REVIEWS
`
`A STRUCTURAL PERSPECTIVE OF
`THE FLAVIVIRUS LIFE CYCLE
`
`Suchetana Mukhopadhyay, Richard J. Kuhn and Michael G. Rossmann
`
`Abstract | Dengue, Japanese encephalitis, West Nile and yellow fever belong to the Flavivirus
`genus, which is a member of the Flaviviridae family. They are human pathogens that cause large
`epidemics and tens of thousands of deaths annually in many parts of the world. The structural
`organization of these viruses and their associated structural proteins has provided insight into the
`molecular transitions that occur during the viral life cycle, such as assembly, budding, maturation
`and fusion. This review focuses mainly on structural studies of dengue virus.
`
`The Flaviviradae is a large family of viral pathogens
`responsible for causing severe disease and mortality in
`humans and animals. The family consists of three genera:
`Flavivirus, Pestivirus and Hepacivirus. This review will
`focus on the largest of the three, the Flavivirus genus,
`which contains more than 70 viruses including dengue
`virus, Japanese encephalitis virus (JEV), tick-borne
`encephalitis virus (TBEV), West Nile virus (WNV) and
`yellow fever virus (YFV). Within the genus, the viruses
`can be further subdivided into antigenic complexes
`according to serological criteria, or into clusters, clades
`and species on the basis of molecular phylogenetics1,2
`(FIG. 1).
`The name ‘flavivirus’ is derived from the Latin word
`flavus meaning yellow, signifying the jaundice caused
`by YFV. The flaviviruses are primarily transmitted by
`arthropods. Symptoms of flavivirus infection can range
`from mild fever and malaise, to fatal encephalitis and
`haemorrhagic fever. Dengue virus, which is transmitted
`by mosquitoes, is responsible for the highest rates of
`disease and mortality among members of the Flavivirus
`genus3. Global epidemics of dengue virus have occurred
`over the past few decades, in part due to decreased
`mosquito control efforts. More than 50 million cases
`of dengue virus are estimated to occur annually3–5.
`Sequential infections by multiple serotypes of dengue
`virus can lead to dengue haemorrhagic fever, of which
`there are an estimated ~500,000 annual cases world-
`wide3. WNV has emerged in temperate regions of
`Europe and North America. In 2002, more than 4,100
`people in the United States were infected with WNV6
`
`and hundreds of humans and thousands of animals
`died. Vaccines are available that have been created from
`live-attenuated YFV and inactivated JEV and TBEV.
`However, disease resulting from these viruses is still
`prominent worldwide2. The World Health Organization
`estimates that each year there are 200,000 cases of
`yellow fever, with approximately 30,000 deaths, world-
`wide7. In addition, more than 50,000 cases of Japanese
`encephalitis were reported in 2001 (REF. 8).
`Flavivirus virions are approximately 500 Å in diameter
`and are composed of a single, positive-strand RNA
`genome that is packaged by virus capsid protein in a
`host-derived lipid bilayer and surrounded by 180 copies
`of two glycoproteins2. The ~10.8-kb genome has one
`open reading frame encoding a single polyprotein. The
`amino terminus of the genome encodes three structural
`proteins — capsid, membrane (M, which is expressed
`as prM, the precursor to M) and envelope (E) — that
`constitute the virus particle. Seven non-structural
`proteins that are essential for viral replication are
`encoded by the remainder of the genome2. The capsid
`protein consists of ~120 amino acids and is involved
`with packaging of the viral genome and forming the
`nucleocapsid (NC) core2. prM (~165 amino acids) and
`E (~495 amino acids) are glycoproteins, each of which
`contains two transmembrane helices. Before it is cleaved
`during particle maturation to yield the pr peptide and
`the M protein (~75 amino acids), the prM protein
`might function as a chaperone for folding and assembly
`of the E protein9. The E protein contains a cellular
`receptor-binding site(s) and a fusion peptide9–11.
`
`Department of Biological
`Sciences, Purdue University,
`915 West State Street,
`West Lafayette, Indiana,
`47907-2054, USA.
