Molecular Evolution of SARS-CoV-2 Structural Genes: Evidence of Positive Selection in Spike Glycoprotein

Abstract Background: SARS-CoV-2 has caused a global pandemic since early 2020 and is still a serious public health issue world-wide. Four structural proteins, envelope (E), membrane (M), nucleocapsid (N) and spike (S) glycoprotein, play a key role in controlling the entry into human cells and virion assembly of SARS-CoV-2. The evolution of these genes may determine the infectivity of SARS-CoV-2, but is largely unknown. Results: We analyzed roughly 3090 SARS-CoV-2 isolates from GenBank database. The distribution of four gene alleles is determined: 16 for E, 40 for M, 131 for N and 173 for S genes. Phylogenetic analysis shows that global SARS-CoV-2 isolates can be clustered into three to four major clades based on the protein sequence. Although intragenic recombination event isn’t detected among different alleles, purifying selection has conducted on the evolution of these genes. By analyzing full genomic sequences of these alleles, it reveals that codon 614 of S glycoprotein has subjected to strong positive selection pressure and a consistent D614G mutation is identified. Additionally, another potential positive selection site at codon 5 in the signal sequence of the S protein is also identified with consistent L5F mutation. The allele containing D614G mutation has undergone significant expansion during SARS-CoV-2 transmission, implying a better adaptability of isolates with the mutation. However, L5F allele expansion is relatively restricted. The D614G mutation is located at the subdomain 2 (SD2) of C-terminal portion (CTP) of the S1 subunit. Protein structural modeling shows that the D614G mutation may cause the disruption of a salt bridge between S protein monomers and increase their flexibility, and in turn promote receptor binding domain (RBD) opening, virus attachment and entry into host cells. Located at the signal sequence of S protein as it is, L5F mutation may facilitate the protein folding, assembly, and secretion of the virus. Conclusions: This is the first evidence of positive Darwinian selection in the spike gene of SARS-CoV-2, which contributes to a better understanding of the adaptive mechanism of this virus and help to provide insights for developing novel therapeutic approaches as well as effective vaccines by targeting on mutation sites.

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Molecular Evolution of SARS-CoV-2 Structural Genes: Evidence of Positive Selection in Spike Glycoprotein

10.1101/2020.06.25.170688 ◽

2020 ◽

Author(s):

Xiao-Yong Zhan ◽

Ying Zhang ◽

Xuefu Zhou ◽

Ke Huang ◽

Yichao Qian ◽

...

Keyword(s):

Positive Selection ◽

Fixed Effects ◽

Signal Sequence ◽

Salt Bridge ◽

Purifying Selection ◽

Host Cells ◽

Intragenic Recombination ◽

S Protein ◽

Codon Substitution ◽

Global Pandemic

AbstractSARS-CoV-2 caused a global pandemic in early 2020 and has resulted in more than 8,000,000 infections as well as 430,000 deaths in the world so far. Four structural proteins, envelope (E), membrane (M), nucleocapsid (N) and spike (S) glycoprotein, play a key role in controlling the entry into human cells and virion assembly of SARS-CoV-2. However, how these genes evolve during its human to human transmission is largely unknown. In this study, we screened and analyzed roughly 3090 SARS-CoV-2 isolates from GenBank database. The distribution of the four gene alleles is determined:16 for E, 40 for M, 131 for N and 173 for S genes. Phylogenetic analysis shows that global SARS-CoV-2 isolates can be clustered into three to four major clades based on the protein sequences of these genes. Intragenic recombination event isn’t detected among different alleles. However, purifying selection has conducted on the evolution of these genes. By analyzing full genomic sequences of these alleles using codon-substitution models (M8, M3 and M2a) and likelihood ratio tests (LRTs) of codeML package, it reveals that codon 614 of S glycoprotein has subjected to strong positive selection pressure and a persistent D614G mutation is identified. The definitive positive selection of D614G mutation is further confirmed by internal fixed effects likelihood (IFEL) and Evolutionary Fingerprinting methods implemented in Hyphy package. In addition, another potential positive selection site at codon 5 in the signal sequence of the S protein is also identified. The allele containing D614G mutation has undergone significant expansion during SARS-CoV-2 global pandemic, implying a better adaptability of isolates with the mutation. However, L5F allele expansion is relatively restricted. The D614G mutation is located at the subdomain 2 (SD2) of C-terminal portion (CTP) of the S1 subunit. Protein structural modeling shows that the D614G mutation may cause the disruption of salt bridge among S protein monomers increase their flexibility, and in turn promote receptor binding domain (RBD) opening, virus attachment and entry into host cells. Located at the signal sequence of S protein as it is, L5F mutation may facilitate the protein folding, assembly, and secretion of the virus. This is the first evidence of positive Darwinian selection in the spike gene of SARS-CoV-2, which contributes to a better understanding of the adaptive mechanism of this virus and help to provide insights for developing novel therapeutic approaches as well as effective vaccines by targeting on mutation sites.

