Glutamate excitotoxicity is involved in the induction of paralysis in mice after infection by a human coronavirus with a single point mutation in its spike protein

Abstract

Human coronaviruses (HCoV) are recognized respiratory pathogens, and some strains, including HCoV-OC43, can infect human neuronal and glial cells of the central nervous system (CNS) and activate neuroinflammatory mechanisms. Moreover, HCoV-OC43 is neuroinvasive, neurotropic, and neurovirulent in susceptible mice, where it induces chronic encephalitis. Herein, we show that a single point mutation in the viral spike (S) glycoprotein (Y241H), acquired during viral persistence in human neural cells, led to a hind-limb paralytic disease in infected mice. Inhibition of glutamate excitotoxicity using a 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl)propranoic acid (AMPA) receptor antagonist (GYKI-52466) improved clinical scores related to the paralysis and motor disabilities in S mutant virus-infected mice, as well as protected the CNS from neuronal dysfunctions, as illustrated by restoration of the phosphorylation state of neurofilaments. Expression of the glial glutamate transporter GLT-1, responsible for glutamate homeostasis, was downregulated following infection, and GYKI-52466 also significantly restored its steady-state expression level. Finally, GYKI-52466 treatment of S mutant virus-infected mice led to reduced microglial activation, which may lead to improvement in the regulation of CNS glutamate homeostasis. Taken together, our results strongly suggest an involvement of excitotoxicity in the paralysis-associated neuropathology induced by an HCoV-OC43 mutant which harbors a single point mutation in its spike protein that is acquired upon persistent virus infection.

Fig. 1.

Schematic representation of the point mutations introduced within the viral spike glycoprotein and associated neurovirulence of the different recombinant viruses in mice. (A) Schematic representation of the main structural domains of the S protein of HCoV-OC43 and the approximate locations of the four point mutations D24Y, S83T, H183R, and Y241H. These mutations were introduced into the viral genome to generate rOC/U_S24-241 (24). Alternatively, only pairs of mutations were introduced to generate rOC/U_S24-83 and rOC/U_S183-241. Finally, one single mutation was introduced at a time to generate rOC/U_S183 and rOC/U_S241. Asterisks indicate recombinant viruses which induced a paralytic disease following infection. RBD, putative cell receptor-binding domain; HVR, hypervariable region; HR, heptad repeat region; aa, amino acids. (B) Survival curves following intracerebral inoculation of the different HCoV-OC43 recombinants harboring various mutations in the S protein compared to that following inoculation with reference rOC/ATCC virus. Whereas infection of mice with the reference virus rOC/ATCC induced 15% mortality, mice infected with rOC/U_S24-241 showed a mortality rate of 80%. The relative survival rates of mice infected by different viruses containing one, two, or four mutations in the spike protein suggest a synergic effect of these mutations on mortality rate. Results are representative of three independent experiments.

Fig. 2.

Infectious-virus production measured in the CNS of infected mice. The kinetics of infectious-virus production in brains and spinal cords was evaluated every 2 days for 22 dpi. Both viruses replicated to similar levels, with the same kinetics in brains, and the highest levels of infectious virions were found at 10 dpi in brains as well as in spinal cords. Recombinant virus rOC/U_S241 was cleared less rapidly from the spinal cord, as infectious virus was repeatedly detected for up to 15 dpi. Results are representative of two independent experiments, and error bars represent standard errors of the means (SEM).

Fig. 3.

AMPA receptor antagonist GYKI-52466 treatment attenuates motor dysfunctions in infected mice without modifying neurovirulence or viral replication. (A) Survival curves of mice infected by rOC/U_S241 or rOC/ATCC and treated with GYKI-52466 or vehicle. GYKI-52466 treatment did not change the rate of survival of mice following infection by either virus. Results with sham-infected mice treated with GYKI-52466 alone illustrated that AMPA treatment was not toxic under the condition used. Results are representative of two independent experiments. (B) Effect of GYKI-52466 treatment on motor clinical scores in mice infected by rOC/U_S241 and treated with GYKI-52466 or vehicle. GYKI-52466 treatment attenuated clinical scores related to mild (CS, 1.5 to 2) or severe (CS, 2.5 to 3.5) paralysis for mice infected by rOC/U_S241, compared to scores for mice treated only with vehicle. Twenty to 30% of mice infected and treated with vehicle presented CS of severe paralysis (CS, 2.5 to 3.5), whereas only 5 to 10% of mice treated with GYKI-52466 fell into this category. The CS of motor dysfunction completely disappeared at 18 dpi in GYKI-52466-treated mice, whereas 20% of mice infected and treated with vehicle only still presented severe paralysis (CS, 2.5 to 3.5) at 21 dpi. Mice that presented mild paralysis (CS, 1.5 to 2) were fewer and recovered more rapidly when treated with GYKI-52466. Results are representative of three independent experiments. (C) Infectious-virus titers in the CNS of mice infected by rOC/U_S241 or rOC/ATCC and treated with GYKI-52466 or vehicle. For both recombinant viruses, viral replication (kinetics and total amount of infectious virus) was not affected following GYKI-52466 treatment. Results are representative of two independent experiments, and error bars represent SEM.

