Coronavirus Spike Protein
and Tropism Changes
R.J.G. Hulswit, C.A.M. de Haan
, B.-J. Bosch
Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands
Corresponding authors: e-mail address: firstname.lastname@example.org; email@example.com
1. Introduction 30
2. Structure of the Coronavirus S Protein 31
2.1 Structure of the S
2.2 Structure of the S
3. Spike–Receptor Interactions 37
3.1 Different Domains Within S
May Act as RBD 37
3.2 CoV Protein Receptor Preference 39
4. S Protein Proteolytic Cleavage and Conformational Changes 39
5. Tropism Changes Associated with S Protein Mutations 41
Receptor Interactions Determining Tropism 42
5.2 Changes in Proteolytic Cleavage Site and Other S
with Altered Tropism 45
6. Concluding Remarks 47
Coronaviruses (CoVs) have a remarkable potential to change tropism. This is particularly
illustrated over the last 15 years by the emergence of two zoonotic CoVs, the severe
acute respiratory syndrome (SARS)- and Middle East respiratory syndrome (MERS)-
CoV. Due to their inherent genetic variability, it is inevitable that new cross-species trans-
mission events of these enveloped, positive-stranded RNA viruses will occur. Research
into these medical and veterinary important pathogens—sparked by the SARS and
MERS outbreaks—revealed important principles of inter- and intraspecies tropism
changes. The primary determinant of CoV tropism is the viral spike (S) entry protein. Tri-
mers of the S glycoproteins on the virion surface accommodate binding to a cell surface
receptor and fusion of the viral and cellular membrane. Recently, high-resolution struc-
tures of two CoV S proteins have been elucidated by single-particle cryo-electron
microscopy. Using this new structural insight, we review the changes in the
S protein that relate to changes in virus tropism. Different concepts underlie these tro-
pism changes at the cellular, tissue, and host species level, including the promiscuity or
Advances in Virus Research, Volume 96 #2016 Elsevier Inc.
ISSN 0065-3527 All rights reserved.
adaptability of S proteins to orthologous receptors, alterations in the proteolytic cleav-
age activation as well as changes in the S protein metastability. A thorough understand-
ing of the key role of the S protein in CoV entry is critical to further our understanding of
virus cross-species transmission and pathogenesis and for development of intervention
Coronaviruses (CoVs) (order Nidovirales, family Coronaviridae, sub-
family Coronavirinae) are enveloped, positive-sense RNA viruses that con-
tain the largest known RNA genomes with a length of up to 32 kb. The
subfamily Coronavirinae, which contains viruses of both medical and
veterinary importance, can be divided into the four genera alpha-,beta-,
gamma- and deltacoronavirus (α-,β-,γ-and δ-CoV). The coronavirus particle
comprises at least the four canonical structural proteins E (envelope
protein), M (membrane protein), N (nucleocapsid protein), and S (spike
protein). In addition, viruses belonging to lineage A of the betacoronaviruses
express the membrane-anchored HE (hemagglutinin–esterase) protein. The
S glycoprotein contains both the receptor-binding domain (RBD) and the
domains involved in fusion, rendering it the pivotal protein in the CoV
Coronaviruses primarily infect the respiratory and gastrointestinal tract of
a wide range of animal species including many mammals and birds. Although
individual virus species mostly appear to be restricted to a narrow host range
comprising a single animal species, genome sequencing and phylogenetic
analyses testify that CoVs have crossed the host species barrier frequently
(Chan et al., 2013;Woo et al., 2012). In fact most if not all human cor-
onaviruses seem to originate from bat CoVs (BtCoVs) that transmitted to
humans directly or indirectly through an intermediate host. It therefore
appears inevitable that similar zoonotic infections will occur in the future.
In the past 15 years, the world witnessed two such zoonotic events. In
2002–2003 cross-species transmissions from bats and civet cats were at
the base of the SARS (severe acute respiratory syndrome)-CoV epidemic
that found its origin in the Chinese Guangdong province (Li et al., 2006;
Song et al., 2005). The SARS-CoV nearly became a pandemic and led to
over 700 deaths, before it disappeared when the appropriate hygiene and
quarantine precautions were taken. In 2012, the MERS (Middle East respi-
ratory syndrome)-CoV emerged in the human population on the Arabian
30 R.J.G. Hulswit et al.
Peninsula and currently continues to make a serious impact on the local but
also global health system with 1800 laboratory confirmed cases and 640
deaths as of September 1, 2016 (WHO jMiddle East respiratory
syndrome coronavirus (MERS-CoV) –Saudi Arabia, 2016). The natural
reservoir of MERS-CoV is presumed to be in dromedary camels from
which zoonotic transmissions repeatedly give rise to infections of the lower
respiratory tract in humans (Alagaili et al., 2014; Azhar et al., 2014; Briese
et al., 2014; Reusken et al., 2013; Widagdo et al., 2016). Besides these two
novel CoVs, four other CoVs were previously identified in humans which
are found in either the alphacoronavirus (HCoV-NL63 and HCoV-229E) or
the betacoronavirus genera (HCoV-OC43 and HCoV-HKU1). Phylogenetic
analysis has shown that the bovine CoV (BCoV) has been the origin for
HCoV-OC43 following a relatively recent cross-species transmission event
(Vijgen et al., 2006). Moreover, HCoV-NL63, HCoV-229E, SARS-CoV,
and MERS-CoV also have been predicted to originate from bats (Annan
et al., 2013; Bolles et al., 2011; Corman et al., 2015; Hu et al., 2015;
Huynh et al., 2012).
