Studies on rabies virus RNA polymerase: 1. cDNA cloning of the catalytic subunit (L protein) of avirulent HEP-flury strain and its expression in animal cells.
To investigate the RNA polymerase of rabies virus, we cloned a cDNA of the catalytic subunit (called L protein because of its large molecular size) of the HEP-Flury strain, an avirulent strain obtained by high frequencies of serial embryonated hen egg passages. Nucleotide sequencing showed that the cDNA encodes a long polypeptide of 2,127 amino acids (Mr. 242,938). A comparison of the deduced amino acid sequence with that of other strains (PV and SAD B19) indicated that the sequence was highly conserved, except for several amino acid substitutions which were accumulated in some limited regions. A fragment of the cDNA was used for expression in Escherichia coli (E. coli) to prepare the L antigen for raising the antibodies in rabbits. Immunoprecipitation studies with the rabbit antiserum showed that the polypeptides produced in the L cDNA-transfected COS-7 cells displayed almost the same electrophoretic mobility as that of authentic L protein. Immunofluorescence studies indicated that both L and P (another subunit of RNA polymerase) proteins displayed colocalized distribution with the nucleocapsid antigen (N) in the cytoplasmic inclusion bodies, where envelope proteins (G and M) were absent. On the other hand, expression of the L protein alone did not cause inclusion body-like granular distribution, suggesting that the inclusion body-like accumulation depends on certain interaction(s) with other viral gene products, probably with the ribonucleoproteins comprising the inclusion bodies.
Morimoto
K
K
Department of Molecular Microbiology, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan.
Akamine
T
T
Takamatsu
F
F
Kawai
A
A
D000595:Amino Acid Sequence; D000818:Animals; D000914:Antibodies, Viral / Q000378:metabolism; D001483:Base Sequence; D019556:COS Cells; D002478:Cells, Cultured; D002642:Chick Embryo; D003001:Cloning, Molecular; D017124:Conserved Sequence; D018076:DNA, Complementary / Q000302:isolation & purification*; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics*; D015870:Gene Expression; D005814:Genes, Viral; D007106:Immune Sera / Q000378:metabolism; D008969:Molecular Sequence Data; D011817:Rabbits; D011820:Rabies virus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D017422:Sequence Analysis, DNA; D014764:Viral Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism
D000914:Antibodies, Viral; D018076:DNA, Complementary; D007106:Immune Sera; D014764:Viral Proteins; C097625:L protein, Rabies virus; D012321:DNA-Directed RNA Polymerases