review article structural differences observed in...

Review ArticleStructural Differences Observed in Arboviruses ofthe Alphavirus and Flavivirus Genera

Raquel Hernandez1 Dennis T Brown1 and Angel Paredes2

1 Department of Molecular and Structural Biochemistry North Carolina State University Raleigh NC 27695 USA2US FDANational Center for Toxicological Research Department of Health and Human Services Jefferson AR 72079 USA

Correspondence should be addressed to Raquel Hernandez raquel hernandezncsuedu

Received 20 May 2014 Revised 28 July 2014 Accepted 18 August 2014 Published 16 September 2014

Academic Editor Trudy Morrison

Copyright copy 2014 Raquel Hernandez et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Arthropod borne viruses have developed a complex life cycle adapted to alternate between insect and vertebrate hosts Thesearthropod-borne viruses belong mainly to the families Togaviridae Flaviviridae and Bunyaviridae This group of viruses containsmany pathogens that cause febrile hemorrhagic and encephalitic disease or arthritic symptomswhich can be persistent It has beenappreciated for many years that these viruses were evolutionarily adapted to function in the highly divergent cellular environmentsof both insect and mammalian phyla These viruses are hybrid in nature containing viral-encoded RNA and proteins whichare glycosylated by the host and encapsulate viral nucleocapsids in the context of a host-derived membrane From a structuralperspective these virus particles are macromolecular machines adapted in design to assemble into a packaging and delivery systemfor the virus genome and only when associated with the conditions appropriate for a productive infection to disassemble anddeliver the RNA cargo It was initially assumed that the structures of the virus from both hosts were equivalent New evidence thatalphaviruses and flaviviruses can exist in more than one conformation postenvelopment will be discussed in this review The dataare limited but should refocus the field of structural biology on the metastable nature of these viruses

1 Background

11 Arbovirus Evolution Thearboviruses are not a taxonomicclassification but rather a grouping based on viral transmis-sion through an insect vector to infection of a vertebratehost The arboviruses contain members of the TogaviridaeFlaviviridae Bunyaviridae Rhabdoviridae Reoviridae andOrthomyxoviridae and are also represented by a single DNAvirus African swine fever virus family Asfarviridae of genusAsfivirus httpictvonlineorgvirusTaxonomyasp Evidenceexists that arboviruses from the alphavirus lineage evolvedfrom plant viruses [1 2] which adapted to growth in insects[3] Hematophagous insect viruses then acquired the abilityto infect vertebrates thus adapting from separate kingdoms(plant to insect) as well as phyla (insect to vertebrate) [4]Members of the Bunyaviridae stillmaintain the plant to insectcycle [5ndash7] as well as the insect only cycle [8ndash10] Arbovirusmembers of the flaviviruses are believed to have emergedabout 1000 years ago in a nonhuman primate to mosquito

cycle [11 12] from predecessors that date at least 85000years [8] It has been suggested that each of the 4 dengueserotypes (DEN1-4) adapted to humans independently onlya few hundred years ago [13] It is believed that this capabilityto diversify so broadly must have arisen from the inherenterror-prone nature of the RNA-dependent RNA polymerases[14] while also limiting the evolution of arboviruses to certainfamilies within the RNA virus class that are highly error-prone [14ndash16] It is thought that the ability of viruses fromeach of these families to use or infect vertebrate hosts aroseindependently [17] For these viruses to be able to cyclebetween insect and vertebrate hosts their genomes must becompatible to hosts of two divergent phyla This has beenachieved by the evolutionary selection of virus that representsa consensus sequence able to function in both hostsThus thearboviruses represent genomes selected by multiple mecha-nisms of adaptation and are exposed to repeated selection

For the families Togaviridae Flaviviridae and Bunyaviri-dae which comprise the bulk of arboviruses the structure of

Hindawi Publishing CorporationAdvances in VirologyVolume 2014 Article ID 259382 24 pageshttpdxdoiorg1011552014259382

2 Advances in Virology

the glycoprotein E1 E and possibly Gc respectively appearto have arisen from an ancient predecessor [3] While thesequences of the E1 cognate glycoproteins have diverged inthe Togaviridae Flaviviridae and Bunyaviridae the functionand structures of these viruses have been retained [18] Evolu-tion of the protein structure has been constrained by adaptingto both arthropod and vertebrate hostsThis difference in therates of genomic divergence has been seen in a nonarbovirusmember of the Togavirus family Rubivirus [19] in which theknown structure of E1 appears to have diverged relative tothe arbovirus members of this family [20] This observationsuggests that the consensus sequence of the arboviral genomeis maintained by eliminating genetic drift which impactsfitness in each host In otherwords the virus sequence evolvesmore slowly when divergent hosts are continuously selectingfor virus fitness Collectively the available information sug-gests that the mosquito-borne viruses acquired the form wenow see from the arthropod vectors and did so concurrentwith becoming hematophagous presumably to optimize eggmaturation [21]

12 Arbovirus Structure Of the seven families of arbovirusesthree (Togaviridae Flaviviridae and Bunyaviridae) are icosa-hedral membrane-containing plus-stranded RNA virusesIt is interesting that though the respective glycoproteinsE1 E and Gc of the alpha flavi and bunyaviruses encodethe same basic protein fold these proteins assemble intodifferent icosahedral structures Alphaviruses are 119879 = 4(Figure 1) [22] flaviviruses are 119879 = 3 (Figure 3) [23] andbunyaviruses (Phlebovirus) are 119879 = 12 [24 25] This capabi-lity of viruses to assemble an inherently similar global foldinto their different structures is dependent on the specificfunctions of their structural proteins Rhabdoviruses areenveloped but assume a rod or bullet shaped structure thatdemonstrates some helical symmetry [26] Reoviruses (119879 =13 l laevorotatory turning toward the left) are icosahedralbut do not contain a membrane [27] Orthomyxovirusesare enveloped viruses that display pleomorphic structuresranging from spherical such as influenza A to filamentous asin influenza C [28] In those influenza structures assuminga spherical shape that shape is not due to icosahedral sym-metry In addition orthomyxoviruses have more lipid-mem-brane relative to their envelopes than do enveloped icosahe-dral viruses resulting in the pleomorphism displayed [29]The Asfarviridae viruses are large double-stranded DNAviruses spherical to pleomorphic and 175ndash215 nm in diam-eter and exhibit icosahedral symmetry (119879 = 189ndash217) Theseviruses are composed of two icosahedral protein shells thatcontain an intervening lipid structure [30] These structuraldifferences illustrate that there is no common virus structureadopted by the arboviruses which by itself would explainhow these viruses infect both arthropods and vertebratesThere are two systems of virus classification currently in useWhile morphology is a useful basis for virus identificationand classification at present two classification systems existThe hierarchical virus classification system the Internationalcommittee on taxonomy of viruses (ICTV) [31] and theBaltimore classification system The Baltimore classificationsystem is based on nucleic acid type which places viruses

into seven groups in hierarchical classification [32]The ICTVuses evolutionary relationships as the classification scheme[33] ICTVclassification places viruses by phenotypemorph-ology protein composition and genotype nucleic acid typesequence mode of replication host organisms infected andthe type of disease they cause Capsid structure has also beenutilized to organize virus groups based on the hypothesis thatonly a limited number of protein folds are available to self-assemble a nucleocapsid [24 34] This review will be limitedto the group arboviral members of the alphaviruses and flavi-viruses for which considerable structural information existsBunyaviruses are less well studied and are proposed to befunctionally analogous to flaviviruses by computational pro-teomic and indirect biochemical analysis [35] Studies onwhole virus particles will be the focus of this discussion

13 Alphaviruses and Flavivirus Virion and Genome StructureThe alphaviruses are a genus in the family Togaviridae Theyare icosahedral viruses of 119879 = 4 geometry and containa +polarity single-stranded RNA genome The genome isorganized with the 4 nonstructural genes found at the 51015840end of the capped RNA genome followed by the structuralproteins capsid (C) PE2 6K and E1 (Figure 2 [Virus DBVBRC genome browser accession VG0000908]) The virus iscomposed of the structural proteins C E2 and E1 that aresynthesized from subgenomic RNA made from an internalpromoter [36] This RNA is then translated as a polyproteinand processed by viral and host enzymes during maturationand comprises the virion The glycoproteins are assembledwithin the ER The 51015840 capsid protein is autoproteolyticallyprocessed from the proprotein and organizes the genomicRNA into a nucleocapsid During virus maturation PE2 isconverted to E3 and E2 by furin [37] In most cases E3does not remain associated with the virus [38] E1 and E2form trimers of heterodimers that envelope the nucleocapsidwhich assembles independently [39] By regulating RNA andprotein synthesis in a temporal manner the Sindbis virusis able to quickly replicate to as many as 106 particlescell[40] Alphaviruses bud from the cell surface in mammaliancells but are assembled in vacuoles in mosquito cells andmature via the exocytic pathway [41] These viruses arehybrid in nature acquiring lipids carbohydrates and othermodifications from the host cell while their proteins arevirus encoded The final assembled structure is that of twonested spheres separated by an interveningmembrane bilayerheld together by protein associations between the E2 proteinendodomain and the capsid protein

Flaviviruses are in the family Flaviviridae and are alsomembrane-containing viruses but they assemble into pseudo119879 = 3 icosahedra The genome is organized with the struc-tural proteins at the 51015840 end of the +strand RNA moleculeand is translated into a single polypeptide which is processedduring maturation by viral and host-encoded enzymes intomultifunctional proteins The three structural and sevennonstructural proteins are cleaved by a series of virus andhost-encoded proteases The structural proteins starting atthe 51015840 end of themonocistronic genome include C preM andE (Figure 4 [Virus DB VBRC genome browser]) As with thealphaviruses the flavivirus polyproteins are inserted into the

Advances in Virology 3

100 A

(a) (b)

Figure 1 Shown in (a) is the cryo-EM reconstruction of the TC-83 strain of Venezuelan equine encephalitis virus (VEE) viewed down thestrict 5-fold axis Resolution is 44 A The trimers consist primarily of E2 with the smoother ldquoskirtrdquo comprised of E1 In (b) is shown a slicedown the 2-fold axis Note the transmembrane domains (arrow) and the extensive organization of the nucleocapsid (circle) compared to thatseen in the flaviviruses (Figure 3(b)) With permission from Zhang et al [22] For full resolution images see EMDATA BANK (EMDB)5275

Nonstructural polyprotein Structural polyprotein

Stopcodon

cEarly

P123

P123 RdRp nsP4

Late

Late

CP E3 E2 6k E1

Subgenomic RNA

Suppressionof termination

By signal peptidaseBy furin

DLP

By capsidBy nsP2 protease

nsP1 RdRp nsP4nsP3nsP2protease

3998400

3998400

5998400

5998400 c

Figure 2 Alphavirus genome organization Alphaviruses are organized with the (nonstructural) ns proteins at the 51015840 end of the genomeThe ns proteins are translated from the genomic RNA while the structural proteins are translated from a subgenomic RNA that includes the31015840 end of the genome The RNA polymerase nsP4 is a read-through protein present early during infection After replication is establishedprotein production switches to the structural proteins C E3 E2 6K and E1TheCprotein autoproteolytically cleaves itself from the remainingpolyprotein Only E1 E2 and C are found in the mature virion In some cases E3 may also be associated with the virus particles [Virus DBVBRC genome browser accession VG0000908]

ER of the host cell However the flaviviruses are also assem-bled in the (endoplasmic reticulum) ER with concurrentassembly of the nucleocapsid RNAC structure [42] preMand E form dimers during protein maturation resulting infurther processing of preM to M as the glycoproteins mature[43] Little is known about the process of encapsidation intothe mature virion but incorporation of the nucleocapsid ofthese viruses is thought to involve nonstructural proteins[42] Flavivirus particles differ from alphavirus particlesin that the nucleocapsid association is more peripheral tothe intervening membrane and no organized structure isobserved [42 44] The particle structure is also nested with

an intervening membrane but no strong contacts with the Mand E membrane domains are formed [45 46]

2 Review

21The Plasticity of Alphavirus Variants as Seen by Ultrastruc-ture The alphavirus virion assembles into a stable structurethat shields the genome from the adverse effects of thesurrounding environment Virus particles are assembled intoa high energy state in which infectious particles are poisedto deliver their genomic cargo after the appropriate stimuliare encountered through specific interaction(s) with the host


(a)

(b)

Figure 3 (a) Dengue 2 New Guinea C cryoEM reconstruction rendered at 35 A EMD 5520 The view is down the icosahedral 5-foldaxis Although this is termed a mature virus because of the smooth appearance preM may still be associated with the virus The surfaceof the structure has few distinctive features however it has been proposed that upon maturation the virus E protein undergoes majorconformational changes as infection is initiated (b)This image is a slice of the virus down the 2-fold axis M (black arrow) and E (red arrow)transmembrane domains are seen below the outer surface Distortion of the lipid membrane is seen where the transmembrane domainspenetrate and no organized capsid structure is detected (circled) Compare to the alphavirus slice in Figure 1 where the nucleocapsid structureis clearly delineated Reprinted with permission from Zhang et al [23] See EM DATA BANK (EMDB)5520 for full resolution images

5998400

Flavivirus genomic RNA

cGenomic polyprotein


C M E NS1 NS2A NS2B NS3 NS4A NS4B NS5

3998400-OH

Signal peptidase

Golgi protease

NS3 protease

Figure 4 The 51015840 terminus of the genome is capped and the polyprotein is expressed as a single polypeptide The 31015840 terminus is notpolyadenylated but rather forms a loop structure The locations of the nsP and structural proteins are switched with respect to the 51015840 endof the RNA compared with the alphaviruses It has been proposed that this switch occurred via recombination in an ancient precursor thusretaining the structural glycoprotein folds Capsid protein preM and E proteins are proteolytically processed as indicated by the enzymesshown by the arrows and arrowheads [Virus DB VBRC genome browser]

cell Thus the infectious virion is a metastable intermediatethat assumes sequentially different conformations dependingon the pH temperature and host environments it encounters(discussed below) The plasticity of the virion structureunderscores the flexibility of the viral proteinsThis flexibilityis most likely not due to global changes in the structuralproteins but rather local changes in the metastable domainsof the proteins [48] For structural stability the virion as awhole must contain protein domains with structural stabilityimparted by the protein sequence itself or via stabiliz-ing protein-protein interactions We describe two examplesof the alphavirus Sindbis laboratory constructed mutants

producing structural variants that illustrate the ability ofpoint mutations in the structural proteins to acquire novelarchitecture

