Mol. Cells 2022; 45(12): 911-922
Published online December 19, 2022
https://doi.org/10.14348/molcells.2022.0130
© The Korean Society for Molecular and Cellular Biology
Correspondence to : jiyook@yuhs.ac (JIY); khs@yuhs.ac (HSK)
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
A structural protein of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), nucleocapsid (N) protein is phosphorylated by glycogen synthase kinase (GSK)-3 on the serine/arginine (SR) rich motif located in disordered regions. Although phosphorylation by GSK-3β constitutes a critical event for viral replication, the molecular mechanism underlying N phosphorylation is not well understood. In this study, we found the putative alpha-helix L/FxxxL/AxxRL motif known as the GSK-3 interacting domain (GID), found in many endogenous GSK-3β binding proteins, such as Axins, FRATs, WWOX, and GSKIP. Indeed, N interacts with GSK-3β similarly to Axin, and Leu to Glu substitution of the GID abolished the interaction, with loss of N phosphorylation. The N phosphorylation is also required for its structural loading in a virus-like particle (VLP). Compared to other coronaviruses, N of Sarbecovirus lineage including bat RaTG13 harbors a CDK1-primed phosphorylation site and Gly-rich linker for enhanced phosphorylation by GSK-3β. Furthermore, we found that the S202R mutant found in Delta and R203K/G204R mutant found in the Omicron variant allow increased abundance and hyper-phosphorylation of N. Our observations suggest that GID and mutations for increased phosphorylation in N may have contributed to the evolution of variants.
Keywords Axin, Delta and Omicron variants, glycogen synthase kinase-3, nucleocapsid, phosphorylation, severe acute respiratory syndrome coronavirus 2
The nucleocapsid (N) protein is the most abundantly expressed structural protein during viral replication of coronavirus (Shah and Woo, 2021; Shan et al., 2021). In addition to its structural role in the ribonucleoprotein complex in virion, the N protein plays key roles in viral RNA and protein synthesis, packaging, and envelope formation (Chang et al., 2014; de Haan and Rottier, 2005). Although the spike protein is only used as an immunogen in current vaccines, serological antibodies against N protein can be used for detection of early and previous infection (Krutikov et al., 2021; Tan et al., 2004). The highly basic N protein of coronavirus consists of about 400 amino acids (~50 kDa) with three distinct domains. Highly conserved N-terminal and C-terminal domains in coronavirus play diverse roles in multimerization and RNA binding (Chang et al., 2014; Shah and Woo, 2021). The central disordered region harbors the Ser/Arg (SR)-rich motif, which is also highly conserved in other coronaviruses, such as OC43, HKU1, and MERS-CoV (Middle East respiratory syndrome-related coronavirus). Interestingly, the SR-rich motif of coronavirus is phosphorylated by glycogen synthase kinase (GSK)-3, and GSK-3 inhibitors suppress viral replication in Vero cells, indicating that GSK-3 mediated N phophosphorylation is a rate-limiting step for viral replication (Peng et al., 2008; Wu et al., 2009). GSK-3 inhibition also selectively reduces genomic RNA and long subgenomic mRNA (Wu et al., 2014), and a clinical trial with a GSK-3 inhibitor, lithium, yielded reduced risk of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) (Liu et al., 2021). Crucially, a recent study revealed that N mutations around the SR-rich motif found in SARS-CoV-2 variants allow significantly increased viral replication compared with ancestral Wuhan Hu-1 N protein (Syed et al., 2021). These results indicate the importance of N phosphorylation by GSK-3 during viral replication and mutational evolution of coronavirus although the underlying molecular mechanism is unclear.
