The Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein Is Phosphorylated and Localizes in the Cytoplasm by 14-3-3-Mediated Translocation

Abstract

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, mitogenactivated 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 (14-3-3), leading to the accumulation of phosphorylated N protein in the nucleus, in the absence of growth factors. Using short interfering RNA specific to 14-3-3 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.

Subcellular Localization of SARS-CoV Structural Proteins

“…14 Several reports indicate that SARSCoV N is transported to the nucleus, whereas another did not observe nuclear localization. [15][16][17][18] Consistent with the latter, we were unable to detect SARS-CoV N in the nucleus. More comprehensive studies are clearly required to fully resolve and understand the trafficking of the N protein.…”

Glycogen Synthase Kinase - 3 Phosphorylates and Regulates the Stability of p27kip1 Protein

“…GFP siRNA was designed using sequence reported earlier. 6 All antibodies were obtained from Santacruz Biotechnology and chemicals from Sigma Chemicals, Roche or Calbiochem Corporation. Radioactivity was obtained from NEN Corporation.…”

“…Labeling with 35 S cys/met promix or 32 P orthophosphate, pulse-chase, immunoprecipitation and immunoblotting was done as described by Surjit et al 6 In vitro phosphorylation assay. Immunoprecipitated cell lysates were washed twice with kinase buffer (25 mM Tris, pH 7.5, 5 mM b glycerolphosphate, 2 mM DTT, 0.1 mM Na3VO4, 10 mM MgCl 2 ) and then incubated with the indicated substrate along with 100mM ATP and 10mCi of g 32 P-ATP for 45 minutes at 30˚C.…”

Emergence of new infections in the 21st century and identification of pathogens using next generation sequencing

“…From November 2002 to July 2003, a total of 8 098 patients in 25 countries contracted SARS; 774 patients died. In some populations [7] and age groups [8] mortality was as high as 40-55 %. Further scattered outbreaks of the infection were reported late in 2003 and early in 2004 in Singapore, Taiwan, Beijing and Guangzhou.…”