Glycogen Synthase Kinase-3 Regulates the Phosphorylation of Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein and Viral Replication

Abstract

Coronavirus (CoV) nucleocapsid (N) protein is a highly phosphorylated protein required for viral replication, but whether its phosphorylation and the related kinases are involved in the viral life cycle is unknown. We found the severe acute respiratory syndrome CoV N protein to be an appropriate system to address this issue. Using high resolution PAGE analysis, this protein could be separated into phosphorylated and unphosphorylated isoforms. Mass spectrometric analysis and deletion mapping showed that the major phosphorylation sites were located at the central serine-arginine (SR)-rich motif that contains several glycogen synthase kinase (GSK)-3 substrate consensus sequences. GSK-3-specific inhibitor treatment dephosphorylated the N protein, and this could be recovered by the constitutively active GSK-3 kinase. Immunoprecipitation brought down both N and GSK-3 proteins in the same complex, and the N protein could be phosphorylated directly at its SR-rich motif by GSK-3 using an in vitro kinase assay. Mutation of the two priming sites critical for GSK-3 phosphorylation in the SR-rich motif abolished N protein phosphorylation. Finally, GSK-3 inhibitor was found to reduce N phosphorylation in the severe acute respiratory syndrome CoV-infected VeroE6 cells and decrease the viral titer and cytopathic effects. The effect of GSK-3 inhibitor was reproduced in another coronavirus, the neurotropic JHM strain of mouse hepatitis virus. Our results indicate that GSK-3 is critical for CoV N protein phosphorylation and suggest that it plays a role in regulating the viral life cycle. This study, thus, provides new avenues to further investigate the specific role of N protein phosphorylation in CoV replication.The causative pathogen for the epidemic severe acute respiratory syndrome (SARS) 2 was identified as the SARS coronavirus (SCoV) in 2003 (1, 2). Its genome consists of a ϳ30-kilobase positive-sense single-stranded RNA which encodes a 3Ј co-terminal set of nine subgenomic mRNAs with a common leader sequence at their 5Ј ends (3, 4). These subgenomic RNAs encode various structural and nonstructural proteins required to produce progeny virions, including the viral nucleocapsid (N) protein.The SCoV N protein is the most abundant viral structural protein. During the viral life cycle multiple copies of the N protein interact with the viral genome to form the ribonucleoprotein complex, which is subsequently packaged by a lipid envelope during viral budding, possibly through its interaction with the viral structure membrane (M) protein (5). In addition to its structural role, the N protein is also implicated in regulating the synthesis of viral RNA and protein (4, 6, 7). Using reverse genetics, the critical role of N protein in the replication of coronaviruses has been identified in HCoV-229E, TGEV (transmissible gastroenteritis coronavirus), and IBV (infectious bronchitis virus) (8 -10). However, the molecular mechanisms in N protein participation in viral replication and the cellular gene(s) involved in regulating the process re...

The Coronavirus Nucleocapsid Is a Multifunctional Protein

“…The flexible LKR is capable of direct interaction with RNA under in vitro conditions [37]. Potential phosphorylation sites have been mapped to the Ser/Arg-rich portion of the LKR of SARS-CoV N [38,39,40]. These LKR phosphorylation sites are thought to function in binding M protein, heteronuclear ribonucleoprotein (hnRNP-A1) and RNA to the N protein with high binding affinity [14,41,42,43].…”

“…Moreover, both the middle and C-terminal IDRs (Figure 1) have been implicated in the oligomerization of the N protein [44,65], with the middle IDR also associated with N protein functionality and N-M interaction [19,39,40,66]. It would be interesting to determine whether the presence of three disordered regions in SARS N, compared to the one disordered region in HCoV-NL63 N for example, would result in SARS N having a higher binding affinity to viral, as well as host cellular proteins.…”

The SARS coronavirus nucleocapsid protein – Forms and functions

“…The middle IDR, which we coined LKR, and C-terminal IDR have both been implicated in the oligomerization of the N protein (He et al, 2004a;Luo et al, 2006). The LKR includes a Ser/Arg-rich region that contains a number of putative phosphorylation sites, which may regulate N protein function (Peng et al, 2008;Surjit et al, 2005;Wu et al, 2009) and N-M interaction (He et al, 2004b). Based on these new findings, Chang et al proposed a structure-based domain arrangement for SARS-CoV N protein where the NTD and CTD are sandwiched between three IDRs.…”

Increased titer and reduced lactate accumulation in recombinant retrovirus production through the down‐regulation of HIF1 and PDK

“…Transcriptome analysis also indicated the glycolytic flux to be partially drained from glycolysis toward UDP-glucose biosynthesis, the first intermediate in glycogen biosynthesis (Fig. Indeed, glycogen biosynthesis has been implied in the replication of several viruses (Guendel et al, 2014;Kehn-Hall et al, 2012;Wu et al, 2009). In line with these results, Coroadinha et al (2006a) reported enhancement of glycogen biosynthesis associated to titer increases for another retroviral vector producer cell line (Te Fly A7).…”