Thefirst example is that of the Sindbis virus capsid proteinmutant Y180SE183G Sindbis virus is a macromolecularstructure composed of RNA protein and lipid The innerprotein shell the nucleocapsid is bound to the outer proteinshell via interactions to the E2 endodomain Genetic andstructural evidence suggest that the nucleocapsid interactswith the E2 endodomain by aromatic amino acid interactionsbetween Y420 in the E2 protein and Y180 and W 247 inthe capsid protein [49 50] Mutations in the capsid protein


1

2

3

4

5

k

h

1 4 9 16 25

1 2 3

3 7 13 21

4 5

T = h2+ (h times k) + k

2

T

Figure 5 A family of icosahedra Shown at the bottom of thelarge triangle in blue are the members of the 119875 = 1 icosa-hedra The triangulation number represents the number of non-or quasiequivalent structural morphological units per asymmetricunit of the icosahedral face 119879 = 1 is the only structure with asingle equivalent morphological unit per icosahedral face shownin blue Neighboring 5-fold vertices of the 3-fold triangular face areconnected to one another by three 2-fold axes giving the structurean overall 5-fold 3-fold and 2-fold symmetry The 119879 number alsodetermined by the formula 119879 = ℎ2 + (ℎ times 119896) + 1198962 indicates therelative position of each 5 fold vertex as a function of the number ofquasiequivalent morphological units per icosahedral face Sindbiswith four quasiequivalent conformations of the E1E2 heterodimer isa 119879 = 4 structure In the capsid mutant Y180SE183G the flexibilityof the capsid protein is altered allowing the mutant protein to adopta wider range of quasiequivalent conformations that increases thetriangular number of the structure Superimposed on the 119875 = 1lattice is a class 3 triangular face outlined in red Flaviviruses arearranged in this 119879 = 3 symmetry and belong to the class 119875 = 3icosahedra shown in black on the 119896 = 1 axis

Y180SE183G result in the assembly of the virus structuralproteins into icosahedra of increasing triangulation numbersThe triangulation numbers calculated for these morpholog-ical variants follow the sequence 119879 = 4 9 16 25 and 36[51ndash53] All fall into the class 119875 = 1 of icosadeltahedra Ithas been suggested that the T = 4 structure of the nucleo-capsid organizes the outer glycoprotein layer [47 54] Theseobservations support models suggesting that the geometry ofthe preformed Sindbis nucleocapsid organizes the assemblyof the virus membrane proteins into a structure of identicalconformation It has been proposed that these two mutationsin the capsid protein endow the protein with the flexibilityto increase the number of capsid proteins incorporated intothe nucleocapsid before formation of the next five-fold vertexThis is seen more readily in the smaller icosahedra examplesof which are shown in Figure 5 The structures of increasingsize and triangulation number also form in mosquito C7-10 cells suggesting that the capsid protein mutation and notthe host cell or the temperature of assembly is involved inthe expression of this phenotype (unpublished data) Thismutant demonstrates the capability of a capsid protein foldto ldquoevolverdquo a new triangulation number and structure These

larger capsid structures can package large RNA molecules[55] While the structures formed by this mutant are notstable they are infectious and it is possible that subsequentmutations could stabilize any of the resulting new icosahedra(Figure 6)

A second example of a Sindbis virus structural proteinmutant able to organize into nonnative structures is thatof the furin mutants in the glycoproteins E1 and E2 shownin Figure 7 [53] Furin protease recognition sites Arg-X-ArgLys-Arg were installed into the E1 sequence at aminoacid positions 392 and E2-341 The numbering scheme refersto the position of the first mutated Arg to create the furinsite The furin double mutant was found to produce virusparticles of normal infectivity and structure when grownin mammalian cells (particles composed of the requisitenumber of wild type proteins see [37]) (Figure 7(A)) as wellas virions that developed long tubular appendages of varyinglengths (virions incorporating aberrant proteins) [53] Virusproduction from insect cells was of insufficient titer to allowmicroscopic examination The tubular structures are 73 nmin diameter consistent with the size of the wild type virionScanning electronmicroscopy of doublemutant infected cellsrevealed that maturing particles were initiated with sphericalstructures that probably contained a capsid (Figure 7(B)arrow)The large amorphous structures as seen in Figure 7(K)may contain multiple capsids with associated membrane Itwas concluded that the tubular structures initiated by orga-nizing around the icosahedral nucleocapsid and dependingon the number of furin processed proteins incorporatedterminated the envelopment process by normal membranefission (budding) or by repeated incorporation of hexagonalprotein arrays In the absence of the quasi-equivalent proteininteractions of the icosahedral five-fold vertex the only latticethat can form is a hexagonal sheet in one dimension or ahexagonal tube in 3 dimensions The geometry of the tubularhelices was determined by calculating the optical contrastreinforcement of the EM images of particles such as thoseseen in Figure 8 Thus when densities are in phase theywill reinforce one another and a repeating distance can becalculatedThiswas seen at 16 and 20 nm distances consistentwith the spacing of the wild type virus 6-fold symmetryshown in Figure 9 indicating that the geometry of themutantparticles is relevant biologically For the furin mutant E1-392E2-341 we see that a loss of function mutation in thestructural glycoproteins E1 and E2 can result in the ability ofthe virion to maintain the protein-protein interactions thatenable the formation of a helical array Again the flexibility ofthese viral proteins to produce variant structures underscoresthe plasticity of these proteins and illustrates the ability ofviruses to explore possible new structures to enable efficientsurvival and facilitate evolutionary divergence

These mutant virus structures help us to understandthe metastable structure of alphaviruses by illustrating thatassembly of the virion proceeds through structural interme-diates In the case of the C mutant this protein can fold intonative or the mutant conformations and assemble multipleforms For this change to occur the mutant protein mustbe able to assume an energetically stable intermediate Thisis thought to happen during the folding of the protein and


(a) (b)

(c) (d) (e) (f) (g) (h)

Figure 6 Electron micrographs of negatively stained virus particles recovered from the supernatant of BHK cells transfected with eitherSindbis virus S420Y ((a) and (c)) or Y180SE183G ((b) (d) (e) (f) (g) and (h)) (a) and (b) a wider view of the fields (c)ndash(h) individualparticles arranged to show examples of the various sizes found for the Y180SE183G mutant ((d)ndash(h)) as compared to the control S420Yparticle (c) Arrows point to morphological units Bars 100 nm Ferreira et al 2003 [47]

assembly in this case of the nucleocapsid The E1-392E2-341 mutant demonstrates that the metastability of the virusextends to both shells and can trap the glycoproteins into aconformation that only allows the formation of the hexagonallattice

22 Protein Structure Protein conformations have beenreported for many of these virus structural proteins as singleproteins and in protein aggregates (httpwwwncbinlmnihgov) The metastable state of the virion however suggestscaution in interpreting structures of the isolated struc-tural proteins Virus particles are macromolecular machinespoised to deliver their genomic cargo once the appropriatestimuli are encountered These particles must also shield thegenome from the adverse effects of the surrounding environ-ment These requirements are accomplished by the virionassembling into intermediates which refold into a highlyenergy rich infectious conformation which is a suffi-ciently stable structure [56] Thus the infectious virion isa metastable intermediate that can exist in more than oneconformational state depending on pH temperature and thecomplex host biochemical environments (discussed below)With the correct stimulus such as interaction with a receptorthe virus undergoes a conformational change that is pro-pagated through the virion to initiate infectionNot all virionsare infectious because the structural integrity of the particle iscompromised during assembly or virus preparation Thus ina field of virus particles the number of noninfectious particlescan outnumber infectious particles by as many as 1000 to1 This large particlepfu ratio can only complicate study ofthe native infectious structure Only stable or transientlystable conformations of these ldquometastablerdquo proteins can be

crystalized or imaged by cryoEM as was done with flu virus[57ndash59] The isolated flu structural proteins comprise limitedconformations of very flexible proteins with multiple func-tions While crystal structures for Sindbis and Chikungunyavirus structural proteins have been reported these may notbe native because of the lack of the transmembrane domainand many more conformations of infectious intermediatesmay exist for these viruses This is evident from bothquasiequivalence and from the different conformations ofE1 that have been identified in Sindbis virus [56 60ndash66]It has been reported that during Sindbis virus infectiondisulfide exchange may occur to enable the formation ofthe fusogenic conformation [67] and is important for theproper assembly of virions [56 68] The conformationalchanges induced by disulfide exchange during infection arenot understood but are known to be required for infectivityof the virus [66] Although there is no independent evidenceto verify that the crystal structure of E1 is a native structurethere is independent evidence that different forms of E1exist in the alphavirus structure when whole infectious virusis analyzed [63] The argument that the crystal structuresldquofit wellrdquo into the cryoEM electron density maps givesmuch leeway to the interpretation of the structure Becauseof quasiequivalence a single high resolution X-ray crystalstructure frequently cannot be fitted into a cryoEM electrondensity map of a high 119879-number virus without distortingthe X-ray structure in order to make the fit For Sindbisvirus a single alphavirus E1 structure is commonly used invirion structural reconstruction analysis at least four otherconformations of the E1 protein are known to exist as theprotein is folding and unfolding [56 60 69] Structuralintermediates of disulfide bridged proteins that are detected


Figure 7 Electron micrographs of negatively stained furin sensitive double mutant E1-392E2-341 containing virus recovered from thesupernatant of transfected BHK-21 cells (A) Low magnification of a typical sample of double mutant E1-392E2-341 Bar 1 120583m ((B)ndash(Q))Selected particles demonstrating the variations seen in one preparation of E1-392E2-341 Arrowheads show points of helical disruption andreinitiation Bars 100 nm Kononchik et al 2009 [53]


(a) (b)

Figure 8 Electron micrograph of a negatively stained furin double mutant E1-392E2-341 (a) A normal E1-392E2-341 particle that showssurface detail (b)The same particle (a) highlighting a hexagonal array clearly visible on the tubular structure Bars 100 nm Kononchik et al2009 [53]

(A) (B)

Figure 9 Helical reconstruction of the tubular section of thefurin sensitive E1-392E2-341 created by 6-fold rotational arrays(highlighted) Black lines illustrate the 16 nm repeat (A) and the20 nm repeat (B) registers seen on the tubular section of themutantsIllustrations are drawn to scale with the average width of thetubular structures The insert illustrates the distances taken from acryoelectron microscopy reconstruction of wild-type Sindbis virusmeasuring across a strict 2-fold axis of a hexagonal array white is16 nm and white with red is 20 nm Kononchik et al 2009 [53]

on nonreducing PAGE after extraction of the E1 proteinfrom infectious virus also form multiple intermediates [56]During alphavirus glycoproteinmaturation E1 and PE2 formintegrated trimers of heterodimers E2 is a molecular scaffoldas well as the escort protein that delivers multimers to the cellsurface [69] Mass spectroscopy analysis of cysteines foundin infectious virus was not the same as those reported for thealphavirus E1 crystal [66] For these reasons it is critical touse infectious virus to assess structure because alphavirus E1infectious conformations only exist in the intact metastable

virus [66] Stated succinctly native intermediates of E1 cannotexist in solution because cysteines will reassort as the highenergy form becomes stable While whole virus analysishas proven difficult because the resolution of most wholevirus reconstructions is not at atomic levels even when theresolution is increased by ldquofittingrdquo a crystal new technologycan address these problems

The cryomicroscopes of today include new advancementssuch as a phase plate used to negate the contrast transferfunction (CTF) of the microscope so that images do notrequire CTF correction The phase plate technology is stillunder development because those thatworkwell contaminatequickly when used [70ndash73] Additionally Charged-coupledevice (CCD) cameras are being replaced with direct (elec-tron) detectors These detectors capture hundreds of imagesper second and once captured the images are averagedtogether to reduce noise and to correct for specimen drift toboost both resolution in the image and signal As a conse-quence data are now being collected that are used to deter-mine structures of biological samples below 4 angstromsresolution This is close enough to atomic resolution that thecarbon backbone of amino acids including the R-side chainscan be followed in the structure Depending on size thistechnology will eliminate the need for pseudocrystal struc-tures for biological samples [74 75]

As seen in Figure 3(b) [23] flaviviruses are more fragileand therefore display a larger particle to pfu ratio thanalphaviruses [76 77] This is the result in part from theweaker association of the nucleocapsid with the structuralproteins Organization of the RNA within the C protein isunclear How the nucleocapsid interacts with the glycopro-teins preM and E during assembly is also vague [78 79]No nonstructural proteins are incorporated into the virionalthough nonstructural proteins have been implicated inencapsidation and budding of the virus from the ER [80 81]The preM protein functions as a chaperone during E folding[42 82 83] During transport of the virion to the cell exterior


preM is cleaved by a furin-like protein resulting in virusparticles with M protein in its mature form [43 84] Theprocessing of preM to M by furin is not as efficient as thatseen for the alphavirus PE2 protein processed to E2 and E3thus particles with preM are detected [85 86] It has beenshown that the lack of preM results in poor protein foldingand poor immunogenicity [87] Because E is the primarya immunogen this implies that the native conformation ofthe E protein can be compromised when expressed in vitroor outside the context of the virus Soluble E protein X-raystructures have been solved for many of the flaviviruses [88ndash91] The structure is divided into 3 domains I II and III[92] Domain I is the N terminal portion of the protein andis centrally located within the crystal structure Domain IIcontains the fusion peptide and the dimerization domainwhile domain III is an immunoglobulin like domain and isthought to contain the receptor binding site [93] This 3Dstructure is similar to that of the alphaviruses with analogousfunctional domains [94] Unlike the alphavirus E1 proteinthe flavivirus TBE and dengue E protein crystalized intoprotein dimers [95] E protein from WNV is crystalizedas a monomer but is fit into cryoEM as a dimer [96] Itis widely held that flavivirus E protein is induced by lowpH to reorganize into a fusogenic trimer which initiatesinfection from an acidified endosome While much indirectevidence has been reported to support thismodel of infectionfor alpha and flaviviruses by comparison to flu [92 97ndash99] a second model of direct penetration by the virus atthe host cell surface determined by direct observation islargely dismissed in favor of the fusion model Ideally aworking model of Togavirus penetration should address allexperimental evidence Two issues will be discussed firstthe use of indirect evidence from structural models whichdescribe a fusion pathway and second direct observation byultrastructural and biochemical analysis that provide empiricevidence that infection by these agents is direct penetration atthe plasma membrane

