S H O R T C O M M U N I C A T I O N
Evolutionary relationships and sequence-structure
determinants in human SARS coronavirus-2 spike
proteins for host receptor recognition
Lalitha Guruprasad
School of Chemistry, University of Hyderabad,
Hyderabad, Telangana, India
Correspondence
Lalitha Guruprasad, School of Chemistry,
University of Hyderabad, Hyderabad,
Telangana 500046, India.
Email: lalitha.guruprasad@uohyd.ac.in
Peer Review
The peer review history for this article is
available at https://publons.com/publon/10.
1002/prot.25967.
Abstract
Coronavirus disease 2019 (COVID-19) is a pandemic infectious disease caused by
novel severe acute respiratory syndrome coronavirus-2 (SARS CoV-2). The SARS
CoV-2 is transmitted more rapidly and readily than SARS CoV. Both, SARS CoV and
SARS CoV-2 via their glycosylated spike proteins recognize the human angiotensin
converting enzyme-2 (ACE-2) receptor. We generated multiple sequence alignments
and phylogenetic trees for representative spike proteins of SARS CoV and SARS
CoV-2 from various host sources in order to analyze the specificity in SARS CoV-2
spike proteins required for causing infection in humans. Our results show that among
the genomes analyzed, two sequence regions in the N-terminal domain “MESEFR”
and “SYLTPG” are specific to human SARS CoV-2. In the receptor-binding domain,
two sequence regions “VGGNY“ and ”EIYQAGSTPCNGV” and a disulfide bridge connecting
480C and 488C in the extended loop are structural determinants for the recognition
of human ACE-2 receptor. The complete genome analysis of representative
SARS CoVs from bat, civet, human host sources, and human SARS CoV-2 identified
the bat genome (GenBank code: MN996532.1) as closest to the recent novel human
SARS CoV-2 genomes. The bat SARS CoV genomes (GenBank codes: MG772933
and MG772934) are evolutionary intermediates in the mutagenesis progression
toward becoming human SARS CoV-2.
K E Y W O R D S
complete genomes, multiple sequence alignment, phylogenetic tree, receptor-binding domain,
severe acute respiratory syndrome coronavirus-2, spike proteins
1 | INTRODUCTION
In the last two decades, zoonotic coronaviruses, severe acute respiratory
syndrome coronavirus (SARS CoV) (2002),1
and Middle East
Respiratory Syndrome coronavirus (MERS CoV) (2012)2
have caused
acute respiratory diseases in humans that have resulted in several
deaths. The present coronavirus disease 2019 (COVID-19) is a pandemic
respiratory disease caused by the novel SARS CoV-2. The initial
infection started in Wuhan, Hubei province, China in December 2019
and very soon became a global outbreak, infecting populations in
almost every country in the world causing a total of 184 248 deaths
and 2 638 852 infections as of 23rd April 2020 (https://www.
worldometers.info/coronavirus/). Within a short span of time, this
pandemic has caused major social and economic disruptions. Compared
to other coronaviruses, the novel SARS CoV-2 appears to be
spreading more rapidly and readily, posing a challenging task before
the administrative and scientific communities. The SARS CoV-2 is
transmitted from person to person contact via respiratory secretions
during coughing and sneezing. Infection of this highly pathogenic virus
can cause acute respiratory distress syndrome which impacts the lung
Received: 24 April 2020 Accepted: 6 June 2020
DOI: 10.1002/prot.25967
Proteins. 2020;88:1387–1393. wileyonlinelibrary.com/journal/prot © 2020 Wiley Periodicals LLC. 1387
and heart functions. The prominent symptoms of this viral infection are flu,
severe respiratory, enteric, and neurological disorders, resulting in increased
white blood cells and kidney failure. There are no vaccines or drugs available
to combat this deadly infectious disease and there is no strategic plan
to treat the infected patients. Hence, there is a need to develop specific
anti-SARS CoV-2 vaccines and drugs to treat infected patients, in order to
reduce viral shedding and further transmission in populations.
