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J Virol, February 1998, p. 910-918, Vol. 72, No. 2
Institute of Virology, University of Würzburg, 97078 Würzburg, Germany1;
M. P. Chumakov Institute of Poliomyelitis and Viral Encephalitides,
Russian Academy of Medical Sciences, 142782 Moscow Region,
Russia2; and
Department of Virology,
Institute of Medical Microbiology, Leiden University, 2300 AH Leiden,
The Netherlands3
Received 25 August 1997/Accepted 16 October 1997
Expression of the coronavirus gene 1-encoded polyproteins, pp1a and
pp1ab, is linked to a series of proteolytic events
involving virus-encoded proteinases. In this study, we used
transfection and immunoprecipitation assays to show that the human
coronavirus 229E-encoded papain-like cysteine proteinase, PCP1, is
responsible for the release of an amino-terminal protein, p9, from
the gene 1-encoded polyproteins. The same protein, p9, has also been
identified in virus-infected cells. Furthermore, using an in vitro
trans-cleavage assay, we defined the proteolytic cleavage
site at the carboxyl terminus of p9 as pp1a-pp1ab amino acids Gly-111
and Asn-112. These results and a comparative sequence analysis suggest
that substrate positions P1 and P5 seem to be the major determinants of
the PCP1 cleavage site and that the latter can occupy a variable position at the amino terminus of the coronavirus pp1a and pp1ab polyproteins. By combining the trans-cleavage assay with
deletion mutagenesis, we were also able to locate the boundaries of the active PCP1 domain between pp1a-pp1ab amino acids Gly-861-Glu-975 and Asn-1209-Gln-1285. Finally, codon mutagenesis was used to show
that Cys-1054 and His-1205 are essential for PCP1 proteolytic activity,
suggesting that these amino acids most likely have a catalytic
function.
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Proteolytic Processing at the Amino Terminus of Human Coronavirus
229E Gene 1-Encoded Polyproteins: Identification of a Papain-Like
Proteinase and Its Substrate
*
Corresponding author. Mailing address: Institute of
Virology, University of Würzburg, Versbacher Str. 7, 97078 Würzburg, Germany. Phone: 49-931-2013966. Fax: 49-931-2013934. E-mail: viro008{at}mail.uni-wuerzburg.de.
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