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Proteolytic processing of a human astrovirus nonstructural protein.
David Kiang1, Suzanne M Matsui1
1Division 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.
The Journal of General Virology
|December 26, 2001
Summary
Human astrovirus serotype 1
Area of Science:
- Virology
- Molecular Biology
- Enzymology
Background:
- Human astrovirus serotype 1 (HAstV-1) encodes a putative 3C-like serine protease in open reading frame (ORF)-1a.
- Understanding the function of this protease is crucial for viral replication mechanisms.
Purpose of the Study:
- To analyze the activity of the HAstV-1 3C-like serine protease.
- To identify the cleavage products and processing sites of the viral protease.
Main Methods:
- In vitro transcription and translation of ORF-1a.
- Immunoprecipitation using specific antibodies and an anti-FLAG antibody.
- Site-directed mutagenesis of the catalytic triad and cleavage site residues.
- N-terminal and C-terminal deletion analyses.
Main Results:
- The full-length ORF-1a product (p101) is processed into p38 and p64 fragments.
- Mutation of the catalytic triad (Ser-551, Asp-489, His-461) abolished p38 production.
- Cleavage occurs at the Gln-567/Thr-568 dipeptide, dependent on the serine protease.
- p38 is localized to the C terminus of ORF-1a.
Conclusions:
- The HAstV-1 ORF-1a encodes a functional 3C-like serine protease essential for viral protein processing.
- The protease cleaves p101 into p64 and p38 at a specific site, indicating its role in viral maturation.