Journal of General Virology (2014), 95, 278–291
Review
DOI 10.1099/vir.0.059634-0
Norovirus gene expression and replication
Lucy G. Thorne and Ian G. Goodfellow
Correspondence
Ian G. Goodfellow
Division of Virology, Department of Pathology, University of Cambridge, Addenbrooke’s Hospital,
Hills Road, Cambridge CB2 2QQ, UK
[email protected]
Received 18 September 2013
Accepted 13 November 2013
Noroviruses are small, positive-sense RNA viruses within the family Caliciviridae, and are now
accepted widely as a major cause of acute gastroenteritis in both developed and developing
countries. Despite their impact, our understanding of the life cycle of noroviruses has lagged
behind that of other RNA viruses due to the inability to culture human noroviruses (HuNVs). Our
knowledge of norovirus biology has improved significantly over the past decade as a result of
numerous technological advances. The use of a HuNV replicon, improved biochemical and cellbased assays, combined with the discovery of a murine norovirus capable of replication in cell
culture, has improved greatly our understanding of the molecular mechanisms of norovirus
genome translation and replication, as well as the interaction with host cell processes. In this
review, the current state of knowledge of the intracellular life of noroviruses is discussed with
particular emphasis on the mechanisms of viral gene expression and viral genome replication.
Introduction
The prototype norovirus, Norwalk virus, was first described
in 1972 as the aetiological agent responsible for an outbreak
of acute gastroenteritis in an elementary school in Norwalk,
OH, USA (Kapikian, 2000). Noroviruses are now accepted
as a leading cause of gastroenteritis in developed and
developing countries (Glass et al., 2009; Hall et al., 2013).
Spread primarily via the faecal–oral route, norovirus
infections are typically an acute self-limiting gastrointestinal
infection. Norovirus gastroenteritis has recently been
identified as a significant cause of morbidity and mortality
in the immunocompromised, and can result in long-term
persistent disease (reviewed by Bok & Green, 2012).
Norovirus infection has also been associated with a number
of more significant clinical outcomes: necrotizing enterocolitis (Turcios-Ruiz et al., 2008), seizures in infants (Medici
et al., 2010), encephalopathy (Ito et al., 2006), pneumatosis
intestinalis (Chan et al., 2010; Kim et al., 2011) and
disseminated intravascular coagulation (CDC, 2002), to
name but a few. In developing countries, an estimated
200 000 deaths in children ,5 years of age are thought to be
due to norovirus infections (Patel et al., 2008) and they have
recently been reported as the second leading cause of
gastroenteritis-related deaths in the USA, typically resulting
in 797 deaths per annum (Hall et al., 2013). Despite their
significant impact, noroviruses remain one of the most
poorly characterized groups of RNA viruses, due largely to
the fact that, despite numerous attempts (Duizer et al., 2004;
Papafragkou et al., 2013; Takanashi et al., 2013), human
noroviruses (HuNVs) have yet to be cultured efficiently in
cell culture.
Noroviruses are members of the family Caliciviridae
of small, positive-sense RNA viruses, which is divided
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currently into five genera: Vesivirus, Lagovirus, Nebovirus,
Sapovirus and Norovirus. Members of the genera Norovirus
and Sapovirus are able to infect humans and cause
gastroenteritis. The genus Norovirus is subdivided into at
least five genogroups (GI–V). Genogroups GI, GII and GIV
infect humans and cause acute gastroenteritis, but
noroviruses have also been isolated from numerous other
species including pigs (GII), cattle and sheep (GIII), and
mice (GV). More recently, a novel norovirus identified in
domestic dogs with diarrhoea has been proposed to
represent a new genogroup, GVI (Mesquita et al., 2010).
To date, zoonotic norovirus infections have not been
reported, but there is clear evidence of the potential for
transmission. For example, HuNVs can infect gnotobiotic
piglets (Cheetham et al., 2006) and there is serological
evidence of HuNV in pigs (Farkas et al., 2005). Whilst
antibodies to GVI noroviruses have been identified in
veterinarians, it has yet to be determined whether infection
results in clinical disease (Mesquita et al., 2013). Additional
studies are required to further elucidate the zoonotic
potential of noroviruses and whether animals represent a
reservoir from which new strains may emerge.
Norovirus genome organization
The norovirus genome is a compact, positive-sense ssRNA
molecule, ranging in size from 7.3 to 7.5 kb across
the genus (Fig. 1). The 59 end of the genomic RNA is
covalently attached to a virus-encoded protein known as
VPg, whilst the 39 end is polyadenylated. The UTRs at
either end of norovirus genomes are typically short, e.g. the
59 and 39 UTRs of murine norovirus (MNV) are 5 and
78 nt, respectively, and the 39 UTR of HuNV is typically
48 nt (Gutiérrez-Escolano et al., 2000a; Karst et al., 2003;
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Norovirus gene expression and replication
(a) Human norovirus
VPg
p48
ORF1
NTPase p22 VPg
N-term
2C-L
ORF2
Pro
VP1
Pol
NS1/2
ORF1
NS3
VP2
AAAA(n)
VP2
AAAA(n)
3A-L
VP1
(b) Murine norovirus
ORF3
NS4 NS5 NS6
ORF2
VP1
NS7
Caspase 3
cleavage sites
VF1
VP1
ORF3
VP2
AAAA(n)
VP2
AAAA(n)
Fig. 1. Organization of the HuNV and MNV genomes. (a) The HuNV genome is covalently attached to VPg (NS5) with a poly(A)
tail and is divided into three ORFs, common to all noroviruses. ORF1 is translated as a polyprotein, which is cleaved by the viral
protease NS6 to produce the NS proteins. ORF2 and ORF3 are translated from a subgenomic RNA. 2C-L, 2C-like; 3A-L, 3Alike. (b) MNV shares a similar genome organization, but has an additional alternative fourth ORF. ORF4 overlaps with ORF2 and
is also translated primarily from the subgenomic RNA into the virulence factor 1 (VF1) protein.
Pletneva et al., 2001). The UTRs contain evolutionarily
conserved RNA secondary structures that extend into the
coding regions and can be found throughout the genome
(Simmonds et al., 2008). These structures are important for
viral replication, translation and pathogenesis (Bailey et al.,
2010b; McFadden et al., 2011; Simmonds et al., 2008).
nomenclature for the norovirus proteins has yet to be
unified in the literature and is summarized in Table 1.
