Download Post-translational regulation and modifications of flavivirus structural

Journal of General Virology (2015), 96, 1551–1569
Review
DOI 10.1099/vir.0.000097
Post-translational regulation and modifications of
flavivirus structural proteins
Justin A. Roby,1,23 Yin Xiang Setoh,1,2 Roy A. Hall1,2
and Alexander A. Khromykh1,2
Correspondence
1
Alexander A. Khromykh
2
[email protected]
Received 4 November 2014
Accepted 18 February 2015
Australian Infectious Diseases Research Centre, The University of Queensland, Australia
School of Chemistry and Molecular Biosciences, The University of Queensland, Australia
Flaviviruses are a group of single-stranded, positive-sense RNA viruses that generally circulate
between arthropod vectors and susceptible vertebrate hosts, producing significant human and
veterinary disease burdens. Intensive research efforts have broadened our scientific understanding
of the replication cycles of these viruses and have revealed several elegant and tightly co-ordinated
post-translational modifications that regulate the activity of viral proteins. The three structural
proteins in particular – capsid (C), pre-membrane (prM) and envelope (E) – are subjected to strict
regulatory modifications as they progress from translation through virus particle assembly and
egress. The timing of proteolytic cleavage events at the C–prM junction directly influences the
degree of genomic RNA packaging into nascent virions. Proteolytic maturation of prM by host furin
during Golgi transit facilitates rearrangement of the E proteins at the virion surface, exposing the
fusion loop and thus increasing particle infectivity. Specific interactions between the prM and E
proteins are also important for particle assembly, as prM acts as a chaperone, facilitating correct
conformational folding of E. It is only once prM/E heterodimers form that these proteins can be
secreted efficiently. The addition of branched glycans to the prM and E proteins during virion transit
also plays a key role in modulating the rate of secretion, pH sensitivity and infectivity of flavivirus
particles. The insights gained from research into post-translational regulation of structural proteins
are beginning to be applied in the rational design of improved flavivirus vaccine candidates and make
attractive targets for the development of novel therapeutics.
Flaviviruses
The genus Flavivirus in the family Flaviviridae comprises
small, enveloped viruses with non-segmented genomes
consisting of single-stranded, positive-sense RNA. These
RNA genomes are flanked by 59 and 39 untranslated regions
(UTRs) and encode a single polyprotein that is cleaved coand post-translationally to generate between 10 and 11 viral
proteins. The 59 UTR contains a type 1 m7GpppNm cap
structure and the 39 UTR lacks a polyadenylated tail.
Structural elements within the UTRs regulate genome
replication, translation and host immune responses. Viral
proteins are divided between structural proteins, which form
the virion, and non-structural proteins, which are involved
in genomic replication and modulating host immunity (Fig.
1a) (Lindenbach et al., 2007; Roby et al., 2012).
Flaviviruses are maintained predominantly in natural transmission cycles between vertebrate reservoir species (normally mammalian or avian) and haematophagous arthropod
vectors (mosquitoes or ticks). Notable exceptions are the
3Present address: Department of Immunology, University of Washington
School of Medicine, Seattle, WA, USA.
000097 G 2015 The Authors Printed in Great Britain
viruses that are maintained solely in mosquito hosts (insectspecific flaviviruses) and viruses with no known invertebrate
vector (Cook et al., 2012; Mackenzie et al., 2004). As of 2013,
the International Committee on Taxonomy of Viruses has
classified 53 different viral species as belonging to the genus
Flavivirus; however, many newly identified flaviviruses are
being isolated each year and remain to be formally described
(Adams et al., 2013; King et al., 2012). Of these 53 separate
species, 40 have been associated with infection in humans
such as dengue virus (DENV), yellow fever virus (YFV),
tick-borne encephalitis virus (TBEV), Japanese encephalitis
virus (JEV) and West Nile virus (WNV). Several flavivirus
species including JEV, WNV and louping ill virus also cause
economically important diseases of livestock and poultry
(Gubler, 2012; van der Meulen et al., 2005). In light of the
heavy disease burden attributed to flaviviruses, the generation of effective vaccines and therapeutics remains a
significant goal within the medical research community.
The replicative cycle of flaviviruses is an elegantly regulated
process and has been described in detail elsewhere (Roby
et al., 2012). Briefly, replication consists of seven discrete
stages (Fig. 2): (i) receptor-mediated virus entry; (ii)
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J. A. Roby and others
(a)
Genomic RNA
5′ UTR
3′ UTR
Structural genes
C ss prM
E
Non-structural genes
NS1
NS2A NS2B
NS3
NS4A 2K NS4B
NS5
Proteins
pr M
(b)
Viral NS2B/3 protease
Unknown protease
Host signal peptidase
Host furin
Envelope protein (E)
Pre-membrane
protein (prM)
to NS1
ER lumen
Cytoplasm
Signal sequence
Proteolytic cleavage points
Viral NS2B/3 protease
Host signal peptidase
Host furin
Capsid protein (C)
Fig. 1. Flavivirus genomic organization and membrane topology. (a) Organization of the flavivirus genome. The positive-sense
ssRNA of ~11 kb encodes a single ORF flanked by 59 and 39 UTRs. Translation of the ORF produces a single polyprotein, which is
cleaved co- and post-translationally to form each of the mature viral proteins. (b) Schematic demonstrating the membrane topology
of flavivirus structural proteins. Crystal structures of the Kunjin virus C (Dokland et al., 2004) and DENV pr peptide and E protein (Li
et al., 2008) have been used to enhance visualization of the polyprotein, rather than to imply that these mature conformations are
adopted prior to cleavage. Sites of proteolytic processing of the polyprotein are indicated, as is the signal sequence (ss) between
C and prM. Trans-membrane domains and membrane-associated helices are represented as cylinders.
low-pH-induced fusion of the viral envelope with the
host endosome, uncoating of the nucleocapsid and initial
polyprotein translation; (iii) asymmetrical semi-conservative replication of genomic RNA within endoplasmic
reticulum (ER)-derived vesicle packets (VPs); (iv) virion
assembly on ER membranes directly opposed to the VP
pore; (v) trafficking of immature virions to the Golgi
apparatus for glycan maturation; (vi) furin cleavage of
pre-membrane protein (prM) within the trans-Golgi
network and low-pH-mediated rearrangement of the
envelope proteins on the virion surface; and (vii)
secretion of mature progeny virus and dissociation of
the cleaved pr peptide.
Throughout this replication cycle, the three flavivirus
structural proteins, capsid (C), prM and envelope (E), are
subjected to strict post-translational control in order to
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regulate efficient virion assembly, secretion and infectivity.
Co-ordination between dual cleavage events at the C–prM
transmembrane junction of the flavivirus polyprotein has
been demonstrated to be important in the effective incorporation of nucleocapsid into nascent virions (Lobigs &
Lee, 2004; Lobigs et al., 2010). Heterodimeric interaction
between prM and E has been revealed as necessary for the
correct conformational folding of the envelope and the
prevention of premature virus fusion during secretory
transit (Mukhopadhyay et al., 2005). Indeed, the protective
role of prM is so efficient that cleavage by the host enzyme
furin is required in the final stages of virion secretion in
order to promote infectivity of progeny virus. The addition
of complex glycans to the envelope proteins prM and E
within the ER and Golgi has also been demonstrated to act
in regulating the rate of virion secretion, sensitivity to low-
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Journal of General Virology 96
Flavivirus structural protein regulation
(vi) Low-pH conformational
change and furin cleavage
of prM
(vii) Secretion of progeny virus
and pr dissociation
(i) Receptor-mediated
virus entry
(v) Trafficking of immature
virus to Golgi
(iv) Virion assembly on
membranes adjacent
to VP
Golgi
(ii) Virus fusion
and uncoating
VP/RC
Genomic RNA
VP/RC
VP/RC
(iii) Replication
of RNA
VP/RC
Nucleus
Endoplasmic
reticulum
Fig. 2. Flavivirus genomic organization and replication cycle. Canonical model of the replication cycle of flaviviruses: (i)
receptor-mediated entry of virus; (ii) low-pH-induced fusion of the viral envelope with endosomes and release of genomic RNA;
(iii) formation of the replication complex (RC) within endoplasmic reticulum (ER)-derived vesicle packets (VPs) and subsequent
asymmetrical semi-conservative replication of genomic RNA; (iv) progeny genomic RNA associates with C and evagination into
the ER lumen generates the viral envelope containing prM and E heterodimer trimeric spikes; this process also occurs
independently of the nucleocapsid to produce empty prM/E particles; (v) immature virions and prM/E particles are trafficked in
individual vesicles to the Golgi apparatus for glycan maturation; (vi) low pH induces conformational rearrangement of prM/E
heterodimers, and cleavage of prM by host furin occurs within the trans-Golgi network; (vii) mature virions and prM/E particles
are secreted from the infected cell by exocytosis, and the associated pr peptide is released.
