Genetic analysis of the SARS-coronavirus spike glycoprotein functional domains involved in cell-surface expression and cell-to-cell fusion.

Abstract

The SARS-coronavirus (SARS-CoV) is the etiological agent of severe acute respiratory syndrome (SARS). The SARS-CoV spike (S) glycoprotein mediates membrane fusion events during virus entry and virus-induced cell-to-cell fusion. To delineate functional domains of the SARS-CoV S glycoprotein, single point mutations, cluster-to-lysine and cluster-to-alanine mutations, as well as carboxyl-terminal truncations were investigated in transient expression experiments. Mutagenesis of either the coiled-coil domain of the S glycoprotein amino terminal heptad repeat, the predicted fusion peptide, or an adjacent but distinct region, severely compromised S-mediated cell-to-cell fusion, while intracellular transport and cell-surface expression were not adversely affected. Surprisingly, a carboxyl-terminal truncation of 17 amino acids substantially increased S glycoprotein-mediated cell-to-cell fusion suggesting that the terminal 17 amino acids regulated the S fusogenic properties. In contrast, truncation of 26 or 39 amino acids eliminating either one or both of the two endodomain cysteine-rich motifs, respectively, inhibited cell fusion in comparison to the wild-type S. The 17 and 26 amino-acid deletions did not adversely affect S cell-surface expression, while the 39 amino-acid truncation inhibited S cell-surface expression suggesting that the membrane proximal cysteine-rich motif plays an essential role in S cell-surface expression. Mutagenesis of the acidic amino-acid cluster in the carboxyl terminus of the S glycoprotein as well as modification of a predicted phosphorylation site within the acidic cluster revealed that this amino-acid motif may play a functional role in the retention of S at cell surfaces. This genetic analysis reveals that the SARS-CoV S glycoprotein contains extracellular domains that regulate cell fusion as well as distinct endodomains that function in intracellular transport, cell-surface expression, and cell fusion.

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Genetic analysis of the SARS-coronavirus spike glycoprotein functional
domains involved in cell-surface expression and cell-to-cell fusion
Chad M. Petita,b, Jeffrey M. Melancona,b, Vladimir N. Chouljenkoa,b, Robin Colgrovec,
Michael Farzand, David M. Knipec, K.G. Kousoulasa,b,*
aDivision of Biotechnology and Molecular Medicine (BIOMMED), School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803, USA
bDepartment of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803, USA
cDepartment of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115, USA
dPartners AIDS Research Center, Brigham and Women’s Hospital, Department of Medicine (Microbiology and Molecular Genetics), Harvard Medical School,
Boston, MA 02115, USA
Received 4 May 2005; returned to author for revision 10 June 2005; accepted 28 June 2005
Available online 15 August 2005
Abstract
The SARS-coronavirus (SARS-CoV) is the etiological agent of severe acute respiratory syndrome (SARS). The SARS-CoV spike (S)
glycoprotein mediates membrane fusion events during virus entry and virus-induced cell-to-cell fusion. To delineate functional domains of
the SARS-CoV S glycoprotein, single point mutations, cluster-to-lysine and cluster-to-alanine mutations, as well as carboxyl-terminal
truncations were investigated in transient expression experiments. Mutagenesis of either the coiled-coil domain of the S glycoprotein amino
terminal heptad repeat, the predicted fusion peptide, or an adjacent but distinct region, severely compromised S-mediated cell-to-cell fusion,
while intracellular transport and cell-surface expression were not adversely affected. Surprisingly, a carboxyl-terminal truncation of 17 amino
acids substantially increased S glycoprotein-mediated cell-to-cell fusion suggesting that the terminal 17 amino acids regulated the S fusogenic
properties. In contrast, truncation of 26 or 39 amino acids eliminating either one or both of the two endodomain cysteine-rich motifs,
respectively, inhibited cell fusion in comparison to the wild-type S. The 17 and 26 amino-acid deletions did not adversely affect S cell-surface
expression, while the 39 amino-acid truncation inhibited S cell-surface expression suggesting that the membrane proximal cysteine-rich motif
plays an essential role in S cell-surface expression. Mutagenesis of the acidic amino-acid cluster in the carboxyl terminus of the S
glycoprotein as well as modification of a predicted phosphorylation site within the acidic cluster revealed that this amino-acid motif may play
a functional role in the retention of S at cell surfaces. This genetic analysis reveals that the SARS-CoV S glycoprotein contains extracellular
domains that regulate cell fusion as well as distinct endodomains that function in intracellular transport, cell-surface expression, and cell
fusion.