The norovirus genome is organized into three conserved
ORFs (Fig. 1a), with the exception of MNV, which has a
fourth alternative ORF (Fig. 1b). The fourth ORF is unique
to the MNV cluster in GV and has not yet been identified in
any other norovirus; the only member of the family
Caliciviridae known to have an equivalent fourth ORF is
human sapovirus (Clarke & Lambden, 2000; McFadden
et al., 2011). For all noroviruses, ORF1 is translated as a large
polyprotein, which is co- and post-translationally cleaved
by the virus-encoded protease (NS6) to release at least
six mature non-structural (NS) proteins, including NS6
(Sosnovtsev et al., 2006) (Fig. 1). The other NS proteins
include the viral RNA-dependent RNA polymerase (RdRp;
NS7), VPg (NS5), the putative NTPase/RNA helicase (NS3),
and NS1/2 and NS4, which have both been implicated in
replication complex formation (Hyde & Mackenzie, 2010;
Hyde et al., 2009). NS1/2 is further processed during the
latter stages of virus infection by cellular caspases activated
by apoptosis and an as-yet-unidentified cellular protease
(Sosnovtsev et al., 2006). ORF2 and ORF3 are translated
from a subgenomic RNA (Fig. 1), and encode the major and
minor capsid proteins, VP1 and VP2, respectively. The
subgenomic RNA is identical to the last 2.4 kb of the
genome and is also attached covalently to VPg at the 59 end
with a poly(A) tail at the 39 end (Herbert et al., 1997). ORF4
of MNV is translated from an alternative reading frame
within ORF2 and encodes the recently identified protein
virulence factor 1 (VF1), a mitochondrially localized novel
innate immune regulator (McFadden et al., 2011). The
As highlighted above, the inability of HuNV to be cultured
in vitro has hampered the characterization of the viral life
cycle. Prior to the discovery of MNV in 2003, numerous
other members of the family Caliciviridae were used widely
as model systems with which to study norovirus biology
(reviewed by Vashist et al., 2009). Feline calicivirus (FCV),
a member of the genus Vesivirus, was the first calicivirus for
which a cell culture and reverse-genetics system was
available (Sosnovtsev & Green, 1995), and has been used
widely to study various aspects of the calicivirus life cycle.
More recently, however, studies have indicated that HuNV
RNA isolated from feacal samples is infectious in cell
culture, as transfection into cells results in RNA replication,
viral protein production and release of virions into the cell
supernatant (Guix et al., 2007). Importantly, this experimental system does not allow multicycle replication as the
virus generated is unable to reinfect the neighbouring cells.
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Model systems for the study of norovirus gene
expression and replication
A number of significant steps in the understanding of
norovirus gene expression and replication have also been
made using MNV, a GV norovirus initially identified as a
lethal infection in immunocompromised mice (Karst et al.,
2003). MNV replicates efficiently in primary or immortalized murine dendritic and macrophage cells (Wobus et al.,
2004), and has a number of reverse-genetics systems
available (Chaudhry et al., 2007; Ward et al., 2007; Yunus
et al., 2010). The development of a Norwalk virus replicon
system (HuNV), where cells stably maintain Norwalk
virus RNA containing an antibiotic selection marker in
the capsid-coding region (Chang et al., 2006), added
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L. G. Thorne and I. G Goodfellow
Table 1. Nomenclature for HuNV and MNV proteins and their functions
MNV
HuNV*
NS1/2
NS3
NS4
NS5
NS6
NS7
VP1
VP2
VF1
p48 (N-term)
NTPase (2C-like)
p22 (3A-like)
VPg
Pro (3C-like)
Pol/3Dpol
VP1
VP2
No equivalent
Function
Replication complex formationD, contributes to persistence in MNV infections
RNA helicaseD/NTPase
Replication complex formationD
Genome-linked protein involved in translation and replication
Protease
RdRp
Major capsid protein
Minor capsid protein
Virulence factor
*Alternative names for the HuNV proteins are given in parentheses, and originate through comparison of the norovirus proteins and genome
organization with those of poliovirus.
DThese functions have been proposed, but have yet to be demonstrated fully.
an additional experimental system. Biochemical studies (e.g.
Belliot et al., 2005) and other cell-based approaches reliant
on the expression of viral proteins in immortalized cells
(Subba-Reddy et al., 2011, 2012) have also been used to gain
insights into mechanisms of viral genome replication. A
discussion of the merits of each experimental system has
been provided previously (Vashist et al., 2009). The data
reviewed below rely on a combination of studies using all
these approaches and, where appropriate, the experimental
system with which the data were obtained is highlighted.
Norovirus binding and entry mechanisms
The interaction of noroviruses with the cell surface is
known to involve carbohydrate structures, which in the
case of HuNVs include the histo blood group antigens
(HBGAs). A detailed description of the interaction of
HuNVs with the cell surface and HBGAs, as well as the role
this interaction plays in norovirus entry and susceptibility,
has been provided in detail recently (Donaldson et al.,
2008; reviewed in Donaldson et al., 2010). MNV, currently
the only norovirus that replicates efficiently in cell culture,
binds host cells via sialic acid moieties, glycolipids and
glycoproteins in a strain-dependent manner (Taube et al.,
2009, 2012) The mechanism by which MNV enters cells has
yet to be elucidated fully, but it is dependent on dynamin
and cholesterol (Gerondopoulos et al., 2010; Perry &
Wobus, 2010). As mentioned above, receptor binding and/
or the entry process appear to be at least one of the limiting
factors for the culture of HuNV in immortalized cells as
viral RNA transfected into cells is able to undergo limited
replication. Additional work in this area may therefore
provide a mechanism with which to further understand the
block to HuNV replication in cell culture.
