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13:04
A Protocol for Analyzing Hepatitis C Virus Replication
Published on: June 26, 2014
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Structural features stabilized by divalent cation coordination within hepatitis E virus ORF1 are critical for viral
Robert LeDesma1, Brigitte Heller1, Abhishek Biswas1
1Department of Molecular Biology, Lewis Thomas Laboratory, Princeton University, Princeton, United States.
Elife
|February 28, 2023
Summary
Hepatitis E virus (HEV) ORF1 polyprotein is multifunctional and not proteolytically processed. Key cysteines in the pPCP domain are vital for HEV replication, highlighting structural roles in viral genome replication.
Area of Science:
- Virology
- Structural Biology
- Molecular Biology
Background:
- Hepatitis E virus (HEV) causes over 20 million infections yearly.
- The HEV open reading frame (ORF)1 polyprotein is crucial for viral genome replication.
- The structural and functional roles of HEV ORF1 subdomains remain largely uncharacterized.
Purpose of the Study:
- To elucidate the structural and functional characteristics of the HEV ORF1 polyprotein.
- To investigate the role of the putative papain-like cysteine protease (pPCP) domain in HEV replication.
- To develop a comprehensive model for HEV ORF1 structure and function.
Main Methods:
- Scanning mutagenesis of the HEV ORF1 protein, focusing on the pPCP domain.
- Analysis of viral replication and genome replication rescue assays.
- Biochemical assays to assess protein processing and domain interactions.
Main Results:
- HEV ORF1 functions as a single, multifunctional polyprotein without proteolytic processing.
- Six cysteine residues within the pPCP domain are essential for HEV replication.
- These cysteines are involved in divalent metal ion coordination, critical for ORF1 structure and interdomain interactions.
- The pPCP domain requires the context of the full-length ORF1 protein to rescue viral genome replication.
Conclusions:
- HEV ORF1 is a structurally integrated, multifunctional protein essential for viral replication.
- The pPCP domain's catalytic activity is likely dependent on its structural context within the full-length ORF1.
- This study provides a novel structural and functional model for HEV ORF1.
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