Abstract
The coronaviruses responsible for severe acute respiratory syndrome (SARS-CoV), COVID-19 (SARS-CoV-2), Middle East respiratory syndrome-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, 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 [0.35-0.74], P = 0.005). 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 an approach to treat COVID-19 and future coronavirus outbreaks.
Highlights
The coronaviruses responsible for severe acute respiratory syndrome (SARS-CoV), COVID-19 (SARS-CoV-2), Middle East respiratory syndrome-CoV, and other coronavirus infections express a nucleocapsid protein (N) that is essential for viral replication, transcription, and virion assembly
The RS domains of N from SARS-CoV and JHM strain of mouse hepatitis virus (JHMV) include repeated motifs (SXXXS) [8] that are frequently associated with glycogen synthase kinase-3 (GSK-3) phosphorylation, in which the C-terminal serine is phosphorylated by a priming kinase [27], which allows GSK-3 to phosphorylate multiple serines or threonines spaced four residues apart in the C- to N-terminal direction (Fig. 1A)
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 [8]
Summary
The coronaviruses responsible for severe acute respiratory syndrome (SARS-CoV), COVID-19 (SARS-CoV-2), Middle East respiratory syndrome-CoV, and other coronavirus infections express a nucleocapsid protein (N) that is essential for viral replication, transcription, and virion assembly. 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 coronavirus-2 (SARS-CoV-2) [1,2,3,4] 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. We further show that GSK-3 is essential for phosphorylation of the SARS-CoV-2 nucleocapsid protein and that GSK-3 inhibition blocks SARS-CoV-2 infection in human lung epithelial cells These findings suggest an antiviral strategy for COVID-19 and new coronaviruses that may arise in the future
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