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Structural Studies of Recombinant Norwalk Capsids
B. V. Venkataram Prasad,1 M. E. Hardy,2,a
and M. K. Estes2
1
Verna and Marrs McLean Department of Biochemistry and
Molecular Biology and 2Department of Molecular Virology and
Microbiology, Baylor College of Medicine, Houston, Texas
Norwalk virus is the major cause of epidemic viral gastroenteritis in humans. Attempts to
grow this human virus in laboratory cell lines have been unsuccessful; however, the Norwalk
virus capsid protein, when expressed in insect cells infected with a recombinant baculovirus,
spontaneously assembles into virus-like particles. The x-ray crystallographic structure of these
recombinant Norwalk particles has been determined to 3.4 Å, using a 22-Å electron cryomicroscopy structure as a phasing model. The recombinant capsids, 380 Å in diameter, exhibit
a T = 3 icosahedral symmetry. The capsid is formed by 90 dimers of the capsid protein, each
of which forms an arch-like capsomere. The capsid protein has two distinct domains—a shell
(S) and a protruding (P) domain—that are connected by a flexible hinge. Although the S
domain has a classical b-sandwich fold, the structure of the P domain is unlike any other
viral protein. One of the subdomains in the P domain formed by the most variable part of
the sequence is located at the exterior of the capsid.
Norwalk virus (NV) and Norwalk-like viruses, which belong
to the family Caliciviridae, cause epidemic acute gastroenteritis
in humans [1]. It is estimated that 96% of outbreaks of acute,
epidemic, nonbacterial gastroenteritis in the United States are
caused by these viruses [2]. NV contains a genome of singlestranded positive-sense RNA of about 7.6 kb [3–7]. The genome
consists of three open-reading frames (ORFs) [3, 5]. The first
ORF encodes a polyprotein precursor to nonstructural proteins. This ORF contains regions analogous to the 2C helicase,
the 3C protease, and a 3D RNA-dependent RNA polymerase
of picornaviruses. The second ORF encodes the capsid protein
with an apparent molecular mass of 58 kDa. The third ORF
codes for a basic protein whose functional properties are still
unclear.
NV and other members of the Caliciviridae are unique among
the animal viruses. The icosahedral capsids of these viruses are
composed of multiple copies of a single structural protein. In
NV, 180 molecules of the capsid protein encoded by the second
ORF form the capsid. Although common among plant viruses,
virus capsids made of a single structural protein are rather
unusual among animal viruses. The only known examples are
viruses in the family Nodaviridae [8]. Consequently, all the functional determinants of structural integrity, immunogenicity, and
infectivity are encoded in one structural protein. Thus, while
caliciviruses possess all the characteristics of animal viruses,
Grant support: NIH (AI-38036, AI-46581) and R. Welch Foundation.
a
Present affiliation: Veterinary Molecular Biology, Montana State University, Bozeman, Montana.
Reprints or correspondence: Dr. B. V. Venkataram Prasad, Verna and
Marrs McLean Department of Biochemistry and Molecular Biology, Baylor
College of Medicine, Houston, TX 77030 ([email protected]).
The Journal of Infectious Diseases 2000; 181(Suppl 2):S317–21
q 2000 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/2000/18105S-0013$02.00
they have the structural simplicity of plant viruses and are
excellent model systems for studying the molecular basis of viral
assembly, genome encapsidation, immunogenicity, and
pathogenesis.
We have undertaken three-dimensional structural studies of
the members of the Caliciviridae, using electron cryomicroscopy
(cryo-EM) and x-ray crystallography techniques to understand
structure-function relationships in these viruses [9–11]. We report here the progress we have made in understanding the structural details of the NV, the prototype human calicivirus. We
have done structural studies of the recombinant Norwalk capsids, in lieu of native virus, which currently cannot be obtained
in large quantities. When expressed using the baculovirus system, the NV capsid protein spontaneously assembles into viruslike particles [4]. These particles can be purified in large quantities, making them amenable for high-resolution structural
analysis.
Materials and Methods
Baculovirus-expressed recombinant NV (rNV) particles were
produced and purified from insect cells infected with a recombinant
baculovirus that contains the second and third ORFs of the viral
genome as previously described [4, 9]. cryo-EM and the threedimensional structure determination of the rNV capsids were done
as described by Prasad et al. [9]. The x-ray crystallographic analysis
of these particles is described in [11].
