanisms used during viral infection is n needed to develop novel approaches to control HRV infections in common cold.
- the complete nucleotide sequence of a cDNA, representing the genomic RNA of HRV-14 and other serotypes of rhinoviruses was determined. Thus far, all attempts have failed to 25 translate the full length HRV RNA in cell free extracts.
- All picornaviruses including poliovirus, cardiovirus, human rhinoviruses and aphthoviruses contain four polypeptide chains: VPl, VP2, VP3, and VP4. These chains are elements of protein subunits called mature Q "protomers". The protomer is defined as the smallest identical subunit of the virus. Traces of a fifth protein, VPO, which is cleaved to VP2 and VP4 are also observed.
- the picornaviral genome consists of a single , strand of messenger-active RNA which can be extracted out - of virions by shaking aqueous suspensions of virus with a equal volume of phenol and chloroform. On phase separation, the proteins dissolve in the phenol-rich phase, while RNA remains in the aqueous phase.
- the specific infectivity of the naked RNA is about 10 ⁇ 6 of th 5 virions.
- the genomic messenger active RNA consists of a "+ ⁇ • strand which is polyadenylated at the 3 ' terminus and carries a small protein, VPg, covalently attached to the 5 1 end.
- the first picornaviral RNA to be completely 0 sequenced and cloned into DNA was that of a type 1 poliovirus.
- the common organizational pattern of picornaviruses may be represented schematically. See Fig 5.
- the number of bases in the RNA range from 7,209 to 8,450.
- the poliovirus does not have a leader (L) protein
- RNA contains single long open reading frame encoding a long polypeptid 0 chain, which is called a ⁇ 'polyprotein".
- the polyprotein is cleaved during translation and is not found as such. cascade of cleavages, carried out by virus-coded proteinases, ultimately generating usually twelve end products in the case of a virus with a leader (L) protein.
- L leader
- Proteinase 3C, or its precursor 3CD mediates most of the cleavages except the early cleavages of the nascent polyprotein and the maturation cleavage of VPO ⁇ VP4 + VP2.
- the maturation cleavage occurs only after the RNA has been packaged in the protein shell. Without the maturation cleavage, infectivity is not observed.
- Picornaviral morphogenesis can formally be described as a tandem three or four stage process in whic monomers are converted to a pentamer. 5 pentamers are then assembled into a provirion consisting of the RNA genome within an unstable immature capsid. The final ste _ during which mature virus is formed involved cleavage of most, if not all, of the VPO chains in the 60 subunit icosahedral capsid.
- Viruses are replicating microorganisms that are among the smallest of all life forms. Because of their
- Fernandez-Tomas et al. (3) also described an attempt to synthesize the poliovirus by incubating cell- free extract that was preinfected with poliovirus.
- the study was conducted using a cytoplasmic extract of poliovirus infected HeLa cells labelled with 3 H-leucine and 1 C-uridine. After incubation for 30 minutes at 37°C, an increase of both leucine and uridine labelling in the poliovirus region and structures which sedimented slower cage than 100S were observed. This is in contrast to the virions which sediment at 155S and the provirions which sediment at 125S.
- EGTA EGTA
- TMV RNA tobacco mosaic virus
- cow pea mosaic virus cow pea mosaic virus
- mice liver t-RNA stimulated the translation of the exogenous mRNA.
- the results showed that the expression products contained j -. larger proteins previously unobtainable in the untreated rabbit reticulocyte system.
- Shih et al. (6) attempted the translation of poliovirus RNA in rabbit reticulocyte lysates (RRL) which were micrococcal nuclease pretreated. Shih et al. found - 3 C that translation was optimal at 60 minutes and leveled off - after 3 hrs. of incubation. No infectious virus could be detected even after 6 hrs. of incubation. The results indicate that nascent viral proteins together with cleavage products VPO, VPl and VP3 were found. However, no VP2 or VP4 were observed. It was postulated that the 5 absence of VP2 and VP4 showed that the conditions of the final assembly of the polio virion are not met by the rabbit reticulocyte lysates he used.
- Brown and Ehrenfeld (7) attempted in vitro translation of poliovirus RNA by using RRL to which was -.
- Q added a ribosomal salt wash of HeLa cell extracts. Again, the presence of VPO, VPl and VP3 and other anomalous proteins were observed after incubation. However, the appearance of anomalous proteins was inhibited by the HeLa ribosomal wash. No VP2 or VP4 were observed and no -i r evidence of formation of provirion or of mature virion was seen.