In general, four major criteria determine cross-species transmission of a
particular virus (Racaniello et al., 2015). The cellular tropism of a virus is
determined by the susceptibility of host cells (i.e., presence of the receptor
needed for entry) as well as by the permissiveness of these host cells to allow
the virus to replicate and to complete its life cycle. A third determinant con-
sists of the accessibility of susceptible and permissive cells in the host. Finally,
the innate immune response may restrict viral replication in a host species-
specific manner. The above-mentioned criteria may play a critical role in the
success of a cross-species transmission event. However, for CoVs, it seems
that host tropism and changes therein are particularly determined by the sus-
ceptibility of host cells to infection. While CoV accessory genes, including
the HE proteins, are thought to play a role in host tropism and adaptation to
a new host, the S glycoprotein appears to be the main determinant for the
success of initial cross-species infection events. In this review, we focus on
the molecular changes in the S protein that underlie tropism changes at the
cellular, tissue, and host species level and put these in perspective of the
recently published cryo-EM structures.
2. STRUCTURE OF THE CORONAVIRUS S PROTEIN
The CoV S protein is a class I viral fusion protein (Bosch et al., 2003)
similar to the fusion proteins of influenza, retro-, filo-, and paramyxoviruses
31Coronavirus Spike Protein and Tropism Changes
(Baker et al., 1999; Bartesaghi et al., 2013; Lee et al., 2008; Lin et al., 2014).
Like other class I viral fusion proteins, the S protein folds into a metastable
prefusion conformation following translation. The size of the abundantly
N-glycosylated S protein varies greatly between CoV species ranging from
approximately 1100 to 1600 residues in length, with an estimated molecular
mass of up to 220 kDa. Trimers of the S protein form the 18–23-nm long,
club-shaped spikes that decorate the membrane surface of the CoV particle.
Besides being the primary determinant in CoV host tropism and pathogen-
esis, the S protein is also the main target for neutralizing antibodies elicited
by the immune system of the infected host (Hofmann et al., 2004).
The S protein can be divided into two functionally distinct subunits: the
subunit is involved in receptor recognition, whereas the S
unit facilitates membrane fusion and anchors S into the viral membrane
(Fig. 1A). The S
domains may be separated by a cleavage site that
is recognized by furin-like proteases during S protein biogenesis in the
infected cell. X-ray crystal structures of several S domains have furthered
our understanding of the S protein in the past. In addition, recent elucidation
of the high-resolution structures of the spike ectodomain of two
betacoronaviruses—MHV and HCoV-HKU1—by single-particle cryo-
electron microscopy (Kirchdoerfer et al., 2016; Walls et al., 2016) has pro-
vided novel insights into the architecture of the S trimer in its prefusion state
(Fig. 1B and C).
2.1 Structure of the S
subunit of the betacoronavirus spike proteins displays a multidomain
architecture and is structurally organized in four distinct domains A–Dof
which domains A and B may serve as a RBD (Fig. 1C). The core structure
of domain A displays a galectin-like β-sandwich fold, whereas domain
B contains a structurally conserved core subdomain of antiparallel β-sheets
(Kirchdoerfer et al., 2016; Li et al., 2005a; Walls et al., 2016; Wang et al.,
2013). Importantly, domain B is decorated with an extended loop on the
viral membrane-distal side. This loop may differ greatly in size and structure
between virus species of the betacoronavirus genus and is therefore also
referred to as hypervariable region (HVR). The cryo-EM structures of
the MHV-A59 and HCoV-HKU1 S trimers show an intricate interlocking
of the three S
subunits (Fig. 1B). Oligomerization of the S protomers results
in a closely clustered trimer of the individual B domains close to the three-
fold axis of the spike on top of the S
trimer, whereas the three A domains are
32 R.J.G. Hulswit et al.
Fig. 1 Spike protein features and structure of the mouse hepatitis coronavirus spike
glycoprotein trimer. (A) Schematic linear representation of the coronavirus S protein
with relevant domains/sites indicated: signal peptide (SP), two proteolytic cleavage
0), two proposed fusion peptides (FP1 and FP2), two heptad repeat
regions (HR1 and HR2), transmembrane domain (TD), and cytoplasmic tail (CT).
(B) Front and top view of the trimeric mouse hepatitis coronavirus (strain A59) spike
glycoprotein ectodomain obtained by cryo-electron microscopy analysis (Walls et al.,
2016; PDB: 3JCL). Three S
protomers (surface presentation) are colored in red,blue,
and green. The S
trimer (cartoon presentation) is colored in light orange. (C) Schematic
representation of MHV spike protein sequence (drawn to scale), the S
B, C, and D are colored in blue,green,yellow, and orange, respectively, and the linker
region connecting domains A and B in gray, the S
region is colored in red, and the
TM region is indicated as a black box.Red-shaded region indicates spike region that was
33Coronavirus Spike Protein and Tropism Changes
ordered more distally of the center. In contrast to domains A and B, the S
C-terminal domains C and D are made up of discontinuous parts of the pri-
mary protein sequence and form β-sheet-rich structures directly adjacent to
stalk core, while the separate S
domains are interconnected by loops
covering the S
surface. Compared to the S
subunit, the S
low level of sequence conversation among species of different CoV genera.
subunits vary considerably in sequence length ranging from
544 (infectious bronchitis virus (IBV) S) to 944 (229-related bat coro-
navirus S) residues in length (Fig. 2), indicating differences in architecture
of the spikes of species from different CoV genera. Structural information
from the spikes of gamma- and deltacoronavirus species is currently lacking.
Two independently folding domains have been assigned in the S
of alphacoronavirus spikes, that can interact with host cell surface molecules,
an N-terminal domain (in transmissible gastroenteritis virus (TGEV) S resi-
dues 1–245) and a more C-terminal domain (in TGEV S residues
506–655). Contrary to betacoronaviruses, these two receptor-interacting
domains in alphacoronavirus spikes are separated in sequence by some 275
residues, which may fold into one or more separate domains. Structural infor-
mation is only available for the C-terminal S
RBD of two α-CoV S proteins,
which differs notably from that of betacoronaviruses. The RBD in the S
CTR of alphacoronaviruses displays a β-sandwich core structure, whereas a
β-sheet core structure is seen for betacoronaviruses (Regueraetal.,2012;
Wu et al., 2009).
