Characterization of SARS-CoV-2 replication complex elongation and proofreading activity.

Area of Science:

• Virology
• Molecular Biology
• Biochemistry

Background:

• The SARS-CoV-2 replication complex (RC) exhibits rapid RNA synthesis, increasing mutation rates.
• Viral genomic variability complicates the development of targeted antiviral therapies.
• Understanding RNA-dependent RNA polymerase (RdRp) fidelity is crucial for antiviral design.

Purpose of the Study:

• To biochemically characterize RNA template recognition and elongation fidelity of the SARS-CoV-2 RdRp.
• To investigate the role of the nsp14 exonuclease in RdRp fidelity.
• To explore the potential of 3' deoxy-terminator nucleotides as antiviral agents.

Main Methods:

• Biochemical assays to study RNA template recognition and elongation.
• Analysis of RdRp fidelity in the presence of modified nucleotides.
• Investigation of nsp10-nsp14 heterodimer activity on RNA substrates.

Main Results:

• The 2'OH group of RNA ribose is critical for RdRp template recognition and elongation.
• 3' deoxy-terminator nucleotides, such as 3'dATP, reduce RdRp fidelity, promoting mismatched base incorporation.
• The nsp10-nsp14 heterodimer cannot degrade RNA lacking free 2'OH or 3'OH groups.

Conclusions:

• The 2'OH group's role in RNA synthesis offers a target for antiviral development.
• 3' deoxy-terminator nucleotides show promise as components of novel SARS-CoV-2 antiviral oligonucleotide inhibitors.
• Targeting the RdRp with modified nucleotides could overcome viral genomic evolution challenges.