- Palmenberg (10) described the in vitro synthesis of encephalomyocarditis virus protein by translation in a . RRL. Viral proteins including capsid proteins capable of assembly into viral capsid intermediate structures were formed. Incubation for 15 hours produced a pentameric association of capsid protomers. Complete virions were not observed.
- 5 Grubman et al. (11) described the in vitro assembly of foot-and-mouth disease virus (FMDV) from structural proteins isolated from FMDV infected cells. The cytoplasmic extract of infected cells were analyzed to show the presence of intermediates of morphogenesis. Full ,- length FMDV RNA was added to RRL and translation was carried out in vitro. The results indicated that the structural protein complexes assembled in the lysate. However, the presence of the mature virus 14OS was not detected. ⁇ Neither Palmenberg or Grubman et al reported formation of infectious viral particles. It was believed that the RRL system lacks components essential for morphogenesis (12) .
- the picornaviruses are one of the largest and most important group of human and agricultural pathogehs. , « Therefore, it is important to be able to harvest the naked virus synthesized in vitro to study its morphogenesis to develop effective anti-viral agents.
- a de novo process for the in vitro synthesis of a picornavirus comprises the following steps: 5 A. Preparing a lysate from mammalian cells selected from the group consisting of kidney cells, epithelial cells, liver cells, cells of the central nervous system, fibroblastic -, cells, transformed or tumorigenic cell lines thereof including HeLa cells, hepatoma cells and L cells; wherein the nuclei and mitochondria were removed; the endogenous mRNA - j 5 deactivated with micrococcal nuclease, calcium chloride and EGTA, B. Preparing an in vitro synthesis medium by mixing: the lysate, about 1 mM ATP, from about 20 ⁇ M to 1000 ⁇ M each of GTP, CTP and UTP, about 10 mM creatine
- the present invention further relates to a cell- free media prepared from a lysate extracted from mammalian cells, from which the nuclei and mitochondria were removed, the endogenous mRNA deactivated with micrococcal , c nuclease, calcium chloride and EGTA; and mixing:
- the lysate (i) about 1 mM ATP, (iii) and from about 20 ⁇ M to 1000 ⁇ M each of GTP, CTP and UTP, (iv) about 10 mM creatine 0 phosphate, (v) about 24 ⁇ g/ml creatine phosphokinase, (vi) about 2 mM dithiothreitol, (vii) about 24 ⁇ g/ml calf liver t- 5
- RNA (viii) about 12 ⁇ M each of 20 amino acids, (ix) about 18 mM Hepes, pH 7.4, 0 (x) about 240 ⁇ M spermidine, (xi) from about 50 mM to 200 mM potassium acetate, and (xii) from about 1 mM to 4 mM of Mg ++ , wherein the amounts specified represent the final concentration in the medium.
- Fig. 1 is a schematic drawing of plasmid pT7 PV1-5 containing poliovirus cDNA.
- Fig. 2 is a photograph of SDS/polyacrylamide gel of the viral proteins formed in the in vitro synthesis of poliovirus over time.
- Fig. 3 is a photograph of the plaque-forming test results using RNA transcribed from plasmid PT7 PV1-5: XL(A) and XL(B), compared with viral RNA: M(A) and M(B) .
- XL(C) and M(C) are plaque formed by native virus for comparison.
- Fig. 4 is a photograph of the gel electrophoresis results obtained in the PCR reaction.
- Fig. 5 is a schematic representation of the organizational pattern of the picornaviruses.
- a process for the de novo, in vitro synthesis of picornaviruses comprises: A. Preparing a lysate from mammalian cells selected from the group consisting of kidney cells, epithelial cells, liver cells, cells of the central nervous system, fibroblastic , ⁇ cells, transformed or tumorigenic cell lines thereof, including HeLa cells, hepatoma cells and L cells; from which the nuclei and mitochondria were removed, the endogenous mRNA -, 5 deactivated with micrococcal nuclease, calcium chloride and EGTA; B. Preparing an in vitro synthesis medium by mixing: (i) the lysate, 0 (ii) about 1 mM ATP,
- RNA 0 (viii) about 12 ⁇ M each of 20 amino acids, (ix) about ;18 mM Hepes, pH 7.4, (x) about 240 ⁇ M spermidine, (xi) from about 50 mM to 200 mM potassium acetate, and (xii) from about 1 mM to 4 mM of
- Mg ++ wherein the amounts of specified represent the final concentration in the medium
- c C Adding viral RNA from virus or in vitro synthesized viral RNA from cDNA to the in vitro synthesis medium
- D Incubating from about 2 to 24 hours at a temperature of from about 30°C to 10 40°C.
