Neddylation of M1 negatively regulates the replication of influenza A virus

Area of Science:

• Virology
• Molecular Biology
• Post-translational Modifications

Background:

• Post-translational modifications are crucial for viral replication.
• Previous research indicated neddylation of influenza A virus (IAV) PB2 inhibits replication.
• NEDD8 overexpression inhibited PB2 K699R mutant viruses, suggesting other IAV proteins are neddylated.

Purpose of the Study:

• To investigate if other IAV proteins are subject to neddylation.
• To identify the specific viral protein and neddylation site.
• To determine the impact of M1 neddylation on viral replication and stability.

Main Methods:

• Identified M1 protein as a target for NEDD8 modification.
• Utilized the E3 ligase HDM2 to promote M1 neddylation.
• Generated and analyzed an IAV M1 K187R mutant in Madin-Darby canine kidney (MDCK) cells.
• Assessed viral replication, M1 protein stability (half-life), and virulence in mice.

Main Results:

• Influenza A virus M1 protein is neddylated by NEDD8, with HDM2 significantly promoting this modification.
• Lysine 187 (K187) on M1 was identified as the primary neddylation site.
• The M1 K187R mutant virus replicated more efficiently than wild-type virus in MDCK cells.
• NEDD8 overexpression more effectively inhibited wild-type WSN replication compared to the M1 K187R mutant.
• The neddylation-deficient M1 mutant (M1 K187R) exhibited increased stability (longer half-life) compared to wild-type M1.
• The M1 K187R mutant demonstrated increased virulence in mice compared to wild-type WSN.

Conclusions:

• Neddylation of the IAV M1 protein by HDM2 negatively regulates M1 stability.
• This reduced M1 stability consequently inhibits viral replication both in vitro and in vivo.
• M1 neddylation represents a potential therapeutic target for controlling influenza A virus infections.