A post-assembly conformational change makes the SARS-CoV-2 polymerase elongation-competent

Area of Science:

• Virology
• Molecular Biology
• Structural Biology

Background:

• Coronaviruses (CoVs) utilize 16 nonstructural proteins (nsps) to form the replication-transcription complex (RTC).
• The RTC core, comprising nsp12 RNA-dependent RNA polymerase (RdRp), nsp7, and nsp8, is crucial for viral RNA synthesis.
• The assembly mechanism and activation of the CoV RTC remain incompletely understood.

Purpose of the Study:

• To elucidate the assembly mechanism of the core RTC.
• To investigate the conformational changes required for RTC processivity.
• To identify potential therapeutic targets within the RTC activation pathway.

Main Methods:

• In vitro assembly assays of the core RTC.
• RNA binding experiments.
• Conformational analysis of the RTC in the presence and absence of RNA.

Main Results:

• The core RTC preferentially assembles with nsp12 binding RNA first, followed by nsp7 and nsp8.
• RNA-bound RTC requires hundreds of seconds for conformational change to achieve processive elongation.
• Apo-RTC (without RNA) requires hours to adopt an elongation-competent conformation.

Conclusions:

• The assembly of the CoV core RTC is RNA-templated.
• An obligatory, time-dependent activation step is required for processive viral RNA synthesis.
• This activation step is a potential target for antiviral therapeutic interventions.