New insights into complex formation by SARS-CoV-2 nsp10 and nsp14

Area of Science:

• * Virology
• * Molecular Biology
• * Biochemistry

Background:

• * SARS-CoV-2 non-structural protein 10 (nsp10) activates nsp14 and nsp16 enzymes.
• * Nsp14 possesses 3'-5' exoribonuclease (ExoN) and N7-methyltransferase (N7-MTase) domains.
• * Nsp10 is key for viral drug resistance and evading host immunity via RNA cap methylation.

Purpose of the Study:

• * To characterize the interaction between SARS-CoV-2 nsp10 and nsp14.
• * To determine the role of nsp10's N- and C-termini in nsp14 binding.
• * To investigate intermediate binding steps between nsp10 and nsp14.

Main Methods:

• * Microscale thermophoresis
• * Multi-detection SEC
• * Hydrogen-deuterium (H/D) exchange mass spectrometry

Main Results:

• * Full-length nsp10 binds nsp14, with N- and C-termini being important for optimal binding.
• * Truncated nsp10 variants show altered binding affinities.
• * H/D exchange mass spectrometry suggests intermediary interactions between nsp10 and nsp14's N7-MTase domain.

Conclusions:

• * The C-terminal region of nsp10 is functionally important for nsp14 binding.
• * The nsp10-nsp14 complex formation involves intermediate steps and transient contacts.
• * These findings offer insights into viral replication mechanisms and potential therapeutic targets.