The SARS CoV-2 comprises positive-sense single-stranded RNA
genome of size 29 to 30 kb and belongs to the coronaviridae family
and betacoronavirus subfamily. Mammals such as bats are the main
reservoir of betacoronaviruses, but due to the zoonotic contacts and
viral genomic mutations, SARS CoV-2 has recently crossed species
and caused infections in humans.3
Research findings have pointed
that previous zoonotic CoV infections such as SARS CoV that first
infected humans in the Guangdong province of southern China in
2002 was transmitted from bats and civets,4-8
the MERS CoV that
originated in bats was first identified from camel to human transmission
in Saudi Arabia in 2012.2,9-12
These coronaviruses have crossed
species and resulted in causing human infections leading to mortality.
It is reported that civet SARS CoV can also infect humans.13,14
The
SARS-like CoVs from some bats and civets are predicted to result in
human infections15,16
due to their changing genomic RNA sequences,
importantly in the spike protein regions.17,18
Since January 2020, several
complete genome sequences of viral SARS CoV-2 isolated from
infected patients belonging to various geographical locations, such as
Australia, China, Denmark, Finland, Hungary, India, Italy, Japan, South
Korea, United States, and Vietnam, have been deposited in the
GenBank (https://www.ncbi.nlm.nih.gov/genbank/). At the genomic
level, the nucleotide sequences of SARS CoV and SARS CoV-2 share
79.6% sequence identity.19
The viral RNA stores the genetic information
and also serves to translate into structural and nonstructural proteins
of SARS CoV-2. The SARS CoV uses angiotensin converting
enzyme-2 (ACE-2) as receptor for entry into human epithelial cells20
to
cause the infection. Virus infectivity studies on HeLa cells have shown
that SARS CoV-2 also uses ACE-2 as receptor for cellular entry.19
It has been reported that the first SARS CoV-2 infection in
Wuhan, China is caused from the original host, bats,19
and in less than
4 months transmitted among the human populations in the entire
world. The initial contact between the SARS CoV/CoV-2 and human
host is via recognition between the heavily glycosylated cell envelope
spike protein of the virus and the ACE-2 receptor of the human host
resulting in infection. In order to understand the specificity, estimate
the extent of similarities and variations in the SARS CoV-2 spike proteins
required for binding the host receptor, we analyzed the representative
spike protein sequences. Furthermore, in order to estimate
the evolutionary progression of the bat SARS CoV genome, such that
it is able to adapt to a human host as a novel coronavirus causing
COVID-19, we have analyzed the complete genomes of the bat, civet,
human SARS CoV, and human SARS CoV-2. We have carried out
computational analyses on the nucleotide and protein sequences by
generating multiple sequence alignments (MSAs), constructing phylogenetic
trees, and analyzing the three-dimensional structures of the
spike proteins to address the above.
2 | MATERIALS AND METHODS
The spike proteins were retrieved from the NCBI database using the
sequence similarity search BLAST program,21
with human SARS
CoV-2 spike protein as the query sequence (NCBI code: QHD43416)
from the genome (GenBank code: MN908947.3).3
The complete
genome nucleotide sequences of SARS CoV from bats, civets, and
SARS CoV and CoV-2 from human host were obtained from NCBI
virus database (https://www.ncbi.nlm.nih.gov/labs/virus/vssi/#/virus?
