Phosphorylation generates different forms of rotavirus NSP5

Area of Science:

• Virology
• Molecular Biology
• Protein Chemistry

Background:

• Rotaviruses are a leading cause of severe diarrheal disease in infants.
• Non-structural protein 5 (NSP5) is crucial for rotavirus replication and assembly.
• NSP5 is known to be post-translationally modified by O-glycosylation and phosphorylation.

Purpose of the Study:

• To investigate the complex post-translational modifications of rotavirus NSP5.
• To identify novel forms of NSP5 arising from phosphorylation.
• To elucidate the role of phosphorylation and O-glycosylation in NSP5 regulation.

Main Methods:

• In vivo and in vitro phosphorylation assays using [gamma-32P]ATP.
• Treatment of NSP5 with phosphatases.
• In vitro translation and phosphorylation of NSP5 precursor.
• Metabolic labeling of NSP5 with [1,6-3H]glucosamine.

Main Results:

• Phosphorylation of NSP5 leads to the formation of previously unidentified forms up to 34 kDa.
• Phosphatase treatment of NSP5 from infected cells yields a single 26 kDa band.
• In vitro phosphorylation produces 28 kDa and 32-34 kDa products, with phosphates on serine and threonine residues.
• NSP5 precursor can be phosphorylated to 28 kDa by infected cell extracts, indicating host cell involvement.
• NSP5 glycosylation and phosphorylation levels regulate protein forms, suggesting autophosphorylation.

Conclusions:

• Rotavirus NSP5 undergoes complex phosphorylation, generating diverse molecular forms.
• Phosphorylation and O-glycosylation are key regulatory mechanisms for NSP5.
• NSP5 likely participates in autophosphorylation, contributing to its modification and function.