pH environments and the infectivity of mature virus
particles (Beasley et al., 2005; Goto et al., 2005; Guirakhoo
et al., 1993; Hanna et al., 2005; Lee et al., 1997, 2010; Li
et al., 2006; Mondotte et al., 2007; Scherret et al., 2001).
Exploitation of these post-translational control measures
has facilitated the continuing development of novel vaccines
and therapeutics for use against flavivirus infections (Goto
et al., 2005; Keelapang et al., 2013; Ohtaki et al., 2010; Pang
et al., 2014; Roby et al., 2011, 2013, 2014; Zhang et al., 2011).
Flavivirus structural proteins
C protein
The flavivirus C protein is a small (approx. 14 kDa)
cytosolic protein with affinity for both lipid membranes
and nucleic acids (Dokland et al., 2004; Ivanyi-Nagy et al.,
2008; Jones et al., 2003; Khromykh & Westaway, 1996; Ma
et al., 2004; Markoff et al., 1997). It is translated in cis at the
N terminus of the flavivirus polyprotein (Fig. 1b) and
http://vir.sgmjournals.org
undergoes maturation to a soluble protein by post-translational cleavage from the prM signal sequence by cytosolic
viral NS2B/3 protease (see ‘Polyprotein processing’ below
and Fig. 3). Nuclear magnetic resonance and X-ray crystallography experiments (Dokland et al., 2004; Ma et al.,
2004) have revealed that the C protein consists of four
structurally conserved a-helices and contains a conserved
internal hydrophobic domain. The C protein forms dimers
in solution mediated by association of the internal hydrophobic regions, and in particular a conserved tryptophan
residue (W69 in WNV) in a-3 (Bhuvanakantham & Ng,
2005; Kiermayr et al., 2004). Recent evidence indicates that
the a-4 helix may also play a crucial role in dimerization
(Teoh et al., 2014). Indeed, this is supported by experiments that demonstrate that WNV can tolerate complete
deletion of the a-2 and a-3 helices (including the
hydrophobic domain and residue 69) and retain the ability
to package RNA (Schlick et al., 2009). Small internal
C-protein deletions in TBEV infectious clones have also
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(b)
(a)
Signallase
site
prM
(c)
Signallase
site
(PQAQA)
prM
Signallase
site
(PQAQA)
Lumen
Lumen
Lumen
Cytosol
Cytosol
Cytosol
C
NS2B/3
site
C
NS2B/3
site
prM
C
NS2B/3
site
(+2 aa+S111Y)
Efficient
signallase
cleavage
Efficient
NS2B/3
cleavage
prM
prM
Lumen
Lumen
Cytosol
Cytosol
Efficient,
simultaneous
NS2B/3 and
signallase
cleavage
C
C
Very
inefficient
NS2B/3
cleavage
Efficient
signallase
cleavage
prM
Lumen
Lumen
Lumen
Cytosol
Cytosol
Cytosol
C
High-titre virions
High-titre prM/E particles
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prM
prM
C
No or low-titre virions
High-titre prM/E particles
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C
Low-titre virions
High-titre prM/E particles
Journal of General Virology 96
Flavivirus structural protein regulation
Fig. 3. Co-ordinated proteolytic cleavages at the C–prM junction. (a) Flavivirus polyprotein cleavage in the natural state.
Cytosolic C protein is first cleaved by the viral NS2B/3 protease. This initial cleavage induces a change at the ER lumenal
signallase cleavage site in prM allowing efficient cleavage. The outcome of the sequential events is a delay in prM accumulation
and concomitant efficient nucleocapsid incorporation. (b) Introduction of the PQAQA mutation upstream of the prM signallase
cleavage site, which removes the dependence on prior cleavage of C. Signallase cleavage of prM renders a change at the
NS2B/3 cleavage site at the end of C, making subsequent C release very inefficient. The outcome of uncoupling signallase
cleavage is that no or very little nucleocapsid incorporation occurs though prM/E particle secretion continues. (c) The
introduction of a GS dipeptide and SAY mutation adjacent to the NS2B/3 cleavage site allows C to be processed irrespective
of prior signallase cleavage of prM. The outcome of completely uncoupling the co-ordinated cleavages is a low titre of
nucleocapsid-containing virions and efficient prM/E particle secretion. Note that crystal structures of the KUNV C (Dokland et
al., 2004) and DENV pr peptide (Li et al., 2008) were used to enhance visualization of the polyprotein.
demonstrated that the a-1 and a-2 helices are dispensable,
as these mutants either were able to functionally package
infectious virions (1–16 aa within a-1 and a-2) (Kofler
et al., 2002) or developed compensatory mutations
elsewhere within C that restored encapsidation (19–30 aa
in a-2) (Kofler et al., 2003).
The a-4 helices of C associate to form a positively charged
surface that has been proposed to act alongside the flexible
N terminus in stabilizing the interaction with RNA
(Dokland et al., 2004; Khromykh & Westaway, 1996; Ma
et al., 2004). The flexible N terminus of DENV C has also
been demonstrated to mediate lipid droplet association
(Martins et al., 2012). Highly charged basic residues found
at the N and C termini (aa 1–32 and 82–105 in WNV)
associate with nucleic acids, particularly with the 59 and 39
UTRs of flavivirus genomic RNA (Khromykh & Westaway,
1996; Khromykh et al., 1998), and nucleic acid interaction
is dependent on dephosphorylation of the C protein
(Cheong & Ng, 2011). Phosphorylation of WNV C has
conversely been shown to promote nuclear localization and
thus removes the protein from sites of genomic RNA
interaction (Bhuvanakantham et al., 2010). The Kunjin virus
(KUNV; a naturally attenuated strain of WNV) and DENV
C structures place a-1 in different orientations; thus, it is
hypothesized that this helix rotates in order to change the
position of the flexible N terminus for optimal RNA
interaction (Dokland et al., 2004; Ma et al., 2004).
The C protein localizes to three distinct cellular compartments:
the ER membrane and lipid droplets in the cytoplasm, as well
as the nucleus (specifically the nucleolus) (Bhuvanakantham
et al., 2010; Bulich & Aaskov, 1992; Colpitts et al., 2011;
Martins et al., 2012; Mori et al., 2005; Samsa et al., 2009;
Westaway et al., 1997). The primary role of the C protein in the
flavivirus life cycle is in the assembly of the nucleocapsid and its
incorporation into nascent virions, whilst other functions of C,
including those performed in the nucleus, are less explored.
prM protein
The prM protein of flaviviruses is an approximately 19–
21 kDa (the M protein is approximately 10 kDa after furin
cleavage) membrane glycoprotein that is translocated
into the ER lumen during polyprotein translation by an
N-terminal signal sequence immediately downstream from
http://vir.sgmjournals.org
C (Fig. 1b) (Lobigs et al., 2010; Lorenz et al., 2002; Setoh
et al., 2012). Liberation of prM from this signal sequence
by host cell signallase is tightly co-ordinated and is dependent on prior cleavage of C by viral NS2B/3 protease (see
‘Polyprotein processing’ below and Fig. 3). Flavivirus prM
also contains two C-terminal transmembrane helices
anchoring the protein to the ER membrane, the second
of which acts as a signal sequence for luminal translocation
of the E protein (Hsieh et al., 2011). Nascent prM is
initially glycosylated in the ER lumen at between one and
three sites within the pr portion of the protein, with glycan
maturation occurring during transit through the Golgi
apparatus (Mackenzie & Westaway, 2001).