- the in vitro synthesis medium comprises: ⁇ c (i) a lysate of mammalian cells selected from the group consisting of kidney cells, epithelial cells, liver cells, cells of the central . « nervous system, fibroblastic cells, transformed or tumorigenic cell lines thereof including HeLa cells, hepatoma cells and L cells; from which the nuclei and mitochondria were removed, the endogenous mRNA deactivated with micrococcal nuclease, calcium chloride Q and EGTA;
- RNA (viii) about 12 ⁇ M each of 20 amino acids, (ix) about 18 mM Hepes, pH 7.4, (x) about 240 ⁇ M spermidine, (xi) from about 50 mM to 200 mM potassium acetate, and (xii) from about 1 mM to 4 mM Mg ++ , wherein the amounts specified represent the final concentration in the medium.
- the cell-free extract is highly efficient for the translation of poliovirus RNA in vitro. Further, by introducing a cycle of freezing at -80°C, thawing to room temperature followed with centrifugation after removal of the nuclei and mitochondria but before deactivation of endogenous mRNA, the efficiency of virus production was found to be increased many folds. Therefore, even though the treatment is not absolutely necessary, it is much preferred.
- Mammalian cells suitable for use in preparing the in vitro cell-free extract includes: kidney cells, epithelial cells, liver cells, cells of the central nervous system, transformed or tumorigenic cell lines thereof including HeLa cells, hepatoma cells and L cells.
- the ⁇ infectivity of the picornaviruses in the various cell lines are known.
- cells of primate origin will be found to be suitable for enteroviruses, rhinoviruses, cardioviruses.
- cell lines from hamsters such as BHK will be found suitable for FMDV.
- the nuclei and mitochondria are removed by know methods.
- cells from a cell line suitable fo the synthesis of the desired picornaviruses are harvested by centrifugation, washed with a suitable buffered solution such as PBS, saline and resuspended in N-2- 5 hydroxylethylpiperazine-N 1 -ethanesulfonic acid (Hepes) pH 7.4 suitably adjusted with KOAc and Mg(OAc) and dithiothreitol (DTT) .
- Hepes N-2- 5 hydroxylethylpiperazine-N 1 -ethanesulfonic acid
- KOAc and Mg(OAc) and dithiothreitol (DTT) suitably adjusted with KOAc and Mg(OAc) and dithiothreitol
- the cells are then swollen and disrupted at 4°C with a Dounce homogenizer.
- the nuclei and mitochondria can be removed by centrifugation.
- lysate by deactivating the endogenous RNA with micrococcal nuclease and calcium chloride followed by treatment with EGTA.
- the lysate can be adjusted to 10% by volume with glycerol and frozen at -80°C.
- the lysate can be adjusted to 10% by volume with glycerol and frozen at -80°C.
- the lysate can be adjusted to 10% by volume with glycerol and frozen at -80°C.
- the in vitro synthesis medium is conveniently prepared in small lots. For example, to make 250 ⁇ l of the in vitro synthesis medium 93 ⁇ l of the cell-free extract can be used. To this can be added the nucleoside 5 triphosphates, ATP, GTP, CTP and UTP to provide energy an precursors for virus production.
- the ATP should generall be present at a higher amount.
- l mM of ATP to a mixture of from about 40 ⁇ M to 250 ⁇ M each of GTP, CTP and UTP is preferred for poliovirus.
- Other component necessary for the synthesis of proteins are also added to the cell-free lysate. These include creatine phosphate, creatine phosphokinase, dithiothreitol, t-RNA, the twenty amino acids, spermidine, potassium acetate, magnesium acetate, magnesium chloride.
- _c concentration of K + and Mg ++ in the mixture is important. .
- the Mg ++ and K + concentrations should be optimized for maximal virus production. Further, when there is an increased amount of each of GTP, CTP and UTP, an increased amount of Mg ++ is needed. It has been found that the concentration of the K + in the form of KOAc should be in the range of about 50 mM to 200 mM, and the concentration of the Mg ++ in the form of Mg(OAc) 2 and/or MgCl 2 should be in the range of about 1 mM to 4 mM in the synthesis medium. The mixture is adjusted
- nucleoside triphosphates represents the final concentration in the mixture.
