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Proteolytic processing of a human astrovirus nonstructural protein

Division of Gastroenterology, Center for Clinical Sciences Research, Room 3115, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305-5187, USA, and VA Palo Alto Health Care System, Palo Alto, CA 94304, USA¹

Author for correspondence: Suzanne Matsui (mail to the Center for Clinical Sciences Research). Fax +1 650 852 3259. e-mail sumatsui{at}stanford.edu

To analyse the activity of the putative 3C-like serine protease encoded in open reading frame (ORF)-1a of human astrovirus serotype 1 (HAstV-1), ORF-1a was transcribed and translated in vitro. Translation products, identified by immunoprecipitation with specific antibodies against recombinant C-terminal ORF-1a fragments, include the full-length 101 kDa (p101) protein and a 38 kDa band (p38). In addition, a 64 kDa protein (p64) was immunoprecipitated by an anti-FLAG antibody when a FLAG epitope was inserted at the N terminus of the ORF-1a product. Mutation of the amino acids predicted to form the catalytic triad of the HAstV-1 3C-like serine protease (Ser-551, Asp-489, His-461) resulted in undetectable levels of p38, supporting the involvement of the HAstV-1 3C-like serine protease and the importance of these amino acids in the processing of p101 into p38 and p64. N-terminal deletions of up to 420 aa of p101 that did not involve the predicted 3C-like serine protease motif did not alter levels of p38 compared to wild-type. C-terminal deletion analysis localized p38 to the C terminus of ORF-1a. Mutation of the P1 residue of the predicted cleavage site, which is conserved among known human and sheep astrovirus sequences, resulted in no detectable p38, supporting cleavage at the Gln-567Thr-568 dipeptide. These results suggest that p101 is cleaved into an N-terminal p64 fragment and a C-terminal p38 product at Gln-567Thr-568 in a process that is dependent on the viral 3C-like serine protease.

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