Lupine Publishers | Alignment of SARS-Cov-2 Spike Protein Compared with Ebola, and Herpes Simplex Viruses in Human Murine and Bats

 Lupine Publishers | LOJ Pharmacology & Clinical Research

Abstract

Our analytical approach consisted in sequence alignment analysis of spike protein in different viruses, followed by construction of phylogenetic tree. Additionally, we investigated some commotional parameters on the protein sequence determining chemical composition as well as estimated PI .Our observation revealed significant difference in S protein between the 3 tested viruses in different species These differences may have significant implications on pathogenesis and entry to host cell.

Introduction

Viruses are inert outside the host cell which are unable to generate energy. As obligate intracellular parasites, during replication, they are fully reliant on the complex biochemical machinery of eukaryotic or prokaryotic cells. The central purpose of a virus is to transport its genome into the host cell to allow its expression by the host cell [1]. Binding of a single surface glycoprotein of virus to its host receptor promotes pH-dependent conformational variations once within the endosome acidic environment, thereby transporting the viral bilayer in closeness with the host cell membrane to promote fusion [2]. The structural physiognomies of virus coats (capsids) are extremely appropriate to virus propagation. The coat must enclose and protect the nucleic acid, flexible against interference be talented of broaching the outer wall of a target cell and provide a confident pathway for attending nucleic acid into the target cell. Hollow spikes on the capsid fulfill the latter two roles, from which it has been deduced that they must have unusually high strength and stiffness in axial compression [3]. The spike protein (S protein) is a type I transmembrane protein, a sequence of amino acids ranging from 1,160 for avian infectious bronchitis virus (IBV) and up to 1,400 amino acids for feline coronavirus [4]. Spike proteins assemble to create the distinctive “corona” or crown-like look in trimers on the surface of the virion. The ectodomain of all CoV spike proteins portions the same organization in two domains: a receptor-binding N-terminal domain called S1 and a fusionresponsible C-terminal S2 domain The variable spike proteins (S proteins) reflect CoV diversity, which have developed into forms that vary in their receptor interactions and their reaction to different environmental triggers of virus-cell membrane fusion [5]. A notable peculiarity between the spike proteins of diverse coronaviruses is whether it is cleaved or not during assembly and exocytosis of virions [6]. The entry Herpesvirus and membrane fusion to the host cell equire three virion glycoproteins, gB and a gH/gL heterodimer, that function as the “core fusion machinery” [7]. The viral envelope of Ebola virus contains spikes consisting of the glycoprotein (GP) trimer. These viral spike glycoprotein docks viral particles to the cell surface, endosomal entry, and membrane fusion [8]. Acute respiratory syndrome coronavirus (SARS-CoV) is a zoonotic pathogens that traversed the species barriers to infect humans. These coronaviruses hold a surface-located spike (S) protein that recruits infection by mediating receptor-recognition and membrane fusion [9]. Bioinformatics plays an important role in all aspects of protein analysis, including sequence analysis, and evolution analysis. In sequence analysis, several bioinformatics techniques can be used to provide the sequence comparisons. In evolution analysis, we use the technique like phylogenetic trees to find homologous proteins and identified the most related taxa. With bioinformatics techniques and databases, function, structure, and evolutionary history of proteins can be easily identified [10]. Several bioinformatics methods can be used in sequence analysis to provide sequence comparisons.

Materials and Methods

Sequences, alignment, and construction of phylogenetic tree

Amino acids sequences for the spike protein of Covid-19, Ebola, and herpes simplex viruses from bats, murine as well as Homo sapiens were taken from the National Center for Biotechnology Information database. The accession numbers of the corresponding database entries and species names are listed in Table 1 and Figure 1. Sequences were aligned with by CLUSTALW [11]. Selection of conserved blocks was performed using GBlocks (version 0.91 to eliminate divergent regions and poorly aligned positions using standard settings according to Castresana [12]. The Akaike information criterion (AIC) were performed, using a maximum-likelihood (ML) starting tree to estimates the quality of each model, relative to each of the other models (Table 2). The most suitable model was WAG+G+F [13]. Then phylogenetic tree was constructed with neighbor-joining and maximum likelihood algorithms using MEGA version 5 [14]. The stability of the topology of the phylogenetic tree was assessed using the bootstrap method with 100 repetitions [15].

