European Review for Medical and Pharmacological Sciences 2021; 25: 5843-5852

SARS-CoV-2: phylogenetic status, mutations and

therapeutic research based on spike protein
Y. ZHU

College of Life Sciences, Northwest A&F University, Yangling, Shaanxi, China

Abstract. – OBJECTIVE: The pandemic of the The viral pathogen responsible SARS-CoV-2,
coronavirus disease 2019 (COVID-19) caused by is an emergent, zoonotic and highly transmis-
severe acute respiratory syndrome coronavi- sible pathogen first identified in China in late
rus 2 (SARS-CoV-2), a highly transmissible and
pathogenic coronavirus, has been represent-
20193,4. According to sequence analysis, SARS-
ing enormous threats to the world in almost all CoV-2 belongs to Betacoronavirus genera and is
aspects. S protein recognizing and binding to thought to inherit from a bat coronavirus (BatCoV
angiotensin-converting enzyme-2 (ACE2) re- RaTG13), with 96.2% identity in the whole ge-
ceptor is the key step of viral infection. This re- nome sequence5. Its S protein specifically recog-
view summarized the structure of S protein, and nizes and binds to ACE2 receptor, leading to mild
the difference among members of the corona- respiratory failure. Phylogenetic analysis based
viridae, especially the different sites between
SARS-CoV-2 and SARS-CoV S protein sequenc- on S protein sequence divided 18 coronavirus-
es. We reconstructed the phylogenetic tree of 18 es into three Clade (Clade I, II and III). SARS-
coronaviruses based on S protein, and detect- CoV, SARS-CoV-2 and RaTG13 make Clade II;
ed the conserved motif. We had a further discus- RaTG13 is still the most identical to SARS-CoV-2
sion on various promising antiviral compounds, in phylogeny. The mutation rate of the virus is so
drugs or approaches for treatment. Considering fast that various kinds of virus mutants spread all
that various virus mutants are rampant around
the world, we introduced some SARS-CoV-2 mu-
over the world. How to develop effective treat-
tants, which are more contagious and spread ment and broad-spectrum therapy is still an ur-
fast. It indicates the limitations of wide spec- gent problem. Despite a flood of SARS-CoV-2
trum therapeutic research. We wish the informa- researches involving in pathology, treatment and
tion provided by this review can be helpful to the prevention improve every day, current knowledge
global battle against SARS-CoV-2 infection. of this novel coronavirus is just the tip of the ice-
berg. Tackling this epidemic is a long-term job
Key Words: and requires efforts of international cooperation
SARS-CoV-2, COVID-19, Variants, Transmission, Vir- by scientists, authorities and the public.
ulence, Neutralization, Antibodies, Vaccines.
S Protein – A Key Structure of Coronavirus
Coronaviridae is composed of four genera (Al-
Introduction pha-, Beta-, Delta-, Gamma-coronaviruses), con-
taining a diverse group of viruses infecting mam-
Since the outbreak of the coronavirus dis- mals and birds that individually cause a variety of
ease 2019 (COVID-19) pandemic, the number diseases, including pneumonia, enteritis, hepati-
of deaths worldwide has continued to rise. By tis, encephalomyelitis, Tracheobronchitis, nephri-
the end of May 2021, the cumulative number of tis and various other diseases6. Alphacoronavirus
confirmed cases has exceeded 170 million, mak- and Betacoronavirus can infect mammals, in-
ing emerging coronaviruses a new public health cluding humans, pigs, cats, dogs and cattle, while
concern in the twenty-first century1. At the end of Gammacoronavirus and Deltacoronavirus usually
April, the virus was rampant in India, and the num- infect birds, although some can infect mammals1.
ber of newly diagnosed cases in a single day even SARS-CoV-2 is clustered into Betacoronavirus
exceeded 360,0002. The viruses spread rapidly all due to the phylogenetic proximity to SARS-CoV
over the world by mutation. Indian mutants have and Middle East Respiratory Syndrome Corona-
spread more than 40 countries, and new mutants virus (MERS-CoV)3,4 and it is the seventh coro-
have been continuously found in other countries2. navirus found to infect human, causing atypical

