key: cord-1028443-fghs1ax3
authors: Liu, X.; Verma, A.; Ramage, H.; Garcia, G.; Myers, R. L.; Lucas, A.; Michaelson, J. J.; Coryell, W.; Kumar, A.; Charney, A.; Kazanietz, M. G.; Rader, D. J.; Ritchie, M. D.; Berrettini, W. H.; Damoiseaux, R.; Arumugaswami, V.; Schultz, D.; Cherry, S.; Klein, P. S.
title: Targeting the Coronavirus Nucleocapsid Protein through GSK-3 Inhibition
date: 2021-02-22
journal: medRxiv : the preprint server for health sciences
DOI: 10.1101/2021.02.17.21251933
sha: a1376fee99a4b03ea4667a91d7316afa08a54a8d
doc_id: 1028443
cord_uid: fghs1ax3

The coronaviruses responsible for severe acute respiratory syndrome (SARS-CoV), COVID-19 (SARS-CoV-2), Middle East respiratory syndrome (MERS-CoV), and other coronavirus infections express a nucleocapsid protein (N) that is essential for viral replication, transcription, and virion assembly. Phosphorylation of N from SARS-CoV by glycogen synthase kinase 3 (GSK-3) is required for its function and inhibition of GSK-3 with lithium impairs N phosphorylation, viral transcription, and replication. Here we report that the SARS-CoV-2 N protein contains GSK-3 consensus sequences and that this motif is conserved in diverse coronaviruses, despite limited overall sequence conservation, raising the possibility that SARS-CoV-2 may be sensitive to GSK-3 inhibitors including lithium. We conducted a retrospective analysis of lithium use in patients from three major health systems who were PCR tested for SARS-CoV-2. We found that patients taking lithium have a significantly reduced risk of COVID-19 (odds ratio = 0.51 [0.34 - 0.76], p = 0.001). We also show that the SARS-CoV-2 N protein is phosphorylated by GSK-3. Knockout of GSK3A and GSK3B demonstrates that GSK-3 is essential for N phosphorylation. Alternative GSK-3 inhibitors block N phosphorylation and impair replication in SARS-CoV-2 infected lung epithelial cells in a cell-type dependent manner. Targeting GSK-3 may therefore provide a new approach to treat COVID-19 and future coronavirus outbreaks.

COVID-19 is exacting a severe toll on personal and community health, healthcare systems, and the global economy. The response to this crisis will require multiple approaches for detection, prevention, and treatment. With three major b-coronavirus epidemics in less than 20 years, it would also be prudent to anticipate new coronavirus outbreaks in the future. In addition to development of effective vaccines, antiviral strategies that target conserved mechanisms in coronavirus replication and transmission may be needed for COVID-19 and potential future coronavirus outbreaks. Recent high throughput screens have identified bioactive compounds that impair viral replication and infectivity in tissue culture models of infection by the severe acute respiratory syndrome coronvavirus-2 (SARS-CoV-2) [1] [2] [3] [4] [5] . However, their mechanisms of action and their clinical efficacy remain to be fully delineated and additional targets may be needed to combat SARS-CoV-2, new SARS-CoV-2 variants, and potential novel coronavirus outbreaks in the future.

Coronaviruses express a nucleocapsid (N) protein that is essential for viral replication, transcription, and assembly [6] [7] [8] [9] [10] . N proteins from the JHM strain of mouse hepatitis virus (JHMV) and from SARS-CoV, which caused the 2002-2004 SARS outbreak, are phosphorylated by glycogen synthase kinase-3 (GSK-3) within an arginine-serine (RS) domain present in N proteins of diverse coronaviruses [7] [8] [9] [11] [12] [13] [14] . Phosphorylation of the JHMV N protein is required for recruitment of the RNA helicase DDX1 and for transcription of long subgenomic RNAs 8 ; inhibition of GSK-3 impairs recruitment of DDX1, binding to viral mRNAs, and viral replication. N These observations suggest that inhibition of GSK-3 could impair coronavirus infections in vivo, including COVID-19 [25] [26] [27] . A recent phosphoproteomic analysis revealed that SARS-CoV-2 N protein is highly phosphorylated within the RS domain 1 , but whether GSK-3 phosphorylates SARS-CoV-2 N protein and whether lithium has any effect against SARS-CoV- 2 have not yet been tested. Here we show that GSK-3 is essential for phosphorylation of the SARS-CoV-2 N protein, that alternative GSK-3 inhibitors impair N phosphorylation and SARS-CoV-2 infection in human lung epithelial cells, and that litihum therapy is associated with significantly reduced risk of COVID-19. Targeting GSK-3 may therefore provide an antiviral therapy for COVID-19 and for coronavirus infections that may arise in the future.