Viral protein translation
Once a norovirus VPg-linked RNA genome is released into
the cytoplasm of a permissive cell, it behaves as an mRNA
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template for the ‘pioneer round’ of viral RNA translation
(Fig. 2). For this to occur, the viral RNA is recognized by
cellular translation initiation factors and is translated into
protein by the cellular translational apparatus. RNA viruses
typically utilize novel mechanisms for viral genome
translation, not only for the initiation of translation, but
also to increase the coding capacity of their relatively short
genomes (Firth & Brierley, 2012) and this is also true also
for noroviruses. Translation of all calicivirus genomes
occurs by a novel mechanism involving VPg that is not
found in many other animal RNA viruses (Herbert et al.,
1997). However, recent data would indicate that a similar
mechanism may be employed by members of the family
Astroviridae, for which the linkage of VPg to viral RNA is
essential for RNA infectivity (Fuentes et al., 2012), as also
described for noroviruses (Chaudhry et al., 2006). Some
aspects of the mechanism of calicivirus RNA genome
translation are similar to that used by the plant viruses in
the family Potyviridae (Léonard et al., 2000). VPg attached
covalently to the 59 end of the genome mediates translation
of viral RNA, acting as a cap substitute, and recruits host
cell translation initiation factors (Fig. 3). The structure of
the MNV VPg protein has recently been described as
containing a compact helical core, flanked by more flexible
intrinsically disordered N- and C-terminal regions (Leen
et al., 2013). This high degree of flexibility is likely to be
due to the numerous roles that VPg plays in the norovirus
life cycle (Goodfellow, 2011). The VPg proteins of HuNV
and MNV have both been shown to interact with
components of the eIF4F translation initiation factor
complex, in particular eIF4E (Chaudhry et al., 2006;
Goodfellow et al., 2005), the cap-binding protein, and
eIF3 (Daughenbaugh et al., 2003), which is recruited to the
complex and in turn helps to recruit the 43S ribosomal
pre-initiation complex (Fig. 3). Functional studies have to
date only been possible with MNV as it is not possible to
obtain a sufficient quantity of authentic VPg-linked HuNV
RNA due to the inability to culture HuNV in immortalized
cells. These studies confirmed that translation of MNV
RNA requires the eukaryotic initiation factor eIF4A, the
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Journal of General Virology 95
Norovirus gene expression and replication
RNA helicase component of eIF4F, potentially for unwinding RNA structures present in the 59 end of the genome, as
both dominant-negative forms of eIF4A and smallmolecule inhibitors of eIF4A inhibited MNV RNA
translation in vitro (Chaudhry et al., 2006). To date only
direct interactions with the norovirus VPg have been
demonstrated for eIF3 and eIF4E, but functions for these
interactions have not been described. Despite the direct
interaction, in vitro studies have indicated that eIF4E is not
required for the translation of MNV RNA in a rabbit
reticulocyte lysate (Chaudhry et al., 2006). Therefore, it is
likely that VPg forms multiple contacts with components
of the translation initiation factor complex, not all of which
are essential for viral RNA translation (Fig. 3). A direct,
high-affinity, functional interaction between the norovirus
VPg and a component of the translation apparatus has yet
to be described.
The extremities of calicivirus genomes contain evolutionarily conserved RNA structures that are known to interact
with host cell factors to promote viral replication and
translation (Simmonds et al., 2008; Vashist et al., 2012).
For example, structures in the Norwalk virus 59 and 39 ends
are thought to interact with the cellular proteins La,
polypyrimidine tract-binding protein (PTB) and poly(A)binding protein (PABP), and these proteins have also been
identified as binding RNA structures in the MNV genome
(Gutiérrez-Escolano et al., 2000b, 2003; Vashist et al.,
2012). The function of these interactions has yet to be
elucidated fully, but studies with MNV have confirmed
that they play a role in virus replication, as is well
established for other RNA viruses (Bailey et al., 2010b;
Simmonds et al., 2008). Many of the host proteins found to
interact with norovirus RNA structures have previously
been identified as regulators of the translation of other
RNA viruses, e.g. La and PTB stimulate picornavirus
internal ribosome entry site (IRES)-mediated translation
(reviewed by Fitzgerald & Semler, 2009). Therefore,
binding of these and other factors may serve to enhance
viral protein translation, perhaps through stabilization of
long-range RNA interactions that promote the circularization of the norovirus genome, as a process of RNA
circularization is known to stimulate host cell mRNA
translation (Wells et al., 1998). In both Norwalk virus and
MNV, the interaction of complementary sequences in the
59 and 39 extremities of the viral genome is stimulated by
cellular proteins (López-Manrı́quez et al., 2013; SandovalJaime & Gutiérrez-Escolano, 2009). Two cellular proteins
in particular, the poly(C)-binding protein PCBP2 and the
heterogeneous nuclear ribonucleoprotein hnRNPA1, are
involved in the circularization of the MNV genome
(López-Manrı́quez et al., 2013). A number of host factors
have also been identified as binding to the extremities of
the MNV genome using a proteomics-based approach and
summary tables of all these interactions can be found in
Vashist et al. (2012). As many of these factors form direct
protein–protein interactions with each other, it is possible
that numerous cellular RNA-binding proteins contribute
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to the circularization of the viral genome during translation and/or replication. In addition to PCBP2 and
hnRNPA1, RNA interference (RNAi)-mediated reduction
of three of the proteins identified in the proteomics
screen, i.e. PTB, DDX3 and La, significantly reduced
MNV replication in cell culture (Vashist et al., 2012).
Importantly, the role of the cellular RNA-binding proteins
in norovirus translation is unknown as currently no
experimental system exists that allows viral RNA translation in cells to be decoupled from viral RNA replication;
therefore, effects on either process have a negative impact
on viral replication. It is possible that these types of RNA–
protein interactions play both positive and negative
regulatory roles in the life cycle of noroviruses as data
from a related calicivirus indicated that binding of PTB to
the viral 59 end impacts negatively virus translation and
may contribute to the switch from RNA translation to
replication (Karakasiliotis et al., 2010).
Translation of the viral proteins VP1 and VP2 (and VF1 in
MNV) occurs primarily from the subgenomic RNA (Fig.
1). This is most likely a strategy to produce higher levels of
the major capsid protein for virus assembly as the
subgenomic RNA is present at higher levels in infected
cells than the viral genomic RNA and each icosahedral
capsid contains 180 copies of VP1 arranged in 90 dimers
(Prasad et al., 1994). Translation of VP2 occurs by a
termination–reinitiation mechanism as the norovirus
subgenomic RNA is polycistronic (Napthine et al., 2009).