`Correspondence to M.G.R.
`e-mail:
`mgr@indiana.bio.purdue.edu
`doi:10.1038/nrmicro1067
`
`NATURE REVIEWS | MICROBIOLOGY
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`R E V I E W S
`
`Virus
`
`Serocomplex
`
`Clade
`
`Cluster
`
`West Nile
`Kunjin
`Japanese encephalitis
`Murray Valley encephalitis
`St Louis encephalitis
`
`Dengue-1
`Dengue-3
`Dengue-2
`Dengue-4
`
`Yellow fever
`
`Central European encephalitis
`Far Eastern encephalitis
`Powassan
`
`Dakar bat
`
`Japanese
`encephalitis
`
`Dengue
`
`None
`
`Tick-borne
`encephalitis
`
`None
`
`XIV
`
`XI
`
`IX
`
`VII
`
`IV
`
`III
`
`Mosquito-
`borne
`
`Tick-borne
`
`No vector
`
`Figure 1 | Flavivirus classification. The relationships between selected flaviviruses are shown in
`the dendrogram on the left. Evolutionary distance is not represented in this figure. The serological
`(serocomplex) and phylogenetical (clade and cluster) classifications of these flaviviruses are shown
`on the right. Reproduced with permission from REF. 2 © (2002) Lippincott Williams and Wilkins.
`
`Box 1 | Flavivirus life cycle
`
`Virions attach to the surface of a host cell and subsequently enter the cell by
`receptor-mediated endocytosis (see the figure). Several primary receptors and
`low-affinity co-receptors for flaviviruses have been identified (as described in this
`review). Acidification of the endosomal vesicle triggers conformational changes in the
`virion, fusion of the viral and cell membranes, and particle disassembly. Once the
`genome is released into the cytoplasm, the positive-sense RNA is translated into a single
`polyprotein that is processed co- and post-translationally by viral and host proteases.
`Genome replication occurs on intracellular membranes. Virus assembly occurs on the
`surface of the endoplasmic reticulum (ER) when the structural proteins and newly
`synthesized RNA buds into the lumen of the ER. The resultant non-infectious,
`immature viral and subviral particles are transported through the trans-Golgi network
`(TGN). The immature virion particles are cleaved by the host protease furin, resulting in
`mature, infectious particles. Subviral particles are also cleaved by furin. Mature virions
`and subviral particles are subsequently released by exocytosis.
`
`Mature
`virion
`
`TGN
`
`Golgi
`
`Virus infection
`
`Fusion and
`virus disassembly
`
`Virus maturation
`
`CAP
`
`vRNA
`
`Polyprotein translation,
`transit to ER and processing
`
`Viral genome
`replication
`
`Virus assembly
`
`ER
`
`Nucleus
`
`Flaviviruses enter host cells by receptor-mediated
`endocytosis (BOX 1). The acidic environment of the endo-
`some triggers an irreversible trimerization of the
`E protein that results in fusion of the viral and cell
`membranes12. Fusion has not been observed at the
`plasma membrane13. After fusion has occurred, the NC
`is released into the cytoplasm, the capsid protein and
`RNA dissociate, and replication of the RNA genome
`and particle assembly is initiated2,14,15. Initially, immature
`particles are formed in the lumen of the endoplasmic
`reticulum (ER). These particles, which contain E and
`prM proteins, lipid membrane and NC, cannot induce
`host-cell fusion, making them non-infectious16,17.
`Subsequently, cleavage of prM occurs in the trans-Golgi
`network, which creates mature, infectious particles10,18.
`Subviral particles are also produced in the ER, but only
`contain the glycoproteins and membrane, and lack
`capsid protein and genomic RNA, making these particles
`non-infectious19. Mature virus and subviral particles are
`released from the host cell by exocytosis.
