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Journal of Virology, February 2000, p. 1810-1814, Vol. 74, No. 4
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Proteolytic Processing of the Astrovirus Capsid

D. M. Bass* and Shiqiang Qiu

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.

* 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.

Journal of Virology, February 2000, p. 1810-1814, Vol. 74, No. 4
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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