GSK-3 is an endogenously abundant kinase which plays a key role in many signaling pathways, including Wnt signaling. In the Wnt pathway, GSK-3 is recruited to a multi-protein complex with adenomatous polyposis coli (APC) and scaffolding protein Axin (Cohen and Frame, 2001; Doble and Woodgett, 2003). Although Axin binds to the C-terminal of GSK-3 apart from its catalytic site, Axin scaffold assembling with APC and β-catenin decreases GSK-3 kinase activity during the regulation of canonical Wnt activity (Hedgepeth et al., 1999). Crystallography of GSK-3 and Axin peptide reveals that hydrophobic helical ridges formed by Axin residues of Phe388, Leu392, and Leu396 in GID pack into the helical groove of the 285-299 loop in GSK-3, at a distance from the ATP binding site (Dajani et al., 2003). This interacting domain is also found in other GSK-3 interacting proteins, such as FRATs, GSK-3 interacting protein (GSKIP), and WWOX (Howng et al., 2010; Wang et al., 2012). The GID in many proteins provides diverse functions for GSK-3 and its substrate. For example, a multi-protein complex of APC and GSK-3 enhances β-catenin phosphorylation and Axin-GSK3 binding provides nuclear export of GSK-3 stabilizing, epithelial-mesenchymal transition (EMT)-inducer Snail in cancer (Dajani et al., 2003; Yook et al., 2006). The typical amino acids in helical GID consist of L/FxxxL/AxxRL (Fig. 1A). In this study, we found GID located next to the SR-rich domain of N protein in SARS-CoV-2 (hereafter N), critical for N protein phosphorylation, suggesting that interaction between N and GSK-3 plays a critical role in viral replication and evolution of SARS-CoV-2. Compared to N protein in other non-pathogenic coronaviruses, N protein of SARS-CoV-2 also harbors a CDK1 phosphorylation site and a flexible linker between GID and the SR-rich region, allowing enhanced phosphorylation of N. Further, we found that N mutations in Delta and Omicron variants provide enhanced phosphorylation and increased abundance of N.
The 293 cells obtained from ATCC (USA) and Vero cells from Korean Cell Line Bank (Korea) were routinely cultured in DMEM and RPMI1640 medium containing 10% fetal bovine serum, respectively. The expression vector pGBW-m4134490 (plasmid No. 152580) having codon optimized (due to quarantine concern rather than protein expression) N of SARS-CoV-2, pGBW-m4134909 (plasmid No. 151901) having N of human coronavirus 229E, pGBW-m4134899 (plasmid No. 151902) having N of human coronavirus OC43 and pGBW-m4134901 (plasmid No. 151922) having N of human coronavirus HKU1 229E, HA-tagged spike (S) of SARS-CoV-2 (plasmid No. 152113), HA-tagged envelop (E) of SARS-CoV-2 (plasmid No. 153661), and HA-tagged membrane (M) of SARS-CoV-2 (plasmid No. 152583) were obtained from Addgene (USA). Those N expression vectors were subcloned into pcDNA3.1 with C-terminal flag or EGFP tag. Mutant expression vectors of N in GID, linker, and CDK1 phosphorylation site were generated by a polymerase chain reaction-based method. The transfection was performed by Lipofectamine 2000 according to the manufacturer’s protocol (Invitrogen, USA). Antibodies against GSK-3β (610202; BD Transduction Laboratories, USA), pS9-GSK-3β (9323S; Cell Signaling Technology, USA), pY216-GSK-3β (612312; BD Transduction Laboratories), Flag (F-3165; Sigma, USA), Snail (L70G2; Cell Signaling Technology), β-catenin (610154; BD Transduction Laboratories), and Tubulin (LF-PA0146; AbFrontier, Korea) were obtained from the commercial vendors. Endogenous GSK-3β kinase activity was determined by commercial kit (V1991; Promega, USA).
For the western blot analyses, protein extracts were prepared in Triton X-100 lysis buffer. Phosphorylation status of N protein was determined by anti-flag antibody and mobility shift on a Phos-tag gel (Wako, Japan) as described previously (Lee et al., 2018).
For immunoprecipitation analysis, whole cell Triton X-100 lysates were incubated with Flag-M2 agarose (Sigma) and washed with lysis buffer three times. The recovered proteins were resolved by SDS-PAGE and subjected to immunoblot analysis. For immunofluorescence study, the cells were washed twice with ice-cold phosphate-buffered saline (PBS) and incubated for 15 min at room temperature with 3% formaldehyde in PBS. The cells were permeabilized with 0.5% Triton X-100 for 5 min and then blocked for 1 h in PBS containing 3% bovine serum albumin followed by incubation with primary antibody overnight at 4°C. Cells were then washed three times with PBS containing 0.1% Tween 20 followed by incubation with anti-mouse-Alexa Fluor-488 (for green) or anti-rabbit-Alexa Fluor-594 (for red) secondary antibody. Cellular fluorescence was monitored using confocal microscopy (Zeiss, Germany).
For TCF/LEF and E-cadherin reporter assay, the cells were transfected with 50 ng of the Super-Top or E-cad(-108)-Luc reporter vector (Kim et al., 2011; Yook et al., 2005) and 1 ng of pSV40-Renilla expression vector in combination with N or N-mutant as indicated. Luciferase and
For split superfolder GFP assay, the GFP1-10 and GFP11 constructs were kindly provided by Professor Hye Yoon Park at Seoul National University. The GFP1-10 fragment was fused into the N-terminus of GSK-3 and GFP11 was fused into the C-terminus of N. The 293 cells were co-transfected with split GFP vectors, and fluorescence intensity was determined by 5 random areas with the same exposure, followed by ImageJ analysis.
