Clustered charge-to-alanine mutagenesis of human respiratory syncytial virus L polymerase generates temperature-sensitive viruses.
Clustered charge-to-alanine mutagenesis was performed on the large (L) polymerase protein of human respiratory syncytial virus to identify charged residues in the L protein that are important for viral RNA synthesis and to generate temperature-sensitive viruses. Clusters of three, four, and five charged residues throughout the entire L protein were substituted with alanines. A minigenome replicon assay was used to determine the functions of the mutant L proteins and to identify mutations that caused temperature sensitivity by comparing the level of reporter gene expression at 39 and 33 degrees C. Charge-to-alanine mutations were introduced into an antigenomic cDNA derived from RSV A2 strain to recover infectious viruses. Of the 27 charge-to-alanine mutations, 17 recombinant viruses (63%) were obtained. Seven mutants (41%) exhibited small plaque morphologies and/or temperature-sensitive growth in tissue culture. To generate mutant viruses with more temperature-sensitive and attenuated phenotypes, several clusters of charge-to-alanine substitutions were combined. Five combination mutants were recovered that exhibited shut-off temperatures ranging from 36 to 39 degrees C in tissue culture and restricted replication in the respiratory tracts of cotton rats.
Tang
Roderick S
RS
MedImmune Vaccines, Mountain View, California 94043, USA.
Nguyen
Nick
N
Zhou
Helen
H
Jin
Hong
H
D000409:Alanine / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002522:Chlorocebus aethiops; D016679:Genome, Viral; D006801:Humans; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D051381:Rats; D011995:Recombination, Genetic; D018113:Respiratory Syncytial Virus, Human / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D016205:Sigmodontinae; D013696:Temperature; D014407:Tumor Cells, Cultured; D014709:Vero Cells; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D014764:Viral Proteins; D000409:Alanine