Human coronaviruses: viral and cellular factors involved in neuroinvasiveness and neuropathogenesis

Abstract

Among the various respiratory viruses infecting human beings, coronaviruses are important pathogens, which usually infect the upper respiratory tract, where they are mainly associated with common colds. However, in more vulnerable populations, such as newborns, infants, the elderly and immune-compromised individuals, these opportunistic pathogens can also affect the lower respiratory tract, leading to pneumonia, exacerbations of asthma, and various types of respiratory distress syndrome. The respiratory involvement of human coronaviruses has been clearly established since the 1960s. Nevertheless, for almost three decades now, data reported in the scientific literature has also demonstrated that, like it was described for other human viruses, coronaviruses have neuroinvasive capacities since they can spread from the respiratory tract to the central nervous system (CNS). Once there, infection of CNS cells (neurotropism) could lead to human health problems, such as encephalitis and long-term neurological diseases. Neuroinvasive coronaviruses could damage the CNS as a result of misdirected host immune responses that could be associated with autoimmunity in susceptible individuals (virus-induced neuroimmunopathology) and/or viral replication, which directly induces damage to CNS cells (virus-induced neuropathology). Given all these properties, it has been suggested that these opportunistic human respiratory pathogens could be associated with the triggering or the exacerbation of neurologic diseases for which the etiology remains poorly understood. Herein, we present host and viral factors that participate in the regulation of the possible pathogenic processes associated with CNS infection by human coronaviruses and we try to decipher the intricate interplay between virus and host target cells in order to characterize their role in the virus life cycle as well as in the capacity of the cell to respond to viral invasion.

Keywords: CNS infection; Human coronavirus; Neuroinvasion; Neurological diseases; Respiratory viral infection.

Fig. 1

Potential route of infection used by HCoV for neuroinvasion into the human central nervous system (CNS) and possible mechanisms of neurovirulence. (A) Following infection of human airways, human coronaviruses may, in some conditions, pass through the epithelium, gain access to the bloodstream and infect monocytes, which are activated by the infection. Among other factors, MMP9, which increases BBB permeability and TNF-alpha, which leads to up-regulation of ICAM-1 expression on endothelial cells, facilitates the passage of infected and activated monocytes into the CNS. Once in the CNS, these cells produce proinflammatory cytokines (such as TNF-alpha) that can damage the oligodendrocytes and/or the neurons. Infiltrated infected monocyte-derived macrophages (or microglia) may produce chemokines (CCL-5, CXCL10, CXCL11), which will induce chemoattraction of activated T cells and/or other monocytes. After sensing the infection, astrocytes may also produce other chemokines (CCL2, CCL5 and CXCL12) that will also participate in the recruitment of more infected leukocytes. Human coronaviruses may therefore initiate an aberrant neuroinflammatory loop which will mediate an immune-mediated neuropathology (adapted from Talbot et al., 2008). (B) Following intranasal infection in human, coronaviruses may infect the olfactory receptor neurons (ORN) and pass through the neuroepithelium of the olfactory mucosa to reach the mitral cells and the olfactory nerve (ON) and gain access to the olfactory bulb (OB) and eventually to the hippocampus and other regions of the brain.

Fig. 2

Human coronavirus transneuronal route of neuroinvasion through the olfactory nerve and spread into the CNS in susceptible mice. Following intranasal infection or 14 day-old susceptible mice, HCoV-OC43 infects first the olfactory bulb (left panel) and then disseminate to other regions of the brain, including the hippocampus (right panel). In both regions of the brain, neurons are the main target of infection.

Fig. 3

Detection of coronaviral RNA in human CNS and of HCoV-myelin antigens cross-reactive T cells in MS patients. (A) Double-blind analysis of ninety human brain autopsy samples revealed the presence of HCoV-229E and HCoV-OC43 RNA in normal controls, patients with other neurological disorders (OND) and patients with multiple sclerosis (MS). The proportion of brain samples from MS patients containing HCoV-OC43 RNA was significantly greater than OND and normal controls. RNA from both HCoV was found more often in Female brains compared to male. (B) More monospecific T-cell clones were isolated from MS patients compared to normal controls and cross-reactive T-cell clones were isolated only from MS patients.

