Enhanced RNA replication and pathogenesis in recent SARS-CoV-2 variants harboring the L260F mutation in NSP6

Area of Science:

• Virology
• Molecular Biology
• Pathogenesis

Background:

• Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants exhibit increased transmission and immune evasion.
• Non-Spike mutations in SARS-CoV-2 variants are accumulating, but their functional impact remains unclear.
• Understanding these mutations is crucial for tracking viral evolution and developing countermeasures.

Purpose of the Study:

• To systematically assess the RNA replication and viral entry capabilities of recent SARS-CoV-2 Omicron variants.
• To investigate the compensatory mechanisms between viral entry and RNA replication.
• To identify specific non-Spike mutations contributing to enhanced viral fitness.

Main Methods:

• Reconstruction of SARS-CoV-2 variant genomes with disabled Spike expression (replicons) to study RNA replication.
• Single-round infection assays using complemented replicons to quantify viral entry.
• In vitro validation of identified mutations using mutant and revertant viral clones.
• Assessment of pathogenesis in a mouse model.

Main Results:

• A negative correlation was observed between viral entry and RNA replication efficiency across variants.
• Multiple non-Spike mutations were identified that enhance viral RNA replication.
• The NSP6 protein, particularly the L260F mutation, was identified as a key driver of enhanced replication and pathogenesis.
• The L260F mutation was shown to reduce host lipid droplet content.

Conclusions:

• SARS-CoV-2 variants employ compensatory evolution, balancing entry and replication.
• NSP6, especially the L260F mutation, plays a critical role in viral RNA replication and pathogenesis.
• These findings provide insights into SARS-CoV-2 evolution and potential therapeutic targets.