Preparation of Recombinant Alphaviruses for Functional Studies of ADP-Ribosylation

Area of Science:

• Virology
• Molecular Biology
• Neuroscience

Background:

• Chikungunya virus (CHIKV) is an alphavirus causing significant human disease.
• The nonstructural protein 3 (nsP3) contains a macrodomain (nsP3MD) with putative ADP-ribosyl hydrolase activity.
• The role of nsP3MD hydrolase activity in CHIKV pathogenesis remains unclear.

Purpose of the Study:

• To characterize the mono(ADP-ribosyl) hydrolase (MAR hydrolase) activity of the CHIKV nsP3 macrodomain.
• To investigate the importance of nsP3MD hydrolase function for viral replication and neurovirulence.
• To establish protocols for generating and analyzing recombinant CHIKV mutants.

Main Methods:

• Site-directed mutagenesis of an infectious CHIKV cDNA clone to create nsP3MD mutants.
• Biochemical assays to characterize MAR hydrolase activity of nsP3MD.
• Cell culture experiments using neuronal cells to assess viral replication.
• Mouse models to evaluate neurovirulence of CHIKV mutants.

Main Results:

• MAR hydrolase activity of nsP3MD was characterized.
• Mutations affecting nsP3MD hydrolase function impaired viral replication in neuronal cells.
• Impaired hydrolase activity correlated with reduced neurovirulence in mice.

Conclusions:

• The MAR hydrolase activity of the CHIKV nsP3 macrodomain is essential for efficient viral replication in neurons.
• nsP3MD hydrolase function is critical for CHIKV neurovirulence in vivo.
• The described methods facilitate further investigation of alphavirus nsP3MD function.