Corresponding Author: Ying Zhu, MD; e-mail: [email protected] 5843

 Y. Zhu

pneumonia that can lead to death; SARS-CoV, of host cells, then S2 protein fuses the membrane
MERS-CoV are also severe disease-related virus of viral and host cells, allowing virus entry. There
and belong to Betacoronavirus, while Alphacoro- are two domains in S1 subunit determine the host
navirus 229E (HCoV-229E), NL63 (HCoV- receptor recognition range, which are N-terminal
NL63), Betacoronavirus HKU1 (HCoV-HKU1) domain of S1 (S1-NTD) and C-terminal domain
and OC43 (HCoV-OC43) are much milder. Phy- of S1 (S1-CTD). These S1 domains can bind to
logenetic analysis of complete genome sequences receptors, usually sugars or protein receptors, and
showed that SARS-CoV-2 shares 79% genome act as receptor binding domains (RBDs).
sequence identity with SARS-CoV, and only 50% X-ray diffraction analyzed the crystal structure
with MERS-CoV7, but interestingly, 96.2% with a of human ACE2 protein complexed with SARS-
bat coronavirus (BatCoV) named RaTG135. CoV-2 S protein RBD. The structure confirmed
Betacoronaviruses shares a similar genome or- that most of the residues that related to recptor
ganisation with six open reading frames (ORFs): binding in SARS-CoV-2 are highly conserved or
replicase (ORF1a/ORF1b), spike(S), envelope(E), similar in characteristics to those in SARS-CoV
membrane(M) and nucleocapsid (N)8. Among RBD13. Among these amino acid residues, the six
these distinctive proteins, the S is responsible RBD amino acids in SARS-CoV-2(Y455, L586,
for recognizing and mediating the virus into the N49, D494, T501) are critical for binding to ACE2
host cell8,9, and usually the S sequences among receptors and for determining the host range. Five
coronaviruses belong to different genera diverse of these six residues vary between SARS-CoV-2
a lot, even SARS-CoV and SARS-CoV-2 diverse and SARS-CoV(L455Y, F486L, Q493N, S494D,
in this region5,7. Coronaviruses can also achieve N501T), but the changed amino acids completely
the host receptor recognition diversity through ge- maintained the original conformation of the inter-
netic mutation and recombination events in their action between S protein and ACE2 receptor14.
S gene. For example, although they all belong to Additionally, SARS-CoV-2 S protein RBD
betacoronavirus, SARS-CoV-2 and HCoV-NL63 not only binds with high affinity to ACE2 from
both recognize human ACE210, while MERS-CoV humans, but also binds to homologous receptors
recognizes dipeptidyl peptidase 4 (DPP4)11. Mu- from ferrets, cats and other species15-17. The es-
rine hepatitis virus (MHV), another betacorona- sence of such homology is to maintain the key
virus mediates cell entry by recognising the car- sites on the interface (Figure 1) between ACE2
cinoembryonic antigen cell adhesion molecules 1 and SARS-CoV-2.
(CEACAM1)12.
S protein observed under the electron micro- Phylogenetic Analysis Based on S Protein
scope is a clove-like trimer, composed of three S1 So far, the phylogenetic analysis of coronavi-
heads and a trimeric S2 stalk5. The S1 subunit rec- ruses is mainly based on the whole genome. Al-
ognizes and binds to the receptor on the surface though S protein is a a key factor in coronaviral

Figure 1. Structure of SARS-CoV-2 RBD complexed with human ACE2. The interface is displayed in the dashed box. The key
sites of SARS-CoV-2 RBD are colored green; the key sites of ACE2 are colored red.