Upon termination of ORF2 translation (VP1), ribosomes
are thought to remain associated with the RNA to reinitiate
at the start of ORF3 (VP2). This is facilitated by
overlapping stop and start codons of ORF2 and ORF3,
respectively (e.g. UAAUG in MNV). An upstream RNA
motif known as the termination upstream ribosomalbinding sequence (TURBS) is also required, which is in
part complementary to 18S rRNA and is thought to tether
the ribosome through direct RNA–RNA base pair interactions (Meyers, 2007; Napthine et al., 2009). There is also
evidence from transfection of bovine norovirus RNA that
translation of VP1 occurs by a similar termination–
reinitiation between ORF1 and ORF2 on the genomic
RNA (McCormick et al., 2008). This was proposed as a
unique translation mechanism for GIII viruses and
although it is feasible that it could contribute to VP1
translation in other genogroups, the mechanism has yet to
be demonstrated within the context of viral infection for
any norovirus. The start codon for translation of ORF4 in
MNV is positioned 13 bases downstream of the start of
ORF2 and may be initiated by leaky scanning and a 22 slip
in the ribosome (McFadden et al., 2011).
Replication complex formation
Translation of the ORF1 polyprotein is followed by co- and
post-translational processing by the viral NS6 protease, and
results in the release of the viral NS proteins ready for
replication complex formation and their precursors, some
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L. G. Thorne and I. G Goodfellow
1. Attachment
factor binding
2. Receptor
binding
VPg
3. Entry
AAAA(n)
4. Uncoating
5. Translation
AAAA(n)
VPg
RdRp
6. Polyprotein
processing
7. Replication complex
formation
8. RNA replication
AAAA(n)
de novo initiation
AAAA(n)
10. Exit
VPg-dependent
initiation
AAAA(n)
AAAA(n)
Genomic
RNA
9. Virion assembly
AAAA(n)
AAAA(n)
AAAA(n)
AAAA(n)
AAAA(n)
AAAA(n)
Subgenomic
RNA
Fig. 2. Outline of the norovirus life cycle. (1) HuNV and MNV are thought to attach to the cell surface using various carbohydrate
attachment factors. This is not sufficient to mediate entry and binding to an unidentified protein receptor is thought to be
required (2). Entry (3) and uncoating (4) proceed through as-yet-undefined pathways. (5) The incoming viral genome is
translated, through interactions with VPg at the 59 end of the genome (red triangle) and the cellular translation machinery. (6)
The ORF1 polyprotein is co- and post-translationally cleaved by the viral protease NS6. (7) The replication complex is formed by
recruitment of cellular membranes to the perinuclear region of the cell (not shown), through interactions in part with NS1/2 and
NS4. (8) Genome replication occurs via a negative-strand intermediate, and genomic and subgenomic RNA are generated by
the viral RdRp (NS7), using both de novo and VPg-dependent mechanisms of RNA synthesis. (9) The replicated genomes are
translated (within the replication complex) or packaged into the capsid, VP1, for virion assembly and exit (10).
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Journal of General Virology 95
Norovirus gene expression and replication
and was adventitiously solved with the C terminus of an
adjacent NS6 molecule bound in the active site as a peptide
substrate, which facilitated identification of the key
residues involved in peptide binding and specificity (Leen
et al., 2012).
(a) Cap-dependent initiation
eIF3
PABP
(n)A
AA
A
eIF4G
eIF4A
eIF4E
m7GTP
(b) VPg-dependent initiation
eIF3
PABP
(n)A
AA
A
eIF4G
eIF4A
eIF4E
VPg
=Biochemically confirmed interactions
Fig. 3. Overview of the norovirus translation initiation complex. (a)
Schematic representation of the eIF4F cap-dependent cellular
translation initiation factor complex formed on the 59 end of capped
cellular mRNAs. The interaction of the eIF4E cap-binding protein
with the methylated cap structure (m7GTP) recruits the eIF4G
scaffold. Binding of eIF4G to eIF4E also enables the recruitment of
the RNA helicase component eIF4A and the eIF3 complex. eIF3 is
responsible subsequently for the recruitment of the 40S small
ribosomal subunit prior to translation initiation. (b) Norovirus VPgdependent translation also requires an interaction between VPg
and translation initiation factors. Thus far only direct interactions
with eIF4E and eIF3 have been documented. Note that the
interaction of VPg with eIF4E is not required for virus translation
initiation in vitro. Biochemically established interactions are
indicated.
of which are thought to be functionally active in replication
(Belliot et al., 2005). The structure and function of the
HuNV protease (Pro, Fig. 1) have been characterized
extensively, and the active site is thought to be a catalytic
triad of H30, E54 and C139, although E54 is thought
largely to determine substrate specificity rather than being
essential for catalysis (Someya et al., 2002, 2008; Zeitler
et al., 2006). HuNV Pro and MNV NS6 share .60 %
amino acid identity (Leen et al., 2012), and sequence
alignments showed that H30 and C139 are conserved,
whereas position 54 is aspartic acid rather than glutamic
acid in MNV NS6, although this implies it has a similar
function. Taken together with structural and mutagenic
studies it is therefore thought that the catalytic triad and
mechanism of catalysis may be conserved between HuNV
3CLPro and MNV NS6 (Leen et al., 2012). The structure of
MNV NS6 has recently been determined to high resolution
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Similar to other positive-strand RNA viruses, norovirus
replication occurs in close association with host-derived
membrane complexes in the cytoplasm (Belov & van
Kuppeveld, 2012; Wobus et al., 2004) (Fig. 2). Evidence for
the replication complex comes from studies with MNV, as
to date the mechanism of replication complex formation
has not been investigated in cells replicating HuNV RNA.
MNV infection induces the formation of membranous
vesicle clusters in infected cells, and proliferation of the
viral RNA polymerase (NS7) and dsRNA, a viral RNA
replication intermediate, occurs at punctate foci in the
perinuclear region (Hyde et al., 2009; Wobus et al., 2004).
