Identification and partial characterization of a rhesus rotavirus binding glycoprotein on murine enterocytes.
In order to assess the possibility that rotavirus binds to a specific cellular receptor on enterocytes, we have used a viral overlay protein blot assay to study viral binding to murine intestinal brush border membranes (BBM). Infectious double-shelled particles of rhesus rotavirus bound specifically to two approximately 300- and 330-kDa glycoproteins from BBM prepared from suckling mice. Significantly less rotavirus binding was observed when adult BBM were examined. Rats have never been shown to harbor natural group A rotavirus infection and correspondingly, rat BBM showed no rotavirus binding activity. In suckling mice, rotavirus was found to bind to villus tip membranes to a much greater extent than to crypt preparations. Rotavirus binding activity was abolished by treatment of membrane preparations with protease. Analysis by glycolytic digestion of BBM with N- and O-glyconases revealed evidence for both N- and O-linked glycosylation of the rotavirus binding protein. Also neuraminidase digestion showed that O-linked sialic acid residues were required for virus binding. Monoclonal antibodies which immunoprecipitate the 300-kDa viral binding glycoprotein react with the apical surface of suckling but not adult enterocytes by Western blot. Baculovirus-expressed vp4, the rotavirus outer capsid spike protein, bound to the 300- and 330-kDa proteins and competed with rotavirus particles for binding sites. The ability of rotavirus to bind via vp4 to large BBM glycoproteins correlates with in vivo rotavirus cell tropism and host range restriction. Specific host cell receptor expression may be important in rotavirus pathogenesis.
Bass
D M
DM
Department of Medicine, Stanford University, California 94305.
Mackow
E R
ER
Greenberg
H B
HB
D000818:Animals; D015153:Blotting, Western; D002213:Capsid / Q000378:metabolism*; D036022:Capsid Proteins; D002460:Cell Line; D006023:Glycoproteins / Q000378:metabolism*; D006168:Guinea Pigs; D015151:Immunoblotting; D007421:Intestine, Small / Q000166:cytology / Q000382:microbiology* / Q000648:ultrastructure; D051379:Mice; D008871:Microvilli / Q000382:microbiology; D008970:Molecular Weight; D009500:Neutralization Tests; D011817:Rabbits; D051381:Rats; D011919:Rats, Inbred Strains; D011991:Receptors, Virus / Q000378:metabolism*; D012401:Rotavirus / Q000378:metabolism*; D012794:Sialic Acids / Q000378:metabolism
D036022:Capsid Proteins; D006023:Glycoproteins; D011991:Receptors, Virus; D012794:Sialic Acids; C060339:VP4 protein, Rotavirus