Whereas the structure of the mature M portion of prM has
yet to be solved beyond cryo-electron microscopy reconstructions (Zhang et al., 2013b), a crystal structure of the
DENV pr peptide has been reported in complex with the
soluble ectodomain of E (Li et al., 2008) (see Figs 1b and
3). The pr peptide forms a tightly folded protein domain
consisting of two small b-sheets linked by disulfide bridges,
with the entire structure positioned at the distal end of E
domain II (DII) within the heterodimer where it obscures
the fusion loop and facilitates pr glycan presentation at the
tip of trimeric spikes (Li et al., 2008; Yu et al., 2008; Zhang
et al., 2003). The C-terminal region of the M protein
ectodomain is also predicted to form a helical domain
(prM-H) that may assist in prM/E heterodimer formation
(Hsieh et al., 2011; Peng & Wu, 2014; Zhang et al., 2012).
Together with the E protein, prM forms an integral part of
the flavivirus envelope. Proposed roles for prM include
assisting in the chaperone-mediated folding of the E protein
(Konishi & Mason, 1993; Lorenz et al., 2002), contributing
to the pH-dependent conformational rearrangement of
trimeric prM/E heterodimer spikes in immature virions via
the influence of the helical stem domain of M (Zhang et al.,
2012), and in the concealment of the fusion loop of E by the
pr peptide to prevent premature fusion during virion egress
(Yu et al., 2009).
E protein
The E protein of flaviviruses is a large (approx. 53 kDa)
membrane-bound glycoprotein that is responsible for virus
fusion and acts as the main antigenic target for the develop-
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ment of neutralizing humoral immunity. Several structures
of the E protein ectodomain have been resolved for DENV,
TBEV, JEV and WNV in a mature conformation (Kanai
et al., 2006; Li et al., 2008; Luca et al., 2012; Modis et al.,
2003; Nybakken et al., 2006; Rey et al., 1995). The E protein
consists of three b-barrel structural domains. The central
domain (domain I; DI) contains the N terminus and appears
to act as an anchor for the hinge point of the large distal
domain (domain II; DII) (Zhang et al., 2004). This second
domain contains the fusion-loop peptide and is involved in
virus fusion during entry (Mukhopadhyay et al., 2005), and
in interactions with prM, which occludes the fusion loop in
immature particles during cellular transit (Zhang et al.,
2003, 2012). The third, immunoglobulin-like domain
(domain III; DIII) on the opposite side of DI is hypothesized
to interact with cellular receptors and is anchored at the C
terminus to the two ‘stem’ helices and the two transmembrane helices (Fig. 1b) (Mukhopadhyay et al., 2005). All three
domains appear to be involved in the dimerization of the E
protein through hydrophilic interactions in mature virions
(Zhang et al., 2004). The E protein is liberated from the
genomic polyprotein at its N and C termini via cleavage with
host signal peptidase in the ER lumen.
The E protein is synthesized immediately downstream of prM
with which it rapidly heterodimerizes, an important interaction associated with the correct folding of E (Lorenz et al.,
2002). These heterodimers coalesce on the lumenal face of the
ER membrane forming trimeric spikes and ultimately bud off
to give rise to either empty prM/E particles, or nucleocapsidcontaining immature virions (Roby et al., 2012). The
observed formation and secretion of prM/E particles upon
ectopic prM and E expression as part of a recombinant gene
cassette (Allison et al., 1995; Goto et al., 2005; Konishi &
Mason, 1993; Ohtaki et al., 2010; Zhang et al., 2011) suggest
that this process is intrinsic to the prM and E proteins. This
hypothesis is supported by a recent analysis of WNV and
DENV membrane curvature induced by the arrangement of
prM and E within the virus particle, suggesting that the
overall spherical shape of the particles may be attributable to
viral protein interaction with lipids without the need of
assistance from host cellular factors (Zhang et al., 2013a).
Low-pH-induced conformational rearrangement of the
prM/E spikes leads to the formation of a ‘herringbone’
pattern of three adjacent prM/E complex homodimers
lying flat on the virion surface, with the pr peptide
obscuring the fusion loop at the DII terminus (Figs 2 and
4) (Kuhn et al., 2002). The pr peptide is subsequently
excised via furin cleavage during virion egress leaving a
smooth, spherical mature particle in which only the E
protein is exposed to solvent (Yu et al., 2008).
Polyprotein processing
Co-ordinated cleavage at the C–prM junction
Of crucial importance to the life cycle of flaviviruses is the
regulated sequence of proteolytic cleavages that occur at
either side of the prM signal sequence in the virus
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polyprotein (Fig. 3). The currently accepted paradigm is
that liberation of mature C by viral NS2B/3 cleavage at the
cytosolic face of the ER membrane induces a change at the
ER lumenal face that promotes efficient signalase cleavage
of prM. This temporal separation of cleavage events is
believed to promote efficient nucleocapsid incorporation
into budding virions (Lobigs & Lee, 2004; Lobigs et al.,
2010).
Early investigations using ectopically expressed flavivirus
structural cassettes in vitro determined that two independent
cleavage sites were recognized on either side of a signal
sequence separating C and prM and that signalase cleavage
of prM appeared dependent on the addition of NS2B/3
protease (Amberg et al., 1994; Lobigs, 1993; Yamshchikov &
Compans, 1993). The parameters controlling this sequential
cleavage of C–prM were demonstrated via a series of
mutations and chimeras in recombinantly expressed Murray
Valley encephalitis virus (MVEV) proteins (Stocks & Lobigs,
1998). Mutation of various residues within C or replacement
of C with ubiquitin in a structural gene cassette did not alter
the dependence of signalase cleavage of prM upon the prior
excision of the cytosolic portion of the polyprotein.
Mutation of the prM signal sequence from GFAAA to
PQAQA and expression in the absence of NS2B/3 allowed
efficient prM cleavage despite the C protein remaining
anchored to the signal sequence. Conversely, however,
replacement of prM downstream of the signal sequence with
E or the mouse H-2Kd MHC antigen showed only partial or
no dependency on NS2B/3 activity for signalase cleavage.
Thus, the regulation of prM cleavage is dependent primarily
upon elements within the C-terminal region of the signal
sequence, with a minor involvement of the prM protein
itself.
This model of sequential cleavage (Fig. 3a) was finally
confirmed to be important in the context of live flavivirus
replication via the mutation of the NS2B/3 cleavage site in
the YFV C–prM junction (Amberg & Rice, 1999). Mutations
that prevented efficient processing of C–prM quickly
reverted to large plaque-forming isolates with efficient polyprotein cleavages. The converse of these mutations was
investigated via the introduction of the foot-and-mouthdisease virus (FMDV) 2A sequence to the C terminus of
WNV and TBEV C inducing an immediate separation of C–
2A from the rest of the polyprotein upon translation
(Schrauf et al., 2009). These mutations were generally
tolerated, as WNV-2A replicated well in mosquito and
mammalian cells (albeit with smaller plaques) and TBEV-2A
was shown to replicate efficiently in mammalian cells,
although it was unable to replicate efficiently in tick cells.
These experiments were able to demonstrate that C
interaction with NS2B/3 was not important for virion
formation per se; rather that it is important for C to be
cleaved from the remainder of the polyprotein.