Results and Discussion
A surface representation of the three-dimensional structure
along the icosahedral 3-fold axes is shown in figure 1a. The
structure has an overall diameter of 380 Å and exhibits T =
3 icosahedral symmetry. The main feature of the three-dimensional structure of rNV particles is the presence of 90 arch-like
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Prasad et al.
capsomeres located at all the local and strict 2-fold axes of the
T = 3 icosahedral lattice. These capsomeres are arranged in
such a way that there are prominent hollows at all the icosahedral 5- and 3-fold axes (figure 1a). The arches begin at a
radius of ∼145 Å and extend to a radius of ∼190 Å. A rectangular platform of a dimension ∼50 3 70 Å is located at the
top of the arch. These arches surround the hollows, which are
about 40 Å deep and 90 Å wide. Each virus particle has 32
such hollows, 12 of which are on the icosahedral 5-fold axes;
the remaining 20 hollows are on the icosahedral 3-fold axes.
The hollows at the 5-fold axes have a small hump at the center
with a moat around it, while the hollows at the icosahedral 3fold positions are rather flat.
The asymmetric unit (1/60th portion of the icosahedral lattice) of the T = 3 icosahedral lattice contains three subunits,
which are designated here as A, B, and C. The 90 capsomeres
that are seen in the Norwalk capsid can be classified into two
types on the basis of their location with respect to the icosahedral axes of symmetry (figure 1a). The first type of capsomeres made of A and B subunits is located at the local 2-fold
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axis (midway between the icosahedral 5- and 3-fold positions).
The second type, made of two C subunits, is located at the
strict 2-fold axes (midway between any two neighboring 5-fold
or 3-fold axes). The A/B dimers surround the icosahedral 5fold axes, while the C/C dimers are located in between the
pentameric rings of A/B dimers. There are 60 A/B dimers and
30 C/C dimers in each particle. Each side of the arch-like capsomere represents a single molecule of the capsid protein. Similar T = 3 icosahedral organization with dimeric capsomeres are
seen in tombus viruses, such as tomato bushy stunt virus
(TBSV) [12] and turnip crinkle virus (TCV) [13].
The protein subunit of NV contains two distinct domains:
the shell (S) domain and a protruding (P) domain. These domains were named following the convention used to describe
the capsid protein structure in TBSV, TCV, and other T = 3
structures [12–14]. From cryo-EM reconstructions, the P domain contains two subdomains: the distal globular domain,
called P2, and a central stem domain, called P1 (figure 1b). The
S domains of these subunits merge together to form the continuous mass density that is found between the radii 105 and
Figure 1. a, Surface representation of 3-dimensional structure of baculovirus-expressed Norwalk virus capsid viewed along icosahedral 3-fold
axes. Icosahedral 5- and 3-fold symmetry axes, 2 types of capsomeres (A/B and C/C), are indicated. A/B capsomeres surround icosahedral 5-fold
axes, while C/C capsomeres are located at icosahedral 2-fold axes. Each virion has 60 A/B and 30 C/C capsomeres. b, Central section along
icosahedral 3-fold axis. S domains associate to form contiguous shell of ∼45-Å thickness from which arches containing subdomains P1 and P2
emanate. Capsid shell extends from radius of ∼105 to 190 Å. Adapted with permission from J Virol 1994; 68:5117–25.
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Structure of Norwalk Capsids
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Figure 2. Crystals of recombinant Norwalk virus particles (∼0.14 mm in overall dimensions) grown using hanging drop method with ammonium
phosphate, at pH 4.8, as precipitant.
145 Å (figure 1b). The arches (P1 and P2) emanate from this
contiguous shell. No significant density is found inside the radius of 105 Å.
While the primary sequences of the capsid protein of NV
and Norwalk-like viruses, along with the other known calicivirus sequences, show identifiable homology among themselves,
these sequences show no homology with the capsid protein
sequences of other viruses. However, similarities in the architectural features between the Norwalk capsid and the other
T = 3 viruses [12–14] prompted us to suggest that the S domain
may fold into an eight-stranded b-barrel domain [9]. Comparison of amino acid sequences of NV and Norwalk-like viruses
[6] indicate that the most conserved region is between residues
30 and 250. While the C-terminal 100 residues are moderately
conserved, the middle portion of the sequence, between residues
280 and 400, are highly variable. On the basis of the secondary
structure prediction algorithms and the three-dimensional profile search methods, we proposed that residues 30 to 250 fold
into an eight-stranded b-barrel motif and the P1 and P2 domains are formed by the residues beyond 250 [9].
Although the three-dimensional structural analysis by cryoEM at a resolution of 22 Å has given a general architectural
description and domain organization, this resolution is not sufficient to address the questions of how the polypeptide chain
is folded; how the various quasi-equivalent subunits (A, B, and
C) differ in their conformations; where the determinants of
strain specificity, antigenicity, and receptor-binding activity are
located in the structure of the capsid protein; and how the
structure of the capsid protein facilitates the icosahedral assembly. To answer such questions, structural analysis at near atomic
resolution is necessary.