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Molecular Evolution of SARS-CoV-2 Structural Genes: Evidence of Positive Selection in the Spike Glycoprotein

10.21203/rs.3.rs-42498/v2 ◽

2020 ◽

Author(s):

Xiao-Yong Zhan ◽

Ying Zhang ◽

Xuefu Zhou ◽

Ke Huang ◽

Yichao Qian ◽

...

Keyword(s):

Molecular Evolution ◽

Positive Selection ◽

Signal Sequence ◽

Phylogenetic Analyses ◽

Host Cells ◽

Public Health Issue ◽

Intragenic Recombination ◽

Structural Genes ◽

S Protein ◽

Spike Gene

Abstract Background: SARS-CoV-2 has caused a global pandemic since early 2020 and remains a serious public health issue worldwide. Four structural genes, envelope (E), membrane (M), nucleocapsid (N) and spike (S), play a key role in controlling entry into human cells and virion assembly of SARS-CoV-2. The evolution of these genes may determine infectivity of SARS-CoV-2, but thus far, little is known about them. Methods: We analyzed 3090 SARS-CoV-2 isolates from the GenBank database to determine the evolutionary patterns of the four structural genes by employing various molecular evolution algorithms. Results: Phylogenetic analyses showed that global SARS-CoV-2 isolates can be clustered into three to four major clades based upon protein sequence. Although intragenic recombination was not detected among different alleles, purifying selection has affected the evolution of these genes. By analyzing full genomic sequences of these alleles, our result revealed that codon 614 of the S glycoprotein has been subjected to a strong positive selection pressure, and a consistent D614G mutation was identified. Additionally, another potentially positive selection site at codon 5 in the signal sequence of the S protein was also identified with a consistent L5F mutation. The allele containing the D614G mutation has undergone significant expansion during SARS-CoV-2 transmission, implying a better adaptability of isolates with the mutation. Nevertheless, L5F allele expansion was found to be relatively restricted. The D614G mutation is located at subdomain 2 (SD2) of the C-terminal portion (CTP) of the S1 subunit. Protein structural modeling showed that the D614G mutation may cause the disruption of a salt bridge between S protein monomers and increase their flexibility, consequently promoting receptor binding domain (RBD) opening, virus attachment, and ultimately entry into host cells. Located at the signal sequence of S protein, the L5F mutation may facilitate protein folding, assembly, and secretion of the virus. Conclusions: This is the first reported evidence of positive Darwinian selection in the spike gene of SARS-CoV-2. This finding contributes to a broader understanding of the adaptive mechanisms of this virus, and provide insight for the development of novel therapeutic approaches, as well as the creation of effective vaccines, through targeting mutation sites.

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D936Y and Other Mutations in the Fusion Core of the SARS-CoV-2 Spike Protein Heptad Repeat 1: Frequency, Geographical Distribution, and Structural Effect

Molecules ◽

10.3390/molecules26092622 ◽

2021 ◽

Vol 26 (9) ◽

pp. 2622

Author(s):

Romina Oliva ◽

Abdul Rajjak Shaikh ◽

Andrea Petta ◽

Anna Vangone ◽

Luigi Cavallo

Keyword(s):