SeqType_s=Nucleotide) in FASTA format. Only complete genome
sequences without any ambiguity in nucleotide composition were
considered for analyses. The redundancy in each data set was
removed using the CD-HIT program.22
The nucleotide and protein sequence homology analyses based
on MSA reveal the substitutions, deletions, and insertions at each
position. To understand the evolutionary relationships between the
members, the MSAs were further processed to generate phylogenetic
trees—a pictorial representation of the evolutionary relationships
between related members of various sequences analyzed. The MSAs
and phylogenetic trees of the spike proteins and complete genomes
were generated using the Next Generation Phylogeny.fr web service
available at https://NGPhylogeny.fr.23
The protocol takes all sequences
(nucleotide or protein) as input in FASTA file format and generates
the MSA and phylogenetic tree. In the NGPhylogeny server, we have
selected FastME 2.0 program that infers phylogenies using a distance
approach since it is capable of handling large data sets.24
Based on
the input FASTA sequences, an MSA is generated that adopts Multiple
Alignment using Fast Fourier Transform (MAFFT)25
with gap
extension penalty 0.123 and gap opening penalty 1.53. The MSA generated
is parsed through Block Mapping and Gathering with Entropy
(BGME) software for selecting regions suitable for phylogenetic infer-
ence.26
This method uses a sliding window size 3, maximum entropy
threshold 0.5, gap rate cutoff 0.5, minimum block size 5, matrix:
PAM250 for DNA, and BLOSUM62 for proteins. FastME estimates
phylogenies that employ distance-based methods from MSAs using
TN93 and LG as substitution models for DNA and proteins, respectively.
In the distance-based methods, pairwise distances between all
pairs of sequences are generated as a square matrix. The sequence
pairs with shorter pairwise distances are clustered together more
closely in the phylogenetic tree. Tree refinement was performed using
Subtree Pruning and Regrafting (SPR) with Balanced version of Minimum
Evolution (BalME), with a decimal precision for branch length
set to 6. Finally, the phylogenetic trees were generated using Interactive
Tree Of Life program (iTOL) v4.27
3 | RESULTS AND DISCUSSION
3.1 | Analyses of the spike proteins of SARS CoV
and SARS CoV-2
The SARS CoV and SARS CoV-2 spike proteins retrieved from various
host sources have a sequence length ranging between 1240 to 1273
1388 GURUPRASAD
amino acids. Structurally, a spike protein is characterized by three
regions: (a) the N-terminal extracellular domain, (b) a transmembrane
anchor domain, and (c) an intracellular segment. The N-terminal extracellular
domain comprises a receptor binding subunit (S1) and a
membrane-fusion subunit (S2). The S1 subunit comprises two
domains: N-terminal domain (NTD) and receptor-binding domain
(RBD). The sequence analyses of spike proteins from the various host
sources are shown in the MSA in Figure S1 and the phylogenetic tree
in Figure 1. From Figure 1, it is observed that the paralogous proteins
from individual host sources are associated with a distinct clade, the
spike proteins from human SARS CoV-2 share highest sequence similarity
according to the least pairwise distances. Also, the orthologous
spike proteins from other host species are highly similar according to
the low pairwise distances. Therefore, it is intriguing to see that
despite high sequence identity between the spike proteins from various
host sources, only some SARS CoV and SARS CoV-2 are able to
bind the human host ACE-2 receptor. In order to understand this, we
have analyzed the MSA of the spike proteins.
From Figure S1, it is observed that the N-terminal 500 amino
acids comprising S1 subunit vary to a moderate extent among all host
sources relative to the later region that share high sequence identity.
The region approximately between 300 and 500 amino acid residues
is crucial in spike proteins as it forms the RBD that recognizes the
ACE-2 receptor which allows entry of the virus into host cells. A
sequence motif “PRRA” from 681P to 684A (amino acid numbering is
according to NCBI code: QHD43416) that is gained only in the human
SARS CoV-2 spike proteins is referred to as furin cleavage site.28,29
In
this work, we identify the sequence regions in human SARS CoV-2; a
six residues insertion sequence “MESEFR” from 153M to 158R and
another six residues insertion sequence “SYLTPG” from 247S to
252G. The bat SARS CoV spike protein QHR63300 (GenBank code:
MN996532.1) also comprises the identical sequence regions as above.
FIGURE 1 Phylogenetic tree of SARS CoV and SARS CoV-2 spike proteins. Human CoV-2 (green), human CoV (dark green), bat CoV (red),
civet CoV (violet), cat CoV (orange), swine CoV (blue), mouse CoV (bright yellow), SARS CoV recombinant spike proteins and from lab adapted
cells (black). SARS CoV-2, severe acute respiratory syndrome coronavirus-2
GURUPRASAD 1389
(A)
(B)
(C)
FIGURE 2 Legend on next page.
