Alternative amino acids at a single site in the Sendai virus L protein produce multiple defects in RNA synthesis in vitro.
Our long-term goal is to define the catalytic domains of the L protein subunit of the Sendai virus RNA polymerase. An aberrant polyadenylation phenotype in the vesicular stomatitis virus tsG16 L protein mutant has recently been identified as a phenylalanine to serine change at amino acid 1488 (Hunt and Hutchinson, Virology 193, 786-793, 1993). To test if functional domains are conserved in the L proteins of negative-strand RNA viruses, we attempted to create a similar polyadenylation defect in the Sendai virus L protein. Nine different amino acid substitutions at the analogous site in the Sendai L protein (cysteine at amino acid 1571) were constructed by site-directed mutagenesis of the gene. Each mutant L protein was synthesized and bound to the Sendai P protein to form the P-L polymerase complex. While none of these L mutants exhibited a change in polyadenylation, the single amino acid changes yielded a variety of activities in vitro. Mutants containing valine, leucine, or phenylalanine at amino acid 1571, amino acids found naturally in the L proteins of other paramyxoviruses, yielded polymerases that had biological activity equal to or better than the wild-type (WT) polymerase. Serine or threonine substitutions in the L protein at this position also resulted in polymerases with nearly WT synthetic activity. In contrast, a glycine substitution significantly decreased overall polymerase activity, whereas a tyrosine substitution gave decreased transcription, but virtually no DI genome replication in vitro. The tyrosine-substituted polymerase may be unable to carry out the packaging step of replication, since DI leader RNA synthesis was normal in this mutant. Mutant L proteins with basic arginine or histidine substitutions were inactive in all viral RNA synthesis in vitro, although the polymerase complexes could bind the nucleocapsid template.
Horikami
S M
SM
Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, Gainesville 32610-0266, USA.
Moyer
S A
SA
D000596:Amino Acids / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D012321:DNA-Directed RNA Polymerases; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010222:Parainfluenza Virus 1, Human / Q000235:genetics* / Q000378:metabolism; D012367:RNA, Viral / Q000096:biosynthesis*; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism
D000596:Amino Acids; D012367:RNA, Viral; D014764:Viral Proteins; C064365:L protein, Sendai virus; D012321:DNA-Directed RNA Polymerases