The SARS-CoV N protein shares 20-30% sequence identity with the N proteins of other coronaviruses 6 , and despite the limited sequence similarity, they each have an arginine-serine rich (RS) domain that lies between N-terminal and C-terminal conserved domains 6 . The RS domains of N from SARS-CoV and JHMV include repeated motifs (SXXXS) 9 that are frequently associated with GSK-3 phosphorylation, in which the C-terminal serine is phosphorylated by a priming kinase 28 , which then allows GSK-3 to phosphorylate multiple serines or threonines spaced 4 residues apart in the C to N terminal direction (Fig. 1A) . The sequence of the RS domain of SARS-CoV-2 N is 90% similar to N from SARS-CoV, and both proteins contain two sets of three SXXXS motifs each (labeled "a" and "b" in Fig. 1A and 1B) . While the N protein sequences of other coronaviruses diverge, they retain SXXXS motifs (Fig. 1B) . In addition, the fourth serine (presumed priming site) is always preceded by an arginine in the -3 position 4 (SRXXS). GSK-3-dependent phosphorylation of RS domains has also been reported for multiple splicing factors and other RNA binding proteins 29, 30 .

A recent phosphoproteomic analysis showed that the RS domain of the SARS-CoV-2 N protein is highly phosphorylated 1 , but whether GSK-3 phosphorylates N protein from SARS-CoV-2 has not been addressed. We expressed SARS-CoV-2 N in human embryonic kidney 293T cells (Fig.   1C ) or mouse lung epithelial MLE12 cells ( Figure S1A ). N phosphorylation was demonstrated by treating cell lysates with alkaline phosphatase, which increased the electrophoretic mobility of N, as observed previously for SARS-CoV N protein 8, 9 . Lithium chloride (LiCl) inhibited N phosphorylation with IC50 ~10mM in 293T cells (Fig. 1C , S1B). Phosphorylation of the GSK-3

substrates Glycogen Synthase (GS; Fig. 1C ) and ß-catenin ( Fig. 2A , lanes 1-3) was also inhibited with an IC50 ~ 10 mM. In contrast, the Ki for LiCl inhibition of GSK-3 in vitro is 1 mM 31, 32 and the effective in vivo concentration for Li + inhibition of GSK-3 in mice and humans is also 1 mM 33,34 . The relatively high Li + concentration needed to inhibit N phosphorylation ex vivo raises the concern that Li + may act through a target other than GSK-3. To examine this possibility rigorously, we tested multiple, selective GSK-3 inhibitors, including bisindolylmaleimide I (BIM-I), CHIR99021, AR-A014418, and Kenpaullone, all of which inhibited N phosphorylation in the low µM range (Fig. 1D , S1C), strongly supporting that GSK-3 is essential for N protein phosphorylation.

However, these compounds, which inhibit GSK-3 by competing for ATP binding, may have offtarget effects. As an alternative and more definitive approach, we used siRNAs and CRISPR/Cas9 to knockdown or knockout (KO) both GSK3A and GSK3B, which encode two highly similar GSK-3 isoforms (GSK-3a and GSK-3b, respectively). KO of GSK3A alone had a minimal effect on phosphorylation of N or b-catenin ( Fig. 2A) , consistent with redundant All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. functions of GSK3A and GSK3B 35 . However, siRNA knockdown of GSK3B in GSK3A KO cells impaired N phosphorylation and increased the sensitivity to LiCl ( Fig. 2A) , with inhibition of N phosphorylation detectable at 1 mM LiCl. Importantly, combined KO of GSK3A and GSK3B completely prevented N protein phosphorylation (Fig. 2B ). In the absence of GSK-3, LiCl had no further effect on N protein mobility, showing that Li + inhibits N phosphorylation solely through inhibition of GSK-3. These pharmacological and genetic data demonstrate unequivocally that GSK-3 is essential for phosphorylation of the SARS-CoV-2 N protein.

