Different substitutions at conserved amino acids in domains II and III in the Sendai L RNA polymerase protein inactivate viral RNA synthesis.
The Sendai virus RNA polymerase is a complex of two virus-encoded proteins, the phosphoprotein (P) and the large (L) protein, where L is believed to possess all the enzymatic activities necessary for viral transcription and replication. The alignment of amino acid sequences of L proteins from negative-sense RNA viruses shows six regions, designated domains I-VI, of good conservation which have been proposed to be important for the various enzymatic activities of the polymerase. To directly address the role(s) of domains II and III, site-directed mutations were constructed by the substitution of multiple amino acids at 13 highly or mostly conserved residues. Analysis of in vitro viral transcription and replication showed that the majority of the mutations completely inactivated the L protein for all aspects of RNA synthesis, thus conservation correlated with the essential nature of the amino acid. At some positions different phenotypes, from inactivation to partial activities, were observed which depended on the nature of the amino acid that was substituted. Two mutants, K543R and K666V, could synthesize some leader RNA, but were defective in mRNA synthesis and replication. K666R and G737E had significantly reduced replication compared to transcription in vitro, but replicated genome RNA much more efficiently in vivo. K666A gave transcription, but no replication. Representative inactive L mutants, however, were still able to bind P protein and the polymerase complex was capable of binding nucleocapsids, so the defect appeared to be in the initiation of RNA synthesis.
Smallwood
Sherin
S
Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, Gainesville, Florida 32610, USA.
Hövel
T
T
Neubert
Wolfgang J
WJ
Moyer
Sue A
SA
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D019251:Nucleocapsid / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000096:biosynthesis; D012367:RNA, Viral / Q000096:biosynthesis*; D029082:Sendai virus / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D017386:Sequence Homology, Amino Acid; D014407:Tumor Cells, Cultured; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
C055859:P protein, Sendai virus; D010750:Phosphoproteins; D012333:RNA, Messenger; D012367:RNA, Viral; D014764:Viral Proteins; C064365:L protein, Sendai virus; D012321:DNA-Directed RNA Polymerases