`Advances in cryo-electron microscopy (cryoEM)
`have provided important insights into the structures
`of enveloped viruses20–22. In addition, many of the
`structural proteins of these viruses have been examined
`at atomic resolution23–29. This has allowed atomic reso-
`lution structures to be fitted into cryoEM density maps,
`resulting in ‘pseudo-atomic’ structures of enveloped
`viruses. By analysing different intermediates in the virus
`assembly and entry pathways, these dynamic processes
`can be understood at a molecular level. This review will
`focus on the structural aspects of flavivirus assembly,
`maturation and fusion.
`
`Processing of structural proteins
`During translation of the polyprotein, the structural
`proteins are translocated and anchored in the ER by
`various signal sequences and membrane anchor domains
`(FIG. 2). The capsid protein contains a hydrophobic sig-
`nal sequence at its carboxyl terminus that translocates
`prM into the lumen of the ER from its site of synthesis
`on the surface of the ER. The prM protein has two
`transmembrane-spanning domains, which contain a
`stop transfer sequence and a signal sequence. As a result,
`the E protein is also translocated into the lumen of the
`ER (FIG. 2). After the appropriate proteolytic cleavages,
`the capsid protein and viral RNA are localized in the
`cytoplasm and the capsid protein remains associated
`with the ER membrane. On the lumenal side of the ER,
`the prM and E proteins form a stable heterodimer
`within a few minutes of translation9,30,31.
`
`Envelope protein
`Structure of E. The X-ray crystallographic structures of
`the ECTODOMAINS (residues 1–395) of TBEV E and
`dengue-2 E proteins (FIG. 3) have been determined24–26.
`Both structures are similar, consisting of a dimer in
`which each monomer has three β-barrel domains. The
`central structural domain (domain I) contains the
`N-terminus and is flanked on one side by an elongated
`dimerization domain (domain II), which contains the
`fusion peptide at its distal end. On the other side of
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`E protein
`
`prM
`
`NSI
`
`ER lumen
`
`Cytoplasm
`
`Capsid
`protein
`
`+
`
`NH3
`
`Viral serine-protease cleavage site
`Host signalase cleavage site
`Furin cleavage site
`
`Figure 2 | Membrane topology of the flavivirus structural proteins. The predicted orientation
`of the structural proteins across the endoplasmic reticulum (ER) membrane is shown.
`Transmembrane helices are indicated by cylinders, arrows indicate the sites of post-translational
`cleavage and the cleavage sites of specific enzymes are indicated by different colours.
`E, envelope; NSI, non-structural protein 1; prM, precursor to membrane protein.
`
`domain I is domain III, which is an immunoglobulin
`(Ig)-like domain that is thought to contain the putative
`receptor-binding sites32–37. Domains I and II are con-
`nected by four polypeptide chains, whereas domains I
`and III are connected by a single polypeptide linker.
`The fusion peptide of one E monomer is buried
`between domains I and III of the adjacent monomer
`within a dimer. The angle between domains I and II
`differs in the three E crystal structures24–26 (FIG. 3).
`Furthermore, one of the crystal structures of dengue-2
`E protein revealed a molecule of N-octyl-β-D-glucoside
`to be positioned at the domain I–II interface24. This
`flexibility might be required to facilitate the large confor-
`mational changes the virus undergoes during maturation
`and fusion (see below). The NMR structure of domain III
`of the JEV E protein has also been determined38 and is
`similar to the crystallographic structures of the TBEV and
`dengue-2 E proteins.
`
`Entry and the E protein. There is ~40% amino acid
`identity among the flavivirus E proteins. The number
`and position of potentially glycosylated residues is not
`conserved among different strains of the same virus and
`it has been suggested that carbohydrate moieties on the
`virus surface might modulate specificity of receptor
`binding39. The glycosylated amino acids map onto the
`E dimer structure spatially near domain III21,24–26,40.
`Furthermore, viruses belonging to the Japanese
`encephalitis serocomplex (FIG. 1) have five extra amino
`acids in domain I that form a surface loop near the
`domain I–III interface40. The proximity of these struc-
`tural changes might indicate that this surface region
`regulates the specificity of flavivirus binding to their
`different cellular receptors.
`DC-SIGN (dendritic-cell-specific ICAM-grabbing
`non-integrin), GRP78/BiP (glucose-regulating protein
`
`ECTODOMAIN
`The part of the protein that is
`exterior to the lipid membrane.