GSK-3 substrates that follow the SXXXS motif require a priming phosphorylation at the Cterminal serine or threonine 36,37 ; mutation of this residue in established GSK-3 substrates including glycogen synthase (GS) and b-catenin prevents phosphorylation of more N-terminal serines and threonines by GSK-3 36,38 . Similarly, mutation of the two putative priming sites in SARS-CoV N blocks phosphorylation by GSK-3 9 . To test whether the SARS-CoV-2 N protein also requires priming site serines, we mutated serine-188 and serine-206 of SARS-CoV-2 N protein to alanines (N S188A,S206A ) and then expressed the single and double mutant N proteins in HEK293T cells. The single mutant form N S188A migrates in the same position as phosphorylated wild-type N protein whereas the N S206A migrates more rapidly, suggesting that it is hypophosphorylated (Fig. 2C ). Mobility of the N S188A,S206A protein is similar to dephosphorylated N protein and is not affected by GSK-3 inhibition LiCl (Fig. 2C ) or treatment with alkaline phosphatase ( Figure S2A ). Additionally, both of the single serine to alanine mutants are more sensitive to LiCl. These data indicate that GSK-3 phosphorylation of SARS-CoV-2 N protein requires the canonical GSK-3 priming site serines.

Although our data show that GSK-3 is required for phosphorylation of N protein at a classical GSK-3 consensus site, it remains formally possible that GSK-3 indirectly regulates N protein phosphorylation. Pharmacological inhibition of GSK-3 also activates mTOR and downstream All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. SRPK1/2 regulate activity of serine/arginine-rich (SR) proteins via phosphorylation of the arginine/serine (RS)-repeat domains. Similar to GSK-3, SRPK1/2 tend to phosphorylate multiple serines within a local domain in a processive manner, and SRPK1 can phosphorylate N in vitro 5, 12 . To determine whether mTOR and SPRK1/2 are involved in N phosphorylation, HEK293T cells expressing SARS-CoV2 N were treated with the mTOR inhibitor Rapamycin or the SRPK1/2 inhibitor SRPIN340. Rapamycin treatment had no effect on N phosphorylation despite potent inhibition of ribosomal protein S6 kinase phosphorylation ( Figure S2B ). The SRPK1/2 inhibitor SRPIN340, either alone or combined with LiCl, also did not affect N protein phoshorylation at sites that affect electrophoretic mobility ( Figure S2C ).

To test whether GSK-3 directly phosphorylates N protein, we performed in vitro kinase assays with recombinant GSK-3b and N protein purified by immunoprecipitation from HEK293T cells.

Use of N protein expressed in mammalian cells was important because the priming site will not be phosphorylated in bacterially expressed recombinant protein and, without the priming phosphorylation, the GSK-3 sites may not be phosphorylated. HEK293T cells expressing myctagged N were treated with the GSK-3 inhibitor LiCl and then immunoprecipitated. As shown in Fig. 2D , N protein lacking phosphorlyation at the GSK-3 dependent sites migrates more rapidly than phosphorlyated N (lane 5). Recombinant GSK-3b was then added to the immunoprecipitate and the reaction was incubated for 30 minutes. N protein phosphorylated by GSK-3 migrated with slower mobility (Fig. 2D, lane 6) , similar to N protein from untreated cells.

N protein expressed in GSK-3 double knock out (DKO) 293T cells, immunoprecipitated, and added to an in vitro kinase reaction was completely phosphorylated by recombinant GSK-3b (Fig. 2E) . These data demonstrate that GSK-3b directly phosphorylates N protein.

All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 

The phosphorylation of a motif within an arginine-rich domain, and especially the high conservation of arginine at the -3 position relative to the priming site (Fig. 1B) , suggested that the priming kinase may be an arginine-directed, or "basophilic", protein kinase. We began to test candidate kinases using inhibitors of MAP/ERK kinases (MEK1/2), casein kinase II (CKII), calmodulin-dependent protein kinase II, and protein kinase C (PKC). Although most inhibitors we tested had no effect on N phosphorylation (data not shown), including the PKC inhibitor Gö6976 ( Figure S3B ), the structurally related PKC inhibitors Enzastaurin, Sotrastaurin, and Gö6983 did inhibit N phosphorylation ( Fig. 3A and S3C ). Unexpectedly, in addition to potently inhibiting phosphorylation of endogenous PKC substrates induced by the PKC activator PMA ( Figure S3A ), these bisindolylmaleimides also inhibited phosphorylation of the endogenous GSK-3 substrate GS. To distinguish whether the inhibition of N phosphorylation was due to inhibition of GSK-3 or inhibition of a priming phosphorylation by PKC, we knocked down expression of PKC-a, PKC-d, and PKC-e, the major PKC isoforms expressed in HEK293T cells.

Single or combined knockdown of PKC had no effect on phosphorylation of N or GS ( Figure   S3D ). To confirm that Enzastaurin and Sotrastaurin directly inhibit GSK-3, we performed in vitro kinase assays. Phosphorylation of the GSK-3 substrate tau and N protein was inhibited by Enzastaurin and Sotrostaurin in vitro (Fig. 3B , 4C, and S3E), confiming that Enzastaurin and Sotrastaurin are direct inhibitors of GSK-3, consistent with a prior report 41 .