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 SARS-CoV-2: phylogenetic status, mutations and therapeutic research based on spike protein

Table I. Description of 18 coronaviruses and clinical characteristics.

recognition and invasion, the phylogeny of S pro- and makes coronavirinae distinctive. However,
tein in mutiple coronavirus remains unclear. the N-terminal of S protein mutated a lot during
We downloaded the S protein sequences of 18 the viral evolutionary history, which offered each
coronaviruses (Table I) from NCBI (https://www. virus peculiar characteristics. At the C-terminal,
ncbi.nlm.nih.gov/), then, we used mafft18 for se- the motif 6, 7 and 9 of Clade I are not found in
quence alignment, and FastTree19 for reconstruct- Clade II and III, and Clade III also lacks motifs
ing the phylogenetic tree based on S protein (Fig- 17 and 18. Clade I is the most conserved motif in
ure 2A).We analyzed the conserved motifs of the S the C-terminal sequence, with 5 or 6 conserved
protein sequences of these coronaviruses (Figure motifs. There are only 2 or 3 motifs in Clade II
2B). The phylogenetic tree can be roughly divid- and 1 or 2 in clade III.
ed into three clades (Clade I, Clade II and Clade It is worth noting that RaTG13 is still the most
III). In general, the C-terminal of the S protein is identical to SARS-CoV-2 based on the S protein
more conservative than the N-terminal, which in- sequence, and RaTG13 is, for now, considered to
dicates that the C-terminal sequence determines be the origin of the SARS-CoV-2. However, even
the conservative characteristics of the S protein if there are a few differences in the S protein,

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 Y. Zhu

Figure 2. Phylogenetic analysis based on S protein. (A) Phylogenetic tree of 18 coronaviruses; (B) Conserved motifs of the vi-
ral S protein. Detailed identification of variable motifs is highlighted with the colored square. Bar with different length represents
the conserved position of each motif in different viruses.

RaTG13 seems not to effectively bind to ACE2 Although there are just several amino acid mu-
receptor16. Deeper Structural and bioinformatics tations , SARS-CoV-2 have evolved adaptively
studies has found two major genomic features of from bat to human and accelerated human-to-hu-
SARS-CoV-2: (i) the S protein of SARS-CoV-2 man transmission.
appears to be optimized for binding to the human
receptor ACE2; and (ii) the S protein of SARS- Viral Mutants of SARS-CoV-2
CoV-2 has a functional polybasic (furin) clearance According to Centers for Disease Control and
site at the S1-S2 boundary through the insertion of Prevention COVID-19 Response, since a G614
12 nucleotides20, which forms a polybasic cleav- substitution in the S protein has emerged; virus
age site (RRAR), that enables effective cleavage containing this substitution has become the pre-
by furin and other proteases21. This furin-cleav- dominant circulating variant in the COVID-19
age site can reduce the stability of SARS-CoV-2 S pandemic. G614 substitution had increased affin-
protein and facilitate the conformational adaption ity to human ACE2, through regulating the stabil-
that is required for binding to the RBD of its recep- ity of S protein trimer. It which may be another
tor22. This explains why the S proteins of RaTG13 mechanism that underlies the increased replica-
and SARS-CoV-2 are highly similar in sequence, tion and transmission of SARS-CoV-2. Some oth-
but RaTG13 can not infect human. The emergen- er mutants have continued to threat. The B.1.1.7
cy of SARS-CoV-2 may have been selected and mutant in the UK has 10 mutational sites on the S
optimized for binding to human ACE2 receptor. protein, of which the key mutation Y501 is in the

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 SARS-CoV-2: phylogenetic status, mutations and therapeutic research based on spike protein

