Profiling Cullin4-E3 Ligases Interactomes and Their Rewiring in Influenza A Virus Infection

Area of Science:

• Virology
• Molecular Cell Biology
• Biochemistry

Background:

• Cullin 4-RING E3 ubiquitin ligase (CRL4) complexes are essential for Influenza A virus (IAV) replication.
• CRL4 complexes, including DDB1, DCAF11, and DCAF12L1, mediate non-degradative ubiquitination of the viral PB2 subunit.
• The impact of IAV infection on the CRL4 interactome is not well understood.

Purpose of the Study:

• To investigate how IAV infection remodels the CRL4 complex interactome.
• To identify cellular proteins that interact with CRL4 components during infection.
• To explore the functional consequences of these interactions for viral replication and potential therapeutic strategies.

Main Methods:

• Affinity Purification coupled with Mass Spectrometry (AP-MS) to identify protein-protein interactions.
• Split-nanoluciferase assay to validate direct CRL4 component partners.
• siRNA-mediated gene silencing to assess the role of identified partners in IAV infection.

Main Results:

• IAV infection significantly alters CRL4 complex associations, decreasing DDB1 and DCAF11 interactions while increasing DCAF12L1-associated proteins.
• The infection-induced rewiring affects cellular proteins involved in protein folding, ubiquitination, translation, splicing, and stress responses.
• Direct partners of CRL4 components were identified and validated as crucial for IAV infection.

Conclusions:

• IAV infection dynamically remodels the CRL4 E3 ubiquitin ligase interactome.
• This remodeling likely contributes to creating a cellular environment favorable for viral replication.
• The identified CRL4-interacting proteins represent potential targets for host-directed antiviral therapies.