`
`R E V I E W S
`
`78), and CD14-associated molecules have been suggested
`as primary receptors for dengue virus39,41–43. DC-SIGN
`is a mannose-specific lectin that is proposed to specifi-
`cally interact with the carbohydrate residues on the
`dengue E protein39. Several other cell surface proteins
`have been proposed to be receptors for dengue44–53,
`WNV54, JEV55 and TBEV56,57. In addition, heparin and
`other glycosaminoglycans are low-affinity co-receptors
`for several flaviviruses32,33,36,58–64. Unlike other viruses65,
`there is very little structural information about the
`mechanisms by which flaviviruses bind to their recep-
`tors. In mosquito-borne flaviviruses (such as YFV, JEV
`and WNV), domain III contains an RGD/RGE
`sequence, which is the recognition motif for integrin
`binding. However, alteration of this motif in YFV did
`not abolish virus–cell binding, which indicates that a
`non-integrin receptor might also be involved in cell
`recognition66.
`
`Fusion and the E protein. The E protein has two con-
`served helices in the ‘stem’ region (residues 398–420 and
`426–448 in dengue-2), between the ectodomain and the
`transmembrane region21,67. In vitro studies with TBEV
`have shown that either full-length E protein or the
`ectodomain of E, which contains the stem region,
`form dimers at neutral pH. On acidification, the
`dimers dissociate into monomers and then reassociate
`irreversibly to form trimers12,68,69. However, a truncated
`E protein that does not contain the stem region
`requires both acidification and the presence of lipids to
`form trimers70.
`X-ray crystallographic structures of the E proteins
`at low pH (representing the post-fusion structure)
`showed a trimeric arrangement, with the long dimen-
`sions of the E proteins positioned approximately parallel
`to each other and with their fusion peptides exposed at
`the tip of the trimer71,72 (FIG. 3). The structures showed
`that the fusion peptides would penetrate only into the
`outer lipid leaflet of the host cell membrane. There is a
`major conformational change in the positioning of
`domain III, which folds back towards domain II by
`~30 Å and rotates ~20°, so that it is no longer extended
`linearly with domains I and II. The C-terminus of the
`ectodomain of the E protein (residue 395) was found
`to be positioned at the end of a hydrophobic groove
`that extends along domain II and towards the fusion
`peptide. The stem helices were predicted to move
`into this groove, bringing the transmembrane region
`into close proximity with the host membrane.
`The E1 protein in alphaviruses (examples include
`Sindbis virus, Semliki Forest virus and Venezuelan
`equine encephalitis virus) is structurally homologous to
`the E protein of flaviviruses in their pre- and post-fusion
`conformations27,73, despite no significant sequence
`similarity between the E1 and E proteins. The distinc-
`tive three domain arrangement with an internal fusion
`peptide that is present in flavivirus and alphavirus
`fusion proteins constitutes a new class of membrane
`fusion — the class II fusion process. The similarities
`between the class I and class II fusion processes are
`discussed below.
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`
`a
`
`Fusion peptide
`
`Amino
`terminus
`
`b
`
`c
`
`Post-fusion
`
`Immature
`
`Pre-fusion
`
`Pre-fusion
`
`Mature
`
`Domain II
`
`Domain I
`
`Domain III
`
`Figure 3 | Structure of the ectodomain of the E protein in pre- and post-fusion
`conformations. a | Dimeric, pre-fusion conformation of the dengue-2 E protein, residues 1–395
`(REF. 26). In one monomer, domains I, II and III are coloured red, yellow and blue, respectively,
`and the fusion peptide is shown in green. The other monomer is coloured grey. b | Trimeric,
`post-fusion conformation of the dengue-2 E protein72. One monomer has the individual domains
`and fusion peptide coloured as in part a, whereas the other two monomers are coloured grey.