- HeLa cell lysate For the synthesis of polioviruses and rhinoviruses, a HeLa cell lysate is found to be
- HeLa S3 cells (5 x 10 5 cells/ml) were harvested by centrif gation, washed 3 times with phosphate buffer saline (PBS), and resuspended in 1.5 packed cell volume of 10 mM Hepes, pH 7.4 containing 10 mM KOAc, 1.5 mM Mg(OAc)2
- DTT dithiothreitol
- S10 extract was dialyzed for 2 hr against 100 vol 10 mM Hepes, pH 7.4, 90 mM KOAc, 1.5 mM Mg(0Ac)2 and 2.5 mM DTT. The retained S10 extract was then centrifuged for 10 min at 10,000 rpm in SS34 rotor and the supernatant was stored at -80°C. On
- the S10 extract was thawed to room temperature, centrifuged in an Eppendorf centrifuge for 10 min at 10,000 rpm.
- the extract was treated with micrococcal nuclease (15 ⁇ g/ml extract) in the presence of CaCl 2 (7.5 ⁇ l of 0.1 M CaCl 2 /ml extract) at 20°C for 15 5 min.
- the reaction was terminated by addition of 15 ⁇ l 20 _ mM EGTA/ml extract (S10 lysate) .
- the S10 lysate was adjusted to 10% glycerol and stored for up to one month i small portions at -80°C or, for longer term storage, at the temperature of liquid nitrogen.
- the in vitro translation reaction mixture was prepared.
- added can be varied from 20 ⁇ M to 1000 ⁇ M by adjusting th concentration of Mg ++ .
- concentration of Mg ++ can be reduced.
- concentration of K + in the synthesis medium is from about 50 mM to 200 mM and the Mg ++ is from about 1 mM to 4 mM.
- the synthesis of the infectious virus is programmed by adding the genomic RNA of the virus or in vitro synthesized viral RNA from cDNA to the cell-free medium.
- the viral RNA programmed cell-free medium is incubated from about 30°C to 40°C for a period of from about 2 to 24 hours.
- the virus so formed can be quantitated and expanded from the cell-free medium by plaque assays and growth in the HeLa cells.
- the virus may be purified from the cell free medium by standard procedures.
- Poliovirus Serotype 1 (Mahoney)
- PV1(M) was used as a model.
- Other picornaviruses, such as rhinoviruses, aphthoviruses, cardioviruses may also be synthesized in like manner in appropriate cell free extracts. This is because picornaviruses have the same type of structures
- RNA was precipitated from the aqueous phase by the addition of 2.5 5 volumes of ethanol and ammonium acetate to a final concentration of 1 M. The precipitate was resuspended in water and the RNA concentration was determined by measuring the optical density at 260 m ⁇ .
- HeLa S3 cells (5 x 10 5 cells/ml) were harvested by centrifugation, washed 3 times with phosphate buffered - saline (PBS), and resuspended in 1.5 packed cell volume of 10 mM Hepes, pH 7.4 containing 10 mM KOAc, 1.5 mM Mg(OAc) 2 and 2.5 mM dithiothreitol (DTT). Cells were left on ice for 10 min and disrupted at 4°C with 15-25 strokes in a Dounce homogenizer. Nuclei were removed by centrifugation c for 5 min at 2000 rpm and the mitochondrial fraction was removed from supernatant by centrifugation for 20 min at 10,000 rpm in SS34 rotor.
- PBS phosphate buffered - saline
- the supernatant (S10 extract) was dialyzed for 2 hr against 100 vol 10 mM Hepes, pH 7.4, 90 mM KOAc, 1.5 mM Mg(0Ac)2 and 2.5 mM DTT. The retained
- 2 Q translation reaction mixtures (250 ⁇ l) contained 93 ⁇ l S10 lysate and the following additions: 1 mM ATP, 125 ⁇ M each of GTP, UTP, CTP, 10 mM creatine phosphate, 6 ⁇ g creatine phosphokinase, 2 mM DTT, 6 ⁇ g calf liver t-RNA, 12 ⁇ M each of 20 amino acid, 18 mM Hepes, pH 7.4, 240 ⁇ M spermidine, 5 0.1 M KOAc, 0.35 mM Mg(OAc) 2 , 0.4 mM MgCl 2 and 2 ⁇ g PV1(M) RNA. The final volume was made to 250 ⁇ l with distilled water. The mixture was incubated for 15 hours at 30°C.