For the VLP experiment, 293 cells were transfected with HA-tagged E, M and S expression vectors with empty or wild type (wt) N or N-L223/227E expression vectors. At 48 h, the culture supernatant was harvested and cleared by centrifuge (800 rpm for 3 min) followed by 0.2 μm syringe filtration. The supernatant was incubated with Lenti-X concentrator (Clontech Laboratories, Japan) for 16 h followed by centrifugation (15,000
Amino acid sequences of N for SARS-CoV-2 (P0DTC9), SARS-CoV (P59595), HKU1 (Q0ZME3), HCoV-OC43 (P33469), HCoV-229E (PP15130), HCoV-NL63 (Q6Q1R8) were obtained from UniProt (https://www.uniprot.org/). Mutational information on N protein in SARS-CoV-2 variants was obtained from PANGO Lineages (https://cov-lineages.org/constellations).
Statistical analysis for reporter, GSK-3 kinase, and split GFP assay was performed with two-tailed Student’s
Phosphorylation of many endogenous and viral proteins by GSK-3 is dependent on GSK-3 binding of protein-protein interaction (Dajani et al., 2003; Fujimuro et al., 2005). In turn, the GID embedded in the GSK-3 substrate provides efficient self-phosphorylation. Because N of coronavirus is phosphorylated by GSK-3 (Peng et al., 2008; Wu et al., 2009)
Because the Axin-GSK-3 binding structure is well-determined (Dajani et al., 2003; Fraser et al., 2002), we next compared the binding site of Axin2 and N with that in wt and mutants GSK-3. Consistently, Y216 and V267/E268 in GSK-3 were critical for Axin2 binding (Supplementary Fig. S2), and N shared those binding sites in GSK-3 (Fig. 2A), indicating that N utilizes a GSK-3 binding mode similar to that of Axin. Previously, we reported that antihelminthic niclosamide interacts with GSK-3 and inhibits Axin-GSK3 binding, resulting in attenuation of Wnt signaling and EMT in APC-mutated colorectal cancer (Ahn et al., 2017a). Niclosamide also inhibits viral replication of SARS-CoV-1 in Vero E6 cells (Wu et al., 2004) although the molecular mechanism of action (MoA) of niclosamide on SARS-CoV is unclear. Given the similar binding modes of N and Axin to GSK-3, we next performed an
Because Axin directly suppresses kinase activity of GSK-3 via GID, thus regulating various signaling pathways (Cohen and Frame, 2001; Doble and Woodgett, 2003), we next examined the role of N in GSK-3 kinase activity and the subsequent Wnt/EMT signaling pathway. Surprisingly, overexpression of N or N-GID mutant did not affect endogenous GSK-3 kinase activity in 293 cells (Supplementary Fig. S3A). Furthermore, overexpression of N or N-GID mutant did not affect GSK-3 abundance or phosphorylation status on Ser9 and Y216 (Supplementary Fig. S3B). Overexpression of N neither increases protein abundance of β-catenin and Snail, nor affects TCF/LEF transcriptional and E-cadherin promoter activity (Supplementary Fig. S3C). These results indicate that N, unlike endogenous GSK-3 scaffolding proteins, utilizes GSK-3 for its phosphorylation but does not affect GSK-3 kinase activity and subsequent signaling pathways.
Phosphorylation of N is essentially required for its phase separation and subsequent viral replication (Carlson et al., 2020; Wu et al., 2009; 2014). Because the N comprises an important structural component of a viral particle, we next examined the role of N or N-L223/227E mutant in VLP production. While the VLP can be produced by the envelope (E), membrane (M) and spike (S) without N, electron microscopic analysis revealed that N protein provides a bigger particle size with solid structure of VLP while the N-L223/227E mutant was similar to the absence of N (Fig. 3A). Interestingly, N-L223/227E mutant loading in VLPs was significantly decreased compared to wt N, while protein abundance of N and other structural components were comparable in cell lysate (Fig. 3B). These results indicate that N phosphorylation is also required for structural assembly of viral particles.
