Dual-Reporter SARS-CoV-2 Replicon for Screening Viral Polymerase Inhibitors

Area of Science:

• Virology
• Molecular Biology
• Drug Discovery

Background:

• The SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) is a critical target for antiviral therapies.
• Existing methods for evaluating RdRp inhibitors may lack specificity or efficiency.
• Developing safe and accurate cellular systems is essential for identifying novel antiviral compounds.

Purpose of the Study:

• To engineer a safe and effective cellular system for assessing SARS-CoV-2 RdRp inhibitors.
• To identify novel nucleoside analogs with potent antiviral activity against SARS-CoV-2.
• To compare the efficacy of identified compounds with existing therapeutic agents like molnupiravir.

Main Methods:

• Engineered a genetic construct lacking structural genes but containing reporter gene blocks (Rluc-GFP and Fluc-RFP) regulated by viral elements.
• Utilized the discontinuous transcription mechanism of coronaviruses for specific detection of viral RdRp activity.
• Established a cell line expressing herpes simplex virus thymidine kinase to facilitate nucleoside phosphorylation.
• Screened various compounds for their ability to inhibit SARS-CoV-2 RdRp activity in the developed cellular system.

Main Results:

• The engineered cellular system accurately differentiated between viral and cellular polymerase activity.
• 2 '-amino-2 '-deoxyadenosine and adenosine-N1-oxide demonstrated significant inhibition of SARS-CoV-2 RdRp.
• The antiviral activity of these novel compounds surpassed that of molnupiravir in the cellular assay.
• The cell line expressing HSV-TK facilitated the screening of nucleoside RdRp inhibitors.

Conclusions:

• The developed cellular system provides a robust platform for evaluating SARS-CoV-2 RdRp inhibitors.
• 2 '-amino-2 '-deoxyadenosine and adenosine-N1-oxide represent promising candidates for COVID-19 therapeutics.
• Further investigation into these compounds could lead to new antiviral strategies against SARS-CoV-2.