RBD and the binding sequence23. Mutation Y501 results. Discussion on these issues will be helpful
can increase the affinity between the viruses and for domestic vaccine research and development,
ACE2 receptor of human cells, thus accelerating and contribute to the fight against COVID-19.
the spread of the virus in the population. In addi- To date, all vaccines being developed can be
tion, the loss of two amino acids on S protein may generally divided into six types: inactivated vac-
help it escape immune response, and the H681 cines, recombinant viral vector vaccines, protein
mutation may enhance its ability to fuse with subunit vaccines, live attenuated vaccines, vi-
cells. The 501.V2 mutant found in South Africa is rus-like particle vaccines and nucleic acid vac-
more serious. There are nine mutations on S pro- cines. There are about a dozen of candidate vac-
tein, three of which are located in the RBD region, cines that are occupying the leading position in
namely Y501, N417 and K48424. K484 is also lo- clinical trials. On the premise of effectiveness and
cated in the binding sequence of RBD. Previous safety, it is of necessity to carry out clinical and
detection showed that this mutation was related to preclinical evaluation, and if possible, consider
the viral ability to escape the complex antibody, the virus mutation and the potential unknown as-
and it may affect the efficacy of existing vaccines sociation between COVID-19 and other diseases.
targeting S protein. In addition, the loss of three Therapies research can start by certain aspects:
amino acids on the S protein of 501.V2 mutant inhibition of virus replication, inhibition of virus
may affect the antigenicity, and then lead to “es- entry, immunomodulatory agents, and immuno-
cape mutant” to escape the immune response of globulin therapy.
monoclonal antibody25. The P.1 in Brazil contains
mutations at T417, K484, Y501 and other sites. Inhibiting the Replication of SARS-CoV-2
According to the SSI report, 5 mutants have been
isolated from Danish minks, among which F453 Chemotherapy-Like Therapy
is of great significance, because it may be more Utilizing quantitative proteomics and transcrip-
suitable for mink host. In addition to F453 mu- tomic to study the host cells infected with SARS-
tation, there were 69-70del, V692, L1147, I1229 CoV-2, the analysis showed that viral infection
and so on24,26-28. reshaped a variety of core regulatory pathways
in host cells, such as translation, splicing, carbon
Advances on Therapeutic Research metabolism and nucleic acid metabolism. Small
In April 2021, the epidemic broke out like a molecule inhibitors targeting these pathways
tsunami in India; the severe epidemic situation can inhibit viral replication in host cells. After
cast a new shadow on the global anti-epidemic testing, drugs targeting these essential signaling
work. According to the data released by World pathways, including cyclohexidine (translation
Health Organization, at present, there are more elongation inhibitor), emetine (inhibition of 40S
than ten countries in the world with mutated ribosomal protein S14), pladienolide, and NMS-
strains from India. 873(small molecule inhibitor of adenosine tri-
By now, inactivated virus is widely used as phosphatase p97), can effectively inhibit the rep-
vaccine; the number of vaccinees in the world lication of SARS-CoV-2 at non-toxic concentra-
has exceeded of the number of people that were tion, which reveals that these drugs can be used as
reported in infection cases. But more than 75% potential therapeutic strategies29. However, these
of vaccinees are concentrated in 10 countries, and small molecule inhibitors not only inhibits virus
2.5 billion people in nearly 130 countries have not replication, but also hinders the core regulatory
received any vaccine yet. It indicates that there is pathways (translation, splicing, carbon metabo-
still a long way to go for the herd immunity. In ad- lism and nucleic acid metabolism) of host cells
dition to accelerate the pace of vaccination, what to a certain extent. Self-damaging may need to be
we also need to pay attention to is that with the ap- considered and avoided in further research.
plication of vaccines, will the virus optimize itself
again and evolve resistance to existing vaccines? Drugs Blocking Viral Replication
Even though the mutation of SARS-CoV-2 in hu- FDA (Food and Drug Administration) de-
mans is a nondeterministic process during which scribed a large-scale screening study to assess
no strong selective pressure is acting on its adapta- nearly 12000 drugs to block the replication of
tion, and virus diversification results mainly from SARS-CoV-2, and eventually found 100 mole-
random genetic drift, we should be aware that cules that can inhibit the replication of the virus,
some base substitutions can lead to undesirable of which 13 drugs showed significant characteris-