The GSK-3 inhibitors CHIR99021 and Enzastaurin were tested for antiviral efficacy (detection of double stranded RNA or spike protein) and for their effects on cell viability at two institutions in two lung epithelial cell lines (Calu-3 and A549-Ace2). Calu-3 cells were treated with drugs at varying concentrations for 1 hour, inoculated with SARS-CoV-2, and cell number and the percent of infected cells were quantified at 48 hours post infection (HPI). CHIR99021 inhibited All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 22, 2021. ; https://doi.org/10.1101/2021.02.17.21251933 doi: medRxiv preprint SARS-CoV-2 infection with an IC50 ~5uM in Calu-3 cells (Fig. 4A) , with marked reduction in frequency of infected cells at 10 µM (Fig. 4B ). CHIR99021 inhibition of SARS-CoV-2 in Calu-3 cells was similar in laboratories at the University of Pennsylvania and UCLA. CHIR98014, a GSK-3 inhibitor that is structurally similar to CHIR99021, was also reported to inhibit SARS-CoV-2 in A549-Ace2 cells at 5 µM 1 , although we did not observe this effect in A549-Ace2 cells.

Similarly, previous work has shown that Enzastaurin inhibits SARS-CoV-2-mediated cytopathic effect in Vero E6 cells at 250 nM 4 and reduces infection (based on qRT-PCR and viral titer) in A549-Ace2 cells at 5 µM 1 ; however, we did not observe an effect of Enzastaurin in A549-Ace2 or Calu-3 cells. The reasons for the cell-type variability in these assays is unclear but has been observed by others as well 1 .

As lithium is a GSK-3 inhibitor that is widely used to treat bipolar disorder, we asked whether patients on lithium have a reduced risk of COVID-19 infection compared to the general population. We included patient data from three health systems in the United States ( The average age of the patients who received lithium was between 42 -48 years.

Given the potential confounding bias for COVID-19 susceptibility with the patient's baseline characteristics, propensity score matching was employed at each site. The matched cohort resulted in 25 patients from UPHS, 50 patients from UIHC, and 10 patients from MSMC who All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. were on lithium treatment and tested positive for COVID-19 (Table 1) . To increase the statistical power, we pooled the data from three sites by conducting a meta-analysis using a random effects model (Table 2) . We found that patients on lithium had reduced risk of COVID-19 infection compared with nonusers (p-value= 0.001, OR=0.51 [0.34 -0.76]). The test for heterogeneity had low variance between the observations from three sites (Q statistic P = 0.45, I 2 = 50.3%). Additionally, a subgroup analysis among patients with bipolar disorder taking lithium compared to nonusers of lithium was conducted. The association was not statistically significant (p > 0.01). There were considerably fewer BPD patients tested for COVID-19 in our study population which may have led to low statistical power to detect association.

Medications that target common features of the coronavirus family could reduce the severity and transmission of COVID-19 as well as other pathogenic coronaviruses. Our analysis of retrospective EHR data on SARS-CoV-2 PCR testing from three major health systems across the US showed a ~50% reduced risk of COVID-19 in patients taking lithium. We show that the SARS-CoV-2 N protein is phosphorylated by GSK-3 and that lithium and other GSK-3 inhibitors block N phosphorylation, as shown previously for JHMV and SARS-CoV 9 . We also show for the first time that GSK-3 is unequivocally essential for N phosphorylation using GSK3A/B double KO. Diverse GSK-3 inhibitors inhibit N phosphorylation and impair SARS-CoV-2 replication in cell culture models. GSK-3 inhibition may therefore allow safe and effective therapy for COVID-19. As we find GSK-3 consensus sites in the N proteins of diverse coronaviruses, GSK-3 inhibitors may also be effective antiviral therapy in other coronavirus infections, including those that may arise in the future.

Lithium has a narrow therapeutic window, however, and the concentration needed to inhibit N phosphorylation and to impair infectivity of SARS-CoV-2 and other coronaviruses in cell All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 22, 2021. ; https://doi.org/10.1101/2021.02.17.21251933 doi: medRxiv preprint culture 7,9,19-24 is above the therapeutic range in humans (peak level ~2 mM, trough levels ~1 mM 17 ) and the level that induces behavioral changes in model organisms. For example, 1 mM lithium is sufficient to inhibit GSK-3 in humans 34 and to alter GSK-3 dependent behaviors in mice 33,42 , whereas the IC50 for inhibition of N phosphorylation and inhibition of SARS-CoV2 replication in cultured cells is ~10 mM. It is not clear why higher concentrations were needed in cell culture, but, given the narrow therapeutic window for lithium in BD, alternative GSK-3 inhibitors may be better tolerated in clinical settings involving coronavirus infection.