While human coronaviruses HKU1, OC43, 229E, and NL63 typically cause upper respiratory infection with relatively minor symptoms, SARS-CoV and SARS-CoV-2 in the
Given the difference between the
The continuous evolution of SARS-CoV-2 through mutational changes has given rise to many variants including Delta (B.1.617.2) and Omicron (B.1.1.529). Given our observations, we further examined the N mutation in those variants focusing on the region between the SR-rich motif and GID (https://cov-lineages.org/constellations). Interestingly, we found many mutations in the variants of SARS-CoV-2 in the proximal N-terminus of the CDK1 phosphorylation site (Fig. 5C). Notably, there was no mutation among the SARS-CoV-2 variants in the GID region, suggesting the evolutionary fitness of GSK-3 binding in the
Highly expressed endogenously in mammals, GSK-3 is an exceptional kinase inactivated by exogenous signaling, such as by insulin and Wnt (Cohen and Frame, 2001; Doble and Woodgett, 2003). Priming phosphorylation by other many kinases allows a strong (500 to 1,000 fold) preference for GSK-3 substrates, which have multiple and serial phosphorylation sites by GSK-3, including EMT-inducer Snail and β-catenin (Kaidanovich-Beilin and Woodgett, 2011; Yook et al., 2005). The importance of GSK-3 has been extensively confirmed in human diseases, including Alzheimer’s, metabolic diseases, and cancer (Cohen and Frame, 2001; Doble and Woodgett, 2003). Notably, many viruses or bacterial genes directly interact with and utilize endogenous GSK-3 for entry, replication and latency (Alfhili et al., 2020). For example, latency-associated nuclear antigen (LANA) in Kaposi’s sarcoma-associated herpesvirus (KSHV) interacts with GSK-3 via GID embedded in LANA. As with Axin, the LANA-GSK-3 interaction is essential to phosphorylation of LANA and inactivation of GSK-3 (Fujimuro et al., 2005). Cytotoxin-associated gene A (CagA) oncoprotein in
SARS-CoV-2 is classified as
While SARS-CoV-2 is currently evolving increased transmissibility, the functional importance of mutations in the variants is technically challenging. Recent observation has revealed that S202R in Lota and R203M in Delta variant provide 166-fold and 51-fold higher viral production, respectively (Syed et al., 2021). In this study, we showed that pS206 priming phosphorylation by CDK1 and Gly-rich linker located between GID and SR-rich motif enhance phosphorylation of N. Given the multiple bands in N of the 229E strain and high density of up to 10 phosphates in the SR-rich motif in various strains of coronavirus, N of SARS-CoV-2 also has multiple phosphorylation sites in the SR-rich motif. Interestingly, emerging variants have mutations near the primed phosphorylation site. Notably, R203K/G204R, found in a highly transmissible Omicron variant, allows greater phosphorylation and protein abundance than N of ancestral and other variants. Because more than fourteen Ser/Thr residues exist in the SR-rich motif in
Previously, we observed that the anthelmintic niclosamide disrupts Axin-GSK3 interaction, providing a repositioned therapeutic for colon cancer and familial adenomatosis coli (Ahn et al., 2017b). While niclosamide is consistently effective in disrupting Axin2-GSK-3 binding, an at least 5-fold concentration of niclosamide was required for disruption of N-GSK-3 interaction in our hands, indicating that N has a stronger interaction with GSK-3 than does Axin2. Conversely, our observations provide an MoA of niclosamide on SARS-CoV replication and N expression, at least in part (Wu et al., 2004), with implications for clinical trials of niclosamide for SARS-CoV-2 (Al-Kuraishy et al., 2021). Although GSK-3 kinase inhibitors can be a therapeutic target for SARS-CoV-2, the endogenous abundance of GSK-3 along with its diverse physiological roles largely limit the therapeutic potential of GSK-3 inhibitors for viral diseases. Thus, further study is required regarding the protein-protein interaction of N-GSK-3 as a therapeutic target for SARS-CoV-2 infection in human.
We thank E. Tunkle for preparation of the manuscript and J. Choi at Dongduk University College of Pharmacy for technical assistance. This work was supported by grants from the National Research Foundation of Korea (NRF-2019R1A2C2084535, NRF-2021R1A2C3003496, NRF-2022R1A2C3004609) funded by the Korean government (MSIP), and a grant from the National Research Foundation of Korea (NRF-2020R1I1A1A01072977) funded by the Korean government (MOE).
J.S.Y. and H.S. performed all experiments. S.Y.C., J.E.L., C.-H.J., S.H.S., S.K., and E.S.C. supported experiments. N.H.K. and K.H.H. performed the split GFP assay. N.H.K., H.S.K., and J.I.Y. planned all experiments, analyzed the data, and wrote the manuscript.
The authors have no potential conflicts of interest to disclose.
Mol. Cells 2022; 45(12): 911-922
Published online December 31, 2022 https://doi.org/10.14348/molcells.2022.0130
Copyright © The Korean Society for Molecular and Cellular Biology.
