Structure and function of a membrane anchor-less form of the hemagglutinin-neuraminidase glycoprotein of Newcastle disease virus.
The hemagglutinin-neuraminidase (HN) glycoprotein of paramyxoviruses is anchored in the virion membrane near its amino terminus, protruding from the virion surface to mediate attachment to cellular receptors. Solubilization of HN spikes can be achieved by treatment of virions with detergent and high salt concentrations. When the solubilized HN protein from the Australia-Victoria (AV) isolate of the virus is incubated at 37 degrees C, a chymotrypsin-sensitive site between residues 112 and 113 is exposed. A chymotrypsin-cleaved soluble form of the protein, named CT-HN, has been prepared using this approach. It is membrane anchor-less, due to removal of a 14-kDa fragment from the NH2 terminus of HN. It retains all potential glycosylation sites and cysteines present in the ectodomain of the native protein. It migrates in nonreducing gels and sediments in sucrose gradients at the rate expected for homodimeric HN. The latter is also consistent with our demonstration by site-directed mutagenesis that cysteine residues at positions 6 and 123, respectively, mediate disulfide-linked homotetramer and homodimer formation. CT-HN retains almost total antigenicity, suggesting that it is conformationally very similar to the intact molecule, as well as receptor recognition function and, at low pH, neuraminidase activity. It should prove to be a useful tool for further studies of the structure and function of this important viral glycoprotein.
Mirza
A M
AM
Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester 01655.
Sheehan
J P
JP
Hardy
L W
LW
Glickman
R L
RL
Iorio
R M
RM
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000956:Antigens, Viral / Q000276:immunology; D002642:Chick Embryo; D002918:Chymotrypsin / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D015707:HN Protein / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008561:Membrane Fusion; D008969:Molecular Sequence Data; D009522:Newcastle disease virus / Q000378:metabolism*; D011487:Protein Conformation; D017510:Protein Folding
D000911:Antibodies, Monoclonal; D000956:Antigens, Viral; D004220:Disulfides; D015707:HN Protein; D002918:Chymotrypsin; D003545:Cysteine