While the association of lithium therapy and reduced risk of COVID-19 across three health systems is both signficant and intriguing, observational studies have many limitations. A variety of factors with potential biases cannot be measured even after comparing the cases and controls in a manner that accounts for known confounding factors using a rigorous matching algorithm. For instance, details on medicine usage were derived from records of prescription orders, but information on compliance before SARS-CoV-2 PCR testing is not available. In addition, the collection of a non-random sample population can create a collider bias and lead to distorted associations. For example, the COVID-19 test was restricted particularly in the early pandemic to symptomatic patients so that many asymptomatic patients in the EHR were not tested. These findings should therefore be interpreted carefully and deeper investigation is required in a cohort with a larger sample size.

Prior work has shown that lithium and the GSK-3 inhibitor Kenpaullone inhibit N phosphorylation and reduce viral titers in SARS-CoV and JHMV infected Vero6 cells 8, 9 and GSK3 knockdown also impairs replication of IBV in Vero cells 15 . We also show that multiple small molecule GSK-3 inhibitors, including CHIR99021, BIM-I, AR-A014418, Enzastaurin, and Sotrastaurin block SARS-CoV-2 phosphorylation. These pharmacological studies are compelling evidence that GSK-3 is a critical host kinase for N protein, but these drugs may have off-target effects. Thus, All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 22, 2021. the complete abrogation of N phosphorylation by the double KO of GSK3A and GSK3B demonstrates unequivocally that GSK-3 is essential for phosphorylation of N at these sites.

The highly selective GSK-3 inhibitor CHIR99021 inhibited SARS-CoV-2 infection in the human lung epithelium-derived cell line Calu-3, an observation that was reproducible in two independent laboratories, and the related compound CHIR98014 was previously reported to inhibit infection in the human lung cancer derived cell line A549-Ace2 1 . Furthermore, the clinically well tolerated drug Enzastaurin was reported to inhibit SARS-CoV-2 infection in A549-Ace2 cells 1 and viral-mediated cytopathic effect in Vero E6 cells 4 . However, the effects of these inhibitors has been variable in different cell lines and in different laboratories. For example, Bouhaddou et al did not observe inhibition with Enzastaurin in Vero6 cells and we did not observe inhibition with Enzastaurin in A549-Ace2 cells or Calu-3 cells. The reasons for this cell type specific effect and variability between laboratories is unclear, but may include differences in the expression and/or activity of targeted signaling pathways in different cell lines that may arise as an adaptation to cell culture conditions and passage number. Nevertheless, it remains clear that GSK-3 is essential for N phosphorylation, as GSK3 knockout abrogates N phosphorylation, and given the essential functions of phosphorylated N in viral transcription, replication, and packaging 8 , developing GSK-3 inhbitors that safely and effectively inhibit N phosphorylation is a promising potential approach to controlling SARS-CoV-2 and other coronavirus infections that may arise in the future.

We propose that inhibition of N phosphorylation underlies the antiviral activity of lithium and other GSK-3 inhibitors; however GSK-3 also regulates inflammatory responses 43 , and lithium has been reported to have antiviral activity against other viruses, notably human herpes viruses 25 . Thus modulation of the inflammatory response by litihum may also contribute to the reduced risk of COVID-19 in patients taking lithium. All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The search for antiviral drugs that target coronaviruses is progressing rapidly. Multiple bioinformatic analyses and high throughput screens have identified an array of promising candidates with efficacy in cultured cells [1] [2] [3] 5, 44 . Our approach is based on a clear mechanism, utilizes clinically-tested and well-tolerated drugs that could be rapidly repurposed for COVID-19, and is supported by clinical data showing an association between treatment with a GSK-3 inhibitor (litihum carbonate) and reduced risk of COVID-19. Inhibition of a host protein required for coronavirus propagation has a potential advantage over inhibiting viral proteins as the (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Iowa Health Care (UIHC). Our main hypothesis was to test for the association between lithium use and COVID-19 susceptibility. We employed several EHR phenotyping algorithms to identify patients who were tested for COVID-19 by RT-PCR, patients with prescriptions for lithium, and patients with bipolar disorder. To account for the possibility that an individual had been tested for COVID-19 within the health system, but otherwise managed their care at a different health system, such that they had no medication or diagnosis information, we restricted the analysis to individuals with at least four encounters within the health system. The analysis workflow is provided in Fig. 5 

Institutional Review Board (IRB) protocol #844360 and for MSMC under IRB#20-00338.