23 Electron Cryomicroscopy As electron optics and electroncryomicroscopy in general have improved it has becomepossible to take images of frozen hydrated viruses fromelectron microscopes and use them to reconstruct the three-dimensional structures of infectious virus to ever higherresolutions by cryoEM (reviewed in [100]) Electron cry-omicroscopy or cryoEM has allowed the placement of thedifferent structural components of the glycoprotein shellsof arboviruses for which structural data is available Thealphavirus Semliki Forest viruswas first imaged byVogel et al[101 102] In 1987 Fuller reported the first cryoEM structureof Sindbis virus to be a 119879 = 4 icosahedron surrounding a119879 = 3 core [103] however the core was later shown to be119879 = 4 [104] The first cryoEM image of Sindbis virus withsufficient resolution to image the trimeric spikes was pro-duced in 1993 by Paredes et al [104] This reconstructionconfirmed a structure that had previously been postulatedgenetically and biochemically [105ndash107]Themost outwardlyprotruding structure in the cryoEM image was a 3-foldtrimer with laterally associated proteins It was not possibleat that time to identify which proteins corresponded to what

part of the structure although it was known that the virusparticles were held together by an E1-E1 protein lattice [106108] Several studies have since used cryoEM to study thestructure of E2 in alphaviruses In 1995 Cheng et al reportedtheir cryoEM reconstruction of Ross River virus in whichthe authors concluded that the capsid protein bound as amonomer to the E1-E2 dimer in a 1 1 stoichiometry [109]The alphavirus E2 density was also probed using Ross Rivervirus and anti-Ross River E2 neutralizing FAbs that blockedattachment In these cryoEM structures the E2 FAb labeledE2 on the outermost density of the tip of the bilobed spikeprotein [110] The position of E2 was later confirmed ina 1998 study by Paredes et al when a mutant of Sindbisvirus defective in processing the N-terminal E3 protein fromthe PE2 precursor was reconstructed using cryoEM Thereconstruction identified the additional E3 density at the tipsof the trimeric spikes and identified the general locations ofE2 in the spike region with E1 involved in the protein latticeat the base of the spike [111] The general location of bothproteins was reported in 2001 by deleting the carbohydratemodification sites from E1 and E2 singly and together inseveral nonglycosylated mutants By comparing the cryoEMdensities of the nonglycosylated mutants to wild type virus itwas possible to determine the relative positions of E1 and E2on the virus surface [112]

In a 1990 study by Flynn et al conformational changesin Sindbis virus E1 and E2 were observed as virus engagedthe plasma membrane at neutral pH in cells that were notacidified [113]These rearrangements were detected by (mon-oclonal antibody) MAb and corresponded to transitionalepitopes These epitopes could also be detected in a timeand temperature dependentmanner Subsequent studies [114]showed that structural rearrangements seen in the previousstudy were closely mimicked by three artificial treatments ofpurified virions Structural rearrangements of virus exposedto brief incubation at 51∘C treatment with 1ndash5mM dithio-threitol or incubation at pH 58 to 60 were probed usinga panel of MAbs specific for Sindbis virus El and E2 glyco-proteins Infectivity was retained after all three treatmentsThese observations were interpreted to suggest that Sindbisvirions are metastable and can exist in at least two infectiousconformations The authors concluded that these interme-diate structures may represent different conformations of acomplex pathway that leads to productive infection and wasan early indication that infection proceeded through proteinstructural intermediates induced by virus-cell interactionsin the absence of low pH at the cell surface None of thesestructural intermediates can be inferred from a single rigidX-ray structure

A high resolution alphavirus structure was recentlyreported by Zhang et al and is shown in Figure 1 [22] At44 A resolution this structure of VEEV was determined by acombination of homology and de novo modeling The finalVEEV model was compared to a pseudomodel in whichCHIKV E1 and E2 X-ray structures were fit separately intothe VEEVmodelThe results indicate that E2 (residues 1ndash341)had a higher RMSD value (42) than E1 (residues 1ndash391) withan RMSD of 18 A representing the difference from identicalmolecules with an RMSD of 00 [115] The low pH SINV


E1-E2 crystal structure was also fit into the VEEV cryo-EMstructure (PDB ID 3MUU chain A) showing an RMSD of24 for E2 and 29 for E1 ectodomain The transmembranedomain and the E2 endodomain were modeled de novo Inaddition the structure of the capsid protein as determinedby cryoEM reveals a predicted 120572 helix at residues 115ndash124that is missing in the capsid structure as revealed by X-raycrystallographyThus this cryoEM reconstruction has servedto refine the structures of viral components by independentlyusing improved methods to define the protein structuresAdvances in single-particle cryo-EM have pushed the limitto near atomic resolution of sim33 A [116ndash119] Methods areimproving and soon it should be possible to build a virusmodel in the absence of structural artifacts without the needto dissociate virions Subtomogram averaging from in situcryo-EMhas the potential of looking at virus proteinswithoutthe need for crystallizationUnlike X-ray crystallography thismethod ensures that the viral proteins imaged in this mannerare in their native conformations While this method yieldslower resolution images than single particle reconstructionsadvancements in this technology will undoubtedly improvethe resolution

24 pH Studies and Viral Penetration A low pH study ofSindbis virus was undertaken by Paredes et al in 2004which hypothesized that low pH triggers the same or similarconformational rearrangements as does contact of the viruswith the cell receptor Sindbis virus treated at low pH wasinvestigated by cryoEM SVHR is a laboratory strain selectedfor heat resistance (Sindbis virus heat resistant) [120] whichalso confers the ability of the virus to produce virus whichis sim100 infectious Infectious BHK-grown SVHR virus of aparticle to pfu sim1 at pH 74 was exposed to pH 53 returned toneutral pH and prepared for microscopyThis study revealedthat low pH treatment triggered a substantial rearrangementof both E1 and E2 spike proteins and there was a significantformation of nobs of E1 density protruding fromeach of the 5-fold axes (see arrow in Figure 10(b))This is the only structureof an alphavirus at a pH which establishes the conditionsrequired for membrane fusion after return to neutral pHReturning to neutral pH did not restore the native structureand resulted in noninfectious virusThe observation that lowpH inactivated alphaviruses had beenmade early on [121] andcan be explained by the inability of the low pH form of thevirus to reorganize to the native conformation

That low pH inactivates Sindbis virus in solution isconsistent with the observation that treated virus does notreturn to its infectious conformation [121ndash124] For thisreorganization of E1 to occur the virus must be taken tothe pH 53 threshold since pH changes above this do notaffect infectivity of SVHR or establish conditions requiredfor membrane fusion [122 123] In another whole virusstudy using SFV clone pSFV4 low pH structures by Haaget al only exposed the virus to pH 59 not taking the virusthrough the requisite 53 pH for producing the low pHstructures and subsequently resulted in very little changein the virus conformation [125] This is because at the pHrequired for fusion concentrated virus samples precipitateThe Paredes et al study in 2004 proposed a new model

for membrane penetration of infectious alphaviruses Giventhat the resolution of the low pH structure was 28 A itwas deemed possible that the knob of density appearing atpH 53 may house a proteinaceous pore (Figure 10(b)) Thisprotruding density is sim52 A in width and sim60 A in length andis generated from the surrounding ldquoskirtrdquo region at the five-fold axis This is of sufficient size to house a pore from whichRNA could be extruded roughly 10 A internal diameter [126]This hypothesis was substantiated by electron micrographsof infectious Sindbis particles interacting with the host cellsurface creating a pore-like structure through which RNAwas seen to extrude (Figure 11) Evidence was also shownthat the virion may be interacting via the five-fold axis assuggested by the cryoEM structure Taken in conjunctionwith emergence of the new structure of E1 at the five-foldaxis and the direct visualization of pore formation at thehost cell plasma membrane using EM it was concluded thatvirus entry may proceed by an ancient pathway proposed forbacteriophage direct cell penetration

A recent study by Cao and Zhang reported an early-stage fusion intermediate of Sindbis virus using cryoEM toreconstruct the low pH intermediate [127] The strain TE12which fuses at pH 65 was used to conduct this researchalthough this strain has a particle to pfu ratio of sim100 Sindbisvirus was then treated to pH 64 and the virus was found toretain its 119879 = 4 structure This is surprising since this pHshould have induced a global conformational change in bothE1 and E2 as was shown previously with SVHR displayingsim100 infectivity [122] This lack of reorganization of theTE12 strain could be a reflection of a large particle to pfu num-ber or too high a pH to induce the conformational changeNeither virus titer nor particle to pfu ratios were reportedfor this study TE12 was then mixed with liposomes at neu-tral or pH 64 At the lower pH virus was seen to interact withliposomes or vice versa via bridge-like densities spanning thedistance between the liposome and the virusThese structuresare sim160 A in length which is a greater span by 10 A than thesoluble low pH structure [127] When the SVHR strain ofSindbis virus was treated at a pH of 52 E1 density at all 12 5-fold vertices formed knobs of density sim60 A in length not thetrimeric spikes seenwith soluble E1However comparisons tothe low pH SVHR structure were not made The authors alsoreport that no specific orientation is required for the virusto bind a target membrane however this lack of orientationis determined with liposomes in the absence of the virusreceptor and without intact particles This was not the casewith the SVHR study which showed EM evidence of SVHRparticles binding to cells at a 5 fold vertex and penetrating thecell at the surface in the absence of low pH [122] Fusogenicproperties of membrane containing virus is often studiedusing liposomes Lipid fusion of alpha- and flaviviruses withliposomes is well documented and occurs with very specificadmixtures of lipid [124 128ndash130] However these liposomesare not representative of the composition or structure ofthe host cell membranes (discussed below) There is directevidence that Sindbis virus can become noninfectious butretain its fusogenic ability [66 131] thus separating the E1fusion function from its infectivity It is also well documentedthat alpha and flaviviruses can undergo low pH mediated


350

290

230

(A)

(a) (b) (c)

350

225

100

(A)

(d) (e) (f)

(A)202

160

(g) (h) (i)

Figure 10 Conformational changes in Sindbis virus after exposure to various pH conditions The three-dimensional structures of Sindbisvirus surface at 28 A resolution viewed along icosahedral threefold axes ((a)ndash(c)) Central cross section of Sindbis virus ((d)ndash(f)) Three-dimensional structure of the Sindbis virus capsid (inside the membrane bilayer) viewed along the icosahedral threefold axes ((g)ndash(i)) Thereconstructions are colored according to a range of radii (key displayed) at different pH conditions ((a) (d) and (g)) Sindbis virus at pH72 ((b) (e) and (h)) Sindbis virus at pH 53 ((c) (f) and (i)) Sindbis virus exposed to pH 53 (5min) and returned to pH 72 Arrows (b)protrusion at the fivefold axis (c) fissure at twofold axis ((d) (e) and (f)) fivefold axis (g) region of cross-section occupied by the membranebilayer Paredes et al 2004 [122]

fusion from within and from without however it is possiblethat the Togaviruses infect cells by amechanismmore similarto that used by polio virus [132] rather than that of influenza[122 131 133]

Cell-mediated endosomal uptake of alpha and fla-viviruses followed by acidification and membrane fusionwith the virus membrane is currently the favored model ofalphavirus penetration and entry [134ndash136] This mechanismis supported by indirect biochemical evidence and structuresof E1 trimers extracted from virus-infected cells [137 138] orexpressed as soluble protein [139ndash141] Current evidence oflow pH structures for E1 includes studies done using lipo-somes to extract the proteins from virus during cofloatation

[142ndash144] While liposomes are useful for studying fusion inother systems in which the virus proteins can be expressedindependently such as with flu these artificial membranesmay not be a suitably stringent reagent for the study ofvirus penetration in the case of alpha- and flavivirusesThis is because (1) no virus receptors are present (2) highconcentrations of cholesterol are required [145ndash147] and (3)the trimer of E1 has not been seen by any other method otherthan extraction or expression of E1 followed by liposomeinteraction Insects are cholesterol auxotrophs [148] and donot contain the amounts of cholesterol required for liposomefusion [149] Finally the fusion of virus with liposomes isa non-leaky process [150] which is not the case with virus


(a)

(b) (c) (d)

(e) (f) (g)

Figure 11 Electron micrographs of thin sections of Sindbis virus-cell complexes at pH 72 (a) Lowmagnification showing ldquofullrdquo and ldquoemptyrdquoparticles and a particle attached by a pore to the cell surface (arrow) (b) A virion attached to the cell surface before pore formation (c) Avirion with an electron dense core attached to the cell surface by a pore structure (arrow) (d) The pore at the vertex (V) of the protein shellpenetrates the cell membrane (arrow) The virion has reduced electron density in the core region (e) Reorganization of virus RNA into thedeveloping pore (f) An empty particle with a possible RNA molecule entering the cell (arrow) (g) An empty virion that has lost structureMagnification scale bar (a) = 1000 A all others = 500 A Paredes et at 2004 [122]

infections of host cells [151] Interestingly we have alsoshown that certain clones of mosquito cells derived fromSinghrsquos original isolate U44 are not susceptible to fusionfrom without Sindbis virus but are readily infected [123 152]Because the details of the fusionmodel of alpha and flaviviruspenetration are predominant in the literature evidence fordirect virus penetration will be further discussed