 

 

Computation of the theoretical pI (isoelectric point) of protein sequences

Estimation of the isoelectric point (pI) based on the amino acid sequence was determined using isoelectric Point Calculator (IPC), a web service and a standalone program for the accurate estimation of protein and peptide pI using different sets of dissociation constant (pKa) values [16]. Models with the lowest BIC scores (Bayesian Information Criterion) are considered to describe the substitution pattern the best. For each model, AICc value (Akaike Information Criterion, corrected), Maximum Likelihood value (lnL), and the number of parameters (including branch lengths) are also presented [1]. Non-uniformity of evolutionary rates among sites may be modeled by using a discrete Gamma distribution (+G) with 5 rate categories and by assuming that a certain fraction of sites is evolutionarily invariable (+I). Abbreviations: TR: General Time Reversible; JTT: Jones-Taylor-Thornton; rtREV: General Reverse Transcriptase; cpREV: General Reversible Chloroplast; mtREV24: General Reversible Mitochondrial.

Results

In order to unravel the phylogenetic relationship of Spike protein between the different taxa, a phylogenetic consensus tree was constructed using the Bayesian Inference (BI) and Maximum Likelihood (ML) methods (Figure 1). The present results revealed that the identity between different taxa was nonsignificant, However alignment of human Coronavirus (Covid-19) and Bat Coronavirus revealed identity equal to 57.98% followed by Murine Coronavirus which displayed 27.61% when compared by human Coronavirus. The chemical composition of the tested protein is illustrated in Table 3. Figures 2 & 3 show the correlation plots between the theoretical isoelectric points for spike protein of different corona virus in different species. The current results displayed that estimated pI of spike protein sequence in three investigated viruses in different species ranges from 5.59 to 8.08. With highest value for spike protein in herpesvirus of bat. The Estimated charge over pH range of the investigated were listed in Table 4. The current results revealed that the behavior of s protein in different species exhibited different estimated charge at different pH ranges. Some S protein reveled low negative charge as pH increases such as Ebola Virus of bat while S protein of Corona virus in murine showed high negative charge as pH elevated.

 

Discussion

One aspect that may provide some insight into the interactions of the S protein is the electrostatic potential it generates. the affinity constant for the receptor-binding domain (RBD) of viron protein potentially contributing to its transmission efficiency [17]. The S protein remains predominantly in the closed conformation to mask its receptor-binding domains (RBDs), thereby impeding their binding. To bind with ACE2, the S protein transforms into its open conformation, revealing its binding interface [18]. The present study relies on a comparative investigation, regarding the identity of spike protein of 3 viruses (Covid-19, Ebola, and herpes simplex) to identify the most related taxa. Our investigation revealed high similarities of Spike protein of coronaviruses in human and bats [19]. The computed amino acid composition of spike protein. Several residues showed a significant difference between the compositions in spike proteins in the three investigated virus in different species. This result reveals the importance of specific residues in these classes of proteins. The polar uncharged residues, especially Serine, Asparagine, and Glycine, have higher occurrence in spike protein of murine, which are important for the folding, stability, and function of such class of proteins [20]. Our analysis revealed that the Coronavirus spike protein of murine may be more efficient in discovering suitable vaccine for Coronavirus. The S protein amino acids variations among different coronaviruses such as (SARS, herpes and ebola). The SARS-CoV-2 virus shares 57.98% of its genome with the other bat coronaviruses. The sequence identity in the S protein bat coronavirus appears to be the closest relative of SARS-CoV-2. Our results in accordance with Rothan [21]. The isoelectric point (pI) is the pH value at which a molecule’s net charge is zero [22]. Information about the isoelectric point is important because the solubility and electrical repulsion are lowest at the pI. Hence, the tendency to aggregation and precipitation is highest. In the case of viruses, the value thus provides information about the viral surface charge in a specific environment [23]. In polar media, such as water, viruses possess a pH-dependent surface charge [23]. This electrostatic charge governs the movement of the soft particle in an electrical field and thus manages its colloidal behavior, which plays a major role in the processes of virus entry. The pH value at which the net surface charge changes its sign is called the isoelectric point and is a characteristic parameter of the virion in equilibrium with its atmosphere in water chemistry [24]. For some viruses the attachment influences that encourage binding to accommodating cells are extremely definite, but the arrangement of actions that activates viral entry is only now establishment to be understood. The charge of attachment protein may play an important role in attachment and entry of virus [25]. The current results revealed that the pH affect the net charge of S protein of different taxa with different behavior. All the investigated taxa exhibit increases in negative charge as the pH increased except for the Ebola virus form Bats which showed unstable behavior regarding the S protein charge.