A purpose for the co-ordinated C–prM cleavages was
finally proposed based upon experiments in which the
PQAQA mutation was introduced into the signal sequences
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Flavivirus structural protein regulation
5
3
5
pH 7.2
3
2
3
2
3
90 × (prM/E)2
furin
90 × (pr + M/E)2 90 × (pr + M/E)2 90 × (M/E)2 + 180 pr released
60 × (prM/E)3
Endoplasmic
reticulum
3
3
2
5
Golgi
cis
pH 6.7
medium
trans
pH 6.0
Secretory
granules
pH 5.7
Extracellular space
pH 7.0
Fig. 4. Low-pH-induced conformational change in prM/E heterodimer organization during virus maturation and secretion. As
immature flavivirus particles are transported from the ER to the Golgi apparatus, they encounter a lower-pH environment. This
induces the rearrangement of prM/E heterodimers from 60 trimeric spikes to an anti-parallel ‘herringbone’ arrangement of 90
dimers that lie flat along the virion surface. This rearrangement exposes the furin cleavage site in prM, facilitating cleavage within
the trans-Golgi network. The liberated pr peptide (shown here as a golden surface model) is then secreted along with the
mature M/E-containing virion. Figure modified from Li et al. (2008).
of YFV and MVEV, effectively removing the requirement for
sequential processing of prM (Fig. 3b) (Lee et al., 2000; Lobigs
& Lee, 2004). Efficient signalase cleavage of prM independent
of prior C excision proved fatal for the replication of YFV,
with only revertant viruses containing mutations in the N
terminus of the signal sequence able to productively replicate
(Lee et al., 2000). In contrast, the PQAQA mutation was
tolerated to some degree in MVEV, culminating in a drastic
reduction in mature C expression and nucleocapsid incorporation whilst maintaining prM/E particle secretion (Lobigs
& Lee, 2004). Thus, two key points were revealed: (i) a delay
in prM cleavage by signalase promotes incorporation of the
nucleocapsid into budding virus particles, and (ii) efficient
cleavage of C by NS2B/3 requires prM to remain attached to
the signal sequence at its N terminus.
The unexpected revelation of a requirement for an intact
signal sequence of prM to facilitate efficient C cleavage was
investigated in PQAQA mutant strains of MVEV and by
utilizing a packaging cell line expressing different Cterminally extended forms of C (Lobigs et al., 2010).
Mutations that enhanced the hydrophobicity of the Cterminal region of the signal sequence, anchored the signal
sequence in membranes with tryptophans or mimicked the
viral protease cleavage site in NS5 failed to enhance the
growth of PQAQA MVEV. However, the introduction of a
GS dipeptide to the C-terminal region of the prM signal
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sequence allowed the selection of an isolate with an S111Y
mutation (two residues downstream of the GS dipeptide
within the signal sequence) that allowed more efficient C
maturation and thus better replication (Fig. 3c). Despite
the mutant virus replicating more efficiently than PQAQA
MVEV, it still displayed a packaging defect compared with
WT MVEV. Analysis of the packaging efficiency of cell lines
expressing various forms of C demonstrated that extension
of C to include the prM sequence up to the furin cleavage
site (pr) improved genomic RNA incorporation compared
with expression of the mature C alone. Thus, it appears
that a cleavable transmembrane anchor at the C terminus
of C promotes efficient packaging of viral RNA
A recent investigation by our group sought to confirm this
hypothesis by comparing the capacity of ectopically
expressed soluble mature C, extended C (containing the
signal sequence and the first 10 aa of prM), and Cpr
(containing the prM sequence up to the furin cleavage site)
to package C-deleted KUNV genomic RNA in trans (Roby
et al., 2014). Both the extC and Cpr constructs were
demonstrated to be able to be cleaved to generate mature C
by the viral protease (albeit incompletely, as uncleaved
forms of the proteins were detectable in cell lysates at
2 days post-transfection) and facilitate encapsidation of Cdeleted KUNV genomic RNA. Thus, the presence of only
10 lumenal residues of prM at the C terminus of the signal
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sequence is sufficient to allow NS2B/3 cleavage of C.
However, this investigation did not confirm a greater
packaging efficiency for anchored forms of C, as the production of infectious particles was determined to be at
efficiencies comparable to soluble C. It is possible that the
use of the stronger EF1a promoter to drive trans-C expression in these experiments (Roby et al., 2014) saturated the
capacity for nucleocapsid formation, thus overcoming any
inherent inefficiencies in mature C. Further investigations
utilizing a limited supply of C protein may better be able to
resolve this phenotype.
Maturation of prM by furin cleavage
An integral process in the maturation of flavivirus particles
is the cleavage of prM within the trans-Golgi network by the
host enzyme furin, liberating the pr peptide and ultimately
facilitating the exposure of the fusion loop within E protein
DII (Fig. 2) (Stadler et al., 1997). Interestingly, in vitro furin
cleavage assays show that maturation of prM only occurs at
low pH, even though the enzymic activity of furin is optimal
at neutral pH (Stadler et al., 1997; Yu et al., 2008). This
suggests that the low-pH-induced structural rearrangement
of the particle is required to expose the furin cleavage site on
prM. Indeed, the currently accepted model of flavivirus
maturation within the trans-Golgi involves the stem region
of the prM protein interacting with the associated E protein
DII in a manner that pulls E down from a trimeric spike into
a flat dimeric conformation parallel to the virion surface
(Zhang et al., 2012, 2013b), with this interaction dependent
upon decreasing pH and originating from one or more
discrete nucleation centres (Plevka et al., 2011, 2014). In
addition, the low-pH environment in the trans-Golgi
compartment allows the pr peptide to remain associated with E (Yu et al., 2009), effectively preventing
premature fusion within the cell.
The release of prM-containing immature particles is consistently observed along with release of mature particles
(Zhang et al., 2007a), as well as partially mature particles
(Junjhon et al., 2010; Plevka et al., 2011), suggesting that
either furin cleavage is not 100 % efficient or the speed of
prM/E conformational rearrangement on the particle is
slower than the rate of cell-driven particle secretion through
the trans-Golgi compartment. This phenomenon appears to
be dependent upon the identity of the virus, as high rates of
immature/partially mature virions are most consistently
observed for DENV (Rodenhuis-Zybert et al., 2011), and
this phenotype has been linked to a DENV-conserved acidic
residue at position 89 in prM (P3 within the furin cleavage
site) (Junjhon et al., 2008; Keelapang et al., 2004). WNV and
TBEV grown in BHK-21 cells have similarly been identified
as partially incomplete in maturity, with faint staining of
uncleaved prM in immunoblots prepared with purified virus
(Elshuber & Mandl, 2005; Moesker et al., 2010), although
complete prM cleavage has also been observed with TBEV in
this same cell line (Stadler et al., 1997).
Although immature flavivirus particles have been demonstrated to be non-infectious in vitro (Elshuber et al., 2003;
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Elshuber & Mandl, 2005; Moesker et al., 2010; Stadler et al.,
1997; Yu et al., 2008, 2009), opsonization of immature
DENV virions with anti-prM antibodies has been demonstrated to enhance the infection of Fc-receptor-positive
cells (Dejnirattisai et al., 2010; Rodenhuis-Zybert et al.,
2010). Bound virions are endocytosed, with acidification of
the endosome allowing furin-mediated cleavage of prM
and subsequent virus fusion. The identification of this prM
antibody-enhanced infection process has led researchers to
postulate that this phenomenon may allow a degree of
escape from the neutralizing host anti-E antibody response
(reviewed by Rodenhuis-Zybert et al., 2011) and may
inform the design of better vaccines (see below). The role
that immature/partially mature DENV virions play during
infection of Aedes spp. mosquito hosts has yet to be fully
resolved.