D 2005 Elsevier Inc. All rights reserved.
Keywords: SARS; Coronavirus; Spike; Heptad repeat; Fusion
Introduction
An outbreak of atypical pneumonia, termed severe acute
respiratory syndrome (SARS), appeared in the Guangdong
Province of southern China in November, 2002. The
mortality rates of the disease reached as high as 15% in
some age groups (Anand et al., 2003). The etiological agent
of the disease was found to be a novel coronavirus (SARS-
CoV), which was first isolated from infected individuals by
propagation of the virus on Vero E6 cells (Drosten et al.,
2003; Ksiazek et al., 2003; Peiris et al., 2003). Analysis of
the viral genome has demonstrated that the SARS-CoV is
phylogenetically divergent from the three known antigenic
groups of coronaviruses (Drosten et al., 2003; Ksiazek et al.,
0042-6822/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.virol.2005.06.046
* Corresponding author. Division of Biotechnology and Molecular
Medicine, School of Veterinary Medicine, Louisiana State University,
Baton Rouge, LA 70803, USA.
E-mail address: vtgusk@lsu.edu (K.G. Kousoulas).
Virology 341 (2005) 215 – 230
www.elsevier.com/locate/yviro

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2003). Analysis of the polymerase gene alone, however, has
indicated that the SARS-CoV may be an early off-shoot
from the group 2 coronaviruses (Snijder et al., 2003).
The coronaviruses are the largest of the enveloped RNA
viruses with a positive-stranded RNA genome of 28 to 32
kb (Holmes, 2003). Coronaviruses possess a wide host
range, capable of infecting mammalian and avian species.
All identified coronaviruses have a common group of
indispensable genes that encode nonstructural proteins
including the RNA replicase gene open reading frame
(ORF) 1ab and the structural proteins nucleocapsid (N),
membrane protein (M), envelope protein (E), and spike
glycoprotein (S), which are assembled into virus particles. A
hemagglutinin-esterase (HE) protein is also encoded by
some coronaviruses. Distributed among the major viral
genes are a series of ORFs that are specific to the different
coronavirus groups. Functions of the majority of these
ORFs have not been determined.
The SARS spike glycoprotein, a 1255-amino-acid type I
membrane glycoprotein (Rota et al., 2003), is the major
protein present in the viral membrane forming the typical
spike structure found on all coronavirions. The S glyco-
protein is primarily responsible for entry of all coronavi-
ruses into susceptible cells through binding to specific
receptors on cells and mediating subsequent virus-cell
fusion (Cavanagh, 1995). The S glycoprotein specified by
mouse hepatitis virus (MHV) is cleaved into S1 and S2
subunits, although cleavage is not necessarily required for
virus-cell fusion (Bos et al., 1997; Gombold et al., 1993;
Stauber et al., 1993). Similarly, the SARS-CoV S glyco-
protein seems to be cleaved into S1 and S2 subunits in Vero-
E6-infected cells (Wu et al., 2004), while it is not known
whether this cleavage affects S-mediated cell fusion. The
SARS-CoV receptor has been recently identified as the
angiotensin-converting enzyme 2 (ACE2) (Li et al., 2003).
Although the exact mechanism by which the SARS-CoV
enters the host cell has not been elucidated, it is most likely
similar to other coronaviruses. Upon receptor binding at the
cell membrane, the S glycoprotein is thought to undergo a
dramatic conformational change causing exposure of a
hydrophobic fusion peptide, which is subsequently inserted
into cellular membranes. This conformational change of the
S glycoprotein causes close apposition followed by fusion
of the viral and cellular membranes resulting in entry of the
virion nucleocapsids into cells (Eckert and Kim, 2001; Tsai
et al., 2003; Zelus et al., 2003). This series of S-mediated
virus entry events is similar to other class I virus fusion
proteins (Baker et al., 1999; Melikyan et al., 2000; Russell
et al., 2001).
Heptad repeat (HR) regions, a sequence motif character-
istic of coiled-coils, appear to be a common motif in many
viral and cellular fusion proteins (Skehel and Wiley, 1998).
