S-palmitoylation of Rotavirus NSP4

Abstract

Rotavirus is the primary cause of gastroenteritis in children. The clinical symptoms of the disease include fever, vomiting and diarrhoea, which can be fatal due to excessive fluid loss. A protein encoded by rotavirus, NSP4, has been linked to the clinical symptoms of the disease acting as an enterotoxin. Further research into NSP4 identified its ability to activate proinflammatory signalling by acting as a ligand of Toll-like Receptor 2 (TLR2). Bacterial lipoproteins are the canonical ligands of TLR2; therefore, this study was designed to identify potential lipid modification of NSP4 and the effect of such modifications on protein function. Post-translational fatty acylation was examined using Acyl Biotin Exchange (ABE) and biorthogonal labelling of NSP4 with the palmitic acid analogue 17-Octadecynoic acid (17- ODYA). Location of potential sites of palmitoylation within the NSP4 sequence was investigated via mutational analysis. Results reveal that NSP4 (both intracellular and secreted forms) produced by bovine rotavirus (UK strain) is S-palmitoylated. Experiments confirm that a cysteine residue conserved throughout all known isolates of rotavirus at position 63 in the amino acid sequence is the site of fatty acylation. Based on the pleiotropic nature of NSP4, the effect of palmitoylation on several known traits of NSP4 was investigated. Results indicate that palmitoylation does not affect the ability of NSP4 to localise to lipid rafts but, governs its incorporation into detergent-resistant membranes (DRMs). Interestingly, a chemical inhibitor of palmitoylation, 2-bromopalmitate, was shown to influence the rate of rotavirus replication, but these experiments did not implicate NSP4.