Initial stages of virion assembly
Formation of the nucleocapsid
Assembly of progeny virions proceeds following initial
replication of positive-strand flavivirus genomic RNA
within replication complex (RC)-containing vesicle packets
(VPs) derived from the ER (Fig. 2) (Roby et al., 2012). The
exact nature of nucleocapsid assembly remains elusive, as
the nucleocapsids themselves display no inherent order to
their structure (Kiermayr et al., 2004; Kuhn et al., 2002;
López et al., 2009; Zhang et al., 2007b) and a physical
interaction of the nucleocapsid with the envelope is not
observed. In accordance, the production of empty prM/E
particles during virus infection (Lindenbach et al., 2007;
Murray et al., 2008) and when these genes are expressed in
the absence of C (Allison et al., 1995, 2003; Konishi &
Mason, 1993; Lorenz et al., 2003; Wang et al., 2009) suggests
that prM/E formation and nucleocapsid assembly are independent processes. However, the efficiency of C dimerization impacts on the overall integrity of the assembled
particle in a thermal stability assay (Patkar et al., 2007),
suggesting that some interaction between C and prM/E does
still occur in the virion. Recent evidence describing the
ability of DENV C to act as a histone mimic and cause
nucleic acid condensation (Colpitts et al., 2011) supports the
hypothesis that the flavivirus nucleocapsid is not an ordered
icosahedral shell surrounding the genome but rather the
RNA condenses around C dimers.
Determination of the precise structure of the flavivirus
nucleocapsid remains elusive despite documentation of
assembly of spherical nucleocapsid-like particles in vitro
following incubation of recombinant C with nucleic acids
(Kiermayr et al., 2004; López et al., 2009; Teoh et al., 2014),
as well as the visualization of purported nucleocapsids
adjacent to VP pores in electron micrographs of DENVinfected cells (Welsch et al., 2009). Regardless of the nature
of flavivirus nucleocapsids, their assembly appears to be
dependent on the association of C with the ER membrane
and lipid droplets, presumably to optimally position the
protein between the VP pore where nascent genomic RNA
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Flavivirus structural protein regulation
exists and the ER sites of prM/E envelope generation
(Samsa et al., 2009; Welsch et al., 2009; Westaway et al.,
1997).
Heterodimerization of prM and E
Heterodimerization between the flavivirus prM and E proteins is an important first step in the generation of
the envelope, with prM purported to play a role in the
chaperone-mediated folding of E (Konishi & Mason, 1993;
Lorenz et al., 2002). Heterodimerization may facilitate the
arrangement of prM/E at the ER membrane forming
heterotrimeric spikes (Zhang et al., 2007a). In turn, the
ordered arrangement of these prM/E spikes may induce
curvature of the ER membrane, resulting in budding of
nascent virions into the ER lumen (Zhang et al., 2013a).
The prM protein is believed to primarily mediate this
heterodimerization, as it folds more rapidly than E and is
required to be present for E to achieve complete antigenic
conformation (Lorenz et al., 2002). As such, detailed investigations have been undertaken to determine specific
residues or domains in prM that may be responsible for
efficient assembly and secretion of virus particles. A
conserved 9 aa region was identified within the prM protein corresponding to residues 62–70 of TBEV and 61–69
of JEV prM (Yoshii et al., 2012). This region (which is
located adjacent to the fusion loop of E in heterotrimeric
spikes) was deemed critical for the assembly and secretion
of prM/E particles from both TBEV and JEV (Yoshii et al.,
2012). The GXXXG motif within the prM protein of JEV
(corresponding to residues 142–146 within the first
transmembrane domain) was also deemed to be important
for prM and E interaction, as alanine insertion mutants
displayed reduced heterodimerization and prM/E particle
secretion (Lin et al., 2010).
The role of the DENV prM-H domain in particle formation
has also been revealed in two thorough proline/alanine
mutation studies (Hsieh et al., 2011, 2014). These investigations identified residues S112, E114, W117, A120, V123,
E124, W126, I127, L128 and R129 as strongly influencing the
assembly of virus particles, with substitutions at M111,
E114, G115, W117, V123, E124, W126, I127, L128 and R129
also impairing particle infectivity. Interestingly, only E114 in
DENV-1 was also demonstrated to be crucial for heterodimeric interactions between prM and E (Hsieh et al., 2014),
indicating that the other residues may play a greater role in
influencing the conformational rearrangement of structural
proteins during virion maturation (Zhang et al., 2012). This
hypothesis is particularly supported by recent evidence
linking residue E125 in the JEV prM-H domain (corresponding to DENV E124) in electrostatic interactions with
the K93 and H246 residues in E protein DII (Peng & Wu,
2014). The flexible linker between prM-H and the first
transmembrane domain in DENV also appears to play a
similar role in the secretion of virus particles, as mutation of
H130 to a non-polar amino acid greatly reduces the recovery
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of either infectious virions or prM/E particles from culture
supernatants (Pryor et al., 2004).
ER retention signals have been identified by mutation
analysis of the transmembrane segments of YFV prM protein and were proposed to be important for prM/E interaction, particle formation and secretion (Ciczora et al.,
2010). Thus, as with the 9 aa motif, the makeup of the
transmembrane regions of prM may influence heterodimerization and hence virion formation in a manner
broadly applicable to diverse flaviviruses. Additionally, a
series of recent investigations (Calvert et al., 2012; Setoh
et al., 2012; Tan et al., 2009) have identified two regions in
WNV prM (residues 20–31 and 72–78) that have been
proposed to be important for the overall structural stability
and function of prM, and thus are likely to influence its
interactions with E.
Heterodimers of prM and E are believed to coalesce at ER
membranes directly opposed to the sites of RNA replication
within the induced membranes of the VP (Roby et al., 2012).
Although this theory has been supported by immunofluorescence assays (Lorenz et al., 2003; Mackenzie & Westaway,
2001), the exact conformation of the prM/E heterodimers
(i.e. trimeric spikes, as in immature virions) at this site has
yet to be ascertained.
Virion budding into the ER lumen
The ER membrane of infected cells is believed to support
the translation of the flavivirus polyprotein, heterodimerization of newly formed prM and E, and formation of
virions and prM/E particles by budding into the lumen
(Roby et al., 2012; Welsch et al., 2009). Although this is the
classical model of flavivirus assembly, it may not be
universal, as at least one report concerning WNV-Sarafend
has demonstrated unusual virion budding at the plasma
membrane (Ng et al., 1994). Although visualization of
these discrete events on the ER is often limited by their
rapid kinetics, co-localization experiments with ER markers
have demonstrated prM and E residing at this organelle by
immunofluorescence microscopy (Lorenz et al., 2003;
Mackenzie & Westaway, 2001) and virion accumulation in
ER cisternae by immunogold electron microscopy (Mackenzie
& Westaway, 2001; Welsch et al., 2009). Recent investigations
utilizing electron tomography have even purported to
demonstrate direct visualization of flavivirus budding into
the ER lumen at membrane sites directly opposite the pores
of VPs (Junjhon et al., 2014; Welsch et al., 2009).
Particle trafficking and secretion
After completion of the viral budding process, an immature
particle pf about 600 Å in diameter is formed in the lumen
of the ER and consists of 60 prM/E heterotrimeric spikes
arranged with icosahedral symmetry (Zhang et al., 2004,
2007a) (Fig. 4). Virions that have accumulated in the ER
cisternae are transported to the Golgi apparatus in
individual vesicles for glycan maturation (Mackenzie &
Westaway, 2001; Welsch et al., 2009). Transition from the
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J. A. Roby and others
ER through to the trans-Golgi compartment involves a
drop in pH from 7.2 to 6.0, which induces a structural
rearrangement from 60 prM/E heterotrimeric spikes to 90
prM/E heterodimers arranged in a herringbone pattern that
is flattened parallel to the virion (Li et al., 2008; Zhang et al.,
2003), a process that is reversible prior to furin cleavage (Yu
et al., 2008) (Fig. 4). As detailed previously, host furinmediated cleavage of prM (to a greater or lesser extent)
occurs in acidified compartments of the trans-Golgi complex,
with liberated pr peptide remaining associated with the
transiting virion or prM/E particle (Yu et al., 2009). Upon
release of virus particles from endosomes, the neutral pH
triggers the dissociation of the pr peptide, forming an
infectious mature virus particle of roughly 500 Å in
diameter, with 90 E homodimers on the surface of the
particle (Yu et al., 2008) (Fig. 4).
