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Construction of a "mutagenesis cartridge" for poliovirus genome-linked viral protein: Isolation and characterization of viable and nonviable mutants

Kuhn, Richard J. and Tada, Hiroomi and Ypma-Wong, Mary Frances and Dunn, John J. and Semler, Bert L. and Wimmer, Eckard (1988) Construction of a "mutagenesis cartridge" for poliovirus genome-linked viral protein: Isolation and characterization of viable and nonviable mutants. Proceedings of the National Academy of Sciences of the United States of America, 85 (2). pp. 519-523. ISSN 0027-8424. PMCID PMC279582. doi:10.1073/pnas.85.2.519.

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By following a strategy of genetic analysis of poliovirus, we have constructed a synthetic "mutagenesis cartridge" spanning the genome-linked viral protein coding region and flanking cleavage sites in an infectious cDNA clone of the type 1 (Mahoney) genome. The insertion of new restriction sites within the infectious clone has allowed us to replace the wild-type sequences with short complementary pairs of synthetic oligonucleotides containing various mutations. A set of mutations have been made that create methionine codons within the genome-linked viral protein region. The resulting viruses have growth characteristics similar to wild type. Experiments that led to an alteration of the tyrosine residue responsible for the linkage to RNA have resulted in nonviable virus. In one mutant, proteolytic processing assayed in vitro appeared unimpaired by the mutation. We suggest that the position of the tyrosine residue is important for genome-linked viral protein function(s).

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Additional Information:© 1988 by the National Academy of Sciences. Communicated by Richard B. Setlow, September 8, 1987 (received for review May 26, 1987). We are grateful to our colleagues for many stimulating discussions. M.F.Y.-W. is a predoctoral trainee supported by a grant from the U.S. Public Health Service (CA 09054), and B.L.S. is supported by a research career development award (AI 00721) from the National Institutes of Health. This work was supported by grants from the U.S. Public Health Service (AI 15122 and CA 28146) to E.W. and (AI 22693) to B.L.S.; research carried out at Brookhaven National Laboratory was sponsored by the U.S. Department of Energy. The publication costs of this 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.
Funding AgencyGrant Number
NIHCA 09054
NIHAI 00721
NIHAI 15122
NIHCA 28146
Department of Energy (DOE)UNSPECIFIED
Issue or Number:2
PubMed Central ID:PMC279582
Record Number:CaltechAUTHORS:KUHpnas88
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:9715
Deposited By: Tony Diaz
Deposited On:10 Mar 2008
Last Modified:08 Nov 2021 21:01

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