`c | E protein X-ray crystal structures showing the variation in the hinge angle between domains I
`and II. Domain II (Cα backbone trace) of the post-fusion E protein structure is in purple72, the
`immature virus in grey26, the dimeric, pre-fusion form in yellow26 and red24, and the protein in the
`mature virus in blue21. The common domain I and III structures are shown diagrammatically in red
`and blue. Part c is reproduced with permission from REF. 26 © (2004) Cell Press.
`
`Capsid protein
`Capsid protein structure. The amino acid identity between
`different flavivirus capsid proteins ranges from 15 to 90%.
`The NMR structure of the dengue-2 capsid protein and
`the X-ray crystal structure of Kunjin capsid protein show
`that both proteins form dimers. Dengue-2 and YFV cap-
`sid proteins readily form dimers in solution74 and a dimer
`has been suggested to be the building block for NC assem-
`bly23,75. The monomer in both structures has four helices
`(α1–α4) that are connected by short loops23,29. Helices α2
`and α4 of one monomer are anti-parallel to helices α2
`and α4 of the neighbouring monomer, respectively23,29.
`
`Comparison of the structures of the dengue-2 and
`Kunjin capsid proteins shows some variability in the
`position of α1 (residues 27–35 in dengue-2)23,29. The
`X-ray crystal structure of the Kunjin capsid protein
`shows the surface to be mostly positively charged and
`consisting of helices α1 and α4. The cryoEM recon-
`structions of both mature and immature dengue virus
`show that in both structures the nucleocapsid lacks a
`well-formed protein shell. So, it is possible that the
`rather basic capsid protein functions like a histone29.
`The N-terminal domains of many of the capsid proteins
`of other positive-strand RNA viruses have been shown
`to function in the same way76,77. However, in the NMR
`structure of dengue-2 capsid protein, the position of
`α1 has moved, exposing a hydrophobic surface created
`by α2. It is possible, as suggested by Ma et al., that the
`capsid protein facilitates binding of the NC to the lipid
`membrane23,78.
`
`Capsid protein and virus assembly. One of the earliest
`events in flavivirus assembly is the formation of the
`NC, which consists of one copy of the genomic RNA
`and multiple copies of the capsid protein. Unlike other
`enveloped viruses, NCs are rarely found in flavivirus-
`infected cells (although they can be assembled in vitro 75),
`which indicates that particle formation is a coordinated
`process between the membrane-associated capsid
`protein and the prM–E heterodimers in the ER.
`Flavivirus capsid proteins contain a conserved
`hydrophobic region spanning helices α2 and α3. NCs
`isolated from TBEV particles tend to aggregate even
`in the presence of detergent, which indicates that the
`conserved hydrophobic region is exposed on the sur-
`face of the NC75. When deletions in the hydrophobic
`regions in helices α1 and α2 were constructed, viable
`virus could still be recovered by second site revertants
`that mapped onto the dimerizing helix α4. Virus
`could not be rescued when these deletions were
`extended into helix α3 (REFS 78,79). These results indi-
`cate that the residues in the hydrophobic region of the
`capsid protein are not an absolute requirement for
`assembly78,79 and that the dimerization might be
`maintained by the α4 helix.
`
`Subviral particles
`Subviral particles are routinely observed in flavivirus
`infections80 and were first observed in virus-infected cell
`cultures. Subviral particles have a smooth exterior and
`most particles have an average diameter of 315 Å,
`although larger particles (~500 Å) have been observed81,82.
`These particles are assembled in the ER and undergo
`the same post-translational modifications as infectious
`particles before being released by the host cell. The
`released particles only contain the E and M proteins
`and a lipid membrane. Recombinant subviral particles,
`also containing only E and M proteins and lipid, can be
`produced by co-expressing E and prM in mammalian
`cells31,81,82, indicating that flavivirus particle formation is
`driven by the interactions between prM–E hetero-
`dimers. Although the organization of the E protein
`homodimer is different in subviral particles compared
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`with the mature virus (see below), these particles have
`similar haemagglutination and fusion activities to
`mature particles and can be used as an immunogen
`with protective capability13,83.