2.2 Structure of the S
The highly conserved S
subunit contains the key protein segments that
facilitate virus-cell fusion. These include the fusion peptide, two heptad
Fig. 1—Cont’dnot resolved in the cryo-EM structure. (Lower panel) Two views on
the structure of the mouse hepatitis virus spike glycoprotein protomer (cartoon repre-
sentation); domains are colored as depicted earlier. (D) Comparison of the S
in its pre- and postfusion conformation. (Lower left) Structure of the MHV S
(cartoon presentation) with four helices of the HR1 region (and consecutive linker
region) and the downstream central helix colored in blue,green,yellow,orange, and
red, respectively. (Upper right) The structure of a single SARS-CoV S HR1 helix of the post-
fusion six-helix bundle structure (PDB: 1WYY)is colored according to the homologous
HR1 region in the MHV S
prefusion structure shown in the lower left panel. Structures
are aligned based on the N-terminal segment of the central helix (in red). Figures were
generated with PyMOL.
34 R.J.G. Hulswit et al.
Fig. 2 Overview of currently known receptors and their binding domains within S
Schematic representation of coronavirus spike proteins drawn to scale. Yellow boxes
indicate signal peptides. Blue boxes indicate the N-terminal regions in alpha- and
betacoronavirus spike proteins, which were mapped based on sequence homology
between viruses within the same genus. Green boxes indicate known receptor-binding
domains in the C-terminal region of S
. Known receptors are indicated in the boxes:APN,
aminopeptidase N; ACE2, angiotensin-converting enzyme 2; CEACAM, carcinoembryonic
antigen-related cell adhesion molecule 1; Sia, sialic acid; O-ac Sia,O-acetylated sialic
acid; DPP4, dipeptidyl peptidase-4. Gray boxes indicate transmembrane domains. Spikes
proteins are shown of PEDV strain CV777 (GB: AAK38656.1), TGEV strain Purdue P115
(GB: ABG89325.1), PRCoV strain ISU-1 (GB: ABG89317.1), Feline CoV strain UU23 (GB:
ADC35472.1), Feline CoV strain UU21 (GB: ADL71466.1), Human CoV NL63 (GB:
YP_003767.1), 229E-related bat CoV with one N domains (GB: ALK28775.1), 229E-related
bat CoV with two N domains (GB: ALK28765.1), Human CoV 229E strain inf-1 (GB:
NP_073551.1), MHV strain A59 (GB: ACO72893), BCoV strain KWD1 (GB: AAX38489),
HCoV-OC43 strain Paris (GB: AAT84362), HCoV-HKU1 (GB: AAT98580), SARS-CoV strain
Urbani (GB: AAP13441), MERS-CoV strain EMC/2012 (GB: YP_009047204), HKU4 (GB:
AGP04928), HKU5 (GB: AGP04943), IBV strain Beaudette (GB: ADP06471), and PDCoV
35Coronavirus Spike Protein and Tropism Changes
repeat regions (HR1 and HR2) and the transmembrane domains which are
well conserved among CoV species across different genera. In the MHV
and HKU1 S prefusion structures, the S
domain consists of multiple α-helical
segments and a three-stranded antiparallel β-sheet at the viral membrane-
proximal end. A 75 A
˚long central helix located immediately downstream
of the HR1 region stretches along the threefold axis over the entire length
of the S
trimer. The HR1 motif itself folds as four individual α-helices along
the length of the S
subunit, in contrast to the 120 A
˚long α-helix formed by
this region in postfusion structures (Duquerroy et al., 2005; Gao et al., 2013;
Xu et al., 2004). A 55 A
˚long helix upstream of the S
0cleavage site runs parallel
to and is packed against the central helix via hydrophobic interactions (Fig. 1C).
The fusion peptide forms a short helix of which the strictly conserved hydro-
phobic residues are buried in an interface with other elements of S
other class I fusion proteins, this conserved fusion peptide (FP1) is not directly
upstream of HR1 but located some 65 residues upstream of this region
(Fig. 1A). Intriguingly, a recent published report provided experimental evi-
dence for the existence of another fusion peptide (FP2) immediately upstream
of the HR1 region (Ou et al., 2016), that had been predicted earlier based on
the position, hydrophobicity profile and amino acid composition canonical for
classIviralfusionpeptides(Bosch and Rottier, 2008; Bosch et al., 2004;
Chambers et al., 1990). The HR2 region locates closely to the C-terminal
end of the S ectodomain, but it appeared to be disordered in both cryo-EM
structures and therefore its prefusion conformation remains unknown.
The metastable prefusion conformation of S
is locked by the cap formed
by the intertwined S
protomers. The distal tip of the S
trimer connects via
hydrophobic interactions with domains B. This distal tip of the S
consists of the C-terminal region of HR1 in the prefusion conformation,
while the entire HR1 rearranges to form a central three-helix coiled coil
in the postfusion structure (Duquerroy et al., 2005; Lu et al., 2014;
Supekar et al., 2004). Interactions between this region of the S
and domain B may therefore prevent premature conformational changes
resulting in the conversion of the prefusion S protein into the very stable
Fig. 2—Cont’dstrain USA/Ohio137/2014 (GB: AIB07807). PSI-BLAST analysis using the
NTR of the HCoV-NL63 S protein (residues 16–196) as a query detected two homologous
regions in the first 425 residues of the 229E-related bat coronavirus spike protein
(GB: ALK28765.1)—designated N1 (residues 32–213) and N2 (residues 246–422) with
32% and 35% amino acid sequence identity, respectively, suggesting a duplication
of the NTR. Spike proteins are drawn to scale and aligned at the position of the con-
served fusion peptide (FP1).
