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Journal of Virology, June 1999, p. 5162-5165, Vol. 73, No. 6
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

A Single Amino Acid Substitution in the Phosphoprotein of Respiratory Syncytial Virus Confers Thermosensitivity in a Reconstituted RNA Polymerase System

Anthony C. Marriott,^* Steven D. Wilson, Jaspal S. Randhawa, and Andrew J. Easton

Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom

Received 28 December 1998/Accepted 23 February 1999

The single amino acid change Gly172 to Ser in the phosphoprotein (P) of respiratory syncytial virus (RSV) has previously been shown to be responsible for the thermosensitivity and protein-negative phenotype of tsN19, a mutant of the B subgroup RSN-2 strain. This single change was inserted into the P gene of the A subgroup virus RSS-2, and the resulting phenotype was observed in a plasmid-driven reconstituted RSV RNA polymerase system. Expression from a genome analogue containing two reporter genes was thermosensitive when directed by plasmids containing the N, L, M2, and mutant P genes cloned under the control of T7 promoters. Analysis of RNA synthesis showed that mutant P protein was unable to produce genome, antigenome, or mRNA at the restrictive temperature. At a semipermissive temperature, genome, antigenome, and mRNA synthesis were all reduced, 6- to 30-fold, relative to synthesis directed by a wild-type P plasmid. Binding of the mutant P protein to N protein in the absence of other viral proteins was unaffected by temperature, indicating that the lesion did not produce a large enough structural change to disrupt this binding. These data suggest that the plasmid rescue system is suitable for investigation of the role of thermosensitive mutations in RSV polymerase components in RNA synthesis.

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^* Corresponding author. Mailing address: Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom. Phone: 44 (0)1203 522696. Fax: 44 (0)1203 523701. E-mail: qm{at}dna.bio.warwick.ac.uk.

Present address: Department of Infection, University of Birmingham Medical School, Edgbaston, Birmingham B15 2TJ, United Kingdom.

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Journal of Virology, June 1999, p. 5162-5165, Vol. 73, No. 6
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

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