Isolation and preliminary characterization of temperature-sensitive mutants of encephalomyocarditis virus

Radloff, Roger

doi:10.1128/jvi.27.1.182-192.1978

Cited by 9 publications

(11 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The results seen after cell-free translation of wild-type and mutant RNA at 38°C (Fig. 1) were analogous to the results obtained in vivo at 39.5°C with EMC wild-type virus and these mutants (17). Cell-free translation of RNA isolated from other cleavage-defective mutants gave similar protein profiles after translation at 37 or 38°C.…”

Section: Resultssupporting

confidence: 79%

“…If the cleavage-defective mutant viral RNA codes for the synthesis of temperature-sensitive capsid precursors, we would expect the following results with in vitro proteolysis experiments: (i) the mutant viral protease should cleave wild-type capsid precursors as completely as wild-type viral protease is capable of cleaving these precursors; and (ii) wild-type viral protease should not cleave mutant capsid precursors as completely as it is capable of cleaving wild-type capsid precursors. Our results indicate that seven of the cleavagedefective mutants previously characterized, temperature-sensitive (ts) mutants ts2, ts3, tslS, ts54, ts83, and tslOO (17), have capsid precursors which are temperature sensitive.…”

mentioning

confidence: 71%

“…Cell-free translation of wild-type and cleavagedefective mutant viral RNA at several temperatures. We did in vitro proteolysis experiments as described in the introduction to this work to functionally characterize seven of the cleavagedefective mutants isolated previously (17). First, it was necessary to establish permissive and nonpermissive temperatures for capsid precursor cleavage during cell-free translation analogous to the respective temperatures of 33 and 39.5°C normally used for growth in vivo of temperature-sensitive mutant EMC viruses.…”

Section: Resultsmentioning

confidence: 99%

“…Slab gel electrophoresis. Slab gel electrophoresis was performed as described previously (17). The gels were stained with Coomassie brilliant blue as described by Swank and Munkres (23) and prepared for autoradiography (11) or fluorography (10).…”

Section: Methodsmentioning

confidence: 99%

“…However, the location of the mutation in these cleavage-defective mutants was not identified. Two possibilities were proposed when this work was begun (17): (i) a mutation in the capsid precursor polypeptide or its intermediates (or both) alters the configura-tion of those proteins sufficiently that proteolytic cleavage cannot occur at the nonpermissive temperature; and (ii) a mutation in the viral protein required for cleavage alters it sufficiently that it cannot participate in the cleavage process at the nonpermissive temperature. In vitro proteolysis of wild-type and cleavage-defective mutant capsid precursor polypeptides with products from cell-free translation of wild-type and cleavage-defective mutant RNA (containing wild-type or mutant viral protease, respectively) provided us with a useful tool for choosing between these two alternatives.…”

mentioning

confidence: 99%

See 4 more Smart Citations

Functional characterization of cleavage-defective mutants of encephalomyocarditis virus

Young¹,

Radloff²

1982

J Virol

Self Cite

View full text Add to dashboard Cite

We characterized seven temperature-sensitive capsid cleavage (cleavage-defective) mutants of encephalomyocarditis virus. Our experimental approach was to monitor in vitro proteolysis reactions of either wild-type or cleavage-defective mutant capsid precursors mixed with cell-free translation products (containing the viral protease) of either wild-type or mutant viral RNA. The cell-free translation reactions and in vitro proteolysis reactions were done at 38°C, because at this temperature cleavage of the capsid precursors was restricted in reactions containing cleavage-defective mutant viral RNA as the message, relative to those reactions containing wild-type viral RNA as the message. Wild-type or cleavagedefective mutant capsid precursors were prepared by adding cycloheximide to cell-free translation reactions primed with wild-type or mutant viral RNA, respectively, 12 min after the initiation of translation. In vitro proteolysis of wildtype capsid precursors with cell-free translation products of either wild-type or cleavage-defective mutant viral RNA led to similar products at 38°C, indicating that the cleavage-defective mutant viral protease was not temperature sensitive. As a corollary to this, at 38°C cleavage-defective mutant capsid precursors were not cleaved as completely as were wild-type capsid precursors by products of cellfree translation of wild-type viral RNA. The results from these in vitro proteolysis experiments indicate that all seven of the cleavage-defective mutants have capsid precursors with a temperature-sensitive configuration. High-molecular-weight precursor polypeptides are cleaved to functional proteins by a series of sequential proteolytic cleavages during the replication of picornaviruses (2-6). With encephalomyocarditis (EMC) virus, the capsid proteins 8, P, y, and a are generated by cleavage of the high-molecular-weight precursor polypeptide A, with polypeptides Dl, e, and possibly B

