The Isolation of Two Enzyme-Ribonucleic Acid Complexes Involved in the Synthesis of Foot-and-Mouth Disease Virus Ribonucleic Acid

Arlinghaus, Ralph B.; Polatnick, Jerome

doi:10.1073/pnas.62.3.821

Cited by 37 publications

(20 citation statements)

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“…Poliovirus RNA polymerase activity is easily measured in crude extracts (6, 7), but the lack of a suitable purification procedure has prevented the identification of the viral polypeptides necessary for the enzymatic activity. Previous attempts to purify the polymerase activity from picornavirus-infected cells have shown that the polymerase activity sediments as a membraneassociated complex of 60-250 S (8)(9)(10)(11)(12)(13)(14) and is associated with many viral polypeptides (15).…”

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confidence: 99%

Isolation of a Viral Polypeptide Associated with Poliovirus RNA Polymerase

Lundquist

Ehrenfeld

Maizel

1974

Proc. Natl. Acad. Sci. U.S.A.

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Poliovirus-infected HeLa cells were labeled with radioactive methionine or phenylalanine and subjected to a new purification procedure for the viral induced RNA polymerase activity. Detergent-solubilized polymerase activity was purified by precipitation with 2 M LiCl and sedimentation through sucrose gradients. Approximately 0.901% of the incorporated amino acid radioactivity sediments with the peak of polymerase activity. Gradient fractions comprising the polymerase activity peak were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and found to contain predominantly one virus-specific polypeptide. Polyacrylamide gel electrophoresis also reveals that this purified polypeptide migrates with a 58,000 molecular weight noncapsid poliovirus polypeptide.At least 14 virus-specific polypeptides can be detected in poliovirus-infected HeLa cells (1), all derived from post-translational proteolytic cleavage of a large precursor molecule (2-4). Although the general relationship of these polypeptides to the cleavage pattern is known (5), no enzymatic function has been assigned to any of the viral polypeptides. Poliovirus RNA polymerase activity is easily measured in crude extracts (6, 7), but the lack of a suitable purification procedure has prevented the identification of the viral polypeptides necessary for the enzymatic activity. Previous attempts to purify the polymerase activity from picornavirus-infected cells have shown that the polymerase activity sediments as a membraneassociated complex of 60-250 S (8-14) and is associated with many viral polypeptides (15).We have found that precipitation with 2 M LiCl results in significant purification of the polio polymerase complex. Details of the purification procedure and properties of the purified polymerase will be reported elsewhere (Lundquist and Maizel, manuscript in preparatron). Here cell of purified Mahoney strain poliovirus type 1. Actinomycin D (4 ,Ag/ml) was added 10 min after infection. The radioactive amino acid (25 ,Ci/ml) was added to infected cells incubated in medium containing normal levels of all other essential amino acids. The medium concentration of the radioactive amino acid was reduced to 2% of the concentration normally present in Eagle's minimum essential medium. The medium was supplemented with 5% fetal calf serum 30 min after infection. Levels of guanidine that have no discernible effect on HeLa cells inhibit the replication of poliovirus, but viral inhibition of host cell protein synthesis is unaffected (1). Virus-specific proteins can then be labeled after reversal of guanidine inhibition. Guanidine (1 mM) was added 15 min after infection with poliovirus. After incubation at 370 for 1.5 hr, the infected cells were harvested by centrifugation, washed with Earle's isotonic salt solution, and resuspended in medium without guanidine, containing 5% fetal calf serum. The radioactive amino acid was added after 15 min, and the infection was stopped 2.5 hr after removal of guanidine.The mechanism of guanidine inhibition is u...

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confidence: 99%

Isolation of a Viral Polypeptide Associated with Poliovirus RNA Polymerase

Lundquist

Ehrenfeld

Maizel

1974

Proc. Natl. Acad. Sci. U.S.A.