Outcome: The primary outcome of our case-control EHR study was COVID-19 susceptibility where cases are defined by positive test results from RT-PCR of nasal samples and controls All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. was also restricted to participants between the ages of 18 and 89.

Exposure: Lithium use was defined using the prescription orders available within the EHR. The medication name and dose generally differ across health systems and it poses a challenge to develop standard selection criteria. To minimize these differences, we used RxNorm -a resource of standardized nomenclature for drug names from the National Library of Medicine 45 .

RxNorm maps branded and generic names, ingredients, drug components, and other drugrelated vocabularies to standard names. The current EHR systems (EPIC) also support RxNorm and there is an existing mapping between drug names and RxNorm concept unique identifiers (RxCUI). We queried RxNorm to extract all the RxCUI linked with lithium carbonate and then extracted prescription orders mapped to RxCUI in the EHR system. The list of RxNorm CUIs can be found in Supplementary Table 2 . A patient was considered on lithium treatment if they had an order placed within 90 days prior to their first positive COVID-19 test (COVID-19 cases) or 90 days before their first negative COVID-19 test (COVID-19 controls). Generally, lithium is prescribed for a longer period of time (> 3 months), so to capture long-term use of lithium we included patients with two or more lithium orders placed within 12 months before their COVID-19 test, using the aforementioned methods for COVID-19 cases and controls.

Statistical Analysis: To minimize potential confounding biases among the population tested for COVID-19, we applied a propensity score matching (PSM) method. For each patient on lithium with a record of COVID-19 testing, we first calculated the propensity score using a multivariate logistic regression model adjusting for age, sex, and race. Then, we applied nearest-neighbor matching (MatchIt R) on the propensity scores to select one matched patient for each patient on lithium. We conducted a meta-analysis on the association between lithium use and COVID-19 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 22, 2021. ; https://doi.org/10.1101/2021.02.17.21251933 doi: medRxiv preprint outcome occurrences using the meta R package. We pooled the effect estimates using fixed effect and random effects models and the Mantel-Haenszel method was used to calculate the fixed effect estimate. Since we have effect estimates from only three sites, a sensitivity analysis of subgroups was not feasible. We assessed the heterogeneity of the meta-analysis through I 2 and Chi 2 statistics.

The following reagents were obtained through BEI Resources, NIAID, NIH: Monoclonal Anti- (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 22, 2021. GSK3B was deleted in GSK3A -/cells using CRISPR (GSK3 DKO). N protein was expressed in both wild-type and DKO cells in the presence of increasing concentrations LiC for 18h as above All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Cells were fixed at 48hpi and total cell count (green) and percent viral infection (blue) detected by immunofluorescence for dsRNA were assessed. B. Calu-3 cells were treated with vehicle or the indicated concentrations of CHIR99021, innoculated with SARS-CoV-2, fixed at 48 hpi, and Spike protein was detected by immunofluorescence (UCLA). Enzastaurin had no effect on viral infection in Calu-3 cells. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. PKCε individually or in combination, as described previously 46 . Cell lysates were immunoblotted for CoV-2 N protein, pGS, or PKC isoforms as indicated in figure. b-actin was a loading control. E. N protein was immunoprecipitated from GSK3 DKO cells as in Fig. 2E and added to an in All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Sotrastaurin, or 2uM CHIR99021. All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 22, 2021. ; Fig. 1 . The SARS-CoV-2 Nucleocapsid protein is phosphorylated by GSK-3 in two conserved consensus sites. A. The RS domains of SARS-CoV-2 (aa176-206) and SARS-CoV N proteins are 90% identical and contain tandem sets of SXXXS motifs, labelled "a" and "b". Consensus site serines and threonines are in bold; red indicates sites shown previously by mass spectroscopy to be phosphorylated 1, 9 . B. Alignment of RS domains in N proteins from pathogenic CoVs showing conservation of repeated SXXXS motifs ("S" in motif represents serine or threonine) and a highly conserved arginine 3 residues before the putative priming sites (SRXXS). Blue indicates b-CoV; orange: a-CoV; green: g-CoV. C. SARS-CoV2 N was expressed in 293T cells, treated with LiCl for 18h, and then subjected to SDS-PAGE and immunoblotting for N protein (CoV2-N) , phosphorylated glycogen synthase (pGS), or b-Actin as a loading control. Alkaline phosphatase (AP) treatment of cell lysates increases electrophoretic mobility. LiCl inhibits N phosphorylation with IC50 ~10 mM. "phos" indicates phosphorylated N protein; "unphos" indicates dephosphorylated N; NT, nontransfected. Ctl, nontreated control. D. SARS-CoV-2 N expressing 293T cells were treated for 18h with LiCl, bisindolylmaleimide I (BIM-I), or CHIR99021 (CHIR) at the indicated concentrations and cell lysates were immunoblotted as in panel C.