The NS proteins, as well as the major and minor capsid
proteins co-localize with NS7 and dsRNA at this location,
forming the replication complex (Hyde & Mackenzie, 2010;
Thorne et al., 2012). More recently the MNV replication
complex was shown to be juxtaposed with the microtubule
organizing centre (MTOC) within the perinuclear region,
suggesting that MNV may use the cytoskeletal network
early in infection to position the complex (Hyde et al.,
2012). This process is mediated potentially by an
interaction between VP1 and acetylated tubulin, although
a direct interaction in infected cells has yet to be
demonstrated.
The host membranes that comprise the MNV replication
complex are derived from components of the secretory
pathway, notably the endoplasmic reticulum (ER), transGolgi network and endosomes, which together serve as a
platform for replication (Hyde et al., 2009). The mechanisms by which noroviruses recruit cellular membranes to
establish the replication complex in the host cell remain
unclear. For MNV, NS1/2 and NS4 have been implicated in
driving complex formation by recruiting cellular membranes, based on their localization to components of the
endocytic and secretory pathways (Bailey et al., 2010a;
Hyde & Mackenzie, 2010). Furthermore, the MNV NS4
protein induces Golgi disassembly and mildly inhibits
protein secretion, demonstrating that it is capable of
driving the rearrangement of cellular membranes (Sharp
et al., 2012). The HuNV NS4 equivalent, p22, inhibits
cellular protein secretion and has a more potent inhibitory
effect on this pathway than MNV NS4 (Sharp et al., 2010,
2012). HuNV p22 contains an ER export signal that is
thought to promote its uptake into COPII vesicles involved
in transport from the ER to the Golgi apparatus, although a
direct interaction between p22 and COPII has not yet
been demonstrated. Nevertheless, uptake of p22 has been
proposed to result in mislocalization of COPII vesicles,
thereby inhibiting proper trafficking to the Golgi, and in
turn leading to Golgi disassembly and loss of protein
secretion (Sharp et al., 2010). MNV NS4 does not contain
the same ER export signal, and the possible involvement of
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COPI and COPII vesicles in MNV replication complex
formation has been excluded (Hyde et al., 2009), suggesting NS4 may act by an alternative mechanism. The HuNV
p48 protein (equivalent to the MNV NS1/2 protein)
promotes Golgi disassembly, and disrupts expression and
trafficking of cell surface proteins in transfected cells
(Ettayebi & Hardy, 2003; Fernandez-Vega et al., 2004).
Disruption by p48 is thought to be mediated through a
direct interaction with VAP-A (SNARE regulator vesicleassociated membrane protein-associated protein A), which
is involved in regulating cellular vesicle transport (Ettayebi
& Hardy, 2003). Studies on HuNV proteins have focussed
largely on the disruption to protein secretion; however, it is
plausible that the observed membrane rearrangements
either contribute to or are a direct consequence of the
recruitment of cellular membranes for the replication
complex.
Genome replication
As for all positive-sense RNA viruses, genome replication
occurs via a negative-sense RNA intermediate and is
performed by the viral RdRp. The norovirus RdRps,
generally referred to as NS7, contain active-site residues
conserved among other positive-strand RNA virus RdRps,
and are conserved structurally and functionally (Högbom
et al., 2009). The replicative properties of the MNV RdRp
(NS7) are comparable in vitro with those of the HuNV
RdRp, supporting the use of MNV for studies of
replication (Bull et al., 2011). Subsequent to the ‘pioneer
round’ of translation of the incoming parental viral RNA
genome, the mRNA template then functions as a template
for the formation of a double-stranded replicative form
(RF). The process of initiation of negative-sense RNA
synthesis on the incoming parental viral RNA is not
understood fully, but the norovirus RdRp has two
mechanisms of initiation that have been demonstrated
both in vitro and in cells: de novo and VPg-dependent
(Rohayem et al., 2006). Unlike for picornavirus replication
where the negative-sense RNA is VPg-linked (Pettersson
et al., 1978), there is no evidence as yet to suggest whether
or not norovirus negative-sense RNA in the doublestranded RF is linked to VPg. However, a recently proposed
model has suggested that de novo initiation is used for the
synthesis of negative-sense genomic and subgenomic RNAs
(Subba-Reddy et al., 2012), both of which can be detected
in calicivirus-infected cells (Green et al., 2002). The de novo
initiation of norovirus RdRps is enhanced through direct
interactions with the shell domain of VP1, in a speciesspecific and concentration-dependent manner (SubbaReddy et al., 2012). This observation was made using
cell-based assays that rely on the co-expression of
norovirus proteins, including the NS7 RdRp in cells. De
novo RNA synthesis is then measured indirectly via the
RIG-I-mediated sensing of 59-triphosphorylated RNA
produced by the RdRp using host cell RNAs as a template,
which subsequently leads to the activation of the IFN-b
promoter linked to luciferase (Subba-Reddy et al., 2011).
284
This assay demonstrated that specific loop sequences in the
shell domain of VP1 interact with the RdRp to specifically
stimulate de novo initiation, without affecting VPgdependent initiation. These observations have resulted in
a model for the initial rounds of viral RNA synthesis,
whereby the shell domain from the incoming parental viral
capsid, or from the early rounds of viral RNA translation,
binds to the RdRp and stimulates negative-sense RNA
synthesis. The model would propose that as the levels of
VP1 increase due to the production of viral positive-sense
RNA, VP1 forms multimeric complexes, preventing the
interaction with the RdRp and leading to capsid assembly
and the formation of new infectious viral particles. This
model would also explain the observation that bovine
noroviruses have a termination–reinitiation sequence
(TURBS) upstream of VP1, similar to that used to produce
VP2 (McCormick et al., 2008). The effect of this strategy is
that a small percentage of ribosomes translating ORF1 will
reinitiate on ORF2 to produce VP1, leading to low levels of
VP1 during the ‘pioneer’ rounds of viral translation.
Whether other noroviruses utilize a similar mechanism for
the synthesis of VP1 during the early stages of the viral life
cycle is unknown currently. Species-specific interactions
with NS1/2 also promote norovirus RdRp activity in this
assay, whereas interactions with VP2 are inhibitory (SubbaReddy et al., 2011). Taken together, this suggests that the
viral proteins may play alternative regulatory roles, perhaps
at different stages in the norovirus life cycle. Recent work
has highlighted that the HuNV RdRp may be regulated by
phosphorylation by the cellular survival kinase Akt (Eden et
al., 2011). Akt phosphorylates HuNV NS7 RdRp on residue
T33, which is conserved among pandemic HuNV GII.4
strains, but absent in non-pandemic strains. However, the
implications of Akt regulation of RdRp activity on infection
have not been investigated fully.
