Pestivirus Npro Directly Interacts with Interferon Regulatory Factor 3 Monomer and Dimer

Area of Science:

• Immunology
• Virology
• Molecular Biology

Background:

• Interferon regulatory factor 3 (IRF3) is a key transcription factor in the innate immune response to viral infections, activating type I interferon production.
• Viruses, including pestiviruses like classical swine fever virus (CSFV), have evolved mechanisms to subvert this response, often by targeting IRF3.
• The CSFV N-terminal protease (N(pro)) facilitates IRF3 degradation, but the molecular basis of this interaction and the targeted IRF3 species were unclear.

Purpose of the Study:

• To elucidate the molecular details of the interaction between CSFV N(pro) and IRF3.
• To determine which form(s) of IRF3 (monomer/dimer, active/inactive) are targeted by N(pro) for degradation.
• To identify the binding site of N(pro) on IRF3.

Main Methods:

• Used recombinant N(pro) and IRF3 proteins for in vitro interaction studies.
• Investigated the interaction with full-length IRF3 and its individual domains.
• Assessed N(pro) binding to both inactive IRF3 monomers and constitutively active IRF3 dimers bound to CREB-binding protein (CBP).

Main Results:

• CSFV N(pro) directly interacts with full-length IRF3 in a 1:1 soluble complex, independent of other cellular proteins.
• The interaction requires the full-length IRF3 protein, not just individual domains.
• N(pro) binds to both inactive IRF3 monomers and constitutively active IRF3 dimers bound to CBP, indicating it targets both species.
• The N(pro) binding site on IRF3 is distinct from the dimer interface and CBP-binding site.

Conclusions:

• CSFV N(pro) directly binds to IRF3, targeting both inactive and activated forms for ubiquitination and proteasomal degradation.
• This broad targeting of IRF3 by N(pro) is a significant mechanism for pestivirus immune evasion.
• The findings provide molecular insights into how viruses disrupt host antiviral defenses.