- the incubated mixture was analyzed by a plaque forming assay to determine the number of infectious Q virions formed.
- the amounts of each of GTP, CTP and UTP were varied in six experiments from 62.5 to 1000 ⁇ M. The results are presented in Table l.
- the amino acid methionine was replaced with 35 S-methionine so that the proteins can be
- SDS gel analysis as shown in Fig. 2 shows the formation of VPO, VPl, VP2, VP3.
- VP2 was not immediately evident in Fig. 2, lane 13, although on longer exposure of the film, a band migrating to the position of VP2 emerged.
- VP4 being a very small protein is not retained on the gel. However, since VP4 and VP2 are cleavage products of VPO, the presence of VP2 shows that VP4 was formed.
- the yield of infectious virus produced was _. further increased by about 60%.
- the total Mg ++ concentration was adjusted with increased ⁇ Ms of NTPs added, the production of virus was maintained at the higher level. For example, when the total Mg ++ concentration was increased to 3.10 mM for 500 ⁇ M each of
- a plasmid pT7XL was constructed in accordance with the procedure described in Van Der Werf et al. (16) for pT7PVl-5. See Fig. 1.
- the cDNA of ., ⁇ PV1(M) was placed under the control of a promoter for T7 RNA polymerase.
- the transcription product is mRNA of PV1(M) .
- the reaction mixture contained 20 ⁇ g/ml of Eco Rl-cut pT7XL DNA, 400 ⁇ M ribonucleoside triphosphates, 8 mM MgCl 2 , 4 mM spermidine, 25 mM potassium phosphate buffer pH 7.5, 10 mM dithiothreitol and 15 ⁇ g of T7 RNA polymerase per ml. After incubation for 30 minutes at 37°C, 10 ⁇ l of 0.5 M EDTA were added. The RNA was extracted by phenol-chloroform and precipitated by the addition of 3 volumes of ethanol with ammonium acetate at a final concentration of 1 M.
- RNAse A (20 ⁇ g/ml)
- RNAse Tl 100 U/ml
- RNA Full length, plus-stranded poliovirus RNA is infectious when added to tissue culture cells, but the specific infectivity is low (10 infectious centers per ⁇ g of purified RNA; see Koch (15). The efficiency of transfection can be increased by a factor of 10 5 , if the host cells are pre-treated with polycations. In view of these properties of viral RNA, the formation of plaques observed here could be the result of transfection of monolayer cells with the RNA used for translation. To test this possibility, we assayed for plaque forming units (PFU) in the complete incubation mixture (cell extract, translation mixture, plus template RNA) (i) before translation, and (ii) after 15 hr. of translation, but only after the mixtures had been treated with RNase A and RNase Tl prior to the addition to cells.
- PFU plaque forming units
- HeLa cell monolayer A lawn of HeLa cells (HeLa cell monolayer) was prepared in petri dishes. Each of the incubation mixture to be tested was diluted to 200 ⁇ l with PBS and separately placed on each monolayer. The following procedure was used. Six well plates of confluent HeLa R19 cells were washed with phosphate buffered saline. A dilution of the virus (200 ⁇ l) were added per well and the plates were rocked gently for 30 min. To each well 2 ml of overlay medium was added which was made up of 22.5 ml of 2X MEM, 25 ml of 2% noble agar and 2.5 ml of calf serum. The plates were incubated at 37°C for 48 hours and the cells were stained with crystal violet (1% crystal violet in 50% methanol) .
- RNAs are, by definition, plus-stranded RNA because it is of messenger-sense polarity. Both virion and transcription-derived RNAs used in translations are therefore plus strands. Since poliovirions are thought 5 not to contain minus strands, the detection of such strands in the incubation mixture programmed with virion RNA would be indicative of virus-specific RNA synthesis. Therefore, we have tested the incubation mixture for poliovirus-specific minus strands by cDNA synthesis and n amplification with the polymerase chain reaction (PCR) . Translation reactions were same as those described in Example 1. After 15 hr incubation, the RNAs were extracted by standard method and resuspended in 20 ⁇ l of water. Generally, 5 ⁇ l samples were used for cDNA
- PV1(M) [plus] (3280-3301) 5' AGTCTGGTGCCCGCGTCCACCG 3' Primer 2 PV1(M) [minus] (3983-4007) 3 1 CCTGAGTGGCCAAGTGGTAGTTGC 5'
- Primer 1 chosen for cDNA synthesis was of plus strand polarity
- primer 2 used for amplification (together with primer 1)
- primer 1 was of minus strand polarity.
