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Journal of Virology, February 2000, p. 1810-1814, Vol. 74, No. 4
Department of Pediatrics, Stanford
University, Stanford, California 94305
Received 16 July 1999/Accepted 22 November 1999
To further characterize the nature of proteolytic processing of the
astrovirus capsid, we infected Caco-2 cells with a high multiplicity of
astrovirus without trypsin in the presence of 5 to 10% fetal calf
serum. These infections were characterized by pulse-chase labeling with
[35S]methionine, electron microscopy, gel electrophoresis
of purified viral particles, and analysis of infectivity of such
particles with and without added trypsin. Pulse-chase experiments
showed that the astrovirus capsid protein was initially translated as an approximately 87-kDa protein. The 87-kDa capsid protein was rapidly
converted intracellularly to a 79-kDa form which was found in smaller
amounts in the cell supernatant. Purification by differential centrifugation yielded particles that appeared quite similar to trypsin-grown astrovirus particles by negatively stained electron microscopy. These particles were antigenically distinct from
trypsin-treated virions as demonstrated by their various reactions with
monoclonal antibodies in a solid-phase immunoassay. The purified
trypsin-free particles were mainly composed of the 79-kDa capsid
protein which was found to have an amino terminus at residue 71 of the
entire open reading frame 2 (ORF2) product. The cleavage site was
identified in a highly conserved region of the astrovirus ORF2 product.
These trypsin-free particles were minimally infectious in cultured
Caco-2 cells but became highly infectious (105-fold
increase) after trypsin but not chymotrypsin treatment. This
trypsin-enhanced infectivity correlated with conversion of the 79-kDa
capsid protein to three smaller peptides of approximately 34, 29, and
26 kDa.
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Proteolytic Processing of the Astrovirus
Capsid
*
Corresponding author. Mailing address: Department of
Pediatrics, Stanford University School of Medicine, Stanford, CA 94305. Phone: (650) 723-5070. Fax: (650) 724-3106. E-mail:
Dorsey.Bass{at}Forsythe.Stanford.edu.
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