Purification and characterization of adult diarrhea rotavirus: identification of viral structural proteins.
Adult diarrhea rotavirus (ADRV) is a newly identified strain of noncultivable human group B rotavirus that has been epidemic in the People's Republic of China since 1982. We have used sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western (immuno-) blot analysis to examine the viral proteins present in the outer and inner capsids of ADRV and compared these with the proteins of a group A rotavirus, SA11. EDTA treatment of double-shelled virions removed the outer capsid and resulted in the loss of three polypeptides of 64, 61, and 41, kilodaltons (kDa). Endo-beta-N-acetylglucosaminidase H digestion of double-shelled virions identified the 41-kDa polypeptide as a glycoprotein. CaCl2 treatment of single-shelled particles removed the inner capsid and resulted in the loss of one polypeptide with a molecular mass of 47 kDa. The remaining core particle had two major structural proteins of 136 and 113 kDa. All of the proteins visualized on sodium dodecyl sulfate-polyacrylamide gel electrophoresis were antigenic by Western blot analysis when probed with convalescent-phase human and animal antisera. A 47-kDa polypeptide was most abundant and was strongly immunoreactive with human sera, animal sera raised against ADRV and against other group B animal rotaviruses (infectious diarrhea of infant rat virus, bovine and porcine group B rotavirus, and bovine enteric syncytial virus) and a monoclonal antibody prepared against infectious diarrhea of infant rat virus. This 47-kDa inner capsid polypeptide contains a common group B antigen and is similar to the VP6 of the group A rotaviruses. Human convalescent-phase sera also responded to a 41-kDa polypeptide of the outer capsid that seems similar to the VP7 of group A rotavirus. Other polypeptides have been given tentative designations on the basis of similarities to the control preparation of SA11, including a 136-kDa polypeptide designated VP1, a 113-kDa polypeptide designated VP2, 64- and 61-kDa polypeptides designated VP5 and VP5a, and several proteins in the 110- to 72-kDa range that may be VP3, VP4, or related proteins. The lack of cross-reactivity on Western blots between antisera to group A versus group B rotaviruses confirmed that these viruses are antigenically quite distinct.
Fang
Z Y
ZY
Viral Gastroenteritis Unit, Centers for Disease Control, Atlanta, Georgia 30333.
Glass
R I
RI
Penaranda
M
M
Dong
H
H
Monroe
S S
SS
Wen
L
L
Estes
M K
MK
Eiden
J
J
Yolken
R H
RH
Saif
L
L
D000328:Adult; D000956:Antigens, Viral / Q000302:isolation & purification*; D015153:Blotting, Western; D002213:Capsid / Q000276:immunology / Q000302:isolation & purification*; D003967:Diarrhea / Q000382:microbiology*; D006023:Glycoproteins / Q000276:immunology / Q000302:isolation & purification; D006031:Glycosylation; D006801:Humans; D008970:Molecular Weight; D012401:Rotavirus / Q000032:analysis / Q000302:isolation & purification* / Q000648:ultrastructure; D014764:Viral Proteins / Q000276:immunology / Q000302:isolation & purification*; D015678:Viral Structural Proteins
D000956:Antigens, Viral; D006023:Glycoproteins; D014764:Viral Proteins; D015678:Viral Structural Proteins