`Subviral particles containing prM and E proteins
`— as opposed to M and E proteins — can be produced
`by treating cells that are co-expressing both these
`glycoproteins with ammonium chloride, or by mutating
`the prM furin cleavage site so that it is not cleaved82.
`In either case, the ratio of larger diameter particles
`(500 Å) to smaller particles (315 Å) is increased81,82
`compared with the ratio of particles that are formed
`after prM cleavage. The endoglycosidase digestion pat-
`terns of the smaller subviral particles and whole virions
`are similar, but differ from those of the larger subviral
`particles. So, the environment of the E protein in the
`larger subviral particles might be different to that of the
`E protein in the smaller subviral particles, making the
`glycoproteins inaccessible to cellular glycan-processing
`enzymes81,82.
`The first insights into whole flavivirus structures
`were using TBEV recombinant subviral particles81.
`Using cryoEM, these particles were shown to contain
`30 copies of the E dimer arranged with icosahedral
`symmetry on the virion surface. Extrapolation of this
`T=1 (T denotes the TRIANGULATION NUMBER) structure to a
`larger T=3 structure produced a particle that is consis-
`tent with the size of mature virions, and was predicted
`to represent the structure of the mature particle.
`However, this structure is not that of the mature virus
`but possibly of an intermediate in the fusion process
`(see below).
`
`Mature virion
`In contrast with other enveloped viruses, such as
`influenza, HIV and measles, mature flavivirus particles
`have a spikeless and smooth surface21,40,84. The outer
`layer of the particle (radii of 220–245 Å) corresponds
`to the E and M proteins. The radii of the outer and
`inner leaflets of the viral membrane are between 165
`and 205 Å. The bilayer is roughly polygonal in shape
`and is exterior to the NC. In both mature and immature
`particles (see below), there is ~30-Å gap between the
`inner lipid leaflet and the NC, and little or no connections
`between them21,40,84,85.
`The ectodomain of the E protein comprises sets of
`three parallel dimers that form 30 rafts over the viral
`surface (FIG. 4). The arrangement of 180 monomers
`lacks the conventional T=3 symmetry that was initially
`predicted86. Furthermore, this arrangement makes the
`lipid bilayer largely inaccessible from the viral exterior.
`Most of the contacts between the individual E
`monomers are hydrophobic26. The hinge angle
`between domains I and II in dengue-2 decreases by
`10–20° relative to the angle that is observed in the
`dengue-2 E X-ray crystal structures24,26 (FIG. 3). This
`conformational change adapts the structure of the E
`protein to the viral surface26. Domain III, the putative
`receptor-binding domain, protrudes from the other-
`wise smooth viral surface, possibly facilitating its
`binding to cellular receptors.
`
`a
`
`Domain II
`
`M - H
`
`M -
`
`T
`
`2
`
`M -
`E-T1
`
`T
`
`1
`
`E-T2
`
`Domain I
`
`E
`
`1
`H
`-
`E-H2
`
`b
`
`Domain III
`
`M-T2
`M - H
`
`1
`
`M - T
`
`E-T1
`E - T 2
`
`E-H 1
`
`CS
`
`E-H2
`
`Domain III
`
`Domain I
`
`Domain II
`Figure 4 | Packing of the E proteins in mature flavivirus virions. a | One raft, consisting
`of three parallel dimers, is highlighted. Domains I, II and III are coloured red, yellow and blue,
`respectively. The fusion peptide is coloured green. Reproduced with permission from REF. 26
`© (2004) Cell Press. b | Diagrams of the dengue virus E protein ectodomain and
`transmembrane domain, side and top views. The stem and transmembrane helices of the
`E (E-H1, E-H2, E-T1 and E-T2) and M (M-H, M-T1 and M-T2) proteins are shown in blue and
`orange, respectively. The conserved amino acid sequence of the region between the two E
`protein stem helices is marked CS. Reproduced with permission from REF. 21 © (2003)
`Nature Structural and Molecular Biology Macmillan Magazines Ltd.