Jun Seop Yun1,2 , Hyeeun Song1,2
, Nam Hee Kim1
, So Young Cha1
, Kyu Ho Hwang1
, Jae Eun Lee1
, Cheol-Hee Jeong1
, Sang Hyun Song1
, Seonghun Kim1
, Eunae Sandra Cho1
, Hyun Sil Kim1,*
, and Jong In Yook1,*
1Department of Oral Pathology, Yonsei University College of Dentistry, Seoul 03722, Korea, 2These authors contributed equally to this work.
Correspondence to:jiyook@yuhs.ac (JIY); khs@yuhs.ac (HSK)
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
A structural protein of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), nucleocapsid (N) protein is phosphorylated by glycogen synthase kinase (GSK)-3 on the serine/arginine (SR) rich motif located in disordered regions. Although phosphorylation by GSK-3β constitutes a critical event for viral replication, the molecular mechanism underlying N phosphorylation is not well understood. In this study, we found the putative alpha-helix L/FxxxL/AxxRL motif known as the GSK-3 interacting domain (GID), found in many endogenous GSK-3β binding proteins, such as Axins, FRATs, WWOX, and GSKIP. Indeed, N interacts with GSK-3β similarly to Axin, and Leu to Glu substitution of the GID abolished the interaction, with loss of N phosphorylation. The N phosphorylation is also required for its structural loading in a virus-like particle (VLP). Compared to other coronaviruses, N of Sarbecovirus lineage including bat RaTG13 harbors a CDK1-primed phosphorylation site and Gly-rich linker for enhanced phosphorylation by GSK-3β. Furthermore, we found that the S202R mutant found in Delta and R203K/G204R mutant found in the Omicron variant allow increased abundance and hyper-phosphorylation of N. Our observations suggest that GID and mutations for increased phosphorylation in N may have contributed to the evolution of variants.
Keywords: Axin, Delta and Omicron variants, glycogen synthase kinase-3, nucleocapsid, phosphorylation, severe acute respiratory syndrome coronavirus 2
The nucleocapsid (N) protein is the most abundantly expressed structural protein during viral replication of coronavirus (Shah and Woo, 2021; Shan et al., 2021). In addition to its structural role in the ribonucleoprotein complex in virion, the N protein plays key roles in viral RNA and protein synthesis, packaging, and envelope formation (Chang et al., 2014; de Haan and Rottier, 2005). Although the spike protein is only used as an immunogen in current vaccines, serological antibodies against N protein can be used for detection of early and previous infection (Krutikov et al., 2021; Tan et al., 2004). The highly basic N protein of coronavirus consists of about 400 amino acids (~50 kDa) with three distinct domains. Highly conserved N-terminal and C-terminal domains in coronavirus play diverse roles in multimerization and RNA binding (Chang et al., 2014; Shah and Woo, 2021). The central disordered region harbors the Ser/Arg (SR)-rich motif, which is also highly conserved in other coronaviruses, such as OC43, HKU1, and MERS-CoV (Middle East respiratory syndrome-related coronavirus). Interestingly, the SR-rich motif of coronavirus is phosphorylated by glycogen synthase kinase (GSK)-3, and GSK-3 inhibitors suppress viral replication in Vero cells, indicating that GSK-3 mediated N phophosphorylation is a rate-limiting step for viral replication (Peng et al., 2008; Wu et al., 2009). GSK-3 inhibition also selectively reduces genomic RNA and long subgenomic mRNA (Wu et al., 2014), and a clinical trial with a GSK-3 inhibitor, lithium, yielded reduced risk of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) (Liu et al., 2021). Crucially, a recent study revealed that N mutations around the SR-rich motif found in SARS-CoV-2 variants allow significantly increased viral replication compared with ancestral Wuhan Hu-1 N protein (Syed et al., 2021). These results indicate the importance of N phosphorylation by GSK-3 during viral replication and mutational evolution of coronavirus although the underlying molecular mechanism is unclear.
