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Avian Reovirus Temperature-Sensitive Mutant tsA12 Has a Lesion in Major Core Protein A and Is Defective in Assembly

Department of Medical Microbiology and Infectious Diseases, University of Manitoba, Winnipeg, Manitoba, Canada

Received 27 February 2004/ Accepted 18 May 2004

Members of our laboratory previously generated and described a set of avian reovirus (ARV) temperature-sensitive (ts) mutants and assigned 11 of them to 7 of the 10 expected recombination groups, named A through G (M. Patrick, R. Duncan, and K. M. Coombs, Virology 284:113-122, 2001). This report presents a more detailed analysis of two of these mutants (tsA12 and tsA146), which were previously assigned to recombination group A. The capacities of tsA12 and tsA146 to replicate at a variety of temperatures were determined. Morphological analyses indicated that cells infected with tsA12 at a nonpermissive temperature produced 100-fold fewer particles than cells infected at a permissive temperature and accumulated core particles. Cells infected with tsA146 at a nonpermissive temperature also produced 100-fold fewer particles, a larger proportion of which were intact virions. We crossed tsA12 with ARV strain 176 to generate reassortant clones and used them to map the temperature-sensitive lesion in tsA12 to the S2 gene. S2 encodes the major core protein A. Sequence analysis of the tsA12 S2 gene showed a single alteration, a cytosine-to-uracil transition, at nucleotide position 488. This alteration leads to a predicted amino acid change from proline to leucine at amino acid position 158 in the A protein. An analysis of the core crystal structure of the closely related mammalian reovirus suggested that the Leu₁₅₈ substitution in ARV A lies directly under the outer face of the A protein. This may cause a perturbation in A such that outer capsid proteins are incapable of condensing onto nascent cores. Thus, the ARV tsA12 mutant represents a novel assembly-defective orthoreovirus clone that may prove useful for delineating virus assembly.

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