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`
`a
`
`b
`
`c
`
`Figure 5 | Immature flavivirus particles. a | Surface-shaded cryoEM reconstruction of immature dengue-2 at 16-Å resolution. b | Superpositioning of the E protein
`onto the cryoEM density. The prM protein that caps the trimeric spike is visible. c | The arrangement of the E protein in the immature particle, which differs markedly
`from the arrangement in the mature particle, shown in FIG. 4a. The E protein is coloured as in FIGS 3,4.
`
`Stiasny et al.69 have shown that the post-fusion E
`homotrimers are more stable than the pre-fusion
`dimeric form. They further suggest that the mature viri-
`ons represent a kinetically trapped METASTABLE assembly
`that undergoes structural rearrangement under acidic
`pH conditions, by a mechanism similar to that of
`influenza virus87. The dimer to trimer transitions could
`not be induced by heating and specific protonation is
`essential for initiating dimer dissociation69, as suggested
`by the putative T=3 structural transition.
`The two transmembrane domains of the E and M
`proteins (residues 452–467 and 473–491 in the E protein
`and residues 40–54 and 58–70 in the M protein of
`dengue-2) form anti-parallel, approximately coiled-
`coil helices21 (FIG. 4). There are no contacts between
`the E and M transmembrane helices. Furthermore,
`neither set of transmembrane helices penetrates past
`the lipid bilayer to make contacts with the NC. The
`first helix of the stem region of the E protein (residues
`398–420 in dengue-2) is angled in the outer lipid
`leaflet, whereas the second helix of the stem region
`(residues 426–448 in dengue-2) lies flat in this
`leaflet21. The stem region makes contact with the side
`of the E protein closest to the lipid, possibly neutraliz-
`ing electrostatic repulsion between the outer phos-
`pholipid bilayer head group and the membrane-facing
`surface of the E protein26 (FIG. 4). The stem region of
`the M protein (residues 22–37 in dengue-2) is also
`helical and is partially buried in the outer lipid leaflet.
`This is one of only a few membrane proteins for
`which structures within cellular membranes have
`been determined in situ 88,89.
`CryoEM density indicates that the maximum height
`of the NC in both the mature and immature virion (see
`below) is ~60% of that observed for the outer glyco-
`protein shell21,40,84,85. In addition, the structure of NC
`appears DISORDERED in both structures, which could be
`because the NC is inherently disordered, the icosa-
`hedral symmetry of the core is not synchronized with
`the glycoprotein layer, or the core is ordered but has
`non-icosahedral symmetry21,85.
`
`Immature virion
`The structure of immature flaviviruses particles, as
`revealed by cryoEM, is markedly different to that of
`mature particles85. The immature particles have 60
`prominent, irregular, trimeric surface spikes (FIG. 5),
`making the external diameter 600 Å, which is somewhat
`larger than mature virions. Like the mature virions,
`these particles do not have classical T=3 QUASI-SYMMETRY85.
`The E protein comprises most of the spike and the
`remaining density has been attributed to the pr pep-
`tide26,85 (FIG. 5). CryoEM has shown the hinge angle
`between domains I and II of each of the three symme-
`try-independent E proteins to differ by ~5–15° from the
`crystal structures24,26 and about 30° from the cryoEM
`structure of the mature particle21,26 (FIG. 3).
`Each spike comprises a trimer of prM–E hetero-
`dimers. The three prM proteins in the spike cap the
`fusion peptides, which are positioned at the external
`tip of the E protein (FIG. 5). Each of the three prM
`proteins extends along the side of an E protein and
`enters the membrane at the point at which each prM
`protein forms two anti-parallel helices. The three pr
`peptides at the top of each spike are the main contacts
`between the prM and E protein heterodimers. Once
`cleavage of the pr peptide from prM occurs during
`particle maturation in the trans-Golgi network,
`the trimer is presumably disrupted, which allows
`rearrangement of the E proteins.
`The distance between the E and M transmembrane
`regions is similar in both the immature and mature
`particles, although their positions, with respect to
`ICOSAHEDRAL REFERENCE AXES, are different26,85.