GSK-3 is an endogenously abundant kinase which plays a key role in many signaling pathways, including Wnt signaling. In the Wnt pathway, GSK-3 is recruited to a multi-protein complex with adenomatous polyposis coli (APC) and scaffolding protein Axin (Cohen and Frame, 2001; Doble and Woodgett, 2003). Although Axin binds to the C-terminal of GSK-3 apart from its catalytic site, Axin scaffold assembling with APC and β-catenin decreases GSK-3 kinase activity during the regulation of canonical Wnt activity (Hedgepeth et al., 1999). Crystallography of GSK-3 and Axin peptide reveals that hydrophobic helical ridges formed by Axin residues of Phe388, Leu392, and Leu396 in GID pack into the helical groove of the 285-299 loop in GSK-3, at a distance from the ATP binding site (Dajani et al., 2003). This interacting domain is also found in other GSK-3 interacting proteins, such as FRATs, GSK-3 interacting protein (GSKIP), and WWOX (Howng et al., 2010; Wang et al., 2012). The GID in many proteins provides diverse functions for GSK-3 and its substrate. For example, a multi-protein complex of APC and GSK-3 enhances β-catenin phosphorylation and Axin-GSK3 binding provides nuclear export of GSK-3 stabilizing, epithelial-mesenchymal transition (EMT)-inducer Snail in cancer (Dajani et al., 2003; Yook et al., 2006). The typical amino acids in helical GID consist of L/FxxxL/AxxRL (Fig. 1A). In this study, we found GID located next to the SR-rich domain of N protein in SARS-CoV-2 (hereafter N), critical for N protein phosphorylation, suggesting that interaction between N and GSK-3 plays a critical role in viral replication and evolution of SARS-CoV-2. Compared to N protein in other non-pathogenic coronaviruses, N protein of SARS-CoV-2 also harbors a CDK1 phosphorylation site and a flexible linker between GID and the SR-rich region, allowing enhanced phosphorylation of N. Further, we found that N mutations in Delta and Omicron variants provide enhanced phosphorylation and increased abundance of N.
The 293 cells obtained from ATCC (USA) and Vero cells from Korean Cell Line Bank (Korea) were routinely cultured in DMEM and RPMI1640 medium containing 10% fetal bovine serum, respectively. The expression vector pGBW-m4134490 (plasmid No. 152580) having codon optimized (due to quarantine concern rather than protein expression) N of SARS-CoV-2, pGBW-m4134909 (plasmid No. 151901) having N of human coronavirus 229E, pGBW-m4134899 (plasmid No. 151902) having N of human coronavirus OC43 and pGBW-m4134901 (plasmid No. 151922) having N of human coronavirus HKU1 229E, HA-tagged spike (S) of SARS-CoV-2 (plasmid No. 152113), HA-tagged envelop (E) of SARS-CoV-2 (plasmid No. 153661), and HA-tagged membrane (M) of SARS-CoV-2 (plasmid No. 152583) were obtained from Addgene (USA). Those N expression vectors were subcloned into pcDNA3.1 with C-terminal flag or EGFP tag. Mutant expression vectors of N in GID, linker, and CDK1 phosphorylation site were generated by a polymerase chain reaction-based method. The transfection was performed by Lipofectamine 2000 according to the manufacturer’s protocol (Invitrogen, USA). Antibodies against GSK-3β (610202; BD Transduction Laboratories, USA), pS9-GSK-3β (9323S; Cell Signaling Technology, USA), pY216-GSK-3β (612312; BD Transduction Laboratories), Flag (F-3165; Sigma, USA), Snail (L70G2; Cell Signaling Technology), β-catenin (610154; BD Transduction Laboratories), and Tubulin (LF-PA0146; AbFrontier, Korea) were obtained from the commercial vendors. Endogenous GSK-3β kinase activity was determined by commercial kit (V1991; Promega, USA).
For the western blot analyses, protein extracts were prepared in Triton X-100 lysis buffer. Phosphorylation status of N protein was determined by anti-flag antibody and mobility shift on a Phos-tag gel (Wako, Japan) as described previously (Lee et al., 2018).
For immunoprecipitation analysis, whole cell Triton X-100 lysates were incubated with Flag-M2 agarose (Sigma) and washed with lysis buffer three times. The recovered proteins were resolved by SDS-PAGE and subjected to immunoblot analysis. For immunofluorescence study, the cells were washed twice with ice-cold phosphate-buffered saline (PBS) and incubated for 15 min at room temperature with 3% formaldehyde in PBS. The cells were permeabilized with 0.5% Triton X-100 for 5 min and then blocked for 1 h in PBS containing 3% bovine serum albumin followed by incubation with primary antibody overnight at 4°C. Cells were then washed three times with PBS containing 0.1% Tween 20 followed by incubation with anti-mouse-Alexa Fluor-488 (for green) or anti-rabbit-Alexa Fluor-594 (for red) secondary antibody. Cellular fluorescence was monitored using confocal microscopy (Zeiss, Germany).