All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 

All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 22, 2021. ; Fig. 2. GSK3 is required for N phosphorylation. A. Control 293T cells (WT), 293T cells with CRISPR/Cas9 KO of GSK3A (GSK3A -/-), and GSK3A -/cells with siRNA knockdown of GSK3B (GSK3A -/--;siGSK3B) were treated with LiCl at indicated concentrations and lysates were immunoblotted for N protein, phospho--catenin, GSK-3a/b, or b-Actin. Combined loss of GSKA and GSK3B impairs phosphorylation of N and b-catenin and enhances sensitivity to LiCl. B. GSK3B was deleted in GSK3A -/cells using CRISPR (GSK3 DKO). N protein was expressed in both wild-type and DKO cells in the presence of increasing concentrations LiC for 18h as above and immunoblotted for N protein, phospho-GS (pGS), total b-catenin, GSK-3a/b, and bactin. N is not phosphorylated in DKO cells and mobility is not affected by LiCl treatment. Total b-catenin protein accumulates in absence of GSK-3. C. Serine-188 and serine-206 were mutated to alanine by site directed mutagenesis and single and double mutant N proteins were expressed in 293T cells in the presence of vehicle or 10mM LiCl and immunoblotted for N protein, pGS, or b-actin. The double mutant N S188A;S206A migrates similar to dephosphorylated wild-type N. Single mutants are more sensitive to LiCl. D/E. N protein was immunoprecipitated from wild-type HEK293T cells treated with or without 10mM LiCl for 18h (indicated by "CTL" or "LiCL" below each lane in panel D) or from GSK3 DKO cells (panel E). Immunoprecipitated N protein was added to an in vitro kinase reaction with recombinant GSK-3b. GSK-3b phosphorylates N from LiCl treated wild-type and DKO cells as indicated by slower electroporetic mobility ("phos" in panel E).

A B C

All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 22, 2021. ; Fig. 3 . Enzastaurin inhibits N phosphorylation: A. N expressing 293T cells were treated with DMSO or increasing doses of Enzastaurin. Enzastaurin inhibited phosphorylation of N and GS in a dose dependent manner. Inhibition of PKC in these samples is described in supplemental figure S3 . B. In vitro GSK-3 kinase assay using tau protein as substrate. Unphosphorylated tau migrates more rapidly ("unphos") than tau phosphorylated by GSK-3 ("phos"). Enzastaurin inhibits GSK-3 activity directly at 0.5 µM. C. N protein was immunoprecipitated from DKO cells as in Fig. 2D /E and added to an in vitro kinase reaction with recombinant GSK-3 . Phosphorylation of N protein was inhibited in the presence of Enzastaurin.

All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 22, 2021. ;

Dose-response analysis of Calu-3 cells treated with GSK-3 inhibitors CHIR99021 or Enzastaurin (UPenn). Cells were treated with drug at the indicated concentrations and then inoculated with SARS-CoV-2. Cells were fixed at 48hpi and total cell count (green) and percent viral infection (blue) detected by immunofluorescence for dsRNA were assessed. B. Calu-3 cells were treated with vehicle or the indicated concentrations of CHIR99021, innoculated with SARS-CoV-2, fixed at 48 hpi, and Spike protein was detected by immunofluorescence (UCLA). Enzastaurin had no effect on viral infection in Calu-3 cells.

