Basic residues within the ebolavirus VP35 protein are required for its viral polymerase cofactor function

Area of Science:

• Virology
• Molecular Biology
• Structural Biology

Background:

• Ebolavirus (EBOV) VP35 protein is crucial for viral replication and immune evasion.
• VP35 binds double-stranded RNA (dsRNA) and inhibits host interferon (IFN) production.
• The C-terminal IFN inhibitory domain (IID) of VP35 has key structural features for dsRNA binding and IFN inhibition.

Purpose of the Study:

• To investigate the role of conserved basic residues in the EBOV VP35 C-terminal IID.
• To determine the function of these residues in dsRNA binding, IFN inhibition, and viral RNA synthesis.
• To elucidate the interaction of VP35 with nucleoprotein (NP) and its role in replication complex formation.

Main Methods:

• Alanine substitution mutagenesis of VP35.
• In vitro assays for dsRNA binding and IFN inhibition.
• Minigenome assays to assess viral RNA polymerase function.
• Coimmunoprecipitation assays to study protein-protein interactions (VP35-NP).

Main Results:

• Basic residues outside the central dsRNA-binding patch are not essential for dsRNA binding or IFN inhibition.
• These residues, particularly within the first basic patch, are critical for viral RNA synthesis.
• The first basic patch is important for VP35 interaction with NP, suggesting a role in replication complex formation.
• VP35 IID interacts with NP, and dsRNA binding can modulate this interaction.

Conclusions:

• Conserved basic residues in the EBOV VP35 C-terminal IID play distinct roles in viral functions.
• Specific basic residues are critical for replication complex assembly via NP interaction.
• The VP35 IID is a validated target for therapeutic intervention against Ebolavirus.