These coiled-coil regions allow the protein to fold back
upon itself as a prerequisite step to initiating the membrane
fusion event. There are usually two HR regions: an N
terminal HR region adjacent to the fusion peptide and a C-
terminal HR region close to the transmembrane region of
the protein. Within the HR segments, the first amino acid (a)
and fourth amino acid (d) are typically hydrophobic amino
acids that play a vital role in maintaining coiled-coil
interactions. Based on structural similarities, two classes
of viral fusion proteins have been established. Class I viral
fusion proteins contain two heptad repeat regions and an N-
terminal or N-proximal fusion peptide. Class II viral fusion
proteins lack heptad repeat regions and contain an internal
fusion peptide (Lescar et al., 2001). The MHV S glyco-
protein, which is similar to other coronavirus S glycopro-
teins, is a class I membrane protein that is transported to the
plasma membrane after being synthesized in the endoplas-
mic reticulum (Bosch et al., 2003). Typically, the ectodo-
mains of the S2 subunits of coronaviruses contain two
regions with a 4, 3 hydrophobic (heptad) repeat the first
being adjacent to the fusion peptide and the other being in
close proximity to the transmembrane region (de Groot
et al., 1987).
In the present study, we investigated the role of several
predicted structural and functional domains of the SARS
spike glycoprotein by introducing specific alterations within
selected S glycoprotein regions. The results show that the
SARS-CoV S glycoprotein conforms to the general struc-
ture and function relationships that have been elucidated for
other coronaviruses, most notably the MHV (Chang et al.,
2000; Ye et al., 2004). However, in contrast to the MHV
endodomain, the carboxyl terminus of the SARS-CoV S
glycoprotein contains multiple non-overlapping domains
that function in intracellular transport, cell-surface expres-
sion, and endocytosis as well as in S glycoprotein-mediated
cell-to-cell fusion.
Results
Genetic analysis of S glycoprotein functional domains
To delineate domains of the S glycoprotein that function
in membrane fusion, intracellular transport, and cell-surface
expression, two types of mutations were introduced within
the S gene: (a) mutations were introduced within and
adjacent to the predicted amino terminal heptad repeat
(HR1) core and the predicted fusion peptide, which are
known to play important roles in membrane fusion (Bosch
et al., 2004; Bosch et al., 2003; Ingallinella et al., 2004;
Tripet et al., 2004); (b) mutations and carboxyl-terminal
truncations of the S glycoprotein were engineered to
delineate S cytoplasmic domains that function in glycopro-
tein synthesis, intracellular transport, and membrane fusion
(Fig. 1). Specifically, to investigate the amino-acid require-
ments of the HR1 of the SARS-CoV S glycoprotein, the a
and d amino-acid positions L(898) and N(901) were both
replaced by lysine residues in the cluster mutation CL2,
effectively collapsing the predicted a-helical structure at the
amino terminal terminus of the HR. This amino-acid
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Fig. 1. Schematic diagram of the SARS-CoV S glycoprotein. (A) Graphical representation of the S glycoprotein showing the approximate location of the
cluster to lysine mutations CL1–CL5 relative to known and indicated functional domains. (B) Shown on the top of the diagram is a graphical representation of
the SARS-CoV S glycoprotein. The predicted fusion peptide and the HR1 region are enlarged below to show the sets of amino acids replaced by lysines in the
cluster mutations. The heptad repeat a and d positions are labeled above the corresponding amino acid. Amino acids changed to lysine are demarcated by
arrows with the name of that particular mutation shown in brackets. (C) Amino-acid sequences of the carboxyl termini of the truncation and acidic cluster
associated mutations. Cysteine clusters (CRM1 and CRM2) are denoted by underlined italicized text as well as a bracket encompassing their respective regions.
The charged cluster is bracketed over the region. Amino acids mutated to alanines for the CL6 and CL7 cluster mutations are in bold.
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sequence is thought to align with the L(1184) of HR2 in the
formation of the HR1/HR2 core complex (Xu et al., 2004).
In addition, cluster-to-lysine mutations CL3 and CL4
replaced the a and d positions within the HR1 region (Fig.