Glycan modification
Flaviviruses secrete three discrete glycoproteins from
infected cells: prM (Calvert et al., 2012; Goto et al., 2005;
Kim et al., 2008), E (Beasley et al., 2005; Goto et al., 2005;
Hanna et al., 2005; Lee et al., 2010; Li et al., 2006;
Mondotte et al., 2007; Scherret et al., 2001; Setoh et al.,
2012; Winkler et al., 1987) and NS1 (Muller & Young,
2012; Muylaert et al., 1996; Somnuke et al., 2011). Asnlinked carbohydrate modifications are always present and
are relatively conserved in prM and NS1 with one to three
modifications in the pr portion of prM (believed to be
important in protecting the fusion peptide in E during
virion egress) (Goto et al., 2005; Hanna et al., 2005; Kim
et al., 2008; Li et al., 2008; Mandl et al., 1988; von Lindern
et al., 2006), and modification at two to three residues in
NS1 (important for multimerization of soluble NS1)
(Muller & Young, 2012; Muylaert et al., 1996; Somnuke
et al., 2011). Glycosylation of the E protein is more
variable, usually occurring at residues 153/154 and at
residue 67 in the four serotypes of DENV (Bryant et al.,
2007; Lee et al., 2010). However, some strains of WNV
(Beasley et al., 2005; Botha et al., 2008; Shirato et al., 2004b;
Wengler et al., 1985), including KUNV (Adams et al.,
1995), as well as strains of St Louis encephalitis virus
(SLEV) (Vorndam et al., 1993), Alfuy virus (May et al.,
2006) and YFV-17D (Post et al., 1992) lack any active
carbohydrate modification in E. Therefore, such glycosylation is not absolutely required for flavivirus replication.
The significance of prM glycosylation in flaviviruses
appears to be twofold, impacting on both particle assembly
and release. Glycosylation of the prM protein (at residue
N15 in WNV) appears to be absolutely required for
authentic virion assembly. Removal of the N-linked
glycoslation site from the prM protein may result in a
complete misfolding of the E protein and a failure to form
prM/E particles efficiently (Goto et al., 2005; Hanna et al.,
2005; Kim et al., 2008; Zai et al., 2013). A T20D mutation
in WNV prM was also shown to reduce (but not completely
prevent) glycosylation of prM, and secretion of prM/E
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particles via ectopic expression of a mutant prME gene
cassette was severely reduced (Calvert et al., 2012). However,
the specific influence conferred on this phenotype by either
the incomplete loss of glycosylation or the nature of the
amino acid substitution remains to be resolved.
In the context of live virus infection, the loss of prM
glycosylation in JEV led to an approximately 20-fold
reduction in the production of extracellular virions, which
had protein compositions and infectivities nearly identical
to those of WT virions. This reduction appears to have
occurred at the stage of virus release, rather than assembly
per se (Kim et al., 2008). Removal of the prM glycosylation
site in a lineage I or II strain of WNV also significantly
decreased prM/E particle release (97–99 % reduction
compared with WT) (Hanna et al., 2005). In a prM
glycosylation-deficient TBEV mutant, the level of secreted
E protein was reduced to 60 % of the WT level. On the
other hand, in E or prM/E protein glycosylation-deficient
mutants, the level of secreted E protein was reduced to
10 % of the WT level (Goto et al., 2005). Thus, it appears
that prM glycosylation may be more significant in regard
to particle secretion than glycosylation of the E protein.
Glycosylation at the E protein of flaviviruses is of particular
interest due to the variety of roles such a post-translational
modification appears to play in the flavivirus life cycle, and
due to the repeated isolation of viruses lacking such
glycosylation at all. E protein glycosylation has been linked
to increased virulence in mammalian and avian models of
infection (Beasley et al., 2005; Brault et al., 2011; Kariwa
et al., 2013; Murata et al., 2010; Shirato et al., 2004b; Totani
et al., 2011) and efficient transmission by mosquitoes
(Moudy et al., 2009). Glycosylation at the E protein is
proposed to potentiate these virulence phenotypes by
facilitating receptor-mediated virus entry (Davis et al.,
2006a, b; Martina et al., 2008; Mondotte et al., 2007;
Pokidysheva et al., 2006; Tassaneetrithep et al., 2003), a
greater efficiency of particle assembly and egress (Goto
et al., 2005; Hanna et al., 2005; Lee et al., 2010; Li et al.,
2006; Scherret et al., 2001), increased pH stability (Beasley
et al., 2005; Guirakhoo et al., 1993; Lee et al., 1997) and
possibly the concealment of immunogenic epitopes (Zhang
et al., 2011).
Two of the best-characterized receptors for flaviviruses rely
almost solely on glycosylation of the E protein for interaction (and prM in immature/partially mature virions): the
type II tetrameric transmembrane proteins containing
Ca2+-dependent carbohydrate recognition domains DCSIGN and DC-SIGNR (Davis et al., 2006a, b; FernandezGarcia et al., 2009; Martina et al., 2008; Mondotte et al.,
2007; Pokidysheva et al., 2006; Tassaneetrithep et al., 2003).
Dendritic cells and macrophages express DC-SIGN in
humans, with homologous Ca2+-dependent (C-type)
lectin receptors (CLRs) in mice displaying distinct patterns
of expression (Koppel et al., 2005; Kwan et al., 2008;
Navarro-Sanchez et al., 2003; Tassaneetrithep et al., 2003).
This lectin receptor recognizes high-mannose glycans and
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Flavivirus structural protein regulation
fructose-containing Lewisx antigens (Koppel et al., 2005)
and has been demonstrated as a primary cellular attachment moiety for DENV and WNV (Davis et al., 2006a, b;
Kwan et al., 2008; Lozach et al., 2005; Martina et al., 2008;
Navarro-Sanchez et al., 2003; Tassaneetrithep et al., 2003;
Wang et al., 2011). Polymorphisms within the promoter
encoding DC-SIGN have also been associated with severe
TBEV infection in humans (Barkhash et al., 2012). Cryoelectron microscopy reconstructions of DENV E bound to
the dependent carbohydrate recognition domain of DCSIGN have demonstrated that the glycans attached to N67
in E protein rather than those on the flavivirus-conserved
N153/4 residue are required for virus binding (Pokidysheva
et al., 2006). This is supported by experiments that
demonstrated increased WNV binding to DC-SIGN when
the E protein was mutated to introduce glycosylation at
residue 67 (Davis et al., 2006a). However, this may not
represent the sole means for glycosylated flaviviruses to
interact with DC-SIGN as it does not account for the
observed impact of polymorphisms on TBEV infection
(Barkhash et al., 2012) or the ability of WT glycosylated
WNV to use it as a receptor (Davis et al., 2006b; Martina
et al., 2008), as both viruses only contain the N154 glycan.
It may be that high-mannose glycosylation of arthropodderived flavivirus virions facilitates better DC-SIGN
interaction with the N153/4 residue (Davis et al., 2006b;
Navarro-Sanchez et al., 2003).
DC-SIGNR is a close homologue of DC-SIGN (77 % amino
acid sequence identity) and is expressed on microvascular
endothelial cells in the human liver, lymph nodes and
placenta (Davis et al., 2006b; Koppel et al., 2005; Pöhlmann
et al., 2001). Evidence from experimental WNV incubation
with K562 cells overexpressing CLRs demonstrated a preferential usage of DC-SIGNR over DC-SIGN for attachment and infection (Davis et al., 2006b). Curiously, whilst
the infection enhancement was strongest with mosquito
cell-derived virions, mammalian cell-derived WNV still
displayed a markedly increased infection of K562-SIGNR
cells compared with K562-SIGN and control K562 cells.
Thus, it seems that DC-SIGNR is able to bind vertebrate
host-generated complex carbohydrates in addition to
the high-mannose pathogen-associated molecular patterns
(PAMPs) recognized by DC-SIGN. In addition, the observed
preference for DC-SIGNR was maintained when infecting
with a lineage II WNV deficient in E glycosylation, indicating
that carbohydrates in the residual prM remaining uncleaved
by furin are likely to take an active part in this interaction.
