Interferon-γ as a Potential Inhibitor of SARS-CoV-2 ORF6 Accessory Protein.

Area of Science:

• Virology
• Immunology
• Molecular Biology

Background:

• SARS-CoV-2 ORF6 protein disrupts host innate immunity by inhibiting interferon pathways.
• ORF6 protein sequesters RAE1, blocking nuclear mRNA export and hindering host cell function.
• The flexible C-terminus of ORF6 is critical for its interaction with host proteins.

Purpose of the Study:

• To investigate the potential of human interferon-gamma (hIFNγ) as an inhibitor of SARS-CoV-2 ORF6 protein activity.
• To evaluate the effect of hIFNγ on ORF6-mediated inhibition of mRNA transport and viral replication.

Main Methods:

• In silico docking simulations to assess hIFNγ binding affinity to ORF6.
• In vitro studies using ORF6-overexpressing cells to analyze RAE1 localization and mRNA export.
• Fluorescence microscopy and qRT-PCR to measure GFP expression as an indicator of mRNA transport.
• Assessment of RNA-DNA hybrids to evaluate effects on DNA replication.

Main Results:

• hIFNγ binds to the SARS-CoV-2 ORF6 protein with high affinity in silico.
• In vitro, hIFNγ treatment restored mRNA export from the nucleus to the cytoplasm by altering RAE1 localization.
• hIFNγ treatment reinstated GFP expression in ORF6-transfected cells, indicating successful mRNA trafficking.
• hIFNγ reduced RNA-DNA hybrids, mitigating ORF6's negative impact on DNA replication.

Conclusions:

• hIFNγ effectively inhibits the SARS-CoV-2 ORF6 protein's biological activity.
• hIFNγ shows potential as a therapeutic strategy to counteract ORF6-mediated immune evasion and replication.
• Targeting ORF6 with hIFNγ offers a promising approach to combat SARS-CoV-2 infection.