Clustered Charge-to-Alanine Mutagenesis of Human Respiratory Syncytial Virus L Polymerase Generates Temperature-Sensitive Viruses

• Published in: Virology (New York, N.Y.) Vol. 302; no. 1; pp. 207 - 216
• Main Authors: Tang, Roderick S., Nguyen, Nick, Zhou, Helen, Jin, Hong
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 10.10.2002
• Subjects: Alanine - genetics; Alanine - metabolism; Animals; Cercopithecus aethiops; Genome, Viral; Humans; Mutagenesis; Mutagenesis, Site-Directed; Phenotype; Rats; Recombination, Genetic; Respiratory Syncytial Virus, Human - enzymology; Respiratory Syncytial Virus, Human - genetics; Respiratory Syncytial Virus, Human - physiology; Sigmodontinae; Temperature; Tumor Cells, Cultured; Vero Cells; Viral Proteins - genetics; Viral Proteins - metabolism
• ISSN: 0042-6822; 1096-0341
• DOI: 10.1006/viro.2002.1596

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°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°C in tissue culture and restricted replication in the respiratory tracts of cotton rats.