25 Ultrastructural Evidence of Direct Virus Penetration Asearly as 1978 Fan and Sefton proposed that virus entry forSindbis andVSV involved amechanismwhich did not requirefusion [153] This evidence gave way to the more popularmodel of membrane fusion [129 136] The a priori beliefthat enveloped virus structures must encode a fusion loopand penetrate cells by fusion now dominates the field to


the exclusion of alternate modes of virus entry E1 of theAlphaviruses and E of the flaviviruses are referred to asgroup II fusion proteins [139 154 155] Indirect evidence hasbeen used to develop a model proposing that these types ofproteins insert a small fusion loop into the host endosomalmembrane after endocytosis and a shift to low pH Thefusion loop is seen in Chikungunya virus [Alphavirus E1(2ALA)] and flavivirus [Dengue E (1TG8)] crystal structuresNotably pestiviruses and hepaciviruses which belong theFamily Flaviviridae do not encode a fusion loop [156 157]and investigators are in search of alternate fusion domainsor fusion mechanisms [158] The Bunyaviridae have beenpredicted to encode a fusion loop and by homology have beenpredicted to be class II fusion proteins [159]

A large volume of work has focused on the ability ofalpha and flaviviruses to fuse artificial membranes and toelucidate the mechanism of low pH-mediated fusion ofvirus The model of infection for these two families positsthat membrane-containing viruses infect cells via low pH-mediated fusion within cell endosomes The membrane-fusion mechanism of virus infection has been studied exten-sively for the influenza (flu) hemagglutinin and has beenshown by direct evidence to form structural intermediatesinvolved in virus penetration [97 160ndash162] Influenza how-ever differs significantly from the alpha and flavivirusesin that the structure of the virus is amorphous with thestructural proteins associated with large areas of exposedlipid Additionally HA and N do not form heterodimericassociations Unlike influenza there is no direct biochemicalor structural evidence for membrane fusion by arbovirusesand no thermodynamics of the fusion process or the induc-tion of the fusion intermediates The ability of alpha andflaviviruses to fuse membranes is not disputed however thismay not result in virus penetration and infection

Our work on virus penetration has shown that SindbisWest Nile virus (WNV) and dengue virus can penetrate cellsat temperatures that do not allow membrane fusion [131 133163 164] Using direct observation and biochemical methodswe have demonstrated that Sindbis and dengue virus infectcells in a time and temperature dependent manner (Figure12)

Recent data show that Sindbis virus can penetratemosquito C7-10 cells even more quickly than what has beenseen in BHK cells By 60min postinfection at 4∘C 90of the virus was empty as compared to the 75 seen inBHK cells (see Figure 12(b)) Cultured insect cells contain lesscholesterol than mammalian cells and are less viscous at 4∘Cwhich may facilitate the process The temperature kinetics ofthis reaction can be fit to Arrhenius plots suggesting that theprocess of entry of the RNA into the cell is not force drivenand that the energy to form the pore structure likely residesin the virus proteins The energy of activation is calculated tobe 27 kcalmoleThe entry process only requires a membranepotential and is affected by the chemistry of the host cellmembrane (Vancini personal communication) The datashow that 70 of Sindbis virions are empty after one hourat 4∘C (Figure 13) Sindbis virus carrying a green fluorescentprotein will infect BHK cells at 15∘C in the absence of fusionor endocytosis producing fluorescent cells without return

through higher temperatures [133] The obvious implicationof these data is that studies in which virus has been allowed toattach on ice for one hour during the infection phase may nothave synchronized the infection as proposed in these studiesbut rather allowed infectious particles to be internalized [122131 133 165] reviewed in [165 166] (Figure 14) However theeffect of 15∘C on formation of the replication complex hasnot been reported for Sindbis virus but it is possible thatsynchronization occurs at the level of RNA synthesis

Strong biochemical support for the model of direct pen-etration at the cell surface comes from studies showing thatcells become permeable to ions and small molecules as theyare infectedwith alphaviruses [126 151] Virus infections leavepores on cell membranes [167] that allow the penetration ofthe cell membrane by small proteins such as the toxin 120572-sarcin (17 kDa) [168]These results show that pores created inthe plasma membrane as entry takes place affect membranepermeability Fusion by contrast is a nonleaky process doesnot compromise membranes and does not leave pores in themembranes [150 169]

Evidence of direct penetration has been presented forboth mammalian and insect cells [165 166] This work hasled to the alternate model that proposes that alpha and fla-viviruses penetrate the host membrane bilayer through hostcell triggered rearrangement of E1 or E proteins at a 5 fold axisresulting in the formation of a proteinaceous pore This poreallows the release of the RNA into the cell cytoplasm thusinitiating the infection process In the 1994 study by Guineaand Carrasco [170 171] it was concluded that an ion gradientwas required for viral entry of vesicular stomatitis virusSemliki Forest virus and influenza virus This observationhas been confirmed for Sindbis virus (Vancini unpublished)We propose that membrane-containing icosahedral virusesincorporated lipid into their structure as an assembly scaffoldallowing the cell exocytic mechanism to process and presentmaturing structural proteins to the nucleocapsid for envelop-mentThis point is crucial for the development of prophylacticsfor pathogenic arboviruses

26 Native Virus Structure Analyzed Using Small Angle Neu-tron Scattering In the paper by He et al [40] we used smallangle neutron scattering to explore the nature of Sindbis virus(alphavirus) particles produced by mammalian and insectcells This method has the advantage over other methodsof observing structure in that lipid and RNA densities areeasily detected through a technique called contrast variation[172] The findings were significantly distinct from whatwas expected because virus particles from these two hostshave important structural differences Using virus particlespurified in deuterium a highly concentrated solution of virussuspension was made from virus grown in mosquito C7-10or mammalian BHK cells The particles were then analyzedusing small angle neutron scattering (SANS) a nondestruc-tive technique [173 174]The119877

119892(radius of gyration) indicated

that the BHK-grown virus is less compact than that grownfrom mosquito cells The diameter of the BHK grown viruswas found to be 689 A compared to that of the insect grownvirus which was 670 A The mass at the center of the BHKparticles was less centrally distributed than that seen in the


100

90

80

70

60

50

40

30

20

10

04 15 24 37

Incubation temperature (∘C)

EmptyFull

Full

empt

y pa

rtic

les (

o

f tot

al)

(a)

1000

900

800

700

600

500

400

300

200

100

0015 30 45 60

Time (min)

EmptyFull

Full

empt

y pa

rtic

les (

o

f tot

al)

(b)

Figure 12 Temperature and time dependence of Sindbis virus genome delivery into BHK cells (a) Sindbis virus-cell complexes incubated andfixed at 15min postinfection at several temperatures were analyzed by electron microscopy The graph shows that as temperature rises from4∘C to 37∘C there is a progressive increase in the population of empty particles at the cell surface (X) representing the total population at37∘C and a decrease in the population of full particles (998771) reaching zero at 37∘C (b) Interaction of Sindbis virus with BHK cells at 4∘C for 15 to60minThe percentage of empty particles increases from 30 at 15min to 666 at 60min (X) and the population of full particles decreasesfrom 70 to 334 over the same time period (998771) Data are shown as the means of triplicate samples of two independent experiments Errorbars are standard errors of the means (SEM) From Vancini et al 2013 [133]

C7-10 virus It was also determined that while the radialposition of the lipid bilayer did not change significantlythe membrane had significantly more cholesterol when thevirus was grown in mammalian cells than in insect cellsThis property has been shown to affect the virus stabilityand with it infectivity [149] Distribution of the densitiesof the particles was modeled using a four shell analysisrepresenting the distribution of the biochemical componentsof the virus RNA capsid protein lipid with protein andglycoprotein Comparing the shell thickness for each virusshowed that the outer protein shell was more extended in themammalian Sindbis virus than that from the insect virusTheSANS data also demonstrated that the RNA and nucleocapsidprotein share a closer interaction in the mammalian cell-grown virus than in the virus from the insect host It ispossible that the nucleocapsid structure from mammaliancells is organized more closely to the virus membrane andthat the center of the virus is mainly solvent The biologicalconsequences of the structural differences uncovered by thisnew technique are not known It may be that the temperatureof assembly may contribute to these differences given thedifferent biochemical environments It is of interest to notethat Zhang et al [23] reported a structure of dengue virusat 35 A resolution when grown from C636 mosquito cellsgrown at 33∘C (see Figure 3) but did not report a difference inthe size of the virusThismay suggest that virus conformation

and size may be the result of the host cell biochemistry andnot temperature This will be discussed further

27 Temperature Studies and Modeling Crystal Structuresinto Cryo-EM Reconstructions Dengue virus particles arestructurally classified as (1) mature (2) partially mature or(3) immature depending on the surface morphology seen incryoEM preparations prior to averaging of the particles [176]The classification of particles intomature partiallymature orimmature status does not correlate with infectivity it is esti-mated that infectious virus contains 30ndash40 uncleaved preM[84 177] Mature dengue has been reported to have a particlediameter of 500 A [94] Two recent studies have reported thattemperature also affects viral conformation for dengue virus 2strains 16681 andNewGuinea C [175 178] In both these DV2studies one by Zhang et al [178] and one by Fibriansah et al[175] it was determined by cryoEM reconstruction of virusgrown in insect C636 cells at 28∘C then heated to 37∘C thatvirus expanded by 40ndash50 A compared to the virus remainingat ambient temperature [175] In each study both heatedand unheated virus was found to have equivalent infectivityindicating that both virus structures represent infectiousintermediates In the Fibriansah study several categories of37∘C treated virus particles were seen in the cryoEM imagesresulting in sorting of the virus into several classes based onthe surfacemorphology shown in Figure 15 Class II particles


(a)

(b)

Figure 13 Thin-section electron microscopy of Sindbis virus-cell complexes at pH 72 A panel of images obtained by TEM illustrates tworepresentative populations of particles at different incubation temperatures High-magnification images show examples of electron-dense fullparticles at the cell surface at 4∘C (row (a)) and empty particles with loss of RNA electron density predominantly at 37∘C (row (b)) In row(b) middle panel there is indication of a stalk connecting the virus and the cell (arrow) Bars 50 nm From Vancini et al 2013 [133]

(a) (b) (c)

Figure 14 Overview of Sindbis virus-cell complexes Low magnifications of Sindbis virus-cell complexes at different temperatures (a)Interaction at 4∘C (b) interaction at 15∘C note the presence of both full and empty (arrows) particles (c) interaction at 37∘C in whichmost particles lose their electron density and their well-defined structure (arrowheads) Bars 100 nm From Vancini et al 2013 [133]


Surfa

ceC

entr

al cr

oss-

sect

ion

E-proteinBilipid layer

IClass II III IV

30 A130 A180 A

240 A260 A280 A


Figure 15 Cryo-EMmaps reconstructed from class I to IV particles present in theDENV2 sample incubated at 37∘CThe surfaces of themaps(above) and their central cross-sections (below) are colored according to their radii (shown in the lower panel) The white triangle representsan icosahedral asymmetric unit and the corresponding 2- 3- and 5-fold symmetry vertices are indicated Maps generated from the classI particles are similar to the previous published DENV-2 maps Class II and III maps showed that the particles in these classes have biggerradii indicating that the virus had expanded There are protruding densities on the virus surface between the 5- and 3-fold vertices (whitearrows) The class IV map showed very poor density on the E protein layer indicating that the E protein had lost its icosahedral symmetryWith permission from Fibriansah et al [175]

have a smooth surface while Class III display a surface withprotrusions Class IV structures are smaller than the otherclasses Class I particles are those grown in mosquito cellsat 28∘C In class II viruses E and M density was seen toreorganize to produce protruding structures between the 5-and 3-fold vertices In the class III structures the protrusionsare more extended at the 2-fold axes and ldquoholesrdquo appearat the strict 3-fold axes While the structure of the classIV viruses was not further investigated by these authorsparticles at 37∘C continued to change conformation reorga-nizing density toward the 5-fold axes until a nonreversibleendpoint was achieved Thus heating of the class I particlesresulted in three different structures In most icosahedralvirus reconstructions from cryoEM the signal is improvedand the noise reduced by imposing icosahedral symmetry onthe image data after refinement For protein density to bevisible after imposing this symmetry the protein structurehas to occupy more than 60 of the icosahedrally relatedpositions [179 180] Conversely structures that occupy veryfew of these positions such as a portal complex are averagedout of 3D reconstructionsThus while the amount of preM isaveraged out during a cryoEM reconstruction the presenceof this protein may still have an important role to play in thestructure infectivity and immunogenicity of the virus It wasnot determinedwhether this structural differencewas a resultof a difference in the host environment or a function of thetemperature of assembly Insect host specific biochemistrywhich functions optimally at 28∘C may be responsible for

assembly of the compact structure which can adopt a lesscompact form at 37∘C As was discussed above virus sizedifferences were seen with Sindbis virus from different hosts(mammalian or insect) [40]

The structure byZhang et al is similar to the one observedby Fibriansah et al in which E andM density is seen to moveaway from the core of the virus after heating insect grownvirus to 37∘C The former structure was solved to 35 A whilethe latter had a resolution of 14 A In the work by Zhanget al the same experiment is performed insect grown virusis heated to 37∘C Both virus structures were found to beequally infectious These authors reconstructed the heatedform to a fit with E protein into trimers however unlikeFibriansah who fit E into the traditional herringbone patternIn the Zhang study the authors propose that this larger 37∘Cstructure represents the prefusogenic form of the virus thatwas previously just a predicted structure [94] In Zhang etal virus expansion was found to be reversible upon heatingto 35∘C and that the end point stable intermediate doesnot occur until the temperature reaches 37∘C after whichthe expansion is not reversible Both studies reported theintroduction of holes in the virus surface at the 31015840 axes notseen in previous reconstructions