For TCF/LEF and E-cadherin reporter assay, the cells were transfected with 50 ng of the Super-Top or E-cad(-108)-Luc reporter vector (Kim et al., 2011; Yook et al., 2005) and 1 ng of pSV40-Renilla expression vector in combination with N or N-mutant as indicated. Luciferase and
For split superfolder GFP assay, the GFP1-10 and GFP11 constructs were kindly provided by Professor Hye Yoon Park at Seoul National University. The GFP1-10 fragment was fused into the N-terminus of GSK-3 and GFP11 was fused into the C-terminus of N. The 293 cells were co-transfected with split GFP vectors, and fluorescence intensity was determined by 5 random areas with the same exposure, followed by ImageJ analysis.
For the VLP experiment, 293 cells were transfected with HA-tagged E, M and S expression vectors with empty or wild type (wt) N or N-L223/227E expression vectors. At 48 h, the culture supernatant was harvested and cleared by centrifuge (800 rpm for 3 min) followed by 0.2 μm syringe filtration. The supernatant was incubated with Lenti-X concentrator (Clontech Laboratories, Japan) for 16 h followed by centrifugation (15,000
Amino acid sequences of N for SARS-CoV-2 (P0DTC9), SARS-CoV (P59595), HKU1 (Q0ZME3), HCoV-OC43 (P33469), HCoV-229E (PP15130), HCoV-NL63 (Q6Q1R8) were obtained from UniProt (https://www.uniprot.org/). Mutational information on N protein in SARS-CoV-2 variants was obtained from PANGO Lineages (https://cov-lineages.org/constellations).
Statistical analysis for reporter, GSK-3 kinase, and split GFP assay was performed with two-tailed Student’s
Phosphorylation of many endogenous and viral proteins by GSK-3 is dependent on GSK-3 binding of protein-protein interaction (Dajani et al., 2003; Fujimuro et al., 2005). In turn, the GID embedded in the GSK-3 substrate provides efficient self-phosphorylation. Because N of coronavirus is phosphorylated by GSK-3 (Peng et al., 2008; Wu et al., 2009)
Because the Axin-GSK-3 binding structure is well-determined (Dajani et al., 2003; Fraser et al., 2002), we next compared the binding site of Axin2 and N with that in wt and mutants GSK-3. Consistently, Y216 and V267/E268 in GSK-3 were critical for Axin2 binding (Supplementary Fig. S2), and N shared those binding sites in GSK-3 (Fig. 2A), indicating that N utilizes a GSK-3 binding mode similar to that of Axin. Previously, we reported that antihelminthic niclosamide interacts with GSK-3 and inhibits Axin-GSK3 binding, resulting in attenuation of Wnt signaling and EMT in APC-mutated colorectal cancer (Ahn et al., 2017a). Niclosamide also inhibits viral replication of SARS-CoV-1 in Vero E6 cells (Wu et al., 2004) although the molecular mechanism of action (MoA) of niclosamide on SARS-CoV is unclear. Given the similar binding modes of N and Axin to GSK-3, we next performed an
Because Axin directly suppresses kinase activity of GSK-3 via GID, thus regulating various signaling pathways (Cohen and Frame, 2001; Doble and Woodgett, 2003), we next examined the role of N in GSK-3 kinase activity and the subsequent Wnt/EMT signaling pathway. Surprisingly, overexpression of N or N-GID mutant did not affect endogenous GSK-3 kinase activity in 293 cells (Supplementary Fig. S3A). Furthermore, overexpression of N or N-GID mutant did not affect GSK-3 abundance or phosphorylation status on Ser9 and Y216 (Supplementary Fig. S3B). Overexpression of N neither increases protein abundance of β-catenin and Snail, nor affects TCF/LEF transcriptional and E-cadherin promoter activity (Supplementary Fig. S3C). These results indicate that N, unlike endogenous GSK-3 scaffolding proteins, utilizes GSK-3 for its phosphorylation but does not affect GSK-3 kinase activity and subsequent signaling pathways.
Phosphorylation of N is essentially required for its phase separation and subsequent viral replication (Carlson et al., 2020; Wu et al., 2009; 2014). Because the N comprises an important structural component of a viral particle, we next examined the role of N or N-L223/227E mutant in VLP production. While the VLP can be produced by the envelope (E), membrane (M) and spike (S) without N, electron microscopic analysis revealed that N protein provides a bigger particle size with solid structure of VLP while the N-L223/227E mutant was similar to the absence of N (Fig. 3A). Interestingly, N-L223/227E mutant loading in VLPs was significantly decreased compared to wt N, while protein abundance of N and other structural components were comparable in cell lysate (Fig. 3B). These results indicate that N phosphorylation is also required for structural assembly of viral particles.
