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Journal of Virology, December 2005, p. 15016-15026, Vol. 79, No. 24
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.24.15016-15026.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

A Point Mutation within the Replicase Gene Differentially Affects Coronavirus Genome versus Minigenome Replication

Carmen Galán, Luis Enjuanes,* and Fernando Almazán

Centro Nacional de Biotecnología, CSIC, Department of Molecular and Cell Biology, Campus Universidad Autónoma, Cantoblanco, Darwin St. 3, 28049 Madrid, Spain

Received 13 June 2005/ Accepted 20 September 2005

During the construction of the transmissible gastroenteritis virus (TGEV) full-length cDNA clone, a point mutation at position 637 that was present in the defective minigenome DI-C was maintained as a genetic marker. Sequence analysis of the recovered viruses showed a reversion at this position to the original virus sequence. The effect of point mutations at nucleotide 637 was analyzed by reverse genetics using a TGEV full-length cDNA clone and cDNAs from TGEV-derived minigenomes. The replacement of nucleotide 637 of TGEV genome by a T, as in the DI-C sequence, or an A severely affected virus recovery from the cDNA, yielding mutant viruses with low titers and small plaques compared to those of the wild type. In contrast, T or A at position 637 was required for minigenome rescue in trans by the helper virus. No relationship between these observations and RNA secondary-structure predictions was found, indicating that mutations at nucleotide 637 most likely had an effect at the protein level. Nucleotide 637 occupies the second codon position at amino acid 108 of the pp1a polyprotein. This position is predicted to map in the N-terminal polyprotein papain-like proteinase (PLP-1) cleavage site at the p9/p87 junction. Replacement of G-637 by A, which causes a drastic amino acid change (Gly to Asp) at position 108, affected PLP-1-mediated cleavage in vitro. A correlation was found between predicted cleaving and noncleaving mutations and efficient virus rescue from cDNA and minigenome amplification, respectively.

* Corresponding author. Mailing address: Department of Molecular and Cell Biology, Centro Nacional de Biotecnología, CSIC, Campus Universidad Autónoma, Cantoblanco. Darwin St. 3, 28049 Madrid, Spain. Phone: 34 91 585 4555. Fax: 34 91 585 4915. E-mail: L.Enjuanes{at}cnb.uam.es.

Journal of Virology, December 2005, p. 15016-15026, Vol. 79, No. 24
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.24.15016-15026.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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