Conclusion

In our study, we have investigated the variation of pHdependent changes in charges of a protein. The current results revealed that the pH affect the net charge of S protein of different taxa with different behavior.

 

https://lupinepublishers.com/pharmacology-clinical-research-journal/fulltext/alignment-of-sars-cov-2-spike-protein-compared-with-ebola-and-herpes-simplex-viruses-in-human-murine-and-bats.ID.000140.php

https://lupinepublishers.com/pharmacology-clinical-research-journal/pdf/LOJPCR.MS.ID.000140.pdf

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Lupine Publishers | A De-Novo SCN2A Mutation Identified in a Chinese Patient With Epilepsy: A Case Report

 Lupine Publishers | LOJ Pharmacology & Clinical Research

Abstract

Objective: To investigate the genetic causes of epilepsy in a 5-year-old Chinese female patient.

Methods: Clinical diagnosis and next-generation sequencing.

Results: The patient carries a de-novo heterozygous missense mutation (c.3686 A>T p.Asp1229Val) in the SCN2A gene. This mutation was evaluated as a pathogenic mutation based on the standards and guidelines of ACMG (American College of Medical Genetics and Genomics) and clinical research publications.

Conclusion: The de-novo heterozygous mutation (c.3686 A>T p.Asp1229Val) in the SCN2A gene is the genetic cause of the epilepsy for the patient. So far, this mutation of SCN2A gene is the first reported in the worldwide overall populations.

Keywords: Chinese; Epilepsy; SCN2A gene

Abbreviations: Whole Exome Sequencing (WES); Sanger sequencing; American College of Medical Genetics and Genomics (ACMG)

Introduction

The SCN2A gene encodes the voltage-gated sodium channel Na(v)1.2, which plays an important role in the initiation and conduction of action potentials. SCN2A is expressed in axon initiation segments and at nodes of Ranvier in myelinated nerve fibers during early development, and is later expressed in unmyelinated axons [1]. Mutations in the SCN2A gene, cause a variety of neuropsychiatric syndromes with different severity ranging from self-limiting epilepsies with early onset to developmental and epileptic encephalopathy with early or late onset and intellectual disability (ID), as well as ID or autism without seizures [2-4]. So far, more than 700 mutations in SCN2A were reported to be associated with epilepy [5]. The aim of the present study was to detect and report genetic causes of a 5-year-old Chinese female patient with epilepsy. The patient was found to have a de-nove mutation (c.3686 A>T p.Asp1229Val) in the SCN2A gene that has not reported in the previous studies.

Materials and Methods

Clinical diagnosis

A 5-year-old female Chinese patient who was born with no family history of seizures or other types of neurological diseases, has experienced epileptic spasms since she was 1.5 years old. The patient had fallen down while she was walking, and her head contacted the ground, but she did not loss consciousness at the time. On the same day, the patient began to nod her head continuously, with each occurance lasting approximately several seconds in length. Since then, her head nods occurred in clusters daily, and up to 30 to 40 times a day at the worst cases. The patient was carried to the hospital and was diagnosed with infantile spasms. Her symptoms of head nodding was relieved after treatment with intravenous immunoglobulin (pH4), Sodium valproate oral solution and Topiramate. The patient took Sodium valproate oral solution and Topiramate after her discharge from the hospital. However, she still had the head nods which occurred at random intervals. After she turned 2-years-old, the head nods did not occur again, but she began to have epilepsy symptoms such as loss of consciousness, twitching of the limbs, spitting out white foam et al. The epilepsy happened approximately once a month and lasted approximately 1 to 2 minutes in length at each occurence. The patient visited the hospital again and the Clonazepam was added to her medications [6]. Her symptoms of epilepsy were being controlled well and did not happen within a 8 month period. However, in April of 2019, when the patient was 5 years old, her head nods occurred again without any obvious causes. This time, the head nods happened in the early morning, and 4～5 times everyday. The patient was admitted to our hospital (the Second Hospital of Shanxi Medical University). The following tests were performed for the patient: physical examinations, brain MRI, and electroencephalogram.

Molecular test

In order to study the cause of the disease, whole exome sequencing was performed for the patient. Furthermore, Sanger sequencing was used to verify the variant for the patient and her parents. Sequencing data was analyzed by using numerous bioinformatics’ softwares, the pathogenicity of the mutation was evaluated based on the standards and guidelines of ACMG (American College of Medical Genetics and Genomics), Clinvar database, OMIM (Online Mendelian Inheritance in Man), HGMD (Human Gene Mutation Database), and clinical research papers published in scientific journals.