`the
`Furthermore, the stem region of the E protein in the
`immature particle is in a different conformation com-
`pared with the mature particle. In the mature particles,
`the two stem helices are approximately co-linear,
`whereas in the immature particles, they are approxi-
`mately anti-parallel. Nevertheless, the first stem helix is
`in the same position relative to domains I and III of the
`E protein in both the mature and immature particles.
`In the immature particle, the second stem helix does
`
`TRIANGULATION NUMBER
`The triangulation (T) number of
`an isometric virus designates the
`quasi-symmetry. In an
`icosahedron there are 60
`asymmetric subunits. In an
`icosahedral particle, there are
`60T protein subunits that
`comprise the structure.
`
`METASTABLE
`A system that is above its
`minimum-energy state, but
`which requires an energy input
`before it can reach a lower-
`energy state. As a result, a
`metastable system functions like
`a stable system provided the
`energy input is below a certain
`threshold.
`
`18 | JANUARY 2005 | VOLUME 3
`
`www.nature.com/reviews/micro
`
`6
`
`

`

`R E V I E W S
`
`Maturation
`
`a
`
`b
`
`5
`
`5
`
`5
`
`3
`
`3
`
`3
`
`3
`
`3
`
`3
`
`Figure 6 | Proposed rearrangement of the E proteins during maturation and fusion. a | The E proteins in the immature
`virus (left) rearrange to form the mature virus particle (right). b | The E protein dimers in the mature virus (left) are shown
`undergoing a rearrangement to form the putative T=3 fusogenic intermediate structure (right) with a possible intermediate
`(centre). The arrows indicate the direction of the E rotation. The solid triangle indicates the position of a quasi three-fold axis.
`This suggested rearrangement would require a ~10% radial expansion of the particle between the intermediate (centre) and
`fusogenic form (right). The right-hand panel of part a is reproduced with permission from REF. 26 © (2004) Cell Press. Part b is
`reproduced with permission from REF. 84 © (2002) Cell Press.
`
`not contact domain II as it does in the mature particle
`and its position relative to the first stem helix and the
`transmembrane helices has changed26. Insertion of
`alanine residues and the replacement of charged
`residues in the transmembrane region of prM and E by
`site-directed mutagenesis does not reduce the stability
`of the prM–E heterodimer, which indicates that few
`interactions occur between the transmembrane regions
`of the two proteins90. This lack of transmembrane
`interaction between the M and E proteins is also
`observed in both the mature and immature structures.
`
`Rearrangements during maturation and fusion
`Flavivirus particles undergo major conformational
`changes during the life cycle. Some of these changes can
`be inferred from structural information available for the
`immature and mature virions as well as the pre-fusion
`dimer and the post-fusion trimer21,24–26,40,71,72,84,85.
`Virus maturation is a two-step process. First, a
`pH-induced irreversible conformational change
`involving the prM and E proteins occurs in the trans-
`Golgi network, which is followed by cleavage of prM
`by furin. During maturation, 60 trimers of prM–E
`heterodimers that project from the virus surface disso-
`ciate and form 90 E homodimers, which lie flat on the
`virus surface (FIG. 6). In addition, the E protein moves
`~30° at the hinge between domains I and II. The furin
`cleavage does not occur unless the virus has been
`
`exposed to low-pH conditions, which indicates that the
`conformational change in prM might be necessary to
`expose the furin cleavage site10,18.
`Second, a major rearrangement occurs when the
`virus undergoes fusion with the host cell. The anti-
`parallel E homodimers dissociate into monomers,
`which then reassociate into parallel homotrimers12,68,69
`(FIG. 6). One hypothesis for the mechanism that under-
`lies this reorganization is that the mature virion forms
`via an intermediate that is similar to the extrapolated
`T=3 structure (see above), either upon receptor binding
`or in the endosome before formation of the E trimers.
`This T=3 structure is composed of trimeric units and
`has extensive regions of exposed membranes, similar to
`mature alphavirus structures22,27,91. Furthermore, the
`proposed intermediate structure has T=3 symmetry, as
`would be expected if the affinity between monomers is
`decreased b