Spike protein (S protein) is the virus “key” to infect cells and is able to strongly bind to the human angiotensin-converting enzyme2 (ACE2), as has been reported. In fact, Spike structure and function is known to be highly important for cell infection as well as for entering the brain. Growing evidence indicates that different types of coronaviruses not only affect the respiratory system, but they might also invade the central nervous system (CNS). However, very little evidence has been so far reported on the presence of COVID-19 in the brain, and the potential exploitation, by this virus, of the lung to brain axis to reach neurons has not been completely understood. In this Article, we assessed the SARS-CoV and SARS-CoV-2 Spike protein sequence, structure, and electrostatic potential using computational approaches. Our results showed that the S proteins of SARS-CoV-2 and SARS-CoV are highly similar, sharing a sequence identity of 77%. In addition, we found that the SARS-CoV-2 S protein is slightly more positively charged than that of SARS-CoV since it contains four more positively charged residues and five less negatively charged residues which may lead to an increased affinity to bind to negatively charged regions of other molecules through nonspecific and specific interactions. Analysis the S protein binding to the host ACE2 receptor showed a 30% higher binding energy for SARS-CoV-2 than for the SARS-CoV S protein. These results might be useful for understanding the mechanism of cell entry, blood-brain barrier crossing, and clinical features related to the CNS infection by SARS-CoV-2.
comparison of SARS-CoV-2 and SARS-CoV S protein sequences, 3D structures, and electrostatic potentials reveals that both proteins have a conserved sequence and structural features but different electrostatic characteristics in both their external surface and their host-interaction interfaces. As previously described, the SARS-CoV-2 S protein is slightly more positively charged in these regions than SARS-CoV is, which will lead to an increased affinity to bind to negatively charged regions of other molecules through nonspecific and specific interactions.
Moreover, some differences in the amino acidic content of the S protein in the RBD-ACE2 interface can lead to the establishment of more specific interactions with the host receptors. Hence, SARS-CoV-2 is more likely to establish interactions with different targets across the human body than SARS-CoV both through nonspecific and specific interactions. All this, ultimately, can increase the capacity of SARS-CoV-2 to enter human cells and bind to the negative charge barriers such as the BBB(32) with respect to SARS-CoV.
In the last months, S protein structure and electrostatic properties have been the subject of much investigation. Previous computer-based experiments have also noted that the SARS-CoV-2 RBD exhibits a more positive electrostatic potential than the SARS-CoV RBD does(31,33−35) and that the electrostatic potential has a particularly important role in the high infection rate of SARS-CoV-2. In agreement with our results, it has previously been observed that SARS-CoV-2 binds with a higher affinity to the human ACE2 receptor than SARS-CoV does.(33) This was also attributed to the enhanced electrostatic interactions between SARS-CoV-2 and ACE2 due to the SARS-CoV-2 RBD having greater electrostatic complementarity with the binding domain of ACE2 than the SARS-CoV RBD.(33) In particular, it has been reported that the increased positive electrostatic potential of the SARS-CoV-2 binding surface is mainly due to an essential mutation of the hydrophobic residue Val404, present in SARS-CoV, to the positively charged residue Lys417 in SARS-CoV-2.(31,34) Amin et al. also identified a complementary negative electrostatic potential on the surface of the binding site of ACE2.(33)
Taking advantage of our previous experience dealing with nanoparticles (NPs) specifically tailored to cross the BBB and target the brain tissue, we can speculate the potential strategies of SAR-CoV-2 to enter into the brain. Indeed, the dimension and the surface properties of the SAR-CoV-2 are similar, in terms of adhesion and cell membrane crossing abilities, to those shown by the nanoparticles specifically designed for BBB crossing.(29,36) So the parallelism between SAR-CoV-2 and the strategies adopted to let nanoparticles cross the BBB can be useful to hypothesize the ways used by the virus to enter into the brain. Therefore, an increase of the number of the positive amino acids of the SAR-CoV-2 envelope might increase in a significant manner the adhesion properties of SAR-CoV-2 crossing the BBB and entering the brain.
In order to quantify the difference in the binding affinity of the two complexes (SARS-CoV-2:ACE2 and SARS-CoV:ACE2), their binding free energy was calculated. The results showed that SARS-CoV-2 S protein binds to the host ACE2 receptor with a 30% higher binding energy than the SARS-CoV S protein does. It has also been observed that the electrostatic contribution to the total binding free energy is the dominant term in the SARS-CoV-2:ACE2 interaction. Hence, this data supports the qualitative analysis of the electrostatic potential of the structures presented above and the quantitative data shown in previous studies.
According to the bioinformatics data regarding the possible interaction between the virus Spike protein and ACE2 protein, it is suggested that it is probable for SARS-CoV-2 to adhere with higher efficiency to the cells through nonspecific interactions which have a major impact on cell adhesion(28) due to (1) SARS-CoV-2 electrostatic properties and (2) binding with higher affinity to the host ACE2 receptor through specific interactions. In fact, our findings revealed that the Spike protein of SARS-CoV-2 binds to the host ACE2 receptor with a significantly higher binding energy than the SARS-CoV S protein does, indicating that the electrostatic contribution to the total binding free energy is the dominant term in the SARS-CoV-2:ACE2 interaction.