It should be noted that experiments with DENV have
demonstrated a heterologous degree of glycan maturation
that may be dependent on the cell type used to grow virus
stocks, as well as, possibly, the degree of prM protein
cleavage (Dejnirattisai et al., 2011). The authors demonstrated that DENV grown in insect (C6/36), immortalized
mammalian (Vero) and primary mammalian cells (monocyte-derived dendritic cells) contained E glycans with
different degrees of complexity (insect-derived virus was
entirely high mannose, DC-derived virus contained only
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complex glycans, and Vero-derived virus was intermediate
beween the two states). Viruses derived from insect and
immortalized mammalian cells were able to utilize DCSIGN as a receptor; however, DENV grown in primary cells
and containing solely complex glycans could only bind to
the liver/lymph node-associated related CLR, L-SIGN. This
phenotype may, however, only be DENV-specific, as WNV
grown in Vero cells has been demonstrated solely to contain
complex glycosylation of the E protein [as demonstrated by a
lack of sensitivity to endoglycosidase H (endoH), which
specifically recognizes glycans containing three or more
terminal mannose residues] (Hanna et al., 2005). Ectopic
expression of prM/E particles of WNV in HEK-293T cells
and DENV-2 in COS cells has also demonstrated insensitivity to endoH digestion and thus complete maturation to
complex glycans (Hanna et al., 2005; Pryor et al., 2004).
Interestingly, the work of Dejnirattisai et al. (2011) also
revealed that utilization of both of the glycosylation sites in
DENV-2 E only occurs in approximately 50 % of cases, as
demonstrated by a faster-migrating band in immunoblots
of untreated WT samples corresponding to E protein with
glycosylation at only a single residue. A previous study
investigating all four serotypes of DENV failed to show the
same heterogeneous population of singly and doubly
glycosylated E in untreated DENV-2 samples (Hacker
et al., 2009). This report did, however, reveal that, for
viruses grown in Vero cells, endoH digestion of DENV-1,
-3 and -4 E protein removed glycosylation at one site only,
whereas endoH digestion of DENV-2 resulted in a mixed
population of E protein with no glycosylation or glycosylation at one site. Thus, it appears that the N67 and N153
glycosylation sites in DENV E are utilized to differing degrees,
and the glycans positioned at each site are differentially
matured. Considering that structural studies of DENV prM
and E have demonstrated partial occlusion of one or both E
glycans by the pr peptide (Li et al., 2008; Yu et al., 2008), and
that DENV has a demonstrated tendency towards incomplete
prM cleavage by furin (Rodenhuis-Zybert et al., 2011), these
results raise the interesting possibility that the degree of prM
processing controls the maturation status of one or both
glycans. Such a hypothesis remains to be properly explored,
although it would be intriguing to investigate whether
partially matured DENV displays a greater capacity to infect
DC-SIGN-expressing cells.
Evidence is also emerging that CLRs recognizing glycosylated flaviviruses may play a role in the infection of vector
mosquito species (Arjona et al., 2011; Cheng et al., 2010).
In both Aedes and Culex mosquitoes, the soluble galactosespecific lectin mosGCTL-1 binds to glycosylated WNV (but
apparently not to DENV) in the haemolymph and then coordinates with a cell-surface CD45 phosphatase homologue
mosPTP-1 to facilitate virus entry (Cheng et al., 2010). As
an evolutionary strategy, the use of CLRs as receptors for
glycosylated flaviviruses appears to be a trade-off. Although
the viruses gain the use of a subset of otherwise unavailable
attachment molecules, they risk potentially triggering host
lectin-based innate immunity such as activation of the com-
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J. A. Roby and others
plement cascade through mannose-binding lectin (Fuchs
et al., 2010, 2011) or inflammasome activation and cytokine
stimulation through CLEC5A (Chen et al., 2008, 2012;
Fernandez-Garcia et al., 2009; Watson et al., 2011; Wu et al.,
2013).
In contrast to the benefit to CLR-mediated infection
conferred by E glycosylation, several groups have reported
that virion or replicon-containing virus-like particle (VLP)
infectivity is reduced in vertebrate and especially insect cell
culture when glycosylation is present. Infection of mammalian, avian and mosquito cells with linage I and II WNV
replicon VLPs or linage II WNV either containing glycosylation motifs or deficient in these sites on prM and E
demonstrated that loss of glycosylation in E was beneficial
for particle infectivity, and that this phenotype was most
pronounced in mosquito C6/36 cells (Hanna et al., 2005).
This observation has since been extended to KUNV, MVEV
and DENV, with E protein glycosylation-null mutants
of each of these viruses displaying less than a twofold
difference in infectivity compared with the glycosylated E
viruses in most mammalian cells, whereas infectivity in C6/
36 cells was greatly enhanced by the lack of E glycosylation
(Alen et al., 2012; Lee et al., 2010). It should be noted,
however, that one study has presented conflicting results
demonstrating that DENV-2 with an N153Q mutation
removing E glycosylation has reduced infectivity in mammalian BHK cells (Mondotte et al., 2007), although this
may in fact be due to the specific mutation (NAQ) rather
than the loss of glycosylation per se. Indeed, a similar
NAQ mutation that eliminated glycosylation of TBEV E
(Yoshii et al., 2013) appeared to demonstrate misfolding of
E protein secreted from mammalian cells. Although
specific infectivity was not measured, the authors attributed this misfolding of E to the attenuated phenotype
observed in mammalian cells (but not observed in tick
cells). Additionally, an NAD mutation within a selected
DENV-2 mutant led to less than a twofold difference in
infectivity of BHK cells but demonstrated a severe reduction in infectivity of DC-SIGN- and L-SIGN-expressing
Raji cells (Alen et al., 2012). This general loss of glycosylation/enhanced infectivity phenotype in cells that are
not positive for CLR expression may be due in part to the
observation that non-glycosylated flaviviruses are more
labile at higher pH. DENV-2 and -3 lacking E glycosylation
at N153 are able to fuse with C6/36 cells at a higher pH
than glycosylated forms (Guirakhoo et al., 1993; Lee et al.,
1997), and WNV with an N154S mutation in E is more
sensitive to low-pH inactivation (presumably through
fusogenic E rearrangement) than WT virus (Beasley et al.,
2005).
Another important biological outcome of E protein
glycosylation is an increase in the rate of flavivirus particle
assembly and secretion, and the resulting enhancement of
virus replication. Investigations utilizing ectopic expression
of WNV and TBEV prM and E proteins have ascertained
that glycosylation of the E protein leads to a more rapid
and efficient secretion of prM/E particles (Goto et al., 2005;
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Hanna et al., 2005). This enhanced secretion is observed
with live DENV (Bryant et al., 2007; Lee et al., 2010) and
WNV (Beasley et al., 2005; Li et al., 2006; Scherret et al.,
2001) and has recently been demonstrated by our group
using non-infectious C-deleted WNV replicons (Roby et al.,
2013). However, an investigation utilizing live TBEV failed
to demonstrate a difference in the degree of E secretion
between glycosylation-mutant and WT viruses in both
mammalian and tick cells (Yoshii et al., 2013).
Glycosylated flaviviruses have in some cases been demonstrated to display enhanced virulence in animal models.
With regard to mammalian virulence, E glycosylation has
been demonstrated to be important for WNV (Beasley
et al., 2005; Shirato et al., 2004b) and MVEV (Prow et al.,
2011) peripheral replication and neuroinvasion in mice.