5847
 Y. Zhu

tics – especially effective at realistic dose levels. for viral replication, large-scale randomized con-
These drugs include: Anti-HIV drug R 82913; ds- trolled trials may be possible to conduct.
6930, a member of the PPAR - γ agonist family
for the treatment of diabetes; Ono 5334, a poten- Inhibition of Virus Entry
tial drug for the treatment of osteoporosis; apili- One of the important ways to prevent SARS-
mod, a potential drug for the treatment of autoim- CoV-2 from entering host cells is blocking the in-
mune diseases such as Crohn’s disease. The three teraction between S protein and ACE2 receptor.
most effective drugs were Ono 5334, apilimod Antibody neutralization is also an effective sub-
and MDL 28170 (it has been previously shown stitute. Antibodies P2C-1F11 and P2B-2F6 have
that MDL 28170 can attenuate Ebola virus infec- nearly 100% binding efficiency to SARS-CoV-2,
tion). Using cultured lung tissue to test the ability even much higher than ACE2, indicating that they
of these three drugs to reduce the replication of can block the interaction between SARS-CoV-2
SARS-CoV-2, the results showed that the num- RBD and ACE2. Although some neutralizing an-
ber of infected cells reduced by 72% (Ono 5334), tibodies were found to have strong binding with
65% (MDL 28170) and 85% (apilimod), respec- SARS-CoV-2 RBD, and may be promising can-
tively. On this basis, it has been shown that apili- didates for prevention and treatment of SARS-
mod is well tolerated in human body and shows CoV-2 interventions, they show no obvious cross
good safety in the dose range that may produce reaction with RBD of SARS-CoV or MERS-CoV.
antiviral effect30. Some of these drugs have been It highlighted the immune difference of RBD
tested in clinical settings, so that can advance the among the three viruses37. In order to elucidate
preclinical evaluation and clinical assessment, the relationship between antibody response and
and promote the understanding of their capacity disease progression, as well as its driving factors
as a therapeutic medicine. and effects, more clinical trials must be studied.
Carmofur is valuable in the treatment of col- There are missense genetic variants, like p.His-
orectal cancer, and inhibit main protease (Mpro) 378Arg, p.Ser19Pro, p.Gly211Arg, p.Asp206Gly,
in the treatment of breast cancer, gastric cancer p.Arg219Cys, p.Arg219His, p.Lys341Arg, p.Ile-
and bladder cancer. Since Mpro is a conserved se- 468Val, and p.Ser547Cys, might affect the struc-
quence in all coronaviruses, SARS-CoV-2 Mpro ture and function of ACE2, and some might affect
are identified as a potential therapeutic target due the binding capacity of SARS-CoV-2, so SARS-
to its involvement in viral replication31-33. So Car- CoV-2 may have different susceptibilities, sever-
mofur as well as drugs based on Carmofur may ity and mortality in those populations with these
be effective against broad-spectrum coronavirus loci38. It means that the variants of ACE2 affects
infection. The X-ray crystal structure of SARS- the binding of SARS-CoV-2 to ACE2, thus slow-
CoV-2 Mpro combined with Carmofur shows that ing down or accelerating the spread of SARS-
Carmofur can directly modify the catalytic el- CoV-2. This reminds us that we can edit ACE2
ement Cys145 of SARS-CoV-2 Mpro34. This and specific sites to prevent it from interacting with
the further details of the interaction between Car- the virus. The distribution of the linear epitopes of
mofur and Mpro revealed by this structure are ex- the S protein of the SARS-CoV-2 was analyzed,
pected to lay a foundation for the design of more and three epitopes might induce neutralizing anti-
powerful derivatives of Carmofur. bodies in the non-RBD region. The validation of
CVL218, an inhibitor of poly-ADP-ribose a larger sample is in progress, and the ability of
polymerase 1 (PARP1), exhibits effective inhibi- each epitope to induce neutralizing antibody is-
tory activity against SARS-CoV-2 replication, and studying and expanding based on animal immuni-
able to suppress the CpG-induced IL-6 production ty. It will provide important support for neutraliz-
in peripheral blood mononuclear cells, suggesting ing antibody and vaccine development39.
that it may also have anti-inflammatory effect that Inhibitors like H014 can competitively bind to
is highly relevant to the prevention immunopa- the SARS-CoV-2 and SARS-CoV, thus preventing
thology induced by SARS-CoV-2 infection35. the virus from attaching to the target cell surface40.
Sequence analysis method named Viral-Track Additionally, It has been known that pH-depen-
was developed in May 2020, based on which, dent viruses, including enveloped coronaviruses
the host response induced by virus and the key (SARS-CoV-2, SARS-CoV and MERS-CoV), in-
host factors needed for virus replication can be fluenza A(H1N1), avian influenza A(H7N9) and
revealed36. For this convenience, more reliable as- uncoated rhinoviruses need endosome acidifica-
sessment of the effectiveness of some inhibitors tion to fuse with host membranes. A defensin an-

