Proteolytic processing of a human astrovirus nonstructural protein.
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-567/Thr-568 dipeptide. These results suggest that p101 is cleaved into an N-terminal p64 fragment and a C-terminal p38 product at Gln-567/Thr-568 in a process that is dependent on the viral 3C-like serine protease.
Kiang
David
D
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.
Matsui
Suzanne M
SM
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.
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D017124:Conserved Sequence; D006801:Humans; D001257:Mamastrovirus / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016366:Open Reading Frames; D011499:Protein Processing, Post-Translational; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*
D017361:Viral Nonstructural Proteins; C446934:3C-like serine protease, human astrovirus serotype 1; D012697:Serine Endopeptidases