Both these flavivirus studies speculated that the change instructure may affect the response of the mammalian host byrevealing previously unexposed epitopes in virus from insectcells This is important for mapping neutralizing epitopesin vitro and it was previously not understood how known


neutralizing Ab (NAb) anti-DV2 epitopes on the virus boundAb to sequences that were occluded in the virus structureThis anomaly was explained by proposing that superficiallyhidden epitopes could be unmasked during thermal flux orldquobreathingrdquo of the virus [181] This quandary is now resolvedby fitting the proteins into a larger expanded structure formedby heating insect virus to 37∘C However the E fold in thecrystal structure may not be native resulting in difficulty witha fit to the smaller structure In addition to epitope exposurewhat is the biological relevance of these observations Amore compact form of insect virus does not affect an invivo mammalian infection because the more compact 28∘Cstructure will only be exposed to the host for one round ofreplication after which the second 37∘C structure will beadopted Because both compact and expanded forms of theviruses from both hosts are infectious these virions representmetastable structural intermediates primed for infection andthese two distinct infectious intermediates may representthe optimal form of the structure from its specific hostbiochemical environment

One interpretation of these observations is that the mam-malian virus is ldquobornrdquo in the prefusogenic-like structure andthe mosquito virus is not While there is no direct evidencefor any function of these predicted intermediate modelsmosquito cells were speculated to be infected by virus adopt-ing the fusogenic form via exposure to low pHor contact withthe receptor thus triggering the rearrangement Again as inthe former study the Zhang study demonstrated that virusincubated at both temperatures is infectious This becomesan important point because arboviruses do not always cyclebetween insect and vertebrate hosts For mosquito-borneviruses there exists a ldquomosquito onlyrdquo zoonotic cycle inthe natural transmission cycle during which mosquito andvirus would only experience ambient temperature [182 183]Arboviruses carried by mosquitoes are transmitted fromvector to vector horizontally by venereal transmission orvertically as infected eggs This mosquito cycle is requiredespecially in temperate climates for the virus to over winterIf fusion is required for infectivity then the ldquoinsect onlyrdquovirus cannot acquire the fusion intermediate in the absenceof invoking other mechanisms of structural alterationWhat-ever the interpretation the present data argue that for dengue2 (1) two infectious dengue virus intermediates are found innature one at 28∘C and another at 37∘C as a result of the tem-perature of assembly or (2) virus architecture is determinedby host components which have a temperature componentresulting in two structural intermediates both of which arebiologically relevant as was reported in He et al [40] Neitherof these alternatives need be mutually exclusive It is of notethat the structure reported by Zhang et al (shown in Figure 3)was made from images of virus grown in insect C636 cells at33∘C At this temperature according to the Zhang et al study50 of the particles should have expanded if temperaturewere the only contributing factor to particle expansionhowever no increase in particle size was reported [178]

In addition to interpreting the high temperature struc-tures these papers modeled crystal structures of isolated Einto the cryoEM reconstructions Both studies used E fromTick Borne Encephalitis virus PDB 1SVB [95] Zhang et

al reconstructed the heated form of the structure to a fitwith E protein in trimers of dimers as seen in [94] to 35 Aresolution Zhang et al propose that the larger 37∘C structurerepresents the prefusogenic form of the virus that reorganizesthe herringbone array into trimers after the virus is exposedto low pH This structure containing the trimer pattern waspreviously only a predicted structure [94] In a separate studyFibriansah reconstructed the DV2 expanded structure into a14 A model by separating the images into three size classesHowever in the Fibriansah study that displayed higher reso-lutionEwasmodeled into the traditional herringbone patternin the larger class III structureThe question arises as towhichof either of the two models represents a biologically relevantstructure Fab 1A1D-2 [175 178] and MAb E 111 epitopes aresuggested to become more exposed and accessible to addweight to each predicted model An additional complicationto the DV2 expansion story is that Kostyuchenko et al havereported that dengue 1 and 4 do not undergo heat-relatedexpansion of the viruswhen insect cell grown virus is exposedto 37∘C [184 185] If virus expansion is required for infectionand the structures are similar in structure and function itis reasonable to assume that it would happen in all fourserotypes It is suggested however that stability of dengue1 and 4 imparted to these serotypes by surface charges onthe virus could explain the lack of conformational changes athigher temperatures This implies that DV2 is more unstableto heat but no biochemical evidence was provided Thepossibility that these viruses may contain unprocessed preMwas not discussed or assessed though preM is known toblock infection It is puzzling that these viruses would notrespond to heat but supposedly respond to low pH inducedconformations to become infectious

3 Conclusions and Comments

As has been discussed the structural proteins of the icosa-hedral arboviruses display a remarkable amount of plasticityFrom the same conserved folds 119879 = 3 symmetry is assem-bled in the flaviviruses and 119879 = 4 for the alphavirusesrepresenting an approximate 200 A increase in virion sizewhile the genome lengths are not significantly larger Asdiscussed for the Sindbis virus C mutant Y180SE183G evenlarger119879 numbers are possible Not discussed but pertinent tothis point is the 119879 = 12 structure found in the bunyavirusesthat deviate to pleomorphic structures in the other membersof the Bunyaviridae These viruses are also presumed to havearisen from an ancient common fold The relationship of thisadaptation is not understood because the 119879 = 12 structuregenome size ranges from 105 to 227 kbp and is composed of atripartite single-stranded negative-sense RNA genome thatcould theoretically be packaged in amuch smaller virion [34]Plasticity has also been documented in virus assembled fromthe insect versus the vertebrate host [40] This was an unex-pected outcomebut in retrospect not surprising because thesehost systems are so biochemically and genetically divergentAlpha- and flaviviruses contain glycoproteins with manycysteines and more than one disulfide bonded form of theseglycoproteins could exist This is difficult to analyzedetectin the intact virus and for the most part has been ignored


and the configuration of the crystal structures accepted asnative Additionally the cryoEM structures of the pH-treatedviruses have been interpreted to assume the same structuralreorganizations of the soluble E protein in the prefusion con-formation which forms a trimer However these structuresare supportive of indirect evidence for trimerizationThus inlight of the conflicting evidence for fusion as the mechanismof virus penetration these data should suggest that thestructure of the isolated protein does not always predict theconformation in the intact metastable virus Even at atomicresolution reconstruction of an infectious virus cannot leadto definitive evidence of the mechanism of virus penetrationwithout direct biochemical and genetic testing of the model

It has been a long quest to solve the atomic structure of amembrane-containing virus As technology improves atomicdetails will certainly be resolved However there are certainimpediments to the process that are not directly related to thetechnology First it has become a canon of some investigatorsthat crystal structures of proteins and their multimeric statesrepresent a native structure This is a huge assumption formetastable virus proteins because in the absence of controlstructures or direct functional assays this assumption cannotbe tested Most virus samples also contain a large particle topfu ratio The most rigorous experiments would not requirethe dissolution of the virus particle [186 187] This is not tosay that structures of proteins are not informative but thatthe process needs to include a caveat that crystals do notrepresent the entirety of the many conformations throughwhich macromolecular metastable dynamic viruses proceedto reach the infectious intermediate and then deliver uponinfection the genomic cargo This is especially important ifthe crystal structure is of a single protein of amacromolecularcomplex that does not naturally exist in solution such asone that contains membrane proteins There is a precept thatif a protein can be expressed by any expression system thesubsequent folds are representative of the native structureThis assumption may be too simplistic because it is wellestablished biochemically and genetically that assembly ofmembrane-containing virus requires both host and viruschaperones including lipids and post translational modifi-cations in a complex and temporal manner to assemble aninfectious virus This exclusive process is not available inany heterologous expression system An example of this isseen in a study of Sindbis virus E1 in which whole infectiousvirions were analyzed by mass spectrometry and it wasdetermined that E1 has cysteines not seen in the alphaviruscrystal structure in which all Cys are found as disulfides [66]The key to these analyses is not the technology employedbut rather the starting infectious material This observationsuggests that other problems may exist with the currentalphavirus E1model as was also suggested above for flavivirusE Very few protein structures are used to build a ldquofitrdquo intoa cryo-EM reconstruction because they are assumed to beinterchangeable If the fit does not work either the processis abandoned or the protein domains are reoriented Anexample of this is found in Fibriansah et al inwhich they statethat E protein could not be fit into the class II particles due tothe lack of structural features required for fitting althoughit is possible that the cryo-EM contains data on a structural

intermediate that is that the structure is expanding in away that does not fit the current methods model or crystalThis implies that there may exist an intermediate structurewhich adopts an unpredicted structure These structuresare models of a very complicated assembly system Cryo-EM of heated and low pH-treated viruses is revealing anextraordinary reorganization of the contour of alphavirusesand flaviviruses at sim20 A resolutions It seems plausiblethat during these conformational reorganizations structuralproteins may proceed through several energetic states untilthe end point state is achievedMuchmorework is required tofully appreciate these intricate structures and theirmechanicsof assembly and infection

Disclaimer

The information in these materials is not a formal dissemina-tion of information by the FDAanddoes not represent agencyposition or policy

Conflict of Interests

The authors declare no conflict of interest

Authorsrsquo Contribution

Raquel Hernandez Dennis T Brown and Angel Paredeswrote this review

Acknowledgments

Authors Raquel Hernandez and Dennis T Brown are sup-ported by the Foundation for Research Carson City Nevadaand the North Carolina Agricultural Research Service

References

[1] E G Strauss R Levinson C M Rice J Dalrymple and J HStrauss ldquoNonstructural proteins nsP3 and nsP4 of Ross Riverand OrsquoNyong-nyong viruses sequence and comparison withthose of other alphavirusesrdquo Virology vol 164 no 1 pp 265ndash274 1988

[2] J H Strauss and E G Strauss ldquoVirus evolution how does anenveloped virus make a regular structurerdquo Cell vol 105 no 1pp 5ndash8 2001

[3] R Goldbach and J Wellink ldquoEvolution of plus-strand RNAvirusesrdquo Intervirology vol 29 no 5 pp 260ndash267 1988

[4] R W Schlesinger ldquoNew opportunities in biological researchoffered by arthropod cell cultures I Some speculations on thepossible role of arthropods in the evolution of arbovirusesrdquoCurrent Topics in Microbiology and Immunology vol 55 pp241ndash245 1971

[5] C A Stafford G P Walker and D E Ullman ldquoInfection with aplant virus modifies vector feeding behaviorrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 108 no 23 pp 9350ndash9355 2011

[6] I C Bezerra R D O Resende L Pozzer T Nagata RKormelink and A C De Avila ldquoIncrease of tospoviral diversityin Brazil with the identification of two new tospovirus speciesone from chrysanthemumand one from zucchinirdquoPhytopathol-ogy vol 89 no 9 pp 823ndash830 1999


[7] S Adkins R Quadt T-J Choi P Ahlquist and T GermanldquoAn RNA-dependent RNA polymerase activity associated withvirions of tomato spotted wilt virus a plant- and insect-infecting bunyavirusrdquoVirology vol 207 no 1 pp 308ndash311 1995

[8] J H-O Pettersson I Golovljova S Vene and T JaensonldquoPrevalence of tick-borne encephalitis virus in ixodes ricinusticks in northern europe with particular reference to southernSwedenrdquo Parasites amp Vectors vol 7 article 102 2014

[9] J J L Lutomiah C Mwandawiro J Magambo and R C SangldquoInfection and vertical transmission of Kamiti river virus inlaboratory bred Aedes aegypti mosquitoesrdquo Journal of InsectScience vol 7 article 55 2007

[10] G Kuno G-J J Chang K R Tsuchiya N Karabatsos and C BCropp ldquoPhylogeny of the genus flavivirusrdquo Journal of Virologyvol 72 no 1 pp 73ndash83 1998

[11] E C Holmes and S S Twiddy ldquoThe origin emergence and evo-lutionary genetics of dengue virusrdquo Infection Genetics and Evo-lution vol 3 no 1 pp 19ndash28 2003

[12] S Cook G Moureau A Kitchen et al ldquoMolecular evolution ofthe insect-specific flavivirusesrdquo Journal of General Virology vol93 no 2 pp 223ndash234 2012

[13] J P Messina O J Brady T W Scot et al ldquoGlobal spread ofdengue virus types mapping the 70 year historyrdquo Trends inMicrobiology vol 22 pp 138ndash146 2014

[14] A T Ciota and L D Kramer ldquoInsights into arbovirus evolutionand adaptation from experimental studiesrdquo Viruses vol 2 no12 pp 2594ndash2617 2010

[15] L L Coffey N Forrester K Tsetsarkin N Vasilakis and SC Weaver ldquoFactors shaping the adaptive landscape for arbo-viruses implications for the emergence of diseaserdquo FutureMicrobiology vol 8 no 2 pp 155ndash176 2013

[16] G D Ebel ldquoToward an activist agenda for monitoring virusemergencerdquo Cell Host amp Microbe vol 15 no 6 pp 655ndash6562014

[17] S C Weaver R Winegar I D Manger and N L ForresterldquoAlphaviruses population genetics and determinants of emer-gencerdquo Antiviral Research vol 94 no 3 pp 242ndash257 2012

[18] M G Rossmann and R R Reuckert ldquoWhat does the molecularstructure of viruses tell us about viral functionsrdquoMicrobiologi-cal Sciences vol 4 no 7 pp 206ndash214 1987

[19] E G Westaway M A Brinton S Y Gaidamovich et alldquoTogaviridaerdquo Intervirology vol 24 no 3 pp 125ndash139 1985

[20] R M Dubois M-C Vaney M A Tortorici et al ldquoFunctionaland evolutionary insight from the crystal structure of rubellavirus protein E1rdquo Nature vol 493 no 7433 pp 552ndash556 2013

[21] P F Mattingly ldquoGenetical aspects of the aedes aegypti problemI Taxonomy and bionomicsrdquo Annals of Tropical Medicine andParasitology vol 51 no 4 pp 392ndash408 1957

[22] R Zhang C F Hryc Y Cong et al ldquo44 A cryo-EM structure ofan enveloped alphavirus venezuelan equine encephalitis virusrdquoEMBO Journal vol 30 no 18 pp 3854ndash3863 2011

