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Journal of Virology, June 2005, p. 7283-7290, Vol. 79, No. 12
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.12.7283-7290.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Proteolytic Processing of Sapovirus ORF1 Polyprotein

Tomoichiro Oka,1* Kazuhiko Katayama,1 Satoko Ogawa,1 Grant S. Hansman,1,2 Tsutomu Kageyama,1 Hiroshi Ushijima,2 Tatsuo Miyamura,1 and Naokazu Takeda1

Department of Virology II, National Institute of Infectious Diseases,1 Department of Developmental Medical Sciences, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan2

Received 1 September 2004/ Accepted 25 January 2005

The genome of Sapovirus (SaV), a causative agent of gastroenteritis in humans and swine, contains either two or three open reading frames (ORFs). Functional motifs characteristic to the 2C-like NTPase (NTPase), VPg, 3C-like protease (Pro), 3D-like RNA-dependent RNA polymerase (Pol), and capsid protein (VP1) are encoded in the ORF1 polyprotein, which is afterwards cleaved into the nonstructural and structural proteins. We recently determined the complete genome sequence of a novel human SaV strain, Mc10, which has two ORFs. To investigate the proteolytic cleavage of SaV ORF1 and the function of protease on the cleavage, both full-length and truncated forms of the ORF1 polyprotein either with or without mutation in 1171Cys to Ala of the GDCG motif were expressed in an in vitro coupled transcription-translation system. The translation products were analyzed directly by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or by immunoprecipitation with region-specific antibodies. The ORF1 polyprotein was processed into at least 10 major proteins: p11, p28, p35, p32, p14, p70, p60, p66, p46, and p120. Seven of these products were arranged in the following order: NH2-p11-p28-p35(NTPase)-p32-p14(VPg)-p70(Pro-Pol)-p60(VP1)-COOH. p66, p46 and p120 were precursors of p28-p35 (NTPase), p32-p14 (VPg), and p32-p14 (VPg)-p70 (Pro-Pol), respectively. Mutagenesis in the 3C-like protease motif fully abolished the proteolytic activity. The cleavage map of SaV ORF1 is similar to those of other heretofore known members of the family Caliciviridae, especially to rabbit hemorrhagic disease virus, a member of the genus Lagovirus.

* Corresponding author. Mailing address: Department of Virology II, National Institute of Infectious Diseases, Gakuen 4-7-1, Musashi-murayama, Tokyo 208-0011, Japan. Phone: 81-42-561-0771. Fax: 81-42-561-4729. E-mail: oka-t{at}nih.go.jp.

Journal of Virology, June 2005, p. 7283-7290, Vol. 79, No. 12
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.12.7283-7290.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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