1B). The CL5 cluster mutation was placed adjacent to the
HR1 region to investigate whether regions proximal to HR1
had any effect on S-mediated cell fusion. Similarly, the role
of the a and d positions within the predicted fusion peptide,
located immediately proximal to the N terminus of HR1,
was investigated by constructing the CL1 cluster mutation
(Fig. 1B).
It has been shown for other viral class I fusion proteins
that the carboxyl terminus plays a regulatory role in
membrane fusion (Bagai and Lamb, 1996; Sergel and
Morrison, 1995; Seth et al., 2003; Tong et al., 2002; Yao
and Compans, 1995). Specifically, for coronaviruses, the
MHV S glycoprotein endodomain has been shown to
contain charged-rich and cysteine-rich regions, which are
critical for fusion of infected cells (Bos et al., 1995; Chang
et al., 2000; Ye et al., 2004). The carboxyl-terminal portion
of the S glycoprotein contains a consensus acidic amino-
acid cluster with a motif that has been predicted by the
NetPhos 2.0 software to be phosphorylated (Blom et al.,
1999). To investigate the potential role of the acidic amino-
acid cluster in synthesis, transport, and cell fusion, serial
truncations of S were constructed. The acidic cluster was
specifically targeted by mutagenizing the predicted phos-
phorylation site embedded within the acidic cluster as well
as by replacing acidic residues of the acidic cluster with
alanine residues. In addition, carboxyl-terminal truncations
of 8, 17, 26, and 41 amino acids were engineered by
insertion of stop codons within the S glycoprotein gene. The
8 aa truncation (T1247) was designed to bring the predicted
charged cluster DEDDSE proximal to the carboxyl terminus
of the mutated S glycoprotein (Fig. 1C). Similarly, the 17 aa
truncation (T1238) was designed to delete the DEDDSE
acidic cluster. The SARS-CoV S glycoprotein endodomain
contains two cysteine residue clusters, a CCMTSCCSC
(CRM1) cluster immediately adjacent to the membrane and
a CSCGSCC (CRM2) downstream of the first cluster. To
address the role of these domains in S glycoprotein-
mediated cell-to-cell fusion, the 26 aa truncation (T1229)
was designed to delete the CRM2 domain, while the 41 aa
truncation (T1214) deleted both the CRM1 and CRM2
domains (Fig. 1C).
Effect of mutations on S synthesis
To investigate the effect of the different mutations on S
synthesis, western immunoblot analysis was used to detect
and visualize all of the constructed mutant glycoproteins as
well as the wild-type S (Fig. 2). Cellular lysates prepared
from transfected cells at 48 h post-transfection were electro-
phoreticallyseparatedbySDS-PAGEandtheSglycoproteins
were detected via chemiluminescence using a monoclonal
antibody specific for the SARS-CoV S glycoprotein.
Carbohydrate addition was shown to occur in at least four
different locations of the SARS-CoV S glycoprotein (Kro-
khin et al., 2003; Ying et al., 2004). Furthermore, transiently
expressed S glycoprotein in Vero E6 cells was proteolyti-
cally cleaved into S1 and S2 components (Wu et al., 2004).
The anti-S monoclonal antibody SW-111 detected a protein
species in cellular extracts from transfected cells, which
migrated with an apparent molecular mass of approximately
180 kDa, as reported previously (Song et al., 2004). All
mutated S glycoproteins produced similar S-related protein
species to that of the wild-type S indicating that none of the
engineered mutations adversely affect S synthesis and
intracellular processing (Fig. 2A). The SARS S glycoprotein
is known to form homotrimers in its native state (Song et al.,
2004). To investigate the effect of the mutations on S
oligomerization, cellular lysates from transfected cells were
electrophoretically separated without prior boiling of the
samples and in the absence of reducing agents (Song et al.,
2004). The different S species were detected via chemilu-
minescence using the monoclonal SW-111 to the SARS S
glycoprotein. An S protein species was detected that had an
approximate apparent molecular mass of 500 kDa, which
was consistent with previously published data (Song et al.,
2004) (Fig. 2B). Although levels of oligomer expression
seemed to vary slightly between mutant forms, all mutated S
glycoproteins produced similar species to the wild type,
indicating that none of the mutations blocked oligomeriza-
tion from occurring.