[23] X Zhang P Ge X Yu et al ldquoCryo-EM structure of themature dengue virus at 35-A resolutionrdquoNature Structural andMolecular Biology vol 20 no 1 pp 105ndash110 2013

[24] D H Bamford ldquoDo viruses form lineages across differentdomains of liferdquo Research in Microbiology vol 154 no 4 pp231ndash236 2003

[25] M B Sherman A N Freiberg M R Holbrook and S JWatowich ldquoSingle-particle cryo-electron microscopy of RiftValley fever virusrdquo Journal of Virology vol 387 no 1 pp 11ndash152009

[26] J C Brown W W Newcomb and G W Wertz ldquoHelical virusstructure the case of the rhabdovirus bulletrdquo Viruses vol 2 no4 pp 995ndash1001 2010

[27] P Metcalf ldquoThe symmetry of the reovirus outer shellrdquo Journalof Ultrastructure Research vol 78 no 3 pp 292ndash301 1982

[28] R Couch Orthomyxoviruses University of Texas MedicalBranch at Galveston Galveston Tex USA 1996

[29] R G Webster W J Bean O T Gorman T M Chambers andY Kawaoka ldquoEvolution and ecology of influenza A virusesrdquoMicrobiological Reviews vol 56 no 1 pp 152ndash179 1992

[30] I Rouiller S M Brookes A D Hyatt M Windsor and TWileman ldquoAfrican swine fever virus is wrapped by the endo-plasmic reticulumrdquo Journal of Virology vol 72 no 3 pp 2373ndash2387 1998

[31] A Lwoff R Horne and P Tournier ldquoA system of virusesrdquo ColdSpring Harbor Symposia on Quantitative Biology vol 27 pp 51ndash55 1962

[32] D Baltimore ldquoExpression of animal virus genomesrdquo Bacterio-logical Reviews vol 35 no 3 pp 235ndash241 1971

[33] A King E Lefkowitz M Adams and E Carstens Virus Taxo-nomyNinth Report of the International Committee onTaxonomyof Viruses Elsevier Waltham Mass USA 1st edition 2012

[34] R V Mannige and C L Brooks III ldquoPeriodic table of virus cap-sids implications for natural selection and designrdquo PLoS ONEvol 5 no 3 Article ID e9423 2010

[35] M Dessau and Y Modis ldquoCrystal structure of glycoprotein cfrom rift valley fever virusrdquoProceedings of theNational Academyof Sciences of the United States of America vol 110 no 5 pp1696ndash1701 2013

[36] J H Strauss and E G Strauss ldquoThe alphaviruses gene expres-sion replication and evolutionrdquo Microbiological Reviews vol58 no 3 pp 491ndash562 1994

[37] S Nelson R Hernandez D Ferreira and D T Brown ldquoIn vivoprocessing and isolation of furin protease-sensitive alphavirusglycoproteins a new technique for producingmutations in virusassemblyrdquo Virology vol 332 no 2 pp 629ndash639 2005

[38] H Garoff K Simons and O Renkonen ldquoIsolation and char-acterization of the membrane proteins of semliki forest virusrdquoJournal of Virology vol 61 no 2 pp 493ndash504 1974

[39] M Carleton H Lee M Mulvey and D T Brown ldquoRole ofglycoprotein PE2 in formation and maturation of the sindbisvirus spikerdquo Journal of Virology vol 71 no 2 pp 1558ndash15661997

[40] L He A Piper F Meilleur et al ldquoThe structure of sindbis virusproduced from vertebrate and invertebrate hosts as determinedby small-angle neutron scatteringrdquo Journal of Virology vol 84no 10 pp 5270ndash5276 2010

[41] J B Gliedman J F Smith and D T Brown ldquoMorphogenesisof sindbis virus in cultured aedes albopictus cellsrdquo Journal ofVirology vol 16 no 4 pp 913ndash926 1975

[42] C L Murray C T Jones and C M Rice ldquoArchitects ofassembly roles of Flaviviridae non-structural proteins in virionmorphogenesisrdquo Nature Reviews Microbiology vol 6 no 9 pp699ndash708 2008

[43] S Elshuber S L Allison F X Heinz and C W MandlldquoCleavage of protein prM is necessary for infection of BHK-21 cells by tick-borne encephalitis virusrdquo Journal of GeneralVirology vol 84 no 1 pp 183ndash191 2003

[44] B D Lindenbach and C M Rice ldquoFlaviviridae the viruses andtheir replicationrdquo in Fields Virology D M Knipe and P MHowley Eds pp 1043ndash1125 Lippincott Williams amp WilkinsPhiladelphia Pa USA 2001


[45] MM Samsa J AMondotte N G Iglesias et al ldquoDengue viruscapsid protein usurps lipid droplets for viral particle formationrdquoPLoS Pathogens vol 5 no 10 Article ID e1000632 2009

[46] W Zhang P R Chipman J Corver et al ldquoVisualization ofmembrane protein domains by cryo-electron microscopy ofdengue virusrdquoNature Structural Biology vol 10 no 11 pp 907ndash912 2003

[47] D Ferreira R Hernandez M Horton and D T Brown ldquoMor-phological variants of sindbis virus produced by a mutation inthe capsid proteinrdquo Virology vol 307 no 1 pp 54ndash66 2003

[48] B V V Prasad andM F Schmid ldquoPrinciples of virus structuralorganizationrdquo Advances in Experimental Medicine and Biologyvol 726 pp 17ndash47 2012

[49] S Lee K E Owen H-K Choi et al ldquoIdentification of a proteinbinding site on the surface of the alphavirus nucleocapsid andits implication in virus assemblyrdquo Structure vol 4 no 5 pp 531ndash541 1996

[50] R Hernandez H Lee C Nelson and D T Brown ldquoA singledeletion in the membrane-proximal region of the Sindbis virusglycoprotein E2 endodomain blocks virus assemblyrdquo Journal ofVirology vol 74 no 9 pp 4220ndash4228 2000

[51] C H VonBonsdorff and S C Harrison ldquoSindbis virus glyco-proteins form a regular icosahedral surface latticerdquo Journal ofVirology vol 28 article 578 1978

[52] D L Caspar R Dulbecco A Klug et al ldquoProposalsrdquo ColdSpring Harbor Symposia on Quantitative Biology vol 27 pp 49ndash50 1962

[53] J P Kononchik Jr S Nelson R Hernandez and D T BrownldquoHelical virus particles formed frommorphological subunits ofa membrane containing icosahedral virusrdquo Journal of Virologyvol 385 no 2 pp 285ndash293 2009

[54] D T Brown ldquoThe assembly of alphavirusesrdquo inTheTogavirusesRW Schlesingerrsquos Ed pp 473ndash501 Academic PressNewYorkNY USA 1980

[55] K Nanda R Vancini M Ribeiro D T Brown and R Her-nandez ldquoA high capacity alphavirus heterologous gene deliverysystemrdquo Journal of Virology vol 390 no 2 pp 368ndash373 2009

[56] M Mulvey and D T Brown ldquoFormation and rearrangementof disulfide bonds during maturation of the Sindbis virus E1glycoproteinrdquo Journal of Virology vol 68 no 2 pp 805ndash8121994

[57] R Xu and I A Wilson ldquoStructural characterization of an earlyfusion intermediate of influenza virus hemagglutininrdquo Journalof Virology vol 85 no 10 pp 5172ndash5182 2011

[58] F Ni X Chen J Shen and Q Wang ldquoStructural insights intothe membrane fusion mechanism mediated by influenza virushemagglutininrdquo Biochemistry vol 53 no 5 pp 846ndash854 2014

[59] J Chen J J Skehel and D C Wiley ldquoN- and C-terminalresidues combine in the fusion-pH influenza hemagglutininHA2 subunit to form an N cap that terminates the triple-stranded coiled coilrdquo Proceedings of the National Academy ofSciences of the United States of America vol 96 no 16 pp 8967ndash8972 1999

[60] M Carleton and D T Brown ldquoDisulfide bridge-mediated fold-ing of Sindbis virus glycoproteinsrdquo Journal of Virology vol 70no 8 pp 5541ndash5547 1996

[61] M Carleton andD T Brown ldquoThe formation of intramoleculardisulfide bridges is required for induction of the sindbis virusmutant ts23 phenotyperdquo Journal of Virology vol 71 no 10 pp7696ndash7703 1997

[62] M Mulvey and D T Brown ldquoInvolvement of the molecularchaperone BiP in maturation of Sindbis virus envelope glyco-proteinsrdquo Journal of Virology vol 69 no 3 pp 1621ndash1627 1995

[63] MMulvey andDT Brown ldquoAssembly of the Sindbis virus spikeprotein complexrdquo Virology vol 219 no 1 pp 125ndash132 1996

[64] B S Phinney K Blackburn and D T Brown ldquoThe surfaceconformation of sindbis virus glycoproteins E1 and E2 atneutral and low pH as determined bymass spectrometry-basedmappingrdquo Journal of Virology vol 74 no 12 pp 5667ndash56782000

[65] B S Phinney and D T Brown ldquoSindbis virus glycoprotein E1 isdivided into two discrete domains at amino acid 129 by disulfidebridge connectionsrdquo Journal of Virology vol 74 no 19 pp 9313ndash9316 2000

[66] C BWhitehurst E J SoderblomM LWest R HernandezMB Goshe and D T Brown ldquoLocation and role of free cysteinylresidues in the Sindbis virus E1 and E2 glycoproteinsrdquo Journalof Virology vol 81 no 12 pp 6231ndash6240 2007

[67] B A Abell and D T Brown ldquoSindbis virus membrane fusion ismediated by reduction of glycoprotein disulfide bridges at thecell surfacerdquo Journal of Virology vol 67 no 9 pp 5496ndash55011993

[68] A J Snyder K J Sokoloski and S Mukhopadhyay ldquoMutatingconserved cysteines in the alphavirus E2 glycoprotein causesvirus-specific assembly defectsrdquo Journal of Virology vol 86 no6 pp 3100ndash3111 2012

[69] M Carleton and D T Brown ldquoEvents in the endoplasmicreticulum abrogate the temperature sensitivity of sindbis virusmutant ts23rdquo Journal of Virology vol 70 no 2 pp 952ndash9591996

[70] R Danev and K Nagayama ldquoTransmission electron micro-scopy with Zernike phase platerdquo Ultramicroscopy vol 88 no4 pp 243ndash252 2001

[71] R Danev R M Glaeser and K Nagayama ldquoPractical factorsaffecting the performance of a thin-film phase plate for trans-mission electron microscopyrdquo Ultramicroscopy vol 109 no 4pp 312ndash325 2009

[72] R Danev andKNagayama ldquoOptimizing the phase shift and thecut-on periodicity of phase plates for TEMrdquo Ultramicroscopyvol 111 no 8 pp 1305ndash1315 2011

[73] K Nagayama ldquoAnother 60 years in electron microscopydevelopment of phase-plate electron microscopy and biologicalapplicationsrdquo Journal of Electron Microscopy vol 60 no 1 ppS43ndashS62 2011

[74] A-C Milazzo A Cheng A Moeller et al ldquoInitial evaluationof a direct detection device detector for single particle cryo-electron microscopyrdquo Journal of Structural Biology vol 176 no3 pp 404ndash408 2011

[75] X Li P Mooney S Zheng et al ldquoElectron counting and beam-induced motion correction enable near-atomic-resolutionsingle-particle cryo-emrdquoNatureMethods vol 10 no 6 pp 584ndash590 2013

[76] H M Van Der Schaar M J Rust B-L Waarts et al ldquoChar-acterization of the early events in dengue virus cell entry bybiochemical assays and single-virus trackingrdquo Journal of Viro-logy vol 81 no 21 pp 12019ndash12028 2007

[77] K M Smith K Nanda C J Spears et al ldquoStructural mutantsof dengue virus 2 transmembrane domains exhibit host-rangephenotyperdquo Virology Journal vol 8 article no 289 2011

[78] Y Zhang J Corver P R Chipman et al ldquoStructures ofimmature flavivirus particlesrdquo EMBO Journal vol 22 no 11 pp2604ndash2613 2003


[79] M Lobigs ldquoInefficient signalase cleavage promotes efficientnucleocapsid incorporation into budding flavivirus mem-branesrdquo Journal of Virology vol 78 no 1 pp 178ndash186 2004

[80] J Y LeungG P PijlmanNKondratieva JHyde JMMacken-zie and A A Khromykh ldquoRole of nonstructural protein NS2Ain flavivirus assemblyrdquo Journal of Virology vol 82 no 10 pp4731ndash4741 2008

[81] J M Mackenzie M K Jones and P R Young ldquoImmunolo-calization of the Dengue virus nonstructural glycoprotein NS1suggests a role in viral RNA replicationrdquo Virology vol 220 no1 pp 232ndash240 1996

[82] M Lobigs ldquoFlavivirus premembrane protein cleavage and spikeheterodimer secretion require the function of the viral pro-teinase NS3rdquo Proceedings of the National Academy of Sciencesof the United States of America vol 90 no 13 pp 6218ndash62221993

[83] I C Lorenz S L Allison F X Heinz andA Helenius ldquoFoldingand dimerization of tick-borne encephalitis virus envelopeproteins prM and E in the endoplasmic reticulumrdquo Journal ofVirology vol 76 no 11 pp 5480ndash5491 2002

[84] I A Zybert H van der Ende-Metselaar J Wilschut and JM Smit ldquoFunctional importance of dengue virus maturationinfectious properties of immature virionsrdquo Journal of GeneralVirology vol 89 no 12 pp 3047ndash3051 2008

[85] P Plevka A J Battisti J Junjhon et al ldquoMaturation offlaviviruses starts from one or more icosahedrally independentnucleation centresrdquo EMBO Reports vol 12 pp 602ndash606 2011

[86] I A Rodenhuis-Zybert J Wilschut and J M Smit ldquoPartialmaturation an immune-evasion strategy of dengue virusrdquoTrends in Microbiology vol 19 no 5 pp 248ndash254 2011