Fig. 4.

AMPA receptor antagonist treatment reduces neuronal dysfunction, as observed by the phosphorylation state of heavy neurofilaments in mice infected with rOC/U_S241. Immunohistochemistry of lumbar spinal cord gray and white matter segments from mice infected with rOC/U_S241 or rOC/ATCC and treated with GYKI-52466 or vehicle at 10 dpi. In the normal spinal cord (a), SMI 311 stained the neurofilament of neuronal soma and dendrites. Following infection with rOC/ATCC or rOC/U_S241 (b or d, respectively), GM presented less SMI 311 neuronal soma staining, illustrating a loss of normal nonphosphorylated NF in GM compared to that for sham-infected mice (a), which was more pronounced following rOC/U_S241 infection than following rOC/ATCC infection (light arrows). Interestingly, spinal cord white matter of mice infected with rOC/ATCC (b) or rOC/U_S241 (d) showed higher levels of abnormal axonal nonphosphorylated NF-H, with abnormal axonal swelling, which was more pronounced following infection by rOC/U_S241 than following sham infection (a) (dark arrows). Staining with SMI 312 showed a low level of phosphorylated NF-H in WM axons following infection by rOC/U_S241 compared to that following rOC/ATCC or sham infection. GYKI-52466 treatment of mice infected with rOC/ATCC (c) or rOC/U_S241 (e) partially restored the physiological NF-H phosphorylation state. (a) Sham-infected mice plus vehicle; (b) rOC/ATCC-infected mice plus vehicle; (c) rOC/ATCC-infected mice plus GYKI-52466; (d) rOC/U_S241-infected mice plus vehicle; (e) rOC/U_S241-infected mice plus GYKI-52466. Magnification, ×400. Results are representative of two independent experiments with three mice per group.

Fig. 5.

Expression of GLT-1 is downregulated in mice infected with rOC/U_S241 and partially restored following treatment with AMPA receptor antagonist. (A) Immunofluorescence analysis of lumbar spinal cord segments of gray matter from mice infected by rOC/ATCC or rOC/U_S241 and treated with vehicle (b and d) or GYKI-52466 (c and e) at 10 dpi. Infected mice (b and d) presented an increased number of activated astrocytes compared to that seen with sham-infected mice (a), as well as a downregulation of GLT-1 staining which was more pronounced following rOC/U_S241 infection (d) than following rOC/ATCC infection (b). Treatment with GYKI-52466 led to partial restoration of GLT-1 expression levels (c and e). Results are representative of two independent experiments with three mice per group. (B) Western blotting strengthened histological data. Mice infected by both recombinants showed significant (###, P < 0.001) GFAP expression compared to that for sham-infected mice. GLT-1 expression was significantly downregulated in mice infected by rOC/U_S241 (d) (##, P < 0.01) compared to the level in mice infected by rOC/ATCC (b) or control (a). Treatment with GYKI-52466 led to significant upregulation of GLT-1 expression in mice infected by rOC/U_S241 (**, P < 0.01) compared to that in mice infected by rOC/ATCC or control. Note that following infection of mice with HCoV-OC43, an additional band of GFAP is detected at 45 kDa, which is suggested to represent a proteolytic fragment derived from the 50-kDa band. Results are expressed as percentages of control (sham-infected mice plus vehicle [a]), and data are represented as means ± SEM (n = 3). (a) Sham-infected mice plus vehicle; (b) rOC/ATCC-infected mice plus vehicle; (c) rOC/ATCC-infected mice plus GYKI-52466; (d) rOC/U_S241-infected mice plus vehicle; (e) rOC/U_S241-infected mice plus GYKI-52466. Results are representative of three independent experiments.

Fig. 6.

Treatment with AMPA receptor antagonist reduced microglial activation. (A) Immunohistochemical staining of activated microglia, using Mac-2 antibody in mice infected with rOC/U_S241 or rOC/ATCC and treated with GYKI-52466 or vehicle at 10 dpi. The spinal cord gray matter ventral horn from infected mice demonstrated a significant activation of microglia/macrophages following infection by both viruses. Treatment with GYKI-52466 reduced microglia/macrophage activation in mice infected by rOC/U_S241. Magnification, ×400. Results are representative of two independent experiments with three mice per group. (B) Western blotting of spinal cord proteins confirmed the histological finding. Infection of mice by rOC/U_S241 led to significant (*, P < 0.05) increased activation of microglial cells (Mac-2 staining) compared to that in mice infected by rOC/ATCC, and GYKI-52466 treatment attenuated microglial cell activation in mice infected by rOC/U_S241 (*, P < 0.05). These results are expressed as percentages of the reference value, with 100% representing mice infected by rOC/ATCC and treated with vehicle (b), as microglial cell activation was undetectable in sham-infected animals (a). Data are represented as means ± SEM (n = 3). (a) Sham-infected mice plus vehicle; (b) rOC/ATCC-infected mice plus vehicle; (c) rOC/ATCC-infected mice plus GYKI-52466; (d) rOC/U_S241-infected mice plus vehicle; (e) rOC/U_S241-infected mice plus GYKI-52466. Results are representative of three independent experiments.