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“…FMDV, as member of the Picornaviridae , has a similar genome organization and is believed to follow a similar replication strategy to poliovirus [22]. In contrast to poliovirus, replication and translation of FMDV RNA occurs in the cytoplasm [23], and a replication complex of FMDV and its possible association with cellular membranes has not yet been described in detail. However, our research with a QD-sdAb probe demonstrates that mature FMD virions are located on one side of the cell nucleus as the time after inoculation increases.…”

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confidence: 99%

Characterization of Asia 1 sdAb from Camels Bactrianus (C. bactrianus) and Conjugation with Quantum Dots for Imaging FMDV in BHK-21 Cells

et al. 2013

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Foot-and-mouth disease (FMD), caused by FMD virus (FMDV), is a highly contagious viral disease affecting cloven-hoofed animals. Camelids have a unique immunoglobulin profile, with the smallest functional heavy-chain antibodies (sdAb or VHH) naturally devoid of light chains with antigen-binding capacity. We screened and characterized five sdAbs against FMDV by immunized library from C. bactrianus with Asia 1 virus-like particles (VLPs). Three of five recombinant sdAbs were stably expressed in E.coli, remained highly soluble, and were serotype-specific for VP1 protein of FMDV Asia 1 by ELISA. These failed to completely neutralize the Asia 1 virus. According to the KD value of binding affinity to three sdAbs, which ranged from 0.44 to 0.71 nm by SPR, sdAb-C6 was selected and conjugated with Zn/CdSe quantum dots (QDs) to form a QDs-C6 probe, which was used to trace and image the subcellular location of FMDV in BHK-21 cells. The results show that FMD virions were observed from 3 h.p.i., and most of virions were distributed on one side of the nucleus in the cytoplasm. We demonstrate the utility of sdAbs as functionalized QDs are powerful tools for FMDV research.

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“…Lodish and Zinder (1966) presented an evidence that two RNA polymerases participated in the synthesis of bacteriophage f2 RNA. Lately, Arlinghaus and Polatnick (1969) isolated two vRNA polymerase-RNA complexes from foot-and-mouth disease virus-infected cells.…”

Section: Discussionmentioning

confidence: 99%

THE GROWTH KINETICS OF AN ATTENUATED POLIOVIRUS TYPE 1 STRAIN, LSc, 2ab AT AN ELEVATED TEMPERATURE

Yamazaki

Totsuka

Ohtaki

1970

JJMSB

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SUMMARY: An appreciable increase in viral RNA polymerase, infectious RNA and infectious virus particles was noticed when HeLa cell cultures infected with an attenuated poliovirus type 1 strain, LSc, 2ab, were maintained at 40 C. The induction of a very small, but significant amount of viral RNA polymerase was always observed in infected cells kept at 40 C. Increase in RNase-resistant infectious RNA was the most remarkable, amounting to from 10 to 30% of that produced at 35 C. The ratio of the amount produced at 40 C to that produced at 35 C was 1 to 3% with total infectious RNA and about 0.01% with infectious virus particles, which could be unmasked only by the use of photosensitive virus as inoculum.The heat sensitivity in situ or in vitro of the LSc, 2ab-induced RNA polymerase and the Mahoney-induced one was compared and both enzymes showed approximately the same rate of inactivation. INTRODUCTIONIt has been reported that the replication of an attenuated strain of type 1 poliovirus, LSc, 2 ab (LSc) is inhibited when infected cell cultures are incubated at 40 C, whereas a virulent strain, Mahoney, can grow at this temperature. This phenomenon has been utilized as one of the in vitro genetic markers of poliovirus closely related to neurovirulence (Sabin and Lwoff, 1959). It is of interest whether or not viral RNA (vRNA) polymerase, infectious RNA (i-RNA) and progeny virus are produced at 40 C, where the replication of LSc virus is largely inhibited.Investigations along this line have been carried out and it was noticed that an appreciable amount of vRNA polymerase activity, i-RNA and viral infectivity increased during the incubation of LSc-infected HeLa cell cultures at 40 C.Results of comparison of the heat sensitivity of vRNA polymerase induced by LSc and Mahoney viruses are also included in this paper. MATERIALS AND METHODS Cells and viruses:HeLa cells have been passaged in our laboratory as monolayer cultures.For the present experiments cells were transferred to Eagle's 382 YAMAZAKI et al.

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The Isolation of Two Enzyme-Ribonucleic Acid Complexes Involved in the Synthesis of Foot-and-Mouth Disease Virus Ribonucleic Acid

Cited by 37 publications

References 13 publications

Isolation of a Viral Polypeptide Associated with Poliovirus RNA Polymerase

Isolation of a Viral Polypeptide Associated with Poliovirus RNA Polymerase

Characterization of Asia 1 sdAb from Camels Bactrianus (C. bactrianus) and Conjugation with Quantum Dots for Imaging FMDV in BHK-21 Cells

THE GROWTH KINETICS OF AN ATTENUATED POLIOVIRUS TYPE 1 STRAIN, LSc, 2ab AT AN ELEVATED TEMPERATURE

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