[87] A T Lehrer and M R Holbrook ldquoTick-borne encephalitisrdquo inVaccines for Biodefense and Emerging and Neglected Diseases AD T Barrett and L R Stanberry Eds vol 1 Academic PressBurlington Mass USA 1st edition 2009

[88] F X Heinz C W Mandl H Holzmann et al ldquoThe flavivirusenvelope protein E isolation of a soluble form from tick-borneencephalitis virus and its crystallizationrdquo Journal of Virologyvol 65 no 10 pp 5579ndash5583 1991

[89] F A Rey ldquoDengue virus envelope glycoprotein structure newinsight into its interactions during viral entryrdquoProceedings of theNational Academy of Sciences of theUnited States of America vol100 no 12 pp 6899ndash6901 2003

[90] L Li S-M Lok I-M Yu et al ldquoThe flavivirus precursor mem-brane-envelope protein complex structure and maturationrdquoScience vol 319 no 5871 pp 1830ndash1834 2008

[91] V C Luca J AbiMansour C A Nelson and D H FremontldquoCrystal structure of the Japanese encephalitis virus envelopeproteinrdquo Journal of Virology vol 86 no 4 pp 2337ndash2346 2012

[92] Y Modis S Ogata D Clements and S C Harrison ldquoStructureof the dengue virus envelope protein after membrane fusionrdquoNature vol 427 no 6972 pp 313ndash319 2004

[93] Y Modis S Ogata D Clements and S C Harrison ldquoA ligand-binding pocket in the dengue virus envelope glycoproteinrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 100 no 12 pp 6986ndash6991 2003

[94] R J Kuhn W Zhang M G Rossmann et al ldquoStructure ofdengue virus implications for flavivirus organization matura-tion and fusionrdquo Cell vol 108 no 5 pp 717ndash725 2002

[95] F A Rey F X Heinz C Mandl C Kunz and S C HarrisonldquoThe envelope glycoprotein from tick-borne encephalitis virusat 2 A resolutionrdquo Nature vol 375 no 6529 pp 291ndash298 1995

[96] G E Nybakken C A Nelson B R Chen M S Diamondand D H Fremont ldquoCrystal structure of the West Nile virusenvelope glycoproteinrdquo Journal of Virology vol 80 no 23 pp11467ndash11474 2006

[97] P A Bullough F M Hughson J J Skehel and D C WileyldquoStructure of influenza haemagglutinin at the pH of membranefusionrdquo Nature vol 371 no 6492 pp 37ndash43 1994

[98] M Kielian ldquoStructural surprises from the flaviviruses andalphavirusesrdquoMolecular Cell vol 9 no 3 pp 454ndash456 2002

[99] M Kielian and S Jungerwirth ldquoMechanisms of enveloped virusentry into cellsrdquoMolecular Biology andMedicine vol 7 no 1 pp17ndash31 1990

[100] J Dubochet ldquoCryo-EMmdashthe first thirty yearsrdquo Journal ofMicroscopy vol 245 no 3 pp 221ndash224 2012

[101] R H Vogel and S W Provencher ldquoThree-dimensional recon-struction from electron micrographs of disordered specimensII Implementation and resultsrdquo Ultramicroscopy vol 25 no 3pp 223ndash239 1988

[102] R H Vogel S W Provencher C-H von Bonsdorff M Adrianand J Dubochet ldquoEnvelope structure of Semliki Forest virusreconstructed from cryo-electron micrographsrdquo Nature vol320 no 6062 pp 533ndash535 1986

[103] S D Fuller ldquoThe T=4 envelope of sindbis virus is organized byinteractionswith a complementary T=3 capsidrdquoCell vol 48 no6 pp 923ndash934 1987

[104] A M Paredes D T Brown R Rothnagel et al ldquoThree-dimensional structure of a membrane-containing virusrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 90 no 19 pp 9095ndash9099 1993

[105] M J Schlesinger S Schlesinger and BW Burge ldquoIdentificationof a second glycoprotein in Sindbis virusrdquo Virology vol 47 no2 pp 539ndash541 1972

[106] R P Anthony and D T Brown ldquoProtein-protein interactions inan alphavirus membranerdquo Journal of Virology vol 65 no 3 pp1187ndash1194 1991

[107] A M Paredes M N Simon and D T Brown ldquoThe mass of theSindbis virus nucleocapsid suggests it has 119879 = 4 icosahedralsymmetryrdquo Virology vol 187 no 1 pp 329ndash332 1992

[108] R P Anthony AM Paredes andD T Brown ldquoDisulfide bondsare essential for the stability of the Sindbis virus enveloperdquoJournal of Virology vol 190 no 1 pp 330ndash336 1992

[109] R H Cheng R J Kuhn N H Olson et al ldquoNucleocapsid andglycoprotein organization in an enveloped virusrdquo Cell vol 80no 4 pp 621ndash630 1995

[110] T J Smith R H Cheng N H Olson et al ldquoPutative receptorbinding sites on alphaviruses as visualized by cryoelectronmicroscopyrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 92 no 23 pp 10648ndash106521995

[111] A M Paredes H Heidner P Thuman-Commike B V VPrasad R E Johnston and W Chiu ldquoStructural localizationof the E3 glycoprotein in attenuated Sindbis virus mutantsrdquoJournal of Virology vol 72 no 2 pp 1534ndash1541 1998

[112] S V Pletnev W Zhang S Mukhopadhyay et al ldquoLocationsof carbohydrate sites on alphavirus glycoproteins show that e1forms an icosahedral scaffoldrdquo Cell vol 105 no 1 pp 127ndash1362001

[113] D C FlynnW J Meyer J MMackenzie Jr and R E JohnstonldquoA conformational change in Sindbis virus glycoproteins E1 andE2 is detected at the plasma membrane as a consequence ofearly virus-cell interactionrdquo Journal of Virology vol 64 no 8pp 3643ndash3653 1990


[114] W J Meyer S Gidwitz V K Ayers R J Schoepp and R EJohnston ldquoConformational alteration of sindbis virion glyco-proteins induced by heat reducing agents or low pHrdquo Journalof Virology vol 66 no 6 pp 3504ndash3513 1992

[115] O Carugo and S Pongor ldquoRecent progress in protein 3D struc-ture comparisonrdquoCurrent Protein and Peptide Science vol 3 no4 pp 441ndash449 2002

[116] Y Zhu B Carragher R M Glaeser et al ldquoAutomatic particleselection results of a comparative studyrdquo Journal of StructuralBiology vol 145 no 1-2 pp 3ndash14 2004

[117] M C Scott C-C Chen M Mecklenburg et al ldquoElectrontomography at 24-angstrom resolutionrdquo Nature vol 483 no7390 pp 444ndash447 2012

[118] E V Orlova andH R Saibil ldquoStructural analysis ofmacromole-cular assemblies by electron microscopyrdquo Chemical Reviewsvol 111 no 12 pp 7710ndash7748 2011

[119] E V Orlova and H R Saibil ldquoStructure determination ofmacromolecular assemblies by single-particle analysis of cryo-electron micrographsrdquo Current Opinion in Structural Biologyvol 14 no 5 pp 584ndash590 2004

[120] B W Burge and E R Pfefferkorn ldquoIsolation and characteriza-tion of conditional-lethal mutants of Sindbis virusrdquo Journal ofVirology vol 30 no 2 pp 204ndash213 1966

[121] J Edwards E Mann and D T Brown ldquoConformationalchanges in Sindbis virus envelope proteins accompanyingexposure to low pHrdquo Journal of Virology vol 45 no 3 pp 1090ndash1097 1983

[122] A M Paredes D Ferreira M Horton et al ldquoConformationalchanges in Sindbis virions resulting from exposure to low pHand interactions with cells suggest that cell penetration mayoccur at the cell surface in the absence of membrane fusionrdquoJournal of Virology vol 324 no 2 pp 373ndash386 2004

[123] J Edwards and D T Brown ldquoSindbis virus-mediated cell fusionfrom without is a two-step eventrdquo Journal of General Virologyvol 67 no 2 pp 377ndash380 1986

[124] L Wessels M W Elting D Scimeca and K Weninger ldquoRapidmembrane fusion of individual virus particles with supportedlipid bilayersrdquo Biophysical Journal vol 93 no 2 pp 526ndash5382007

[125] L Haag H Garoff L Xing L Hammar S-T Kan and R HCheng ldquoAcid-induced movements in the glycoprotein shell ofan alphavirus turn the spikes into membrane fusion moderdquoEMBO Journal vol 21 no 17 pp 4402ndash4410 2002

[126] A Koschinski G Wengler and H Repp ldquoThe membrane pro-teins of flaviviruses form ion-permeable pores in the targetmembrane after fusion identification of the pores and analysisof their possible role in virus infectionrdquo Journal of GeneralVirology vol 84 no 7 pp 1711ndash1721 2003

[127] S Cao andW Zhang ldquoCharacterization of an early-stage fusionintermediate of Sindbis virus using cryoelectron microscopyrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 110 no 33 pp 13362ndash13367 2013

[128] A Salminen J M Wahlberg M Lobigs P Liljestrom and HGaroff ldquoMembrane fusion process of Semliki Forest virus IIcleavage-dependent reorganization of the spike protein com-plex controls virus entryrdquo The Journal of Cell Biology vol 116no 2 pp 349ndash357 1992

[129] J White J Kartenbeck and A Helenius ldquoFusion of Semlikiforest virus with the plasma membrane can be induced by lowpHrdquoThe Journal of Cell Biology vol 87 no 1 pp 264ndash272 1980

[130] S W Gollins and J S Porterfield ldquopH-dependent fusionbetween the flavivirus West Nile and liposomal model mem-branesrdquo Journal of General Virology vol 67 part 1 pp 157ndash1661986

[131] G Wang R Hernandez K Weninger and D T Brown ldquoInfec-tion of cells by Sindbis virus at low temperaturerdquo Virology vol362 no 2 pp 461ndash467 2007

[132] B Brandenburg L Y Lee M Lakadamyali M J Rust XZhuang and JMHogle ldquoImaging poliovirus entry in live cellsrdquoPLoS Biology vol 5 article e183 2007

[133] R Vancini G Wang D Ferreira R Hernandez and D TBrown ldquoAlphavirus genome delivery occurs directly at theplasma membrane in a time- and temperature-dependentprocessrdquo The Journal of Virology vol 87 no 8 pp 4352ndash43592013

[134] J M Smit R Bittman and J Wilschut ldquoLow-pH-dependentfusion of Sindbis virus with receptor-free cholesterol- andsphingolipid-containing liposomesrdquo The Journal of Virologyvol 73 no 10 pp 8476ndash8484 1999

[135] M Kielian and F A Rey ldquoVirus membrane-fusion proteinsmore than one way to make a hairpinrdquoNature Reviews Microbi-ology vol 4 no 1 pp 67ndash76 2006

[136] A Helenius J Kartenbeck K Simons and E Fries ldquoOn theentry of Semliki Forest virus into BHK-21 cellsrdquo The Journal ofCell Biology vol 84 no 2 pp 404ndash420 1980

[137] J MWahlberg R Bron JWilschut andH Garoff ldquoMembranefusion of Semliki Forest virus involves homotrimers of thefusion proteinrdquo Journal of Virology vol 66 no 12 pp 7309ndash7318 1992

[138] J M Wahlberg and H Garoff ldquoMembrane fusion process ofSemliki Forest virus I low pH-induced rearrangement in spikeprotein quaternary structure precedes virus penetration intocellsrdquo The Journal of Cell Biology vol 116 no 2 pp 339ndash3481992

[139] J Lescar A RousselMWWien et al ldquoThe fusion glycoproteinshell of Semliki Forest virus an icosahedral assembly primed forfusogenic activation at endosomal pHrdquo Cell vol 105 no 1 pp137ndash148 2001

[140] S W Metz C Geertsema B E Martina et al ldquoFunctionalprocessing and secretion of Chikungunya virus E1 and E2glycoproteins in insect cellsrdquoVirology Journal vol 8 article 3532011

[141] G Roman-Sosa and M Kielian ldquoThe interaction of alphavirusE1 protein with exogenous domain III defines stages in virus-membrane fusionrdquo Journal of Virology vol 85 no 23 pp 12271ndash12279 2011

[142] K Stiasny S L Allison J Schalich and F X Heinz ldquoMembraneinteractions of the tick-borne encephalitis virus fusion proteinE at low pHrdquo Journal of Virology vol 76 no 8 pp 3784ndash37902002

[143] A Roussel J Lescar M-C Vaney G Wengler and F A ReyldquoStructure and interactions at the viral surface of the envelopeprotein E1 of semliki forest virusrdquo Structure vol 14 no 1 pp75ndash86 2006

[144] C Sanchez-San Martın H Sosa and M Kielian ldquoA stableprefusion intermediate of the alphavirus fusion protein revealscritical features of class II membrane fusionrdquo Cell Host ampMicrobe vol 4 no 6 pp 600ndash608 2008

[145] D L Gibbons M-C Vaney A Roussel et al ldquoConformationalchange and protein-protein interactions of the fusion proteinof semliki forest virusrdquo Nature vol 427 no 6972 pp 320ndash3252004


[146] J White and A Helenius ldquopH-Dependent fusion between theSemliki Forest virus membrane and liposomesrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 77 no 6 pp 3273ndash3277 1980

[147] R Bron J MWahlberg H Garoff and JWilschut ldquoMembranefusion of semliki forest virus in a model system correlationbetween fusion kinetics and structural changes in the envelopeglycoproteinrdquo EMBO Journal vol 12 no 2 pp 693ndash701 1993

[148] A Luukkonen M Brummer-Korvenkontio and O RenkonenldquoLipids of cultured mosquito cells (Aedes albopictus) com-parison with cultured mammalian fibroblasts (BHK 21 cells)rdquoBiochimica et Biophysica Acta vol 326 no 2 pp 256ndash261 1973

[149] A Hafer R Whittlesey D T Brown and R Hernandez ldquoDif-ferential incorporation of cholesterol by sindbis virus grown inmammalian or insect cellsrdquo Journal of Virology vol 83 no 18pp 9113ndash9121 2009