36 R.J.G. Hulswit et al.
postfusion structure. Also domains C and D of the betacoronavirus S
subunit and the linker region connecting domain A and B interact with
the surface of the adjacent S
protomer and may hence play a role in stabi-
lizing the prefusion S
trimer. Domain A appears to play a minor role in
this respect in view of its relatively small a surface area that interacts with
3. SPIKE–RECEPTOR INTERACTIONS
3.1 Different Domains Within S
May Act as RBD
Over the past decades, molecular studies on the CoV S glycoprotein have
shown that both the N-terminal region (NTR, domain A in β-CoV) and
the C-terminal region of S
(CTR, comprising domain B, C, and D in
β-CoV) can bind host receptors and hence function as RBDs (Fig. 2)
(Li, 2015). The CTR of alpha- and betacoronaviruses appears to bind
proteinaceous receptors exclusively. The α-CoV HCoV-229E, serotype
II feline CoV (FCoV), TGEV, and porcine respiratory coronavirus use
the human aminopeptidase N (APN) of their respective hosts as recep-
tors (Bonavia et al., 2003; Delmas et al., 1992; Reguera et al., 2012).
The HCoV-NL63 (α-CoV) and SARS-CoV (β-CoV) both utilize
angiotensin-converting enzyme 2 (ACE2) as a functional receptor (Li
et al., 2005b; Wu et al., 2009), whereas the β-CoVs MERS-CoV and
BtCoV-HKU4 recruit dipeptidyl peptidase-4 (DPP4) as a functional recep-
tor (Lu et al., 2013; Mou et al., 2013; Raj et al., 2013; Wang et al., 2014;
Yang et al., 2014).
The receptor-binding motifs (RBMs) in the S
CTRs of alpha- and
betacoronavirus spike proteins are presented on one or more loops exten-
ding from the β-sheet core structure. Within alpha- and betacoronavirus
genera the RBD core is structurally conserved yet the RBM(s) that deter-
mine receptor specificity may vary extensively. For instance, the CTR
of the α-CoVs PRCoV and HCoV-NL63 has a similar core structure
suggesting common evolutionary origin but diverged in their RBMs rec-
ruiting different receptors (APN and ACE2, respectively). A similar situa-
tion is seen for the CTRs of β-CoVs SARS-CoV and MERS-CoV that
bind ACE2 and DPP4, respectively (Li, 2015). Conversely, the CTRs of
the α-CoV HCoV-NL63 and β-CoV SARS-CoV both recognize ACE2,
yet via distinct molecular interactions (ACE2 recognition via three vs
one RBM, respectively), which suggested a convergent evolution path-
way for these viruses in recruiting the ACE2 receptor (Li, 2015). The core
37Coronavirus Spike Protein and Tropism Changes
structures of the CTRs in α- and β-CoVs provide a scaffold to present
RBMs from extending loop(s), which may accommodate facile recep-
tor switching by subtle alterations in or exchange of the RBMs via
Contrary to the CTR, the NTR appears to mainly bind glycans. The
NTR of the α-CoV TGEV and of the γ-CoV IBV S proteins binds to sialic
acids (Promkuntod et al., 2014; Schultze et al., 1996), while the NTR
of betacoronaviruses including BCoV and HCoV-OC43 was shown to
bind to O-acetylated sialic acids (K€unkel and Herrler, 1993; Peng
et al., 2012; Schultze et al., 1991; Vlasak et al., 1988). Only the NTR of
MHV (domain A) is known to interact with a protein receptor, being
mCEACAM1a (Peng et al., 2011), while lacking any detectable sialic acid-
binding activity (Langereis et al., 2010). However, as the NTR of MHV
displays the β-sandwich fold of the galectins, a family of sugar-binding pro-
teins, it probably has evolved from a sugar-binding domain (Li, 2012).
The presence of RBDs in different domains of the S protein that can bind
either proteinaceous or glycan receptors illustrates a functional modularity of
this glycoprotein in which different domains may fulfill the role of binding
to cellular attachment or entry receptors. The CoV S protein is thought to
have evolved from a more basic structure in which receptor recognition was
confined to the CTR within S
(Li, 2015). The observed deletions of the
NTR in some CoV species in nature are indicative of a less stringent require-
ment and integration of this domain with other regions of the spike trimer
compared to the more C-terminally located domains of S
and support a sce-
nario in which the NTR has been acquired at a later time point in CoV evo-
lutionary history. For example, the NTR of MHV, which displays a human
galectin-like fold, was suggested to originate from a cellular lectin acquired
early on in CoV evolution (Peng et al., 2011). Acquisition of glycan-binding
domains and fusion thereof to the ancestral S protein may have resulted in a
great extension of CoV host range and may have caused an increase in CoV
diversity. The general preference of the NTR and CTR to bind to, respec-
tively, glycan or protein receptors may be related to their arrangement in the
S protein trimer. In contrast to the CTR, which is located in the center of
the S trimer, the NTR is more distally oriented (Fig. 1B). As protein–glycan
interactions are often of low affinity, the more distal orientation of domain
A may allow multivalent receptor interactions, thereby increasing avidity.
Interestingly, some CoVs appear to have a dual receptor usage as they
may bind via their NTR and CTR to glycan and protein receptors, respec-
tively (Fig. 2).