- the DNA product to be expected from such reaction is 178 nucleotides long.
- cDNA transcripts were prepared by incubation (42 °C, 2 hr) of RNA templates in 50 ⁇ l reaction mixtures containing 50 mM Tris-HCl, pH 8.3, 70 mM KCl, 5 mM MgCl 2 , 10 mM DTT, PCR primers (50 p oles) , 200 ⁇ M each of dATP, dCTP, dGTP, dTTP, 25 U RNasin, and 40 U avian myeloblastosis virus
- the samples were incubated at 65°C for 1 hr and were neutralized by the addition of 12.5 ⁇ l 2 M Tris-HCl, o pH 8.0 and 5 ⁇ l 5 M HCl.
- the samples were extracted with phenol/chloroform and cDNA was separated from unincorporated dNTPs by gel filtration using push column, precipitated with ethanol, dried and dissolved in 20 ⁇ l water.
- Five ⁇ l cDNA products were diluted to 100 ⁇ l reaction mixtures containing PCR primers (50 pmoles each) and 2.5 U of thermostable DNA polymerase from Thermus aquaticus (Taq DNA polymerase) and overlaid with mineral oil.
- lane 2 a band of the same chain length was generated from total RNA isolated after 15 hr of translation but no band was seen at 0 hr of incubation (lane 4) .
- the latter is a control confirming that virion r RNA used in this translation does not contain minus strands (note that we were unable to use plasmid-derived RNA for this experiment because we found it difficult to remove all plasmid DNA prior to PCR) .
- no band was seen when the reaction was carried out with total RNA
- Guanidine hydrochloride (Gua HCl) is a compound known to strongly inhibit poliovirus RNA replication in HeLa cell at a concentration of 2 mM. At this concentration, the drug has no adverse effect on the metabolism and growth of HeLa cells themselves for several
- HCl can be readily selected; their mutation(s) map to a specific locus in the non-structural polypeptide 2C, a locus highly conserved amongst 2C polypeptides of all picornaviruses. The involvement of 2C in RNA replication
- Poliovirus occurs in three distinct serotypes that share the same receptor on HeLa cells. Treatment of the monolayer HeLa cells with monoclonal antibodies D171, -,£- which completely blocks the uptake of poliovirus into host ⁇ cells, prior to addition of the incubation mixture completely inhibited plaque formation (Table 3) . Similarly, no plaques were observed when the incubation mixture was treated with anti-PVl(M) rabbit hyperimmune serum (Table 3) .
- RNA used for translation is specific for type 1 (Mahoney) ; accordingly, anti-poliovirus type 3 (Leon) , PV3(L) , hyperimmune serum did not reduce the titer in the cell-free extract (Table 3) .
- the results suggest that intact poliovirions of serotype 1 (Mahoney) were synthesized in the cell-free extract, and that on subsequent incubation of HeLa cells, these virions entered the cells by a poliovirus receptor- mediated pathway.
- the samples were diluted to 200 ⁇ l in the presence of 1 ⁇ g/ml of anti PV1(M) (D171) monoclonal antibody (Ab) or 25 ⁇ l each of PV1(M) or PV1(L) hyperimmunesera, preincubated at room temperature for 20 min and then plaque assays were 5 performed as described in Example 4.
- infectious poliovirus type 1 (Mahoney) was synthesized de novo in an in vitro process.
- the product was as infectious as the natural virus.
- specific tests by guanidine HCl and antibodies specific to PV1(M) shows that the virus synthesized is the PV1(M) .
- RNA of Rhinovirus 14 was obtained.
- the RNA of the HRV-14 was incubated at a temperature of 30°C in a translation mixture of a cell-free extract of HeLa S3 cells except
- ribonuclease was added to destroy the naked, non- . t- encapsidated viral RNA.
- the mixture was then added to a monolayer of HeLa cells.
- the monolayer of HeLa cells was incubated at 35°C. 10-40 plaque forming units, detected as defined areas of destroyed HeLa cells, were observed.