Following the generation of a double-stranded RF, the
synthesis of positive-sense genomic and subgenomic RNA
occurs (Fig. 2). It is accepted generally that this process
occurs in a VPg-dependent manner as positive-sense
infectious norovirus RNA isolated from MNV-infected cells
is linked to VPg (Chaudhry et al., 2006). For this process to
occur, the NS7 RdRp uses VPg as a protein primer for RNA
synthesis, initiating at the 39 end of the negative-sense RNA.
This mechanism is also employed by other caliciviruses and
members of the family Picornaviridae of positive-sense RNA
viruses (Liu et al., 2009; Rohayem et al., 2006). In the case of
noroviruses, the NS7 RdRp initiates RNA synthesis by
covalently attaching VPg via a phosphodiester bond to the
initiating nucleotide, which is invariably guanine in all
members of the family Caliciviridae. This process is referred
to as VPg nucleotidylation or guanylation. The linkage is
formed between the guanine at the 59 end of the genome and
a conserved tyrosine residue in VPg (Y26 in MNV and Y27
in HuNV) (Subba-Reddy et al., 2011). This linkage is
essential for infectivity as enzymic removal of VPg from
purified norovirus RNA significantly reduces infectivity
(Chaudhry et al., 2006; Guix et al., 2007). Biochemical and
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Journal of General Virology 95
Norovirus gene expression and replication
(a) Premature termination
(b) Internal initiation
3′
AAAA(n)
5′(+)
VPg
3′
AAAA(n)
5′(+)
VPg
Pol/NS7
Pol/NS7
Termination signal
5′(+)
3′
AAAA(n)
UUUU(n)
5′(–)
Termination of
RNA synthesis
5′(+)
3′
AAAA(n)
UUUU(n)
5′(–)
SG promoter
Internal initiation
on SG promoter
3′
5′(+)
3′
UUUU(n)
5′(–)
3′
AAAA(n)
UUUU(n)
5′(–)
5′(+)
3′
AAAA(n)
5′(+)
3′
AAAA(n)
UUUU(n)
5′(–)
3′
3′
3′
UUUU(n)
5′(–)
3′
AAAA(n)
UUUU(n)
5′(–)
Fig. 4. Mechanisms of norovirus subgenomic RNA synthesis. Schematic representation of the two proposed models for
norovirus subgenomic RNA synthesis. (a) The presence of a termination signal upstream of the VP1-coding region (termination
signal) results in premature termination during negative-sense RNA synthesis by the viral RNA polymerase (NS7/Pol). The VPglinked positive-sense RNA template is drawn in black and initiation occurs de novo. The resulting negative-sense subgenomic
RNA (blue) is then used for VPg-dependent RNA synthesis to produce a subgenomic dsRNA. The newly synthesized positivesense ‘daughter’ subgenomic RNA (green) can then be used as a template for the synthesis of negative-sense subgenomic
RNA. These in turn function as templates for additional rounds of ‘daughter’ subgenomic RNA synthesis. (b) The internal
initiation model relies on the VPg-dependent subgenomic RNA synthesis occurring on a promoter sequence present
downstream of the VP1-coding region on the negative-sense RNA (blue, SG promoter). The viral RNA polymerase initiates RNA
synthesis, presumably in a VPg-dependent manner, to produce new ‘daughter’ VPg-linked subgenomic RNA (green), which
may in turn function as a template for additional rounds of subgenomic RNA synthesis as shown in the premature termination
model.
structural studies have indicated that the conformation of
VPg and its helical core is central in facilitating the VPg–RdRp
interaction, but have also highlighted that VPg is likely to
unfold during the process of guanylation to enable the
positioning of the tyrosine in the active site of the RdRp (Leen
et al., 2013). This process of VPg nucleotidylation has been
studied widely in the picornaviruses where a small RNA
structure, first identified in the 2C-coding region of poliovirus
(Goodfellow et al., 2000, 2003) was used as a template to
produce VPg-pUpU that was then used to initiate viral RNA
synthesis at the termini of the template RNAs (Liu et al.,
2009). A similar structure has been proposed for noroviruses,
http://vir.sgmjournals.org
located in the NS7-coding region (Victoria et al., 2009);
however, this structure is not required for in vitro nucleotidylation of the norovirus VPg. There is in fact evidence than an
element at the 39 end of the genome enhances the efficiency of
nucleotidylylation, at least in vitro (Belliot et al., 2005).
Based on the observation that both positive- and negativesense forms of the viral genomic and subgenomic RNAs
have been detected in calicivirus-infected cells (Green
et al., 2002), two models have been put forward for
the mechanism of norovirus subgenomic RNA synthesis
(Fig. 4). The first involves premature termination during
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285
L. G. Thorne and I. G Goodfellow
synthesis of the negative-sense genomic RNA, arising from
a termination signal. The resulting negative-sense subgenomic RNA would then serve as a template for the
production of positive-sense subgenomic RNA. The second
model is that an RNA secondary structure upstream of
ORF2 in the negative-sense genomic RNA acts as a
promoter for synthesis of positive-sense subgenomic
RNA. In agreement with this model, bioinformatic analysis
has indicated the presence of a highly conserved RNA
stem–loop structure downstream of the VP1-coding region
on the negative-sense RNA, 6 nt from the start of the
subgenomic RNA in all members of the family Caliciviridae
(Simmonds et al., 2008). Mutational analysis of this
structure confirmed its essential role in the norovirus life
cycle, but whether it plays a direct role in viral subgenomic
RNA synthesis is unknown. In vitro biochemical studies
with a related member of the genus Lagovirus, rabbit
haemorrhagic disease virus, suggests that the calicivirus
RdRp can utilize antisense genomic RNA as a template for
the production of subgenomic-like RNA in vitro, at least
(Morales et al., 2004). Further studies in this area are
warranted to determine if this model is correct, but it is
worth noting that the models are not mutually exclusive as
positive-sense subgenomic RNA produced in the internal
initiation model could be used as a template for replication
by NS7. Once the synthesis of positive-sense viral genomic
and subgenomic RNAs is initiated, multiple rounds of
translation of the newly synthesized RNAs ensues, followed
by additional rounds of RNA synthesis.