38 R.J.G. Hulswit et al.
3.2 CoV Protein Receptor Preference
Although the number of currently known CoV receptors is limited, receptor
usage does not appear to be necessarily conserved between closely related
virus species such as HCoV-229E (APN) and HCoV-NL63 (ACE2),
whereas identical receptors (ACE2) can be targeted by virus species from
different genera such as HCoV-NL63 and SARS-CoV. It seems that CoVs
prefer certain types of host proteins as their entry receptor, with three out of
four of the so far identified proteinaceous receptors being ectopeptidases
(APN, ACE2, and DPP4), although enzymatic activity of these proteins
was shown not to be required for infection by their respective viruses
(Bosch et al., 2014). Possibly, the localization to certain membrane micro-
domains and efficient internalization of two of these proteins in polarized
cells (APN and DPP4) may contribute to their suitability to function as entry
¨t-Slimane et al., 2009). In the case of MERS-CoV, the region
of DPP4 that is bound by the S protein coincides with the binding site for its
physiological ligand adenosine deaminase (Raj et al., 2014). Employment of
conserved epitopes such as these may also contribute to the cross-species
transmission potential of viruses (Bosch et al., 2014), as is exemplified by
MERS-CoV being able to use goat, camelid, cow, sheep, horse, pig, mon-
key, marmoset, and human DPP4 as entry receptor (Barlan et al., 2014;
Eckerle et al., 2014; Falzarano et al., 2014; M€uller et al., 2012; van
Doremalen et al., 2014). Similarly, this may apply for the ability of feline,
canine, porcine, and human CoVs to use fAPN as entry receptor, at least
in vitro (Tresnan et al., 1996).
4. S PROTEIN PROTEOLYTIC CLEAVAGE AND
Coronavirus entry is a tightly regulated process that appears to be
orchestrated by multiple triggers that include receptor binding and proteo-
lytic processing of the S protein and that ultimately results in virus-cell
fusion. It is initiated by virion attachment mediated through interaction
of either the NTR or CTR (or both) in the S
subunit of the spike protein
with host receptors. Upon attachment, the virus is taken up via receptor-
mediated endocytosis by clathrin- or caveolin-dependent pathways
(Burkard et al., 2014; Eifart et al., 2007; Inoue et al., 2007; Nomura
et al., 2004) although other entry routes have also been reported (Wang
et al., 2008). Prior to and/or during endocytic uptake the CoV S protein
39Coronavirus Spike Protein and Tropism Changes
is proteolytically processed. The spike protein may contain two proteolytic
cleavage sites. One of the cleavage sites is located at the boundary between
cleavage site), while the other cleavage site is
located immediately upstream of the first fusion peptide (S
Although not irrevocably proven, it is expected that all CoVs depend on
proteolytic cleavage on or close to S
0for fusion to occur. Virus-cell fusion
thus not only critically depends on the conformational changes following
spike–receptor engagement, and perhaps on acidification of endosomal ves-
icles (Eifart et al., 2007; Matsuyama and Taguchi, 2009; Zelus et al., 2003),
but also on proteolytic activation of the S protein by proteases along the
endocytic route (Burkard et al., 2014; Simmons et al., 2005). Indeed, inhi-
bition of intracellular proteases has been shown to block virus entry and
virus-cell fusion (Burkard et al., 2014; Frana et al., 1985; Simmons et al.,
2005; Yamada and Liu, 2009). The specific proteolytic cleavage require-
ments of the S protein at the S
boundary and particularly at the S
may furthermore determine the intracellular site of fusion (Burkard et al.,
2014). In agreement herewith, it has become evident that the protease
expression profile of host cells may form an additional determinant of the
host cell tropism of coronaviruses (Millet and Whittaker, 2015).
Analysis of the CoV S prefusion conformation suggests that relocation
(or shedding) of the S
subunits that cap the S
subunit is a prerequisite
for the conformational changes in S
that ultimately result in fusion. Shed-
ding of S
probably requires receptor binding as well as proteolytic
processing at S
. The cryo-EM structure indicates that the S
lytic cleavage site is accessible to proteases prior to spike–receptor interac-
tion, and depending on the particular cleavage site present may already be
processed in the cell in which the virions are produced. As indicated earlier,
the conformational changes in the S protein that result in virus-cell fusion
most likely also require cleavage at the S
0site immediately upstream of
the fusion peptide. Interestingly, the S
0cleavage site is located within an
α-helix exposed on the prefusion S structure which prevents efficient pro-
teolytic cleavage (Robertson et al., 2016). This indicates the necessity for
preceding conformational changes induced by receptor binding and subse-
quent shedding of S
, upon which the secondary structure of the S
transforms into a cleavable flexible loop. Following proteolytic cleavage
activation at the S
0site, hydrophobic interactions between the fusion pep-
tide and the adjacent S
helices are disturbed which allows the four α-helices
and the connecting regions that make up the HR1 region in the prefusion
S protein to refold into a long trimeric coiled coil (Fig. 1D). This coiled coil
40 R.J.G. Hulswit et al.
forms an N-terminal extension of the central helix projecting the fusion
peptide(s) toward the target membrane. Successively, the fusion peptide(s)
will be inserted into the limiting membrane of the host cell endocytic com-
partment. Next, as a consequence of S
rearrangements, the two HR regions
will interact to form an antiparallel energetically stable six-helix bundle
(Bosch et al., 2003, 2004), enabling the close apposition and subsequent
fusion of the viral and host lipid bilayers.
5. TROPISM CHANGES ASSOCIATED WITH S PROTEIN
Changes in the S protein may result in an altered host, tissue, or cel-
lular tropism of the virus. This is clearly exemplified by genomic recombi-
nation events that result in exchange of (part of ) the S protein and in a
concomitant change in tropism. The propensity of CoVs to undergo homol-
ogous genomic recombination has been exploited for the genetic manipu-
lation of these viruses (de Haan et al., 2008; Haijema et al., 2003; Kuo et al.,
2000). To this end, interspecies chimeric coronaviruses were generated,
which carried the spike ectodomain of another CoV and which could be
selected based on their altered requirement for an entry receptor. Exchange
of S protein genes may also occur in vivo, resulting in altered tropism as is
illustrated by the occurrence of serotype II feline infectious peritonitis virus
(FIPV). This virus results from a naturally occurring recombination event
between feline and canine CoVs (CCoVs) in which the feline virus acquires
a CCoV spike gene (Herrewegh et al., 1995; Terada et al., 2014). As a result
of the acquisition of this new S protein, the rather harmless enteric feline
CoV (FECV) turns into a systemically replicating and deadly FIPV. As
FECV has a strict feline tropism (Myrrha et al., 2011), while CCoV has been
shown to infect feline cells (Levis et al., 1995), it is likely that serotype II
FIPVs arise in cats coinfected with serotype I FECV and CCoV. Further-
more, as different recombination sites have been observed for each serotype
II FIPV, while serotype II FECVs have not been observed, it appears that
serotype II FIPVs exclusively result of reoccurring recombination events
(Terada et al., 2014). In addition to these feline–CCoV recombinants, a chi-
meric porcine coronavirus with a TGEV backbone and a spike of the por-
cine epidemic diarrhea virus (PEDV) was recently isolated from swine fecal
samples in Italy and Germany, likely also resulting from a recombination
event (Akimkin et al., 2016; Boniotti et al., 2016). Moreover, the α-CoV
HKU2 BtCoV probably resulted from genomic recombination as it encodes
41Coronavirus Spike Protein and Tropism Changes
an S protein that resembles a betacoronavirus S protein except for its N-ter-
minal region that is similar to that of alphacoronaviruses (Lau et al., 2007).