Fig. 4

HCoV infection induces increased production of proinflammatory cytokines and neuronal degeneration as a consequence of glutamate excitotoxicity. In physiological conditions, glutamate is mainly synthesized by neurons and released in the synaptic cleft as the primary excitatory neurotransmitter of the CNS that activates the ligand-dependant receptor AMPAr (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionoc acid receptor), which allows the entry of sodium ions and the passage of the nerve impulse in the post-synaptic neuron, leading to activation of the NMDA receptor (N-methyl-D-aspartate receptor) that allows the entry of calcium ions. During infection of neurons by HCoV-OC43, microglial cells detect the presence of virus and produce pro-inflammatory cytokines (TNF-alpha, IL-1 beta and IL-6) that down-regulate the astrocytic receptor GLT-1 (glutamate transporter 1) and prevent the efficient recapture of glutamate. This situation disturbs the regulation of glutamate homeostasis and the excess of this neurotransmitter in the synaptic cleft leads to excitotoxicity associated with a massive entry of calcium which eventually leads to degeneration of and death of neuronal cells.

Fig. 5

Pathways of neuronal degeneration and programmed cell death (PCD) activated or potentially inhibited after HCoV-OC43 infection of neuronal cells. Hallmarks of apoptosis, including the relocalization of the activated pro-apoptotic protein BAX (Bcl-2 associated protein X) from the cytosol to the mitochondrial membrane, cytochrome C release from mitochondria toward the cytosol, DNA fragmentation and activation of caspases-3 and -9, are observed during infection of human neurons. However, even though virus induces Bax relocalization, it may inhibit classical apoptosis by blocking Bax pro-apoptotic function at the mitochondria and/or downstream of caspase activation, suggesting a caspase-independent type of apoptosis. Relocalization of the mitochondrial protein AIF (apoptosis-inducing factor) toward the nucleus (truncated tAIF) is observed after infection and participates in DNA fragmentation. AIF is known to be activated during caspase-independent apoptosis. However, AIF is also involved in Parthanatos, another form of PCD potentially associated with neurodegeneration. As they are synthesized by the poly(ADP-ribose) polymerase (PARP) during a neuronal stress, including during HCoV-OC43 infection, polymers of ADP-ribose (PAR) may relocalize toward mitochondria and participate in the activation and relocalization of AIF toward the cytosol before it reaches the nucleus. Cyclophilin D (CypD) inhibition decreases AIF release from mitochondria and abrogates cell death induced by infection. AIF release from mitochondria may be induced through its truncation (tAIF) by activated calpain, which is usually activated by a rise in the mitochondrial calcium concentration. This increase in calcium concentration may be linked with either an important entry from the extracellular milieu (for instance during glutamate excitotoxicity) or with a release of calcium from the ER following induction of ER stress. However HCoV-OC43 may inhibit the ER stress-related pathway in infected neurons. Infection induces RIP1 gene expression and the knock-down of the receptor interacting protein kinase-1 (RIP-1), significantly increases cell survival, suggesting that necroptosis, a third form of PCD which involves RIP-1 and RIP-3 downstream of the death receptor family, may play a role in HCoV-OC43-induced neuronal death. Solid arrows indicate experimental data and dashed arrows represent possible pathways based on the literature (see text for details).

Fig. 6

Importance of HCoV-OC43 non-structural accessory proteins as neurovirulence factors in infected mice depends of the mouse strain. Groups of 20 mice (A) of three different strains were infected by the intracerebral route with reference wild-type HCoV-OC43 (ATCC) or with a mutant deleted for the ns2 accessory protein (ns2-KO) and (B) with reference wild-type HCoV-OC43 (ATCC) or with a mutant deleted for the ns5 accessory protein (ns5-KO). Infection with ten times (ns5-KO-10×) or even a hundred times (ns5-KO-100×) more ns5 mutant virus led to a reduced neurovirulence compared to wild type virus (ATCC). Survival was evaluated daily over a period of 21 days. Results are representative of two independent experiments.