Interactions with host cell factors and pathways
Interactions with host cell proteins and cellular pathways
are essential for achieving the productive replication of all
positive-strand RNA viruses (Nagy & Pogany, 2011).
Knowledge of host cell interactions involved in the
norovirus life cycle currently lags behind that of other
RNA viruses. As mentioned above, several studies have
demonstrated interactions between host cell proteins and
structures in the viral RNA, which play a role in the viral life
cycle (Bailey et al., 2010b; Gutiérrez-Escolano et al., 2000b;
Sandoval-Jaime & Gutiérrez-Escolano, 2009; Vashist et al.,
2012). The host cell proteins DDX3, La and PTB were all
found subsequently to at least partially localize to the viral
replication complex during infection, and RNAi-mediated
knockdown of these factors reduced MNV replication in
cell culture, indicating that these interactions play a role in
replication, although their specific functions are not known
(Vashist et al., 2012). Another study identified an
interaction between PTB and the polypyrimidine tract in
a stem–loop structure at the 39 end of the MNV genome
(Bailey et al., 2010b). Mutation of the polypyrimidine tract
reduced PTB binding and had no effect on replication in
cell culture, but was sufficient to attenuate MNV in vivo,
demonstrating a role of RNA–protein interactions in
norovirus replication and as possible determinants of
pathogenesis (Bailey et al., 2010b).
286
To create a beneficial cellular environment for replication,
noroviruses are thought to disrupt a number of host cell
pathways. As described above, the host cell protein
secretion pathway is one such target that is disrupted
during replication of both HuNV and MNV to modify and
recruit membranes for replication (Sharp et al., 2010, 2012;
Wobus et al., 2004). It is possible, however, that disruption
of this pathway and abrogation of cell surface protein
expression confers additional advantages during norovirus
infections, such as interfering with antigen presentation or
cytokine secretion, as has been reported for other RNA
viruses that disrupt the host cell secretion pathway (Dodd
et al., 2001). The HuNV replicon system has facilitated a
number of studies into HuNV replication in the absence of
an efficient cell culture system. It consists of a human
hepatoma cell line (Huh-7) expressing self-replicating
Norwalk virus genomic RNA and has been used to
investigate cellular pathways that are altered by HuNV
replication (Chang, 2009; Chang et al., 2006). In this way,
both cholesterol and carbohydrate biosynthesis pathways
were found to be altered in replicon-bearing cells by DNA
microarray analysis. Cholesterol biosynthesis was downregulated and the use of statins to lower cholesterol was
found to promote replication (Chang, 2009). Likewise,
simvastatin has been found to increase replication in
gnotobiotic pigs infected with HuNV (Jung et al., 2012)
and epidemiological data has since raised concerns
regarding the use of statins as an increased risk factor
in the elderly (Rondy et al., 2011). The enhanced
replication resulting from treatment with statins in vivo is
thought in part to be mediated through suppression of
innate immunity (Jung et al., 2012); however, the direct
molecular basis for the interaction between norovirus and
cholesterol synthesis has yet to be determined.
Studies on related positive-sense RNA viruses suggest an
extensive modification of the cellular transcriptional and
translational apparatus occurs with infection, which is
beneficial to virus replication. In addition to the modification
of the cholesterol pathway, in vitro biochemical studies have
indicated that the norovirus NS6 protease possess the ability
to cleave components of the cellular translation apparatus,
namely PABP (Kuyumcu-Martinez et al., 2004). Cleavage of
PABP, as well as other components of the eIF4F complex, has
been observed during FCV infection (Willcocks et al., 2004),
but whether or not this occurs during authentic norovirus
replication has yet to be determined.
Modification of the nuclear–cytoplasmic shuttling pathways has also been reported in other positive-sense RNA
viruses and several observations would suggest that this
may also occur during norovirus infection of permissive
cells. (i) Infection with FCV results in the movement of the
PTB protein from the nucleus to the cytoplasm where it
negatively regulates FCV translation (Karakasiliotis et al.,
2010). Importantly, this appears to occur prior to a global
effect on nuclear export. (ii) During MNV infection of cells
the nuclear–cytoplasmic shuttling protein hnRNPA1,
predominantly nuclear in uninfected macrophage cells,
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Norovirus gene expression and replication
redistributes to the cytoplasm upon infection, where, as
highlighted above, it is thought to play a role in norovirus
genome circularization (López-Manrı́quez et al., 2013).
Antagonistic interactions with the innate immune system are
a well-known feature of RNA virus infections. Given the short
time course of HuNV, innate immunity has been implicated
highly in the control of infection, which has been supported
by studies with HuNV and MNV. Treatment with IFN-a/b
reduced HuNV RNA replication in the replicon system in
gnotobiotic pigs and decreased replication of MNV in vitro
(Chang & George, 2007; Jung et al., 2012). The role of the
signal transducer and activator of transcription STAT1
signalling in the IFN-mediated innate immune response has
also been well documented to control the appearance of
clinical disease and viral dissemination during MNV infection
in vivo (Karst et al., 2003; Mumphrey et al., 2007; Wobus et al.,
2004). The initial detection of norovirus RNA is thought to
occur primarily by the cellular helicase MDA-5, which senses
dsRNA, rather than the RIG-I RNA sensor, which did not
confer resistance to HuNV replication in transfected Huh-7
cells (Guix et al., 2007; McCartney et al., 2008). Accordingly,
MNV replicates to higher titres in MDA-52/2 mice and also,
to a smaller extent, higher in TLR32/2 mice, suggesting it may
also contribute to detection of dsRNA (McCartney et al.,
2008). The signalling cascade initiated by MDA-5 is mediated
by the mitochondrial antiviral signalling (MAVS) protein,
which results in activation of the transcription factors IFNregulatory factor (IRF)-3, IRF-7 and NF-kB. This in turn
results in the production of type I IFNs and the upregulation
of IFN-stimulated genes (ISGs) (Kawai et al., 2005; Seth et al.,
2005). Both IRF-3 and IRF-7 are required for IFN-mediated
control of MNV (Thackray et al., 2012). MNV is able to
interfere with innate immune signalling at the cellular level at
least partially through the actions of VF1, the product of
ORF4 (McFadden et al., 2011). In vitro, VF1 was found
recently to delay the upregulation of IFN-b and other ISGs,
and loss of VF1 resulted in a fitness cost in vitro and
attenuation in vivo (McFadden et al., 2011). Interestingly
attenuation was observed in a STAT12/2 model of infection,
suggesting that STAT1-independent signalling pathways may
also be involved in, but are not sufficient to control, MNV
infection alone. Given its mitochondrial localization, VF1 is
expected to interfere with signalling through the MAVS
complex or a downstream component affecting IRF-3 and
IRF-7 activation, although further studies are required to
elucidate the direct target. As HuNV does not express a
homologue to VF1, it is unknown at present if it has the same
capacity as MNV to antagonize the innate immune response;
however, functional duplication of VF1 remains a possibility.