Thus, such genomic recombination events are not necessarily restricted to
occur between viruses of the same genus.
Receptor Interactions Determining Tropism
Several changes in the amino-terminal domain of S
have been associated
with changes in the tropism of the virus. For example, for several α-CoVs,
loss of NTR of the S protein appears to be accompanied with a loss of
enteric tropism. While the porcine CoV TGEV displays a tropism for both
the gastrointestinal and respiratory tract, the closely related PRCoV, which
lacks the sialic acid-binding N-terminal region (Krempl et al., 1997), only
replicates in the respiratory tract. The loss of sialic acid-binding activity
by four-amino acid changes in the NTR of its S protein resulted in an
almost complete loss of enteric tropism (Krempl et al., 1997). Similar to
TGEV, enteric serotype I FCoVs also have been reported to bind to sialic
acids (Desmarets et al., 2014). Large deletions within the S
corresponding to the N-terminal region have been found in variants of
the systemically replicating FIPV (strains UU16, UU21, and C3663) after
intrahost emergence from enteric FECV (Chang et al., 2012; Terada
et al., 2012). Also FIPVs seem to have lost the ability to replicate in the
enteric tract (Pedersen, 2014). Clinical isolates of human coronavirus
229E as well as of the related alpaca coronavirus, both of which cause respi-
ratory infections, encode relatively short spike proteins that lack the NTR
(Crossley et al., 2012; Farsani et al., 2012). In contrast, closely related bat
coronaviruses with intestinal tropism contain S proteins with a NTR or
sometimes even two copies of the NTR (Corman et al., 2015)(Fig. 2).
Overall, these observations suggest that the alphacoronavirus spike
NTR—in particular its sialic acid-binding activity—may contribute to
the enteric tropism of these alphacoronaviruses, while it is not required
for replication in the respiratory tract or in other extraintestinal organs. It
has been hypothesized that the sialic acid-binding activity of the spike pro-
tein can allow virus binding to (i) soluble sialoglycoconjugates that may pro-
tect the virus from hostile conditions in the stomach or (ii) to mucins that
may prevent the loss of viruses by intestinal peristalsis and allow the virus to
pass the thick mucus barrier, thereby gaining access to the intestinal cells to
initiate infection (Schwegmann-Wessels et al., 2003).
42 R.J.G. Hulswit et al.
Besides deletions of entire domains of the S protein, more subtle changes
consisting of amino acid substitutions in S
NTR may also suffice to alter the
virus’ tropism. For example, MHV variants have been observed that
acquired the ability to use the human homologue of their murine
CEACAM1a receptor to enter cells as a result of mutations in their RBD
that is located in S
NTR (Baric et al., 1999).
As the CTR of the S
subunit contains the protein RBD for most CoVs, also
mutations in this part of S have been associated with changes in the virus’
tropism. Perhaps the most well-known example of viral cross-species trans-
mission involves the SARS-CoV. Studies support a transmission model in
which a SARS-like CoV was transmitted from Rhinolophus bats to palm
civets, which subsequently transmitted the palm civet-adapted virus to
humans at local food markets in southern China (Li et al., 2006). According
to this model, SARS-like viruses adapted to both the palm civet and human
host, which was reflected in the rapid viral evolution observed for these
viruses within these species (Song et al., 2005). Two-amino acid substitu-
tions within the RBD were elucidated that are of relevance for binding
to the ACE2 proteins of palm civets and humans (Li et al., 2005b, 2006;
Qu et al., 2005). From these studies it appears that due to strong conserva-
tion of ACE2 between mammalian species only a few amino acid alterations
within the RBD are needed to change coronavirus host species tropism.
Indeed serial passage of SARS-CoVs in vitro or in vivo can rapidly lead
to adaptation to new host species (Roberts et al., 2007). SARS-like viruses
isolated from bats displayed major differences including a deletion in the
ACE2 RBM compared to human SARS-CoV (Drexler et al., 2010; Ren
et al., 2008) and as a consequence were unable of using human ACE2 as
an entry receptor (Becker et al., 2008). However, recently a novel SARS-
like BtCoV was identified, which could use ACE2 of Rhinolophus bats, palm
civets as well as of humans as a functional receptor (Ge et al., 2013). These
findings not only provide further evidence that bats are indeed the natural
reservoir for SARS-like CoVs, but also that these bat coronaviruses can
directly include human ACE2 in their receptor repertoire. The detection
of sequences of SARS-CoV-like viruses in palm civets and raccoon dogs
(Guan et al., 2003; Tu et al., 2004) therefore probably reflects the unusually
wide host range of these viruses. A similar promiscuous receptor usage is also
observed for MERS-CoV which binds to DPP4 of many species (Barlan
43Coronavirus Spike Protein and Tropism Changes
et al., 2014; Eckerle et al., 2014; Falzarano et al., 2014; M€uller et al., 2012;
van Doremalen et al., 2014) as indicated earlier.