We have crystallized the rNV capsids suitable for x-ray crystallographic analysis. These crystals, grown using hanging drop
methods (figure 2), diffract to a resolution higher than 3.2 Å.
Data were collected on crystals at room temperature and on a
frozen crystal at liquid nitrogen temperature (21707C), using
the synchrotron radiation source at Cornell High Energy Synchrotron Radiation Source. Analysis of the oscillation photographs indicated that the unit cell dimensions are 606 Å 3 606
Å 3 467 Å with a tetragonal space group [11]. Using cryo-EM
structure as an initial phasing model, the x-ray crystallographic
structure of the rNV capsid was determined to 3.4-Å resolution
[11]. These recent x-ray crystallographic studies have confirmed
our earlier prediction from cryo-EM studies regarding the do-
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Prasad et al.
main organization of the capsid protein (figure 3). The N-terminal 225 residues of the capsid protein form an eight-stranded
antiparallel b-sandwich. Residues from 225 onward form a protruding P domain, which contains two subdomains. The highly
variable region of the sequence (residues 279–406) forms the
externally located P2 subdomain, whereas the central subdomain, P1, located between the S and P2, is formed by residues
from 226–278 and the C-terminal 124 residues. The S and P
domains are connected by a flexible hinge that is present around
residue 225. This flexible hinge appears to facilitate changes in
the relative orientations between the P and the S domains of
the three quasiequivalent subunits.
To adapt to the three quasi-equivalent locations of the T =
3 icosahedral lattice, the three subunits, A, B, and C, must
undergo appropriate conformational changes. Although the
overall conformations of the three subunits in the asymmetric
unit of the T = 3 structure are essentially the same, there are
three main differences. The first difference is that the relative
orientation between the S and P domains in the C subunit is
slightly different from that seen in A or B, which show similar
relative orientations. The second difference is in the N-terminal
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arms of these subunits. In the B subunit, only the first 9 residues
are disordered, whereas in the A and C subunits, the first 28
residues are disordered. This ordered arm of the B subunit
interacts with the F-strand in the S domain of the neighboring
C subunit in the T = 3 icosahedral lattice. The third difference
is that in both A and B subunits, residues 505 and 506 in the
P1 subdomain are hydrogen bonded to residues 64 and 65 in
the S domain, whereas in the C subunit, these interactions are
not present. These conformational changes confer appropriate
curvatures for the A/B and the C/C dimers in the formation
of the closed shell.
Conclusions
Despite difficulties in adapting NV to a tissue culture system,
the recombinant particles have made possible detailed atomic
resolution of the structural characteristics of the virus capsid.
The architecture of the NV capsid is similar to that in other
animal caliciviruses, such as primate calicivirus [10], rabbit
hemorrhagic disease virus [15], feline calicivirus, and San Mi-
Figure 3. a, x-ray crystallographic structure of recombinant Norwalk virus (rNV) capsid at 3.4 Å resolution, as viewed along icosahedral 2fold axis. Only backbone atoms of 180 subunits are depicted. Structure is depth-cued, with deeper blue at lower radii and lighter blue at higher
radii. b, Ribbon representation [16] of structure of rNV capsid protein (C subunit). N-terminal arm, S domain, P1, and P2 subdomains are colored
in green, yellow, red, and blue, respectively. N- and C-termini of capsid protein are indicated. C-terminus faces hollow, whereas N-terminal arm
faces interior of capsid and P2 subdomain faces exterior of capsid.
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Structure of Norwalk Capsids
guel sea lion virus (Rong C, Liu M, Neill J, Prasad BVV, unpublished results).
It is quite likely that many of the details seen in the atomic
resolution structure of the NV capsid are present in other caliciviruses as well. However, some structural variations, particularly in the P2 subdomain, are to be expected as this subdomain
is likely to be responsible for host specificity. The overall structural similarity between the caliciviruses and plant tombus viruses may indicate an evolutionary relationship between these
two classes of viruses.
Knowledge of the atomic resolution structure of the Norwalk
capsid now provides a firm foundation for future mutational
analyses aimed toward establishing structure-function relationships in human caliciviruses. With further biochemical characterization of these viruses, including identification of receptors
and the locations of the antigenic epitopes, the structure of the
Norwalk capsid will be helpful for formulating antiviral strategies for both human caliciviruses and other animal caliciviruses that cause acute or persistent infections.
Acknowledgment
We thank Dr. M. Vyas for valuable help during crystallization of
recombinant Norwalk virus capsids.
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