Assembly and exit
The processes behind viral assembly, encapsidation and the
exit of noroviruses are largely unknown. The ability of VP1
to self-assemble into virus-like particles (VLPs) indistinguishable morphologically and antigenically from native
virions suggests that it may be sufficient to drive capsid
http://vir.sgmjournals.org
assembly during virus replication (Bertolotti-Ciarlet et al.,
2002), although the involvement of cellular proteins cannot
be excluded. This self-assembly property of the norovirus
VP1 protein has been utilized to generate a recombinant
VLP-based vaccine that has proven efficacious in human
volunteer studies (Atmar et al., 2011). Whilst VP2 is not
required for VLP assembly, it is thought to promote the
stability of VP1 and is essential for the production of
infectious virions (Bertolotti-Ciarlet et al., 2002; Sosnovtsev
et al., 2005). The highly basic nature of VP2 has formed the
basis of a long-held theory that it may be involved in
encapsidation via an interaction with the acidic viral RNA
(Sosnovtsev et al., 2005). In support of this, VP1 and VP2
have been shown to interact, and the binding of VP2 was
mapped to a conserved motif in the shell domain of VP1
(Vongpunsawad et al., 2013). This places VP2 in the interior
of the capsid, consistent with a role in encapsidation.
However, to date, a direct interaction between VP2 and viral
genomic RNA has not been demonstrated. A possible
interaction between the VPg protein linked covalently to the
viral RNA and the capsid protein VP1 has also been
observed in yeast two-hybrid studies with the related FCV
(Kaiser et al., 2006). Such an interaction would provide a
mechanism for the specific encapsidation of replicated viral
RNA in preference to cellular mRNAs, although further
studies are required to validate such a model.
There have been no direct studies on the exit and release of
the assembled norovirus virion to complete the viral life cycle.
One proposed exit strategy for other caliciviruses involves the
induction of apoptosis (Alonso et al., 1998; Bok et al., 2009;
Sosnovtsev et al., 2003). Whether noroviruses employ a
similar strategy is unknown; however, accumulation of
apoptotic epithelial cells has been observed in intestinal
biopsies from infected patients (Bok et al., 2009; Furman et al.,
2009; Troeger et al., 2009). Furthermore, as highlighted
above, apoptosis is induced with active MNV replication and
plays a role in the processing of NS1/2 during the latter stages
of the viral life cycle. The induction is associated with
downregulation of survivin, a pro-survival factor, followed by
caspase and cathepsin B activation and cytochrome c release
(Bok et al., 2009; Furman et al., 2009). Whether the
downregulation of survivin occurs by a direct interaction
with a viral protein or as a response to indirect effects of MNV
on the host cell is as yet unknown. Instead, VF1 has been
shown to delay the onset of apoptosis through its localization
at the mitochondria (McFadden et al., 2011), although it is
possible that this may serve to prolong the window for
replication prior to the final stages of exit and release.
Inhibition of apoptosis results in reduced MNV production,
either way demonstrating a clear requirement for apoptosis
for an aspect of the norovirus life cycle (Furman et al., 2009).
Concluding remarks
Although our understanding of the norovirus life cycle is
far from complete, the past decade has seen a dramatic
increase in publications relating to various aspects of
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norovirus biology, including the molecular mechanisms of
norovirus genome translation and replication. This is in
part due to increased awareness of the prevalence and
clinical importance of noroviruses, but also due to the
significant efforts in the development of tools and
resources for the study of norovirus biology. A lack of an
efficient culture system for HuNVs is a major hurdle that
researchers in the field have yet to overcome, but steps
towards this are already under way (Asanaka et al., 2005;
Guix et al., 2007). Work with related viruses has provided
novel insights into the biology of members of the family
Caliciviridae, but mechanistic differences between the
various members of the family are apparent. For example,
whilst further processing of the MNV NS1/2 protein occurs
during virus replication, whether the HuNV p48 (N-term)
protein is further processed in not known. Also, whilst FCV
translation initiation is clearly dependent on eIF4E, the
only study to date on norovirus translation initiation
found that eIF4E is in fact dispensable (Chaudhry et al.,
2006). With this in mind, continued efforts and the use of
novel approaches to norovirus culture will be required to
provide the breakthrough needed. One such approach has
found recently that mice deficient in aspects of the immune
response can be infected by intraperitoneal injection of
HuNV (Taube et al., 2013). This experimental system, which
does not recapitulate norovirus gastrointestinal disease,
does, however, provide a useful tool for the study of aspects
of norovirus pathogenesis. More importantly, this system,
combined with the advances in other areas, provides a
starting block from which to move forward to the ‘Holy
Grail’ of norovirus biology, i.e. a cell culture system that
enables HuNVs to undergo a full replication cycle. Such a
system, when combined with a reverse-genetics system to
enable viral genome manipulation and a small, preferably
inexpensive and genetically defined, animal model, would
provide the complete toolset with which to fill in the
remaining gaps in our understanding of norovirus biology.
Acknowledgements
The authors are supported by funding from the Wellcome Trust as a
PhD Studentship to L. G. T. and a Senior Fellowship to I. G. G. I. G. G.
is also supported by funding from the Biotechnology and Biological
Sciences Research Council.
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