Ability of mutant S glycoproteins to be expressed on the cell
surface
To determine if the mutant S glycoproteins were
expressed on the surface of cells, immunohistochemical
analysis was used to label cell-surface-expressed S under
live cell conditions that restrict antibody binding to cell
surfaces. In addition, immunohistochemistry was used to
detect the total amount of S expressed in cells by fixing and
permeabilizing the cells prior to reaction with the antibody.
A recombinant S protein having the 3xFLAG added in-
frame to the carboxyl terminus of S was used as a negative
control, since it would not be stained by the live cell
reaction conditions (see Materials and methods). Both wild-
type versions of S having the 3xFLAG, either at the amino
or carboxyl terminus of S, caused similar amounts of fusion
(Fig. 3), which also was similar to that obtained with the
untagged wild-type S (not shown). The relative amounts of
cell-surface versus total cellular expression of S were
obtained through the use of an ELISA. A ratio between
the cell-surface localized S and total cellular S expression
was then calculated and normalized to the corresponding
ratio obtained with the wild-type S glycoprotein (see
Materials and methods) (Fig. 4). The CL1 (95%), CL2
(78%), CL3 (86%), CL4 (80%), CL5 (92%), CL6 (86%)
mutants as well as the T1229 (91%), T1238 (94%), and
T1247 (79%) truncations were expressed on the cellular
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surface by the percentages indicated when compared to the
cell-surface expression of the wild-type protein. In contrast,
the 1214T mutant expressed 77% less S on cell surfaces in
comparison to the wild-type S (Fig. 4).
Effect of mutations on S-mediated cell-to-cell fusion
Transiently expressed wild-type S causes extensive cell-
to-cell fusion (syncytial formation), especially in the
presence of the SARS-CoV ACE2 receptor (Li et al.,
2003). To determine the ability of each mutant S glyco-
protein to cause cell-to-cell fusion and the formation of
syncytia, fused cells were labeled by immunohistochemistry
using the anti-FLAG antibody (Fig. 5). The extent of cell-to-
cell fusion caused by each mutant glycoprotein was
calculated by obtaining the average size of approximately
300 syncytia. The average syncytium size for each mutant
was then normalized to that found in wild-type S-transfected
cells (see Materials and methods). The CL1 (73%), CL2
(75%), CL3 (68%), CL4 (71%), CL5 (76%), CL6 (51%) as
well as the T1214 (86%) and T1247 (66%) mutants
inhibited the formation of syncytia by the percentages
indicated. The T1229 truncation and the cluster mutant CL7
produced syncytia, which were on the average 22% and
15% smaller, respectively, than that of the wild-type S. In
contrast, the T1238 mutant produced on the average 43%
larger syncytia than that of the wild-type S (Fig. 5).
Comparison of the membrane fusion and cell-surface
expression results allowed the grouping of the different
mutant S phenotypes into four distinct groups (Table 1): (1) S
mutant forms in Group I (CL1–CL6 and T1247) resulted in
high levels of cell-surface expression (78–95% of the wild-
type S); however, the average size of syncytial formed by
these mutated S glycoproteins was reduced substantially in
comparison to the wild-type S (23–48% of the wild-type).
CL1affectsthepredictedfusionpeptide,CL2–CL4affectthe
HR1 domain, and CL5 affects a region downstream of the
HR1 domain. The CL6 mutation is located within the S
carboxyl-terminal acidic cluster. The T1247 mutation trun-
cates the S carboxyl terminus by 8 amino acids; (2) S mutant
forms in Group II produced high levels of S cell-surface
expression and an average size of syncytia slightly smaller
than that of the wild-type S. These mutations included CL7,
which modified the acidic cluster and the T1229 truncations
that deleted the cysteine-rich motif CRM2; (3) the single S
mutant in Group III, T1214, produced significantly less cell-
Fig. 2. Western blot analysis of the expressed mutant SARS-CoV S mutant glycoproteins. (A, B) Immunoblots of wild-type [3xFLAG So (WT)], cluster to
lysine, cluster to alanine, and carboxyl truncation mutant S glycoproteins probed with monoclonal anti-SARS S antiserum. ‘‘Cells only’’ represents a negative
control in which Vero cells with no protein transfected into them were probed with the monoclonal antibody to SARS S glycoprotein. (B) In order to detect
trimer formation more efficiently, the protein extracts of the mutants were neither boiled nor subject to treatment with beta mercaptoethanol.
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