[150] J M Smit G Li P Schoen et al ldquoFusion of alphaviruses withliposomes is a non-leaky processrdquo FEBS Letters vol 521 no 1ndash3pp 62ndash66 2002

[151] V Madan M A Sanz and L Carrasco ldquoRequirement of thevesicular system for membrane permeabilization by Sindbisvirusrdquo Virology vol 332 no 1 pp 307ndash315 2005

[152] J Edwards and D T Brown ldquoSindbis virus induced fusionof tissue cultured Aedes albopictus (mosquito) cellsrdquo VirusResearch vol 1 no 8 pp 703ndash711 1984

[153] D P Fan and B M Sefton ldquoThe entry into host cells of Sindbisvirus vesicular stomatitis virus and Sendai virusrdquo Cell vol 15no 3 pp 985ndash992 1978

[154] D J Schibli and W Weissenhorn ldquoClass I and class II viralfusion protein structures reveal similar principles in membranefusionrdquoMolecular Membrane Biology vol 21 no 6 pp 361ndash3712004

[155] M Kielian ldquoClass II virus membrane fusion proteinsrdquo Journalof Virology vol 344 no 1 pp 38ndash47 2006

[156] D Moradpour and F Penin ldquoHepatitis C virus proteins fromstructure to functionrdquo Current Topics in Microbiology andImmunology vol 369 pp 113ndash142 2013

[157] K El Omari O Iourin K Harlos J M Grimes and D I StuartldquoStructure of a pestivirus envelope glycoprotein E2 clarifies itsrole in cell entryrdquo Cell Reports vol 3 no 1 pp 30ndash35 2013

[158] T Krey H-J Thiel and T Rumenapf ldquoAcid-resistant bovinepestivirus requires activation for pH-triggered fusion duringentryrdquo Journal of Virology vol 79 no 7 pp 4191ndash4200 2005

[159] M Rusu R Bonneau M R Holbrook et al ldquoAn assemblymodel of Rift Valley fever virusrdquo Frontiers in Microbiology vol3 article 254 2012

[160] PA Bullough FMHughsonAC Treharne RWHRuigrokJ J Skehel and D CWiley ldquoCrystals of a fragment of influenzahaemagglutinin in the low pH induced conformationrdquo Journalof Molecular Biology vol 236 no 4 pp 1262ndash1265 1994

[161] X Han J H Bushweller D S Cafiso and L K Tamm ldquoMem-brane structure and fusion-triggering conformational changeof the fusion domain from influenza hemagglutininrdquo NatureStructural Biology vol 8 no 8 pp 715ndash720 2001

[162] J J Skehel and D C Wiley ldquoReceptor binding and membranefusion in virus entry the influenza hemagglutininrdquo AnnualReview of Biochemistry vol 69 pp 531ndash569 2000

[163] J Lippincott-Schwartz T H Roberts and K HirschbergldquoSecretory protein trafficking and organelle dynamics in livingcellsrdquo Annual Review of Cell and Developmental Biology vol 16pp 557ndash589 2000

[164] R Hernandez C Sinodis M Horton D Ferreira C Yang andD T Brown ldquoDeletions in the transmembrane domain of asindbis virus glycoprotein alter virus infectivity stability andhost rangerdquo The Journal of Virology vol 77 no 23 pp 12710ndash12719 2003

[165] J P Kononchik R Hernandez and D T Brown ldquoAn alternativepathway for alphavirus entryrdquo Virology Journal vol 8 article304 2011

[166] D T Brown and R Hernandez ldquoInfection of cells by alpha-virusesrdquo Advances in Experimental Medicine and Biology vol726 pp 181ndash199 2012

[167] M A Sanz L Perez and L Carrasco ldquoSemliki forest virus6K protein modifies membrane permeability after inducibleexpression in Escherichia coli cellsrdquo The Journal of BiologicalChemistry vol 269 no 16 pp 12106ndash12110 1994

[168] A Munoz J L Castrillo and L Carrasco ldquoModification ofmembrane permeability during Semliki Forest virus infectionrdquoVirology vol 146 no 2 pp 203ndash212 1985

[169] H J Risselada G Bubnis and H Grubmuller ldquoExpansion ofthe fusion stalk and its implication for biological membranefusionrdquo Proceedings of the National Academy of Sciences of theUnited States of America 2014

[170] R Guinea and L Carrasco ldquoConcanamycin A a powerfulinhibitor of enveloped animal-virus entry into cellsrdquo Biochem-ical and Biophysical Research Communications vol 201 no 3pp 1270ndash1278 1994

[171] R Guinea and L Carrasco ldquoRequirement for vacuolar proton-ATPase activity during entry of influenza virus into cellsrdquo TheJournal of Virology vol 69 no 4 pp 2306ndash2312 1995

[172] W T Heller and K C Littrell ldquoSmall-angle neutron scatteringfor molecular biology basics and instrumentationrdquoMethods inMolecular Biology vol 544 pp 293ndash305 2009

[173] G Zaccaı and B Jacrot ldquoSmall angle neutron scatteringrdquoAnnual Review of Biophysics and Bioengineering vol 12 pp 139ndash157 1983

[174] W THeller ldquoSmall-angle neutron scattering and contrast varia-tion a powerful combination for studying biological struc-turesrdquo Acta Crystallographica D Biological Crystallography vol66 no 11 pp 1213ndash1217 2010

[175] G Fibriansah T-S Ng V A Kostyuchenko et al ldquoStructuralchanges in dengue virus when exposed to a temperature of37∘Crdquo Journal of Virology vol 87 no 13 pp 7585ndash7592 2013

[176] Y ZhangW Zhang S Ogata et al ldquoConformational changes ofthe flavivirus E glycoproteinrdquo Structure vol 12 no 9 pp 1607ndash1618 2004

[177] H M van der Schaar M J Rust B-L Waarts et al ldquoChar-acterization of the early events in dengue virus cell entry bybiochemical assays and single-virus trackingrdquo Journal of Viro-logy vol 81 no 21 pp 12019ndash12028 2007

[178] X Zhang J Sheng P Plevka R J Kuhn M S Diamond andM G Rossmann ldquoDengue structure differs at the temperaturesof its human and mosquito hostsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 110 no17 pp 6795ndash6799 2013

[179] R Hernandez A Paredes and D T Brown ldquoSindbis virusconformational changes induced by a neutralizing anti-E1monoclonal antibodyrdquo Journal of Virology vol 82 no 12 pp5750ndash5760 2008

[180] R Hernandez and A Paredes ldquoSindbis virus as a modelfor studies of conformational changes in a metastable virusand the role of conformational changes in in vitro antibody


neutralisationrdquo Reviews in Medical Virology vol 19 no 5 pp257ndash272 2009

[181] S-M Lok V Kostyuchenko G ENybakken et al ldquoBinding of aneutralizing antibody to dengue virus alters the arrangement ofsurface glycoproteinsrdquoNature Structural andMolecular Biologyvol 15 no 3 pp 312ndash317 2008

[182] S Cook G Moureau R E Harbach et al ldquoIsolation of a novelspecies of flavivirus and a new strain of Culex flavivirus (Flavi-viridae) from a natural mosquito population in UgandardquoJournal of General Virology vol 90 no 11 pp 2669ndash2678 2009

[183] M Calzolari L Ze-Ze D Ruzek et al ldquoDetection of mosquito-only flaviviruses in Europerdquo Journal of General Virology vol 93pp 1215ndash1225 2012

[184] V A Kostyuchenko Q Zhang J L Tan T-S Ng and S-MLok ldquoImmature and mature dengue serotype 1 virus structuresprovide insight into thematuration processrdquo Journal of Virologyvol 87 no 13 pp 7700ndash7707 2013

[185] V A Kostyuchenko P L Chew T S Ng and S M Lok ldquoNear-atomic resolution cryo-electron microscopic structure of den-gue serotype 4 virusrdquo Journal of Virology vol 88 no 1 pp 477ndash482 2014

[186] J A G Briggs ldquoStructural biology in situ-the potential of subto-mogram averagingrdquo Current Opinion in Structural Biology vol23 no 2 pp 261ndash267 2013

[187] T A M Bharat J D Riches L Kolesnikova et al ldquoCryo-elec-tron tomography of marburg virus particles and their morpho-genesis within infected cellsrdquo PLoS Biology vol 9 no 11 ArticleID e1001196 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


the glycoprotein E1 E and possibly Gc respectively appearto have arisen from an ancient predecessor [3] While thesequences of the E1 cognate glycoproteins have diverged inthe Togaviridae Flaviviridae and Bunyaviridae the functionand structures of these viruses have been retained [18] Evolu-tion of the protein structure has been constrained by adaptingto both arthropod and vertebrate hostsThis difference in therates of genomic divergence has been seen in a nonarbovirusmember of the Togavirus family Rubivirus [19] in which theknown structure of E1 appears to have diverged relative tothe arbovirus members of this family [20] This observationsuggests that the consensus sequence of the arboviral genomeis maintained by eliminating genetic drift which impactsfitness in each host In otherwords the virus sequence evolvesmore slowly when divergent hosts are continuously selectingfor virus fitness Collectively the available information sug-gests that the mosquito-borne viruses acquired the form wenow see from the arthropod vectors and did so concurrentwith becoming hematophagous presumably to optimize eggmaturation [21]

12 Arbovirus Structure Of the seven families of arbovirusesthree (Togaviridae Flaviviridae and Bunyaviridae) are icosa-hedral membrane-containing plus-stranded RNA virusesIt is interesting that though the respective glycoproteinsE1 E and Gc of the alpha flavi and bunyaviruses encodethe same basic protein fold these proteins assemble intodifferent icosahedral structures Alphaviruses are 119879 = 4(Figure 1) [22] flaviviruses are 119879 = 3 (Figure 3) [23] andbunyaviruses (Phlebovirus) are 119879 = 12 [24 25] This capabi-lity of viruses to assemble an inherently similar global foldinto their different structures is dependent on the specificfunctions of their structural proteins Rhabdoviruses areenveloped but assume a rod or bullet shaped structure thatdemonstrates some helical symmetry [26] Reoviruses (119879 =13 l laevorotatory turning toward the left) are icosahedralbut do not contain a membrane [27] Orthomyxovirusesare enveloped viruses that display pleomorphic structuresranging from spherical such as influenza A to filamentous asin influenza C [28] In those influenza structures assuminga spherical shape that shape is not due to icosahedral sym-metry In addition orthomyxoviruses have more lipid-mem-brane relative to their envelopes than do enveloped icosahe-dral viruses resulting in the pleomorphism displayed [29]The Asfarviridae viruses are large double-stranded DNAviruses spherical to pleomorphic and 175ndash215 nm in diam-eter and exhibit icosahedral symmetry (119879 = 189ndash217) Theseviruses are composed of two icosahedral protein shells thatcontain an intervening lipid structure [30] These structuraldifferences illustrate that there is no common virus structureadopted by the arboviruses which by itself would explainhow these viruses infect both arthropods and vertebratesThere are two systems of virus classification currently in useWhile morphology is a useful basis for virus identificationand classification at present two classification systems existThe hierarchical virus classification system the Internationalcommittee on taxonomy of viruses (ICTV) [31] and theBaltimore classification system The Baltimore classificationsystem is based on nucleic acid type which places viruses

into seven groups in hierarchical classification [32]The ICTVuses evolutionary relationships as the classification scheme[33] ICTVclassification places viruses by phenotypemorph-ology protein composition and genotype nucleic acid typesequence mode of replication host organisms infected andthe type of disease they cause Capsid structure has also beenutilized to organize virus groups based on the hypothesis thatonly a limited number of protein folds are available to self-assemble a nucleocapsid [24 34] This review will be limitedto the group arboviral members of the alphaviruses and flavi-viruses for which considerable structural information existsBunyaviruses are less well studied and are proposed to befunctionally analogous to flaviviruses by computational pro-teomic and indirect biochemical analysis [35] Studies onwhole virus particles will be the focus of this discussion

13 Alphaviruses and Flavivirus Virion and Genome StructureThe alphaviruses are a genus in the family Togaviridae Theyare icosahedral viruses of 119879 = 4 geometry and containa +polarity single-stranded RNA genome The genome isorganized with the 4 nonstructural genes found at the 51015840end of the capped RNA genome followed by the structuralproteins capsid (C) PE2 6K and E1 (Figure 2 [Virus DBVBRC genome browser accession VG0000908]) The virus iscomposed of the structural proteins C E2 and E1 that aresynthesized from subgenomic RNA made from an internalpromoter [36] This RNA is then translated as a polyproteinand processed by viral and host enzymes during maturationand comprises the virion The glycoproteins are assembledwithin the ER The 51015840 capsid protein is autoproteolyticallyprocessed from the proprotein and organizes the genomicRNA into a nucleocapsid During virus maturation PE2 isconverted to E3 and E2 by furin [37] In most cases E3does not remain associated with the virus [38] E1 and E2form trimers of heterodimers that envelope the nucleocapsidwhich assembles independently [39] By regulating RNA andprotein synthesis in a temporal manner the Sindbis virusis able to quickly replicate to as many as 106 particlescell[40] Alphaviruses bud from the cell surface in mammaliancells but are assembled in vacuoles in mosquito cells andmature via the exocytic pathway [41] These viruses arehybrid in nature acquiring lipids carbohydrates and othermodifications from the host cell while their proteins arevirus encoded The final assembled structure is that of twonested spheres separated by an interveningmembrane bilayerheld together by protein associations between the E2 proteinendodomain and the capsid protein

Flaviviruses are in the family Flaviviridae and are alsomembrane-containing viruses but they assemble into pseudo119879 = 3 icosahedra The genome is organized with the struc-tural proteins at the 51015840 end of the +strand RNA moleculeand is translated into a single polypeptide which is processedduring maturation by viral and host-encoded enzymes intomultifunctional proteins The three structural and sevennonstructural proteins are cleaved by a series of virus andhost-encoded proteases The structural proteins starting atthe 51015840 end of