show abstract

Section: Resultssupporting

confidence: 79%

mentioning

confidence: 71%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Functional characterization of cleavage-defective mutants of encephalomyocarditis virus

Young¹,

Radloff²

1982

J Virol

Self Cite

View full text Add to dashboard Cite

show abstract

Temperature-sensitive RNA polymerase mutants of a picornavirus.

Lowe¹,

King²,

McCahon³

et al. 1981

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

View full text Add to dashboard Cite

Temperature-sensitive (ts) RNA polymerase mutants of a picornavirus are reported, Two foot-and-mouth disease virus (FMDV) mutants designated ts 22 and to 115 have been characterized. As judged by isoelectric focusing, both have charge alterations in P56a, the FMDV RNA polymerase protein. Virus RNA synthesis in cells infected with the mutants is severely impaired at the nonpermissive temperature. RNA polymerase purified from baby hamster kidney cells infected with these mutants exhibits a marked to transcribing activity in vitro. Spontaneous revertants of both mutants have P56a polypeptides that are indistinguishable from the parental proteins on the basis of charge. The revertants regain the ability to synthesize virus RNA in vivo at the nonpermissive temperature. RNA polymerase purified from the revertants remains transcriptionally active at the nonpermissive temperature.The genome of foot-and-mouth disease virus (FMDV) is a single-stranded "positive"-sense RNA molecule of approximately 8 kilobases with a small protein termed VPg covalently attached to its 5' end, an internal poly(C) tract, and a poly(A) sequence at the 3' end (1) (Fig. 1) (14,15). Hence, a tentative alignment of the genetic and biochemical maps has been constructed (15). We describe how a modified EF technique was used to screen mutants with ts loci at the 3' end of the FMDV genetic map for an altered P56a polypeptide. Two candidate RNA polymerase mutants (ts 22 and.ts 115) were identified by these criteria. Cells infected with these mutants synthesize only trace amounts of virus RNA at the nonpermissive temperature. Furthermore, FMDV RNA polymerase purified from the cytoplasm of baby hamster kidney cells infected with the mutants showed a ts transcribing activity in vitro when compared with the enzyme extracted from cells infected with the parental virus. MATERIALS AND METHODSViruses. The parental FMDV strain from which all mutants were derived was 0 Pacheco (ts+). Mutants mapping at the 3' end of the FMDV genome (5-fluorouracil-induced ts 22 and spontaneous ts 115) have been described (12, 13). The isolation ofa revertant ofts 22 (designated ts 22R) also has been described (15 The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

show abstract

Defective Interfering Particles of Encephalomyocarditis Virus

Radloff

Young

1983

Journal of General Virology

View full text Add to dashboard Cite

SUMMARYEncephalomyocarditis (EMC) virus and five temperature-sensitive mutants of EMC virus were serially passed numerous times in HeLa, L929 and BHK-21 cells. Equilibrium centrifugation in CsC1 density gradients of virus and electrophoresis of virus RNA from many of the high number passages indicated the presence of standard EMC virus and particles containing RNA of lower molecular weight. Three virus isolates with high passage number were able to interfere with the replication of the standard virions in mixed infections. We believe our results show the generation of defective interfering particles of EMC virus.

show abstract

Isolation and preliminary characterization of temperature-sensitive mutants of encephalomyocarditis virus

Cited by 9 publications

References 27 publications

Functional characterization of cleavage-defective mutants of encephalomyocarditis virus

Functional characterization of cleavage-defective mutants of encephalomyocarditis virus

Temperature-sensitive RNA polymerase mutants of a picornavirus.

Defective Interfering Particles of Encephalomyocarditis Virus

Contact Info

Product

Resources

About