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EP92914053A EP0591382A4 (en) | 1991-06-24 | 1992-06-16 | De novo cell-free synthesis of picornavirus. |
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US6461815B1 (en) | 1998-05-22 | 2002-10-08 | North Carolina State University | Antibacterial agents and methods of screening for the same |
US6264940B1 (en) | 1998-08-05 | 2001-07-24 | The Research Foundation Of State University Of New York | Recombinant poliovirus for the treatment of cancer |
RU2169154C2 (en) | 1999-03-25 | 2001-06-20 | Институт белка РАН | Method of synthesis of polypeptides in cell-free system |
AUPQ425699A0 (en) * | 1999-11-25 | 1999-12-23 | University Of Newcastle Research Associates Limited, The | A method of treating a malignancy in a subject and a pharmaceutical composition for use in same |
CA2409190A1 (en) | 2000-04-14 | 2001-10-25 | The Uab Research Foundation | Poliovirus replicons encoding therapeutic agents and uses thereof |
AU2002306709A1 (en) * | 2001-03-14 | 2002-09-24 | Replicon Technologies, Inc. | Oncolytic rna replicons |
WO2003064672A1 (en) * | 2002-01-31 | 2003-08-07 | Yaeta Endo | Cell extract for cell-free protein synthesis and process for producing the same |
JP4441170B2 (en) * | 2002-11-28 | 2010-03-31 | 独立行政法人理化学研究所 | Escherichia coli cell extract having mutation in S12 ribosomal protein and cell-free protein production method using the same |
AU2002953436A0 (en) * | 2002-12-18 | 2003-01-09 | The University Of Newcastle Research Associates Limited | A method of treating a malignancy in a subject via direct picornaviral-mediated oncolysis |
JP4590249B2 (en) * | 2004-11-17 | 2010-12-01 | 独立行政法人理化学研究所 | Cell-free protein synthesis system for glycoprotein synthesis |
JP4868731B2 (en) * | 2004-11-17 | 2012-02-01 | 独立行政法人理化学研究所 | Cell-free protein synthesis system derived from cultured mammalian cells |
JP4787488B2 (en) * | 2004-11-19 | 2011-10-05 | 独立行政法人理化学研究所 | Cell-free protein synthesis method using linear template DNA and cell extract therefor |
EP2213747B1 (en) * | 2007-11-05 | 2021-06-30 | Riken | Method for producing membrane protein |
WO2017022696A1 (en) | 2015-07-31 | 2017-02-09 | 国立研究開発法人理化学研究所 | Method of manufacturing membrane protein and utilization thereof |
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US4403035A (en) * | 1981-06-19 | 1983-09-06 | Regents Of The University Of Minnesota | In vitro DNA-Protein viral assembly and gene cloning system |
-
1992
- 1992-06-16 AU AU22520/92A patent/AU2252092A/en not_active Abandoned
- 1992-06-16 EP EP92914053A patent/EP0591382A4/en not_active Withdrawn
- 1992-06-16 WO PCT/US1992/005177 patent/WO1993000442A1/en not_active Application Discontinuation
- 1992-06-16 CA CA002115991A patent/CA2115991A1/en not_active Abandoned
- 1992-06-22 NZ NZ243248A patent/NZ243248A/en unknown
-
1994
- 1994-06-30 US US08/268,679 patent/US5674729A/en not_active Expired - Fee Related
Non-Patent Citations (3)
Title |
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Journal of Virology, Volume 50, No. 2, issued May 1984, A.J. DORNER et al., "In Vitro Translation of Poliovirus RNA: Utilization of Internal initiation Sites in Reticulocyte Lysate", pages 507-514, see especially "Materials and Methods", and Figure 3. * |
Proceedings of the National Academy of Sciences (USA), Volume 75, No. 12, issued December 1978, D.S. SHIH et al., "Cell-free synthesis and processing of the proteins of poliovirus", pages 5807-5811, especially Figures 1 and 2. * |
See also references of EP0591382A4 * |
Also Published As
Publication number | Publication date |
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AU2252092A (en) | 1993-01-25 |
CA2115991A1 (en) | 1993-01-07 |
NZ243248A (en) | 1994-11-25 |
EP0591382A4 (en) | 1995-01-18 |
EP0591382A1 (en) | 1994-04-13 |
US5674729A (en) | 1997-10-07 |
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