Results and Analysis

Clinical data analysis

The patient was admitted to our hospital in April of 2019. The patient appeared normal under physical examinations. She has a clear mind, but only can pronunce simple words. She had poor orientation, cognition, memory, calculation, and attention span. Brain MRI showed that the symmetrical brain hemispheres on both sides, and the structures of gray and white matter were normal. No other abnormalities were found in the brain parenchyma (Figure 1). The electroencephalogram of the patient showed that the slowwave activity were in the awake period (Figure 2A). Total epileptic discharge during sleep period (Figures 2B & 2C). The patient was given intravenous human immunoglobulin and Dexamethasone for immunotherapy. The medications she has had were also adjusted to Levetiracetam tablets, Clonazepam, Topiramate, and Sodium valproate oral liquid. No recurrence of the head nods after 3 month of the treatment. Figure 2D showed that no obvious abnormal discharge was found in the patient’s return visit on Oct. 2020.

 

 

Molecular biological data analysis

In order to identify the causes of the patient’s seizures, we conducted Whole Exome Sequencing (WES) based on Nextgeneration sequencing for the patient. a heterozygous missense mutation in SCN2A gene (c.3686 A>T p.Asp1229Val) reference transcript, NM_001040143) was detected in the patient. This variant was further confirmed by Sanger Sequencing, and not detected in either of the healthy parents, which indicated that this variant was de novo (Figure 3A). No other pathogenic variants were detected in more than approximately 800 genes that were defined by the OMIM database as related with epilepsy syndromes for this patient (Figures 3B & 3C). The mutation c.3686 A>T (p.Asp1229Val) either not being recorded in any clincial disease-related database (Clinvar and HGMD), nor in Human genome databases (1000 Genome and Genome mutation frequency database). The function prediction databases (SIFT and polyphen, etc.) predicted this mutation to be damaging. This variant was evaluated as a pathogenic mutation based on the standards and guidelines of ACMG.

 

Discussion

Pathogenic variants in SCN2A are reported in a spectrum of neurodevelopmental disorders including developmental and epileptic encephalopathies, benign familial neonatal-infantile seizures, episodic ataxia, and autism spectrum disorder and intellectual disability with and without seizures [1]. More than 1000 mutaions of SCN1A gene were reported in the Clinvar database, including deletion, duplication, indel, insertion and single nucleotide types. Around 70% of those variations was evaluated as responsible for the occurences of Benign familial infantile seizures or early infantile epileptic encephalopathy 11. The single nucleotide mutations were the most frequently reported mutations and were 84% in the total mutation types. However, only about 8.5% of the single nucleotide mutations were due to de-nove mutations in the SCN2A gene. Comparing with the 65% of the de-novo rate in the SCN1A gene, the rate of de-novo in the SCN2A gene is significantly lower [6]. We evaluated WES data from 332 Chinese patients with epilepsy and identified the one de-nove missense mutation in the SCN2A in this patient and the mutation was evaluated as the cause of the disease. Generally, the de-novo mutations of SCN2A gene often lead to severe phenotype with developmental delay [3]. The patient in our case reported here is only 5 years old, but she had epilepsy syndromes for more than three years and had more severe symptoms such as poor orientation, cognition, memory, calculation, and attention et al.
The mutation of the SCN2A gene we detected (c.3686 A>T p.Asp1229Val) has not been recorded in the Clinvar database as of today [6]. Our report provided further evidence for the cause of epilepsy from a genetic level. We predict the result will be immediately useful for the clinical interpretation of SCN2A variants, and also provides deeper insights for SCN2A mutations associated with the broad clinical spectrum of seizures.

Declarations

The experimental protocol was established, according to the ethical guidelines of the Helsinki Declaration and was approved by the Human Ethics Committee of the Second Hospital of Shanxi Medical University. Written informed consent was obtained from individual or guardian participants. This study was supported in the part by grants from the Epilepsy Research Fund (UCB No. 2014007) from China antiepileptic Association.

 https://lupinepublishers.com/pharmacology-clinical-research-journal/pdf/LOJPCR.MS.ID.000139.pdf

https://lupinepublishers.com/pharmacology-clinical-research-journal/fulltext/a-de-novo-scn2a-mutation-identified-in-a-chinese-patient-with-epilepsy-a-case-report.ID.000139.php

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