Reuer, Quentin and Kuhn, Richard J. and Wimmer, Eckard (1990) Characterization of poliovirus clones containing lethal and nonlethal mutations in the genome-linked protein VPg. Journal of Virology, 64 (6). pp. 2967-2975. ISSN 0022-538X http://resolver.caltech.edu/CaltechAUTHORS:REUjvir90
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Viral RNA synthesis was assayed in HeLa cells transfected with nonviable poliovirus RNA mutated in the genome-linked protein VPg-coding region. The transfecting RNA was transcribed in vitro from full-length poliovirus type 1 (Mahoney) cDNA containing a VPg mutagenesis cartridge. Hybridization experiments using ribonucleotide probes specific for the 3' end of positive- and negative-sense poliovirus RNA indicated that all mutant RNAs encoding a linking tyrosine in position 3 or 4 of VPg were replicated even though no virus was produced. VPg, but no VPg precursor, was found to be linked to the 5' end of the newly synthesized RNA. Encapsidated mutant RNAs were not found in transfected-cell lysates. After extended maintenance of transfected HeLa cells, a viable revertant of one of the nonviable RNAs was recovered; the revertant lost the lethal lesion in VPg by restoring the wild-type amino acid, but it retained all other nucleotide changes introduced during construction of the mutagenesis cartridge. Mutant RNA encoding phenylalanine or serine rather than tyrosine, the linking amino acid in VPg, was not replicated in transfected cells. A chimeric mutant containing the VPg-coding region of coxsackievirus within the poliovirus genome was viable but displayed impaired multiplication. A poliovirus-coxsackievirus chimera lacking a linking tyrosine in VPg was nonviable and replication-negative. The results indicate that a linkage-competent VPg is necessary for poliovirus RNA synthesis to occur but that a step in poliovirus replication other than initiation of RNA synthesis can be interrupted by lethal mutations in VPg.
|Additional Information:||Copyright © 1990 by the American Society for Microbiology. Received 27 October 1989; Accepted 22 March 1990. We thank Sung K. Jang and Jonathan Bradley for helpful suggestions and discussions, Aniko Paul and Christopher Hellen for critical reading of the manuscript, and Peter Kissel for synthesis of oligonucleotides. This work was supported in part by Public Health Service grants AI-15122 and CA-28146 to E.W. from the National Institutes of Health. Q.R. is supported by training grant 5T32 CA-09176 from the National Institutes of Health.|
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|Deposited By:||Tony Diaz|
|Deposited On:||01 Jun 2006|
|Last Modified:||26 Dec 2012 08:53|
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