As previously described, Spike protein and ACE2 represent the key, but not the exclusive, site of entry of the virus into the cell; thus, non-ACE2 pathways for virus infection of neural cells cannot be excluded.(37) Whether COVID-19 infects neurons and astroglial cells and enters astrocytes by endocytosis remains to be studied. Overall, considering the computational assays that have been performed in this study, we suggest that the Spike protein dependent pathway is thought to be more important than clathrin-dependent endocytosis for cell entry and BBB crossing. Therefore, the Spike dependent pathway should be taken into account in therapeutic strategies for specific antibodies or vaccine production research.
Regardless of how the virus enters the brain, there are some CNS complications in patients with COVID-19 that should be taken into consideration. The presence of the virus in the brain stem may affect chemosensing neural cells related to respiration as well as respiratory center neurons, thus damaging the lung ventilatory function.(37) It has been shown that SARS-CoV downregulates ACE2 protein expression in a replication dependent manner.(38) Supporting these findings, it has been revealed that SARS-CoV infections and the Spike protein of SARS-CoV reduced ACE2 expression and the injection of SARS-CoV Spike into mice worsened acute lung failure in vivo, which was attenuated by blocking the renin-angiotensin pathway.
Considering the high similarity of SARS-CoV and SAR-CoV-2, and higher binding energy of SAR-CoV-2 than the SARS-CoV S protein to bind ACE2, it has been hypothesized that SARS-CoV-2 also can downregulate ACE2 in different organs including the brain.(40,41) This downregulation might be a part of this complicated story; inhibition of ACE2 activity reduces the sensitivity of the baroreceptor reflex control of the heart rate as well as increases sympathetic tone, eventually resulting in blood pressure elevation and cardiac dysfunction. On the other hand, an increase of inflammatory cytokines during lung injury, hypoxemia, and elevation of sympathetic tone through ACE2 downregulation leads to CNS hyperactivation which might play a crucial role in the etiopathogenesis of neurogenic pulmonary edema (NPE),(42) a life-threatening complication following a neurologic insult,(43) and finally leading to deterioration with the respiratory and cardiovascular complications in these patients
Supporting the idea of brain infection, more recently, in a case report, one patient with no past medical history showed frequent seizures probably due to COVID-19 infection.(44) Several mechanisms for the etiology of seizure have been taken into consideration, including the direct infiltration of brain tissue, production of toxins by the virus, or increase of inflammatory cytokines by the brain.(45) Recently, It has been reported that COVID-19 initiates the inflammatory cascade and, as a result, releases inflammatory cytokines which is called cytokine storm syndrome.(47) Consecutively, these cytokines can drive neuronal hyperexcitability via activation of glutamate receptors and play a role in the development of acute seizures.
In addition, in a case report study, a case of self-limited encephalitic associated with SARS-CoV-2 was presented. The authors suggested that, with the clearance of the virus and the use of mannitol, CSF pressure might gradually decrease and the patient’s consciousness will improve.
In a recent study, neurologic features in severe COVID-19 patients who were admitted to the hospital have been reported. Magnetic resonance imaging (MRI) of the brain was performed in 13 patients in this evaluation. Although these patients did not have focal signs that suggested stroke, they underwent MRI because of unexplained encephalopathic features. Two of the 13 patients who underwent brain MRI showed single acute ischemic strokes. The authors concluded that their data were not enough to recognize which of these features were due to critical illness-related encephalopathy, cytokines, or the effect or withdrawal of medication and which features were directly due to SARS-CoV-2 infection. Postviral anosmia, which is also named olfactory dysfunction,(52,53) and ageusia(54) are other neurologic symptoms that have been reported in patients with COVID-19. More recently, in a cross-sectional study in Iran of 10,069 cases, the coincidence of COVID-19 epidemic and olfactory dysfunction has been reported.(55) In this context, recently, Lechien et al. reported that olfactory and gustatory dysfunctions are prevalent in patients with mild-to-moderate COVID-19 infection.(56) Some mechanisms have been raised to explain this association including (1) injury at the level of the neuroepithelium of olfactory receptor cells in the nasal roof or in the central olfactory processing system,(55) (2) damage of the central olfactory routes and other regions of the brain,(57−59) and (3) inflammation or possible damage to the nasal epithelium cells that are required for normal olfactory function.(60) Therefore, both epithelial damage and CNS involvement have been reported as the possible causes; however, the exact pathophysiology remains yet to be elucidated.
In accordance with the neurotrophic mechanism proposed by Baig et al.,(10) which hypothesizes SAR-CoV-2 brain access via the transcribrial route, as documented for other CNS targeting pathogens, we suppose a possible entry of the virus from the olfactory bulb and, exploiting the blood microcirculation, SAR-CoV-2 may have access to the cerebral circulation and interact with ACE2 receptors expressed on neuronal cells.
Conclusion Considering the neurological manifestations of patients with COVID-19 and in light of the bioinformatics findings of this study indicating more positive charged Spike protein structure and higher binding free energy of the SARS-CoV-2:ACE2 interaction, it is expected that SAR-CoV-2 possesses higher efficiency than SARS-CoV to enter the cells and reach the brain. This neuroinvasive characteristic should be taken into account in basic and clinical research as well as prioritization and individualization of therapeutic approaches.
Reference & Source information: https://pubs.acs.org/
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