Just as SARS like and MERS-CoVs are able to use entry receptors of
different host species, also several α-CoVs display promiscuity to ortho-
logous receptors. For example, the feline APN molecule can be used as a
receptor by feline (serotype II FIPV), canine (CCoV), porcine (TGEV),
and human (HCoV-229E) α-CoVs in cell culture (Tresnan and Holmes,
1998; Tresnan et al., 1996). Conversely, serotype II FIPV can only enter
cells expressing feline APN (Tresnan and Holmes, 1998). The ability of
TGEV and CCoV to use feline APN as a receptor probably results from
strong conservation of the viral-binding motif (VBM) among APN
orthologs in combination with the RBDs recognizing APN in a similar
fashion (Reguera et al., 2012). Though recruiting the same receptor,
HCoV-229E binds another domain within APN, which apparently is also
conserved in feline APN (Kolb et al., 1997; Tusell et al., 2007). Conserva-
tion of the VBM obviates the need for large adaptations within the RBD of
these viruses to orthologous receptors allowing more facile cross-species
Other mutations in the S
CTR associated with altered tropism have
been described for the β-CoV MHV. Similar to the humanized
CEACAM1a-recognizing MHV variant, serial passaging of virus-infected
cells resulted in the selection of viruses with an extended host range, which
were subsequently shown to be able to enter cells in a heparan sulfate-
dependent and CEACAM1a-independent manner (de Haan et al., 2005;
Schickli et al., 1997). Two sets of mutations in the S protein were shown
to be critically required for this phenotype, both of which resulted in the
occurrence of multibasic heparan sulfate-binding sites. While one heparan
sulfate-binding site was located in the S
subunit immediately upstream of
the fusion peptide, the other was located in the S
CTR. The presence
of this latter, but not of the former, domain resulted in MHV that depended
on both heparan sulfate and CEACAM1a for entry. Additional introduction
of the second heparan sulfate-binding site enabled the virus to become
mCEACAM1a independent (de Haan et al., 2006). In addition, a mutation
of the HVR of S
may affect CoV tropism as was demonstrated for the MHV
strain JHM (MHV-JHM). The spike protein of MHV-JHM may induce
receptor-independent fusion (Gallagher et al., 1992, 1993). However, dele-
tion of residues in HVR of MHV-JHM resulted in the spike protein being
entirely dependent on CEACAM1a binding for fusion (Dalziel et al., 1986;
Gallagher and Buchmeier, 2001; Phillips and Weiss, 2001).
44 R.J.G. Hulswit et al.
5.2 Changes in Proteolytic Cleavage Site and Other S
Mutations Associated with Altered Tropism
5.2.1 Changes in Proteolytic Cleavage Sites
Although the S
subunit does not appear to contain any RBDs, several
mutations in this subunit have been associated with changes in the virus’ tro-
pism. Some of these changes affect the cleavage sites in the S protein that are
located at the S
boundary or immediately upstream of the fusion peptide
0cleavage site). As these cleavages appear to be essential for virus-cell
fusion, the availability of host proteases to process the S protein is of critical
importance for the virus’ tropism. The importance of S protein cleavage at
boundary for the tropism of the virus is exemplified by the BtCoV
HKU4, which is closely related to the MERS-CoV. Although domain B of
the HKU4 S protein can interact with both bat and human DPP4, it is only
in the context of bat cells, but not human cells, that the virus can utilize these
molecules as entry receptors (Yang et al., 2014). In contrast, MERS-CoV
can enter cells of human and bat origin via both DPP4 orthologues. This
difference results from host restriction factors at the level of proteolytic
cleavage activation. Two-amino acid substitutions (S746R and N762A)
in the S
boundary of the S protein were shown to be crucial for the
adaptation of bat MERS-like CoV to the proteolytic environment of the
human cells (Yang et al., 2015).
Although probably not directly responsible for the tropism change asso-
ciated with the enterically replicating FECV evolving into the systemically
replicating FIPV, loss of a furin cleavage site at S
junction is observed in
the majority of the FIPVs, whereas this furin cleavage site is strictly con-
served in the parental FECV strains (Licitra et al., 2013). Apparently, con-
servation of this furin cleavage site is not required for efficient systemic
replication. However, as FIPV is generally not found in the feces of cats,
it may well be that loss of the furin cleavage site at S
—as well as muta-
tions in other parts of the genome, such as the accessory genes—may prevent
efficient replication of FIPV in the enteric tracts.
Besides the influence of the S
cleavage site, virus tropism may also
depend on the S
0cleavage site upstream of FP1. In contrast to wild-type
MHV strain A59, a recombinant MHV carrying a furin cleavage site at this
position was shown to no longer depend on lysosomal proteases for efficient
entry to occur (Burkard et al., 2014). As a consequence, this virus was able to
infect cells in which trafficking to lysosomes was inhibited. Cleavage at the
0site may also be important for the tropism of PEDV, which causes major
damage to the biofood industry in Asia and the Americas (Lee, 2015;
45Coronavirus Spike Protein and Tropism Changes
Song et al., 2015). PEDV replication in cell culture is strictly dependent
on trypsin-like proteases, a requirement which is expected to limit its tro-
pism in vivo to the enteric tract. The trypsin dependency of PEDV entry
was shown, however, to be lifted after introduction of a furin cleavage
site at the S
0cleavage site by a single-amino acid substitution. Such muta-
tions may potentially affect the spread of this virus in the pig by allowing it to
replicate in nonenteric tissues in the absence of trypsin-like proteases
(Li et al., 2015).
5.2.2 Other S
Mutations Associated with Altered Tropism
Mutations in other parts of the S
subunit than those affecting the pro-
teolytic cleavage sites may also influence the tropism of different CoVs.
