Deubiquitination activity associated with hepatitis E virus putative papain-like cysteine protease

Area of Science:

• Virology
• Molecular Biology
• Biochemistry

Background:

• Hepatitis E virus (HEV) is a significant human pathogen.
• The HEV ORF1 protein (pORF1) is essential for viral replication and possesses multiple functional domains.
• The LXGG motif within viral proteins is known to be recognized by deubiquitinating enzymes.

Purpose of the Study:

• To investigate the deubiquitinating activity of the HEV pORF1 protein, specifically the methyltransferase (MetT) and papain-like cysteine protease (PCP) domains.
• To determine if HEV pORF1 can process ubiquitinated substrates and interfere with cellular antiviral mechanisms.

Main Methods:

• HEV pORF1 MetT-PCP domain construct was expressed and purified.
• Deubiquitinating activity was assessed using fluorogenic substrates: ubiquitin-AMC, ISG15-AMC, Nedd8-AMC, and SUMO-AMC.
• DeISGylation of ISG15-conjugated cellular proteins was analyzed.

Main Results:

• The HEV pORF1 MetT-PCP domain exhibited robust deubiquitinating activity, efficiently cleaving ubiquitin, ISG15, Nedd8, and SUMO substrates.
• Processing of ISG15-AMC was particularly pronounced.
• The RNA helicase (Hel) and RNA-dependent RNA polymerase (RdRp) domains, despite containing an LXGG site, did not show processing activity.

Conclusions:

• The MetT-PCP domains of HEV pORF1 possess significant deubiquitinating enzyme activity.
• HEV pORF1 can perform deISGylation, indicating a potential mechanism for antagonizing host antiviral responses.
• This enzymatic activity may contribute to HEV pathogenesis by modulating host innate immunity.