The Global Phosphorylation Landscape of SARS-CoV-2 Infection

Drug repurposing screens reveal FDA approved drugs active against SARS-Cov-2

Discovery of SARS-CoV-2 antiviral drugs through large-scale compound repurposing

Antiviral Drug Screen of Kinase inhibitors Identifies Cellular Signaling Pathways Critical for SARS-CoV-2 Replication. bioRxiv

SRSF protein kinases 1 and 2 are essential host factors for human coronaviruses including SARS-CoV-2. bioRxiv

The SARS coronavirus nucleocapsid protein--forms and functions

The SARS-CoV nucleocapsid protein: a protein with multifarious activities

Nucleocapsid phosphorylation and RNA helicase DDX1 recruitment enables coronavirus transition from discontinuous to continuous transcription

Glycogen synthase kinase-3 regulates the phosphorylation of severe acute respiratory syndrome coronavirus nucleocapsid protein and viral replication

A contemporary view of coronavirus transcription

The severe acute respiratory syndrome coronavirus nucleocapsid protein is phosphorylated and localizes in the cytoplasm by 14-3-3-mediated translocation

Phosphorylation of the arginine/serine dipeptide-rich motif of the severe acute respiratory syndrome coronavirus nucleocapsid protein modulates its multimerization, translation inhibitory activity and cellular localization

Phosphorylation and subcellular localization of transmissible gastroenteritis virus nucleocapsid protein in infected cells

Identification of mouse hepatitis coronavirus A59 nucleocapsid protein phosphorylation sites

The cellular interactome of the coronavirus infectious bronchitis virus nucleocapsid protein and functional implications for virus biology

A molecular mechanism for the effect of lithium on development

 Figure S1 . A. SARS-CoV2 N was expressed in MLE12 cells, treated with LiCl for 18h, and then subjected to SDS-PAGE and immunoblotting for N protein (upper panel) and phosphorylated b-catenin (lower panel). Alkaline phosphatase (AP) treatment of cell lysates increases electrophoretic mobility. "P" indicates slower migrating, phosphorylated N and "un-P" indicates dephosphorylated species. B. N was expressed in 293T cells, treated with 10 mM LiCl, and cell lysates were harvested at different time points for Western blot analysis. CoV-2 N protein (upper panel); b-actin (lower panel) is loading control. C. Alternative GSK-3 inhibitors Kenpaullone and AR-A014418 inhibit N protein phosphorylation. SARS-CoV2 N expressing 293T cells treated for 18h with Kenpaullone or AR-A014418 at the indicated concentrations were immunoblotted as in panel B.Supplementary Figure S2 . A. Serine-188 and serine-206 of SARS-CoV-2 N protein were mutated to alanine and single and double mutant N proteins were expressed in 293T cells. Alkaline phosphatase (AP) treatment of cell lysates increases electrophoretic mobility. B. mTOR inhibitor Rapamycin had no effect on N phsohorylation. SARS-CoV2 N expressing 293T cells were treated with Rapamycin (50 and 200 nM) in the presence of LiCl for 18h and then subjected to SDS-PAGE and immunoblotting for N protein (upper panel) and phosphorylation of -catenin (middle panel) and S6 (lower panel). Rapamycin inhibited S6 phosphorylation but had no effect on N phosphorylation. "P" indicates phosphorylated N; "Un-P" indicates dephosphorylated species. C. N expressing 293T cells were treated with LiCl or the SRPK inhibitor SRPIN340 separately (left panel) or in combination (right panel) and cell lysates were then harvested for Western blot analysis as in panels A and B. Figure S3 . A. N expressing 293T cells were treated with DMSO or increasing doses of Enzastaurin and then stimulated with 200nM phorbol 12-myristate 13-acetate (PMA) for 30 mins prior to lysis. PKC activity was assessed with an antibody that detects phosphorylated PKC substrates. b-actin is shown as a loading control. B. N expressing 293T cells were treated with DMSO or increasing doses of the PKC inhibitors Enzastaurin and Gö6976 and stimulated with PMA as in panel A. Cell lysates were immunoblotted for N protein (upper panel), phospho-GS (middle panel), or phosphorylated PKC substrates (lower panel). Gö6976 does not affect phosphorylation of N or GS. C. The structurally related PKC inhibitors Sotrastaurin and Gö6983 inhibit N phosphorylation. N expressing cells were treated with Sotrastaurin or Gö6983 at the indicated concentrations and then lysates were immunotted for N and p-GS as above. D. Global PKC knockdown does not affect phosphorylation of N protein or GS. N expressing 293T cells were treated with vehicle or LiCl, then transfected with siRNAs directed against PKCα, PKCδ, or PKCε individually or in combination, as described previously 46 . Cell lysates were immunoblotted for CoV-2 N protein, pGS, or PKC isoforms as indicated in figure. b-actin was a loading control. E. N protein was immunoprecipitated from GSK3 DKO cells as in Fig. 2E and added to an in vitro kinase reaction with recombinant GSK-3b. Phosphorylation of N protein assessed by electrophoretic mobility shift was inhibited in the presence of 5uM Enzastaurin, 10uM Sotrastaurin, or 2uM CHIR99021.