Three Dimensional ◽

Salt Bridge ◽

Structural Effect ◽

Host Cells ◽

Heptad Repeat ◽

Genomic Sequences ◽

Strong Interactions ◽

Virus Infectivity ◽

S Protein ◽

Global Threat

The crown of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is constituted by its spike (S) glycoprotein. S protein mediates the SARS-CoV-2 entry into the host cells. The “fusion core” of the heptad repeat 1 (HR1) on S plays a crucial role in the virus infectivity, as it is part of a key membrane fusion architecture. While SARS-CoV-2 was becoming a global threat, scientists have been accumulating data on the virus at an impressive pace, both in terms of genomic sequences and of three-dimensional structures. On 15 February 2021, from the SARS-CoV-2 genomic sequences in the GISAID resource, we collected 415,673 complete S protein sequences and identified all the mutations occurring in the HR1 fusion core. This is a 21-residue segment, which, in the post-fusion conformation of the protein, gives many strong interactions with the heptad repeat 2, bringing viral and cellular membranes in proximity for fusion. We investigated the frequency and structural effect of novel mutations accumulated over time in such a crucial region for the virus infectivity. Three mutations were quite frequent, occurring in over 0.1% of the total sequences. These were S929T, D936Y, and S949F, all in the N-terminal half of the HR1 fusion core segment and particularly spread in Europe and USA. The most frequent of them, D936Y, was present in 17% of sequences from Finland and 12% of sequences from Sweden. In the post-fusion conformation of the unmutated S protein, D936 is involved in an inter-monomer salt bridge with R1185. We investigated the effect of the D936Y mutation on the pre-fusion and post-fusion state of the protein by using molecular dynamics, showing how it especially affects the latter one.

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D936Y and Other Mutations in the Fusion Core of the SARS-Cov-2 Spike Protein Heptad Repeat 1 Undermine the Post-Fusion Assembly

10.1101/2020.06.08.140152 ◽

2020 ◽

Cited By ~ 2

Author(s):

Luigi Cavallo ◽

Romina Oliva

Keyword(s):

Three Dimensional ◽

Point Mutations ◽

Protein Sequences ◽

Salt Bridge ◽

Host Cells ◽

Heptad Repeat ◽

Genomic Sequences ◽

Protein Conformations ◽

S Protein ◽

Global Threat

AbstractThe iconic “red crown” of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is made of its spike (S) glycoprotein. The S protein is the Trojan horse of coronaviruses, mediating their entry into the host cells. While SARS-CoV-2 was becoming a global threat, scientists have been accumulating data on the virus at an impressive pace, both in terms of genomic sequences and of three-dimensional structures. On April 21st, the GISAID resource had collected 10,823 SARS-CoV-2 genomic sequences. We extracted from them all the complete S protein sequences and identified point mutations thereof. Six mutations were located on a 14-residue segment (929-943) in the “fusion core” of the heptad repeat 1 (HR1). Our modeling in the pre- and post-fusion S protein conformations revealed, for three of them, the loss of interactions stabilizing the post-fusion assembly. On May 29th, the SARS-CoV-2 genomic sequences in GISAID were 34,805. An analysis of the occurrences of the HR1 mutations in this updated dataset revealed a significant increase for the S929I and S939F mutations and a dramatic increase for the D936Y mutation, which was particularly widespread in Sweden and Wales/England. We notice that this is also the mutation causing the loss of a strong inter-monomer interaction, the D936-R1185 salt bridge, thus clearly weakening the post-fusion assembly.

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On the origin and evolution of SARS-CoV-2

Experimental & Molecular Medicine ◽

10.1038/s12276-021-00604-z ◽

2021 ◽

Author(s):

Devika Singh ◽

Soojin V. Yi

Keyword(s):

Mammalian Cells ◽

Genetic Recombination ◽

Disease Outbreaks ◽

Purifying Selection ◽

Host Cells ◽

Human Populations ◽

S Protein ◽

Origin And Evolution ◽

Multiple Species ◽

The Impact

AbstractThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the ongoing global outbreak of a coronavirus disease (herein referred to as COVID-19). Other viruses in the same phylogenetic group have been responsible for previous regional outbreaks, including SARS and MERS. SARS-CoV-2 has a zoonotic origin, similar to the causative viruses of these previous outbreaks. The repetitive introduction of animal viruses into human populations resulting in disease outbreaks suggests that similar future epidemics are inevitable. Therefore, understanding the molecular origin and ongoing evolution of SARS-CoV-2 will provide critical insights for preparing for and preventing future outbreaks. A key feature of SARS-CoV-2 is its propensity for genetic recombination across host species boundaries. Consequently, the genome of SARS-CoV-2 harbors signatures of multiple recombination events, likely encompassing multiple species and broad geographic regions. Other regions of the SARS-CoV-2 genome show the impact of purifying selection. The spike (S) protein of SARS-CoV-2, which enables the virus to enter host cells, exhibits signatures of both purifying selection and ancestral recombination events, leading to an effective S protein capable of infecting human and many other mammalian cells. The global spread and explosive growth of the SARS-CoV-2 population (within human hosts) has contributed additional mutational variability into this genome, increasing opportunities for future recombination.

