The severe acute respiratory syndrome coronavirus nucleocapsid protein is phosphorylated and localizes in the cytoplasm by 14-3-3-mediated translocation.
The severe acute respiratory syndrome coronavirus(SARS-CoV) nucleocapsid (N) protein is one of the four structural proteins of the virus and is predicted to be a 46-kDa phosphoprotein. Our in silico analysis predicted N to be heavily phosphorylated at multiple residues. Experimentally, we have shown in this report that the N protein of the SARS-CoV gets serine-phosphorylated by multiple kinases, in both the cytoplasm and the nucleus. The phosphoprotein is stable and localizes in the cytoplasm and coprecipitates with the membrane fraction. Also, using specific inhibitors of phosphorylation and an in vitro phosphorylation assay, we show that the nucleocapsid protein is a substrate of cyclin-dependent kinase (CDK), glycogen synthase kinase, mitogen-activated protein kinase, and casein kinase II. Further, we show that the phosphorylated protein is translocated to the cytoplasm by binding to 14-3-3 (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein). 14-3-3 proteins are a family of highly conserved, ubiquitously expressed eukaryotic proteins that function primarily as adapters that modulate interactions between components of various cellular signaling and cell cycle regulatory pathways through phosphorylation-dependent protein-protein interactions. Coincidentally, the N protein was also found to downregulate the expression of the theta isoform of 14-3-3 (14-3-3theta), leading to the accumulation of phosphorylated N protein in the nucleus, in the absence of growth factors. Using short interfering RNA specific to 14-3-3theta we have inhibited its expression to show accumulation of phosphorylated N protein in the nucleus. Thus, the data presented here provide a possible mechanism for phosphorylation-dependent nucleocytoplasmic shuttling of the N protein. This 14-3-3-mediated transport of the phosphorylated N protein and its possible implications in interfering with the cellular machinery are discussed.
Surjit
Milan
M
Virology Group, International Centre for Genetic Engineering & Biotechnology, New Delhi, India.
Kumar
Ravinder
R
Mishra
Rabi N
RN
Reddy
Malireddy K
MK
Chow
Vincent T K
VT
Lal
Sunil K
SK
D048948:14-3-3 Proteins / Q000378:metabolism*; D000818:Animals; D047390:Casein Kinase II / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D000086462:Coronavirus Nucleocapsid Proteins; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D038341:Glycogen Synthase Kinases / Q000378:metabolism; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D019590:Nucleocapsid Proteins / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D020033:Protein Isoforms / Q000378:metabolism; D045473:Severe acute respiratory syndrome-related coronavirus / Q000378:metabolism*
D048948:14-3-3 Proteins; D000086462:Coronavirus Nucleocapsid Proteins; D019590:Nucleocapsid Proteins; D020033:Protein Isoforms; D038341:Glycogen Synthase Kinases; D047390:Casein Kinase II; D018844:Cyclin-Dependent Kinases; D020928:Mitogen-Activated Protein Kinases