Several studies report a correlation between mutations in the HR1 region
of FCoVs and the conversion of FECV into FIPV (Bank-Wolf et al.,
2014; Desmarets et al., 2016; Lewis et al., 2015). Such a correlation
appeared even more convincing for mutations found in the recently
identified FP2 (Chang et al., 2012; Ou et al., 2016). While these corre-
lations suggest an important role for the S protein in the transition of
FECV into FIPV, the causal relationship between these mutations in
S and FIP remains to be determined. It is plausible, however, that such
mutations may play a role in the acquired ability of FIPVs to infect mac-
rophages. Indeed, for serotype II FCoV, the ability to replicate in
macrophages was shown to be determined by residues located in the
C-terminal part of the S
subunit, although the responsible residues were
not identified (Rottier et al., 2005).
Also for other CoVs, mutations in the S
subunit have been linked to
changes in the virus’ tropism. A serially passaged MHV-A59 virus was
shown to obtain mutations (M936V, P939L, F948L, and S949I) in and
adjacent to the HR1 region which conveyed host range expansion of the
mutant virus to normally nonpermissive mammalian cell types in vitro
(Baric et al., 1999; McRoy and Baric, 2008). Contrary, Krueger et al.
reported three mutations in the S
subunit of MHV-JHM (V870A located
upstream of the S
0cleavage site and A994V and A1046V located in the
HR1 region) all of which reduced the CEACAM1a-independent
fusogenicity of this virus (Krueger et al., 2001). Many studies on MHV-
JHM point to a crucial role of a leucine at amino acid position 1114 in
S protein fusogenicity. The MHV S cryo-EM structure demonstrates
that the L1114 residue is located in the central helix and contributes to inter-
protomer interactions. A L1114F substitution in the MHV-JHM S protein
46 R.J.G. Hulswit et al.
was observed in a mutant strain of JHM and correlated with an increased
stability and the loss of the ability to induce CEACAM1a-
independent fusion (Taguchi and Matsuyama, 2002), while a substitution
of the same residue to an Arg (L1114R) reduced the neurotropism of this
virus (Tsai et al., 2003). Mutants resistant to a monoclonal antibody
(Wang et al., 1992) and soluble receptor (Saeki et al., 1997) also correlate
with substitutions at this specific residue, illustrating the importance of this
residue in S fusogenicity. For the MERS-CoV, mutations in HR1 have
been identified that are thought to be associated with its adaptive evolution
(Forni et al., 2015). Among these sites, position 1060 is particularly interest-
ing, as it appears to correspond to substitutions found in MHV and IBV that
modify the tropism of these viruses (MHV: E1035D; IBV: L857F; Navas-
Martin et al., 2005; Yamada et al., 2009). Substitution E1035D in HR1 of
MHV was shown to restore the hepatotropism of an otherwise non-
hepatotropic MHV, the latter resulting from mutations in the S
and the S
cleavage site. These studies collectively indicate that mutations
in and close to the HR regions may affect CoV tropism, possibly by affecting
the metastability and consequently fusogenicity of the S protein and/or the
formation of the postfusion six-helix bundle.
6. CONCLUDING REMARKS
It appears that changes in the S protein associated with altered tropism
can be found in several regions of the spike protein. These regions obviously
include the NTR and CTR of S
that are involved in the interaction with
attachment and/or entry receptors. Substitutions within the S
convey an altered viral tropism by adaptation of the virus to new or
orthologous entry receptors. In addition, the S protein cleavage sites are
important for host tropism as the processing of these sites by host proteases
will critically affect the removal of the S
-mediated locking of the S
prefusion conformation by shedding of S
cleavage site) and the
release of the fusion peptide(s) (S
0cleavage site). Finally, changes in S
ticularly in the HR regions) may compensate for yet suboptimal spike bind-
ing to orthologous receptors by which low relative affinity interactions
suffice to induce the required conformational changes of the S protein that
ultimately result in the formation of the postfusion six-helix bundle and
The observation that the different domains of the S protein all contribute
to the tropism of CoVs is indicative of a coordinated interplay between these
47Coronavirus Spike Protein and Tropism Changes
domains. This interplay has also been inferred from several studies, which
reported changes in one S protein subunit often to be accompanied by adap-
tations in the other subunit (Saeki et al., 1997; Wang et al., 1992). In addi-
tion, the interplay between S
has also been shown to be important
for changes in the tropism of the virus as indicated earlier (de Haan et al.,
2006; Navas-Martin et al., 2005). The recently published cryo-EM struc-
tures of CoV spike proteins (Kirchdoerfer et al., 2016; Walls et al., 2016)
now provide structural evidence for the complex interplay between the sub-
units and domains of the S protein.
From all these studies, a picture arises in which the S protein is progres-
sively destabilized through receptor engagement and proteolytic activation.
In this process the S
subunits serve as a safety pin that stabilizes the fusogenic
trimer. The safety pin is discharged upon interaction with a specific recep-
tor and processing by host cell proteases and thereby gives way to confor-
mational changes of the instable S
subunit. Subsequent release of the
fusion peptide may resemble the pulling of the trigger which inevitably
results in fusion of viral and host membranes through interaction of the hep-
tad repeats regions.
Based on the presented data we propose a model in which the ability of a
CoV to cross the host species barrier is critically dependent on the interplay
between the different regions of the S proteins. In this model, the probable
low affinity of the S
RBD for a novel receptor must be compensated by
sufficiently low S
metastability, which depends on both proteolytic cleav-
age of the S protein and the S
interprotomer interactions. These required
S protein characteristics may be generated during naturally occurring
quasispecies variation and may result in the ability of the virus to replicate
in and adapt to a new host.
This study is supported by TOP Project Grant (91213066) funded by ZonMW and as part of
the Zoonotic Anticipation and Preparedness Initiative (ZAPI project; IMI Grant Agreement
No. 115760), with the assistance and financial support of IMI and the European Commission.
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