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Modified FGF4 Signal Peptide Inhibits Entry of Herpes Simplex Virus Type 1

Journal of Virology ◽

10.1128/jvi.75.6.2634-2645.2001 ◽

2001 ◽

Vol 75 (6) ◽

pp. 2634-2645 ◽

Cited By ~ 30

Author(s):

Hermann Bultmann ◽

James S. Busse ◽

Curtis R. Brandt

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Virus Type ◽

Herpes Simplex Virus Type ◽

Signal Sequence ◽

Host Cells ◽

Virus Attachment ◽

Simplex Virus ◽

Hsv 1

ABSTRACT Entry of herpes simplex virus type 1 (HSV-1) into host cells occurs through fusion of the viral envelope with the plasma membrane and involves complex and poorly understood interactions between several viral and cellular proteins. One strategy for dissecting the function of this fusion machine is through the use of specific inhibitors. We identified a peptide with antiviral activity that blocks HSV-1 infection at the entry stage and during cell-to-cell spreading. This peptide (called EB for “entry blocker”) consists of the FGF4 signal sequence with an RRKK tetramer at the amino terminus to improve solubility. The activity of EB depends exclusively but not canonically on the signal sequence. Inhibition of virus entry (hrR3) and plaque formation (KOS) strongly depend on virus concentrations and serum addition, with 50% inhibitory concentrations typically ranging from 1 to 10 μM. Blocking preadsorbed virus requires higher EB concentrations. Cytotoxic effects (trypan blue exclusion) are first noted at 50 μM EB in serum-free medium and at ≥200 μM in the presence of serum. EB does not affect gC-dependent mechanisms of virus attachment and does not block virus attachment at 4°C. Instead, EB directly interacts with virions and inactivates them irreversibly without, however, disrupting their physical integrity as judged by electron microscopy. At subvirucidal concentrations, EB changes the adhesive properties of virions, causing aggregation at high virus concentrations. This peptide may be a useful tool for studying viral entry mechanisms.

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Amino Acids 1055 to 1192 in the S2 Region of Severe Acute Respiratory Syndrome Coronavirus S Protein Induce Neutralizing Antibodies: Implications for the Development of Vaccines and Antiviral Agents

Journal of Virology ◽

10.1128/jvi.79.6.3289-3296.2005 ◽

2005 ◽

Vol 79 (6) ◽

pp. 3289-3296 ◽

Cited By ~ 59

Author(s):

Choong-Tat Keng ◽

Aihua Zhang ◽

Shuo Shen ◽

Kuo-Ming Lip ◽

Burtram C. Fielding ◽

...

Keyword(s):

Amino Acids ◽

Severe Acute Respiratory Syndrome ◽

Viral Entry ◽

Neutralizing Antibodies ◽

Antiviral Agents ◽

Host Cells ◽

Heptad Repeat ◽

Antigenic Determinants ◽

Amino Acid Residues ◽

S Protein

ABSTRACT The spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) interacts with cellular receptors to mediate membrane fusion, allowing viral entry into host cells; hence it is recognized as the primary target of neutralizing antibodies, and therefore knowledge of antigenic determinants that can elicit neutralizing antibodies could be beneficial for the development of a protective vaccine. Here, we expressed five different fragments of S, covering the entire ectodomain (amino acids 48 to 1192), as glutathione S-transferase fusion proteins in Escherichia coli and used the purified proteins to raise antibodies in rabbits. By Western blot analysis and immunoprecipitation experiments, we showed that all the antibodies are specific and highly sensitive to both the native and denatured forms of the full-length S protein expressed in virus-infected cells and transfected cells, respectively. Indirect immunofluorescence performed on fixed but unpermeabilized cells showed that these antibodies can recognize the mature form of S on the cell surface. All the antibodies were also able to detect the maturation of the 200-kDa form of S to the 210-kDa form by pulse-chase experiments. When the antibodies were tested for their ability to inhibit SARS-CoV propagation in Vero E6 culture, it was found that the anti-SΔ10 antibody, which was targeted to amino acid residues 1029 to 1192 of S, which include heptad repeat 2, has strong neutralizing activities, suggesting that this region of S carries neutralizing epitopes and is very important for virus entry into cells.