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 SARS-CoV-2: phylogenetic status, mutations and therapeutic research based on spike protein

alogue, P9R, has strong antiviral activity against Future Perspectives

pH-dependent viruses, depending on the direct SARS-CoV-2 is the third highly pathogenic
binding to viruses and the inhibition of virus-host human coronavirus disease after SARS- CoV and
endosomal acidification41. MERS-CoV. Since the epidemic continued for
more than one year, some countries and regions
IFN-I Response and Antiviral Drugs have carried out large-scale vaccination in order
Induction of Type I Interferon (IFN-I) is a cen- to achieve herd immunity. However, the precip-
tral event of the immune defense against viral itous epidemic situation in India this April posed
infection42. Upon exposure to RNA viruses, an challenges to the efficiency, the insufficient sup-
intracellular antiviral response is initiated by ac- ply and uneven distribution of vaccines. Virus
tivation of retinoic acid inducible gene I (RIG-I) genetic drift occurs all the time. Although this is
like receptors. Particularly, when RIG-I/MDA5 a random result, we has to realize that the mu-
(melanoma differentiation-associated gene 5) de- tants reserved by natural selection is often more
tects viral RNA, they trigger a signaling complex virulent and spreads more rapidly. The mutation
on the mitochondrial outer membrane, including rate is far faster than that of vaccine research and
the adapter proteins mitochondria antiviral sig- development. Therefore, it is necessary to further
naling protein/TNF receptor associated factors 3/ discover the origin of animal SARS-CoV-2, the
TNF receptor associated factors 6/ translocase of cross species infection pathway, the pathogenesis
the outer membrane70 (TOM70). This process ul- of SARS-CoV-2 infection and the interaction be-
timately leads to IFN-β production and induction tween virus and host. In view of these biological
of a host antiviral state43,44. The most prominent processes, we should design treatment strategies
feature of SARS-CoV-2, in terms of immune re- that can still play an effective role in the case of
sponses as compared to that of other viruses such viral mutation.
as influenza A, is that it strongly inhibits the lev-
el of IFN-I in patients45,46. In addition, it has also
been found that the chemical, Liquiritin, can in- Acknowledgments
hibit SARS-CoV-2 by mimicking IFN-I46. SARS- This work was supported by College of Life Sciences,
CoV-2 ORF9b inhibits IFN-I response by bind- Northwest A&F University, and we thank the editor and
the reviewers for their useful feedback that improved this
ing to TOM70, thereby significantly inhibits the paper.
induction of IFN-I in cells, and the overexpres-
sion of TOM70 can largely offset the inhibition
of IFN-I mediated by ORF9b48. Based on this, a Authors’ Contribution Statement
potential drug design strategy is developing inhib- Designed the experiments: YZ Analyzed the data and plot:
itors targeting the interaction interface between YZ. Wrote the paper: YZ. All authors read and approved the
ORF9b and TOM70 to restore IFN-I response. final manuscript.
SARS-CoV, MERS-CoV and other respiratory
viruses such as rhinovirus and influenza influ-
enza A can up-regulate ACE2 in host cells, and Conflict of Interest
cytokines such as IFN - γ can also stimulate the The Authors declare that they have no conflict of interests.
expression of ACE249,50. It is speculated that vi-
rus infection can activate the immune system and
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