Identification and baculovirus expression of the VP4 protein of the human group B rotavirus ADRV.
A complete cDNA copy of the fourth RNA segment of the human group B rotavirus adult diarrheal rotavirus (ADRV) has been cloned into lambda phage and excised into plasmid pSK Bluescript. Gene segment 4 contains 2,303 bases and encodes one long open reading frame beginning at base 16 and terminating at base 2263. The encoded protein contains 749 amino acids, with a calculated molecular mass of 84.4 kDa and a pI of 6.1. Gene 4 cDNA was inserted into a recombinant baculovirus via homologous recombination. The gene 4 polypeptide migrates at 84 kDa when expressed either by a recombinant baculovirus or in vitro in a rabbit reticulocyte lysate. The gene 4 protein is immunoprecipitable by hyperimmune serum to ADRV, human ADRV convalescent-phase serum, a porcine group B rotavirus infection serum, and a monoclonal antibody made to ADRV virion. Guinea pig hyperimmune serum to the baculovirus-expressed ADRV VP4 protein recognizes virus and immunoprecipitates an 84-kDa protein from in vitro translations of total ADRV mRNA. In addition, the gene 4-encoded protein shares significant amino acid identity and similarity with the group A rotavirus VP4 protein. This information, together with our previous identification of an 84-kDa protein present on iodinated intact virion but not EDTA-treated ADRV, suggests that gene 4 encodes the VP4 protein equivalent present on the outer capsid of ADRV. The ADRV VP4 protein is also 58% identical to the IDIR rat group B rotavirus gene segment 3 protein. The substantial differences between these two group B VP4 proteins suggests that they are distantly related and likely to define two different group B rotavirus VP4 serotypes. The baculovirus-expressed VP4 protein should be useful for developing serotyping reagents and tests for human and animal group B rotaviruses as well as for addressing the role of VP4 in ADRV neutralization.
Northport Veterans Administration Medical Center, New York 11768.
E R
ER
Mackow
R
R
Werner-Eckert
M E
ME
Fay
H
H
Tao
G
G
Chen
D000595:Amino Acid Sequence; D000914:Antibodies, Viral / Q000097:blood; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002213:Capsid / Q000096:biosynthesis / Q000235:genetics*; D036022:Capsid Proteins; D003001:Cloning, Molecular; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011994:Recombinant Proteins / Q000096:biosynthesis; D012401:Rotavirus / Q000145:classification / Q000235:genetics* / Q000254:growth & development; D017386:Sequence Homology, Amino Acid; D012703:Serotyping
D000914:Antibodies, Viral; D036022:Capsid Proteins; D011994:Recombinant Proteins; C060339:VP4 protein, Rotavirus