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New Perspectives for SARS-CoV-2 Rapid Detection

Coronaviruses ◽

10.2174/2666796702666210308114737 ◽

2021 ◽

Vol 02 ◽

Author(s):

Latifa Khattabi ◽

Mustapha Mounir Bouhenna ◽

Feriel Sellam

Keyword(s):

Rapid Detection ◽

High Sensitivity ◽

The Other ◽

Host Cells ◽

Rt Pcr ◽

Human Immune System ◽

Virus Attachment ◽

Testing Procedures ◽

Diagnostic Kits ◽

Human Immune

: The present paper elucidates the conceivable application of two key molecules in SARS-CoV-2 detection of suspected infected persons. These molecules were selected from the basis of ACE-2 and S protein strong interaction that allows virus attachment to its host cells, on the other hand specific immunocompetant effectors generated by human immune system during the infection. Several testing procedures are already used to diagnose SARS-CoV-2 infection, particularly RT-PCR technique. ELISA and LFIA are possible assays for the employment of shACE-2/ hAc-anti-S (the molecules of interest) as the main agents of the test and confer a dual principal functions (capture and detection). The future diagnostic kits involving shACE-2 and hAc-anti-S will have the particularity of high sensitivity and rapid detection in addition to its advantage of relatively easy conception. It could be largely considered as a technical advanced kits in regards to the current SARS-CoV-2 diagnostic immunoassays.

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SARS-CoV-2 S protein:ACE2 interaction reveals novel allosteric targets

eLife ◽

10.7554/elife.63646 ◽

2021 ◽

Vol 10 ◽

Cited By ~ 1

Author(s):

Palur V Raghuvamsi ◽

Nikhil Kumar Tulsian ◽

Firdaus Samsudin ◽

Xinlei Qian ◽

Kiren Purushotorman ◽

...

Keyword(s):

Cleavage Site ◽

Proteolytic Processing ◽

Host Cells ◽

Heptad Repeat ◽

S Protein ◽

Exchange Mass Spectrometry ◽

Central Helix ◽

Interaction Interface ◽

Therapeutic Development ◽

Dynamics Simulations

The Spike (S) protein is the main handle for SARS-CoV-2 to enter host cells via surface ACE2 receptors. How ACE2 binding activates proteolysis of S protein is unknown. Here, using amide hydrogen-deuterium exchange mass spectrometry and molecular dynamics simulations, we have mapped the S:ACE2 interaction interface and uncovered long-range allosteric propagation of ACE2 binding to sites necessary for host-mediated proteolysis of S protein, critical for viral host entry. Unexpectedly, ACE2 binding enhances dynamics at a distal S1/S2 cleavage site and flanking protease docking site ~27 Å away while dampening dynamics of the stalk hinge (central helix and heptad repeat) regions ~130 Å away. This highlights that the stalk and proteolysis sites of the S protein are dynamic hotspots in the pre-fusion state. Our findings provide a dynamics map of the S:ACE2 interface in solution and also offer mechanistic insights into how ACE2 binding is allosterically coupled to distal proteolytic processing sites and viral-host membrane fusion. Our findings highlight protease docking sites flanking the S1/S2 cleavage site, fusion peptide and heptad repeat 1 (HR1) as alternate allosteric hotspot targets for potential therapeutic development.

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Current Potential Therapeutic Approaches against SARS-CoV-2: A Review

Biomedicines ◽

10.3390/biomedicines9111620 ◽

2021 ◽

Vol 9 (11) ◽

pp. 1620

Author(s):

Dharmendra Kumar Yadav ◽

Desh Deepak Singh ◽

Ihn Han ◽

Yogesh Kumar ◽

Eun-Ha Choi

Keyword(s):

Wide Spectrum ◽

Host Cells ◽

Hiv Protease ◽

Hiv Protease Inhibitors ◽

Therapeutic Approaches ◽

S Protein ◽

Angiotensin Converting Enzyme 2 ◽

Nucleotide Analogue ◽

Infection Treatment ◽

Current Potential

The ongoing SARS-CoV-2 pandemic is a serious threat to public health worldwide and, to date, no effective treatment is available. Thus, we herein review the pharmaceutical approaches to SARS-CoV-2 infection treatment. Numerous candidate medicines that can prevent SARS-CoV-2 infection and replication have been proposed. These medicines include inhibitors of serine protease TMPRSS2 and angiotensin converting enzyme 2 (ACE2). The S protein of SARS-CoV-2 binds to the receptor in host cells. ACE2 inhibitors block TMPRSS2 and S protein priming, thus preventing SARS-CoV-2 entry to host cells. Moreover, antiviral medicines (including the nucleotide analogue remdesivir, the HIV protease inhibitors lopinavir and ritonavir, and wide-spectrum antiviral antibiotics arbidol and favipiravir) have been shown to reduce the dissemination of SARS-CoV-2 as well as morbidity and mortality associated with COVID-19.

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