WNV strains lacking glycosylation have also been demonstrated to be attenuated for replication in birds (Murata
et al., 2010; Totani et al., 2011) and in Culex mosquitoes
(Moudy et al., 2009). This last point at first appears
counterintuitive, considering the apparent severe detriment
to mosquito cell infectivity conferred by WNV E glycosylation (Hanna et al., 2005). Perhaps this is merely a
reflection of differential selective pressures imposed by
the mosquito midgut infection barrier (ability for initial
infection to occur, probably reliant on virion infectivity)
and midgut escape barrier (ability for virus to disseminate
from the midgut, possibly reliant on degree of virion
secretion) (Olson & Blair, 2012). This is supported by the
observation that there was no apparent change in the
replication of a DENV-2 N67Q mutant compared with the
WT virus when the viruses were inoculated intrathoracically
into Aedes mosquitoes (Bryant et al., 2007). Glycosylation of
the E protein, however, was shown to be non-essential for
virulence, as strains of SLEV (Monath et al., 1980) and WNV
(Shirato et al., 2004a) lacking E glycosylation are not severely
attenuated in mice.
It is plausible that E glycosylation may represent an evolutionary strategy that assists viruses in expanding into new
territories. Increased virion secretion leads to higher
viraemias in vertebrate hosts (Beasley et al., 2005; Murata
et al., 2010; Shirato et al., 2004b; Totani et al., 2011) and
thus may improve the likelihood that novel vector species
become exposed to and infected with the virus. This may
explain why North American (emergent) WNV is predominantly glycosylated (Beasley et al., 2005; Charrel et al.,
2003), whereas isolates of WNV (including KUNV) that
circulate in the Old World (with long-established vector–
host relationships) have been demonstrated to lack E
glycosylation (Adams et al., 1995; Botha et al., 2008).
It is important, therefore, to note that, as the emergent and
most virulent strains of WNV display glycosylation at N156
of E, vaccines that are produced to target these viruses
should probably also be glycosylated to ensure an appropriate immune response. This statement does not at first
appear justified by the literature. Reports have demonstrated that aspects of the antigenic profile of TBEV E
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Journal of General Virology 96
Flavivirus structural protein regulation
protein are quite stable when Asn-linked carbohydrates
are removed (Winkler et al., 1987) and that removal of
glycosylation may enhance JEV E immunogenicity (Zhang
et al., 2011). However, E glycosylation still appears to be
important to generate an authentic immune response
against virulent WNV in horses, as the carbohydrate modification has been demonstrated to be crucial for the correct
presentation of an antibody-inducing epitope (HobsonPeters et al., 2008).
Post-translational modification of structural
proteins in vaccine design
A greater understanding of the degree of post-translational
modification enacted upon flavivirus structural proteins
during the course of an infection has led to increasing
exploitation of such events in the development of novel
vaccines (Table 1). Three primary aspects of post-translational modification have been targeted: (i) the coordinated cleavage at the C–prM junction; (ii) furin cleavage
maturation of prM; and (iii) glycosylation of the E protein.
C-deleted flavivirus genomes have emerged as an attractive
approach for the design of vaccines against flaviviruses
(Mandl, 2004; Roby et al., 2011). They rely on the effective
production and secretion of prM/E particles from selfreplicating RNA in the absence of genome packaging from
functional C. Thus, in essence, each of these constructs
relies on co-ordinated C–prM cleavage, engineering the Cdeletion to maintain efficient liberation of the truncated C
by NS2B/3 and thus proper prM processing (although
these events have not been examined directly). The latest
iterations of these vaccines developed in our group also
provide a functional C gene in trans in order to allow
packaging of nucleocapsids into particles for a single round
of infection (Roby et al., 2011). Rationally, extension of the
trans-C gene to include the prM signal sequence should
target its translation to the ER (the proposed site of
polyprotein translation and virus particle formation). Thus,
recent investigations by our and other groups have built on
the discoveries of Lobigs et al. (2010) to create constructs in
which an anchored form of C should still be cleavable by
NS2B/3 in the absence of downstream prM. Constructs with
trans-C genes extended to either include the signal sequence
and the first 10 codons of prM (Roby et al., 2014), or the
entire sequence of the pr peptide (Pang et al., 2014), were
generated and shown to be highly efficient at both infectious
and non-infectious particle secretion, thus representing
attractive candidates for future vaccine development.
Exploitation of the maturation state of prM has also been
investigated in the development of better flavivirus vaccines.
By purifying their prM/E particle vaccine using sucrose
gradients, Ohtaki et al. (2010) found that they could separate
fully mature, M-containing particles [fast-sedimenting VLPs
(F-VLPs)] from immature, prM-containing particles [slowsedimenting VLPs (S-VLPs)]. Subsequently, it was discovered that F-VLPs with fully matured M elicited a more
robust immune response than S-VLPs with uncleaved prM.
Thus, this simple purification step was able to improve the
efficacy of their vaccine candidate. In addition to purification to remove uncleaved prM, furin cleavage has also been
improved in a vaccine through the use of mutation
(Keelapang et al., 2013). By introducing the E203A mutation
into prM in their chimaeric DENV-1/2 live-attenuated
vaccine (DENV-1 prM and E in a DENV-2 genomic
background), Keelapang et al. (2013) were able to enhance
furin cleavage and improve the immune response in
vaccinated rhesus macaques. Such enhanced furin cleavage
mutations may be particularly beneficial for DENV vaccines,
as antibodies to prM have been demonstrated to lead to
antibody-dependent enhancement of infection (Dejnirattisai
Table 1. Exploitation of the post-translational modification of flavivirus structural proteins
Aspect of structural protein
modification
Application
Strategy
Co-ordinated cleavage of C–prM Single-round infectious
Utilized an extended form of trans-encoded C
particle (SRIP)-producing that includes regions of prM in order to promote
vaccines
targeted expression at the rough ER membrane
Furin-mediated maturation
Live-attenuated chimaeric Mutations to enhance furin cleavage of prM
of prM
DENV vaccine
producing a greater proportion of mature virus
enhances immunogenicity
WNV prM/E particle
Purification of mature particles to remove
vaccine
immature prM enhances immunogenicity
Glycosylation of prM and E
WNV SRIP-producing
Mutation of KUNV E to introduce glycosylation
proteins
vaccine
significantly improves prM/E particle and SRIP
secretion
TBEV prM/E particle
Mutations that remove prM or E glycosylation
vaccine
reduce particle secretion, but addition of N66
glycosylation mimicking DENV enhances secretion
JEV prM/E particle
Mutations to eliminate E glycosylation enhance
vaccine
immunogenicity of vaccine
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Reference(s)
Pang et al. (2014);
Roby et al. (2014)
Keelapang et al. (2013)
Ohtaki et al. (2010)
Roby et al. (2013,
2014)
Goto et al. (2005)
Zhang et al. (2011)
1563
J. A. Roby and others
et al., 2010; Luo et al., 2013; Rodenhuis-Zybert et al., 2010),
and indeed the presence of prM as an antigen may prevent
the development of an efficient neutralizing antibody
response against DENV E (Rodenhuis-Zybert et al., 2011).
Glycosylation of the E protein has been demonstrated to be
an effective means of enhancing particle secretion in flavivirus vaccines, thus increasing their potential to stimulate
an immune response. This has been applied by our group
to C-deleted KUNV-based vaccines that normally lack
glycosylation at N154 (Roby et al., 2013, 2014) and by
others to add DENV-like N66 glycosylation to a TBEV
prM/E particle vaccine (Goto et al., 2005). Conversely,
however, elimination of E protein glycosylation in a DNAbased JEV prM/E particle-producing vaccine led to the
development of a more robust immune response in
vaccinated mice (Zhang et al., 2011) and addition of E
glycosylation to C-deleted KUNV-based vaccines did not
appear to significantly enhance its immunogenicity (J. A.
Roby, R. A. Hall & A. A. Khromykh, unpublished data). It
appears that the glycosylation state of the E protein is a
parameter worth investigating in the construction of all
particle-secreting flavivirus vaccines due to these potentially opposing outcomes.
Acknowledgements
The authors would like to acknowledge the advice and helpful
discussion provided by Dr Mario Lobigs, who has been instrumental
in broadening scientific understanding of C–prM cleavage coordination and the role of E glycosylation in flavivirus infections.
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