1390 GURUPRASAD
In two bat spike proteins, AVP78042 (GenBank code: MG772934)
and AVP78031 (MG772933), a six residues sequence “SIREFA” and a
three residues sequence “GDP” are present at equivalent positions,
respectively. The insertion sequence regions present in human SARS
CoV-2, 153MESEFR158, and 247SYLTPG252 are associated with the
NTD and are located distant from the ACE-2 binding site. From the
three-dimensional structure of human SARS CoV-2, we infer that
these regions are likely to be exposed toward the surface of the spike
protein in NTD. In human SARS CoV-2, there are two insertion
regions: a five residues insertion “VGGNY” from 445V to 449Y, and a
13 residues insertion “EIYQAGSTPCNGV” from 471E to 483V. The
bat SARS CoV spike protein QHR63300 has also gained equivalent
insertions with the sequences; “EGGNF” and “EIYQAGSKPCNGQ.” It
is interesting to note that the bat SARS CoV QHR63300 has already
acquired a 13 residues sequence region, whereas all other spike proteins
that recognize the ACE-2 receptor in SARS CoV comprise a
12 residues sequence region. The Figure 2A was generated by editing
the MSA in Figure S1 to depict the sequence regions discussed above
in representative spike proteins from the four host sources (bat, civet,
human SARS CoV, and human SARS CoV-2).
The two sequence regions, “VGGNY” and “EIYQAGSTPCNGV,”
are part of the RBD and involved in recognition of the ACE-2 receptor
in human host. Their absence in the bat SARS CoV at equivalent positions
may be responsible for their inability to bind human ACE-2. To
study this, we have analyzed the three-dimensional structures of
human SARS CoV (PDB code: 6ACG)30
and the RBD domain of
human SARS CoV-2 (6M17) complexed with ACE-2 receptor.31
The
structures were superimposed and amino acid residues within 5 Å distance
from the ACE-2 receptor were identified and highlighted in the
pairwise sequence alignment of RBD (Figure 2B). Despite amino acid
mutations in these regions in both proteins, the structures are highly
superimposable. The location corresponding to the deletions of the
two sequence regions in bat SARS CoV with respect to human SARS
CoV-2 matches with the ACE-2 binding region (Figure 2C). The second
insertion sequence region, that is, 471EIYQAGSTPCNGV483 in
the RBD domain forms an extended loop in human SARS CoV-2 and
is stabilized by a disulfide bond between 480C and 488C (Figure 2C).
This disulfide bond is conserved in all spike proteins that comprise the
insertion sequence regions in RBD. In bat SARS CoV spike proteins,
the position equivalent to 488C is replaced by a conserved glycine
residue and therefore the above disulfide bond will be absent. Interestingly,
the bat SARS CoV spike protein QHR63300 which has
acquired the insertion sequences in RBD also possesses the disulfide
bond. We believe that the presence of the above sequence regions in
RBD and the disulfide bond that stabilizes the conformation of the
extended loop are required for the recognition of human ACE-2.
The sequence regions identified in this work serve as potential
candidate epitopes for the design of antibodies specific for human
SARS CoV-2 recognition. Our analyses suggest that the bat spike protein
QHR63300 has undergone significant evolutionary changes such
that it resembles the human SARS CoV-2 spike protein more than the
bat SARS CoV which may have led to the transmission of SARS CoV
from bat to human as the novel SARS CoV-2. Our results also suggest
that the bat SARS CoV spike proteins—AVP78042 and AVP78031—
are in the progression of acquiring mutations toward becoming SARS
CoV-2-like proteins. The phylogenetic tree in Figure 1 showing the
proteins—QHR63300, AVP78042, and AVP78031 close to the human
SARS CoV-2—is in support of our hypothesis.