While human coronaviruses HKU1, OC43, 229E, and NL63 typically cause upper respiratory infection with relatively minor symptoms, SARS-CoV and SARS-CoV-2 in the
Given the difference between the
The continuous evolution of SARS-CoV-2 through mutational changes has given rise to many variants including Delta (B.1.617.2) and Omicron (B.1.1.529). Given our observations, we further examined the N mutation in those variants focusing on the region between the SR-rich motif and GID (https://cov-lineages.org/constellations). Interestingly, we found many mutations in the variants of SARS-CoV-2 in the proximal N-terminus of the CDK1 phosphorylation site (Fig. 5C). Notably, there was no mutation among the SARS-CoV-2 variants in the GID region, suggesting the evolutionary fitness of GSK-3 binding in the
Highly expressed endogenously in mammals, GSK-3 is an exceptional kinase inactivated by exogenous signaling, such as by insulin and Wnt (Cohen and Frame, 2001; Doble and Woodgett, 2003). Priming phosphorylation by other many kinases allows a strong (500 to 1,000 fold) preference for GSK-3 substrates, which have multiple and serial phosphorylation sites by GSK-3, including EMT-inducer Snail and β-catenin (Kaidanovich-Beilin and Woodgett, 2011; Yook et al., 2005). The importance of GSK-3 has been extensively confirmed in human diseases, including Alzheimer’s, metabolic diseases, and cancer (Cohen and Frame, 2001; Doble and Woodgett, 2003). Notably, many viruses or bacterial genes directly interact with and utilize endogenous GSK-3 for entry, replication and latency (Alfhili et al., 2020). For example, latency-associated nuclear antigen (LANA) in Kaposi’s sarcoma-associated herpesvirus (KSHV) interacts with GSK-3 via GID embedded in LANA. As with Axin, the LANA-GSK-3 interaction is essential to phosphorylation of LANA and inactivation of GSK-3 (Fujimuro et al., 2005). Cytotoxin-associated gene A (CagA) oncoprotein in
SARS-CoV-2 is classified as
While SARS-CoV-2 is currently evolving increased transmissibility, the functional importance of mutations in the variants is technically challenging. Recent observation has revealed that S202R in Lota and R203M in Delta variant provide 166-fold and 51-fold higher viral production, respectively (Syed et al., 2021). In this study, we showed that pS206 priming phosphorylation by CDK1 and Gly-rich linker located between GID and SR-rich motif enhance phosphorylation of N. Given the multiple bands in N of the 229E strain and high density of up to 10 phosphates in the SR-rich motif in various strains of coronavirus, N of SARS-CoV-2 also has multiple phosphorylation sites in the SR-rich motif. Interestingly, emerging variants have mutations near the primed phosphorylation site. Notably, R203K/G204R, found in a highly transmissible Omicron variant, allows greater phosphorylation and protein abundance than N of ancestral and other variants. Because more than fourteen Ser/Thr residues exist in the SR-rich motif in
Previously, we observed that the anthelmintic niclosamide disrupts Axin-GSK3 interaction, providing a repositioned therapeutic for colon cancer and familial adenomatosis coli (Ahn et al., 2017b). While niclosamide is consistently effective in disrupting Axin2-GSK-3 binding, an at least 5-fold concentration of niclosamide was required for disruption of N-GSK-3 interaction in our hands, indicating that N has a stronger interaction with GSK-3 than does Axin2. Conversely, our observations provide an MoA of niclosamide on SARS-CoV replication and N expression, at least in part (Wu et al., 2004), with implications for clinical trials of niclosamide for SARS-CoV-2 (Al-Kuraishy et al., 2021). Although GSK-3 kinase inhibitors can be a therapeutic target for SARS-CoV-2, the endogenous abundance of GSK-3 along with its diverse physiological roles largely limit the therapeutic potential of GSK-3 inhibitors for viral diseases. Thus, further study is required regarding the protein-protein interaction of N-GSK-3 as a therapeutic target for SARS-CoV-2 infection in human.
We thank E. Tunkle for preparation of the manuscript and J. Choi at Dongduk University College of Pharmacy for technical assistance. This work was supported by grants from the National Research Foundation of Korea (NRF-2019R1A2C2084535, NRF-2021R1A2C3003496, NRF-2022R1A2C3004609) funded by the Korean government (MSIP), and a grant from the National Research Foundation of Korea (NRF-2020R1I1A1A01072977) funded by the Korean government (MOE).
J.S.Y. and H.S. performed all experiments. S.Y.C., J.E.L., C.-H.J., S.H.S., S.K., and E.S.C. supported experiments. N.H.K. and K.H.H. performed the split GFP assay. N.H.K., H.S.K., and J.I.Y. planned all experiments, analyzed the data, and wrote the manuscript.
The authors have no potential conflicts of interest to disclose.
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