3.2 | Complete genome analyses of SARS CoV and
SARS CoV-2
The representative complete genomes of nucleotide sequences from bat,
civet, human CoV, and human CoV-2 were analyzed. The MSA is shown
in Figure S2 and the phylogenetic tree in Figure 3. From Figure 3, it is
observed that the human SARS CoV-2 genomes cluster into one clade
(pairwise distance is lower than 0.002) revealing high identity that suggest
their recent evolution. The bat SARS CoV genome (GenBank code:
MN996532.1) is also member of this clade (pairwise distance between
0.042 and 0.043) indicating that it is the closest homolog to the human
SARS CoV-2 among the bat genomes. The two bat SARS CoV genomes
(GenBank codes: MG772933.1 and MG772934.1) are also close to the
human SARS CoV-2 clade. The human and civet SARS CoV genomes
cluster into another distinct clade. The members of bat SARS CoV clade
have undergone maximum evolutionary changes as observed in Figure 3.
Based on these results, we propose that the bat SARS CoV genomes
have diverged the most during the last 18 years (since its detection in
2002) and have evolved closer to civet and human SARS CoV genomes.
The bat SARS CoV genome (GenBank code: MN996532.1) has diverged
significantly into the recent novel human SARS CoV-2 genomes, whereas
the bat SARS CoV genomes (GenBank codes: MG772933 and
MG772934) are intermediates during the evolution of bat SARS CoV
into human novel SARS CoV-2. We believe that the bat SARS CoV
genomes such as these are likely to undergo further evolutionary mutations
and become adaptable to infecting human populations.
FIGURE 2 A, Portions of the alignment of spike proteins extracted from the multiple sequence alignment (Figure S1). Insertion sequence regions
and their locations within the NTD, RBD, and furin cleavage sites for human SARS CoV-2 (*), bat SARS CoV (@), civet SARS CoV (#), human SARS CoV
($). B, Pairwise sequence alignment corresponding to the RBD from human SARS CoV (6ACG) and human SARS CoV-2 (6M17). The residues that lie
within 5 Å in RBD from ACE-2 are highlighted in 6ACG (green) and 6M17 (magenta). The start and end amino acid residues numbers in RBD are
shown. “*” indicates identical residues; “:” indicates conservative amino acid residue substitutions; “.” indicates weakly conserved amino acid residue
substitutions in the alignment. C, Structural superposition of human SARS CoV (6ACG, green) and human SARS CoV-2 (6M17, magenta) and the long
H1 helix in ACE-2 (blue). The side chains of amino acid residues in RBD that lie within 5 Å from ACE-2 are shown. The deletion region in RBD of bat
SARS CoV is shown in the structure of 6M17 (red). The C480-C488 disulfide bond (yellow) connects the extended loop in 6M17. ACE-2, angiotensin
converting enzyme-2; NTD, N-terminal domain; RBD, receptor-binding domain; SARS CoV-2, severe acute respiratory syndrome coronavirus-2 [Color
figure can be viewed at wileyonlinelibrary.com]
GURUPRASAD 1391
4 | CONCLUSIONS
Two sequence regions “MESEFR” and “SYLTPG” in the NTD of spike
protein in human SARS CoV-2 and two sequence regions “VGGNY”
and “EIYQAGSTPCNGV” in the RBD that interact with ACE-2 may be
exploited as potential candidates for antibody design. The phylogenetic
analyses of the bat, civet, human SARS CoV, and human SARS
CoV-2 genomes show that the bat SARS CoV genome (GenBank
code: MN996532.1) is closest homolog of human SARS CoV-2. The
two other bat SARS CoV genomes (GenBank codes: MG772933 and
MG772934) are intermediates in the evolution of bat genomes into
human SARS CoV-2.
ACKNOWLEDGMENT
L. G. thanks School of Chemistry and CAS, UGC for providing research
facilities.
CONFLICT OF INTEREST
The authors declare no potential conflict of interest.
ORCID
Lalitha Guruprasad https://orcid.org/0000-0003-1878-6446
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SUPPORTING INFORMATION
Additional supporting information may be found online in the
Supporting Information section at the end of this article.
How to cite this article: Guruprasad L. Evolutionary
relationships and sequence-structure determinants in human
SARS coronavirus-2 spike proteins for host receptor
recognition. Proteins. 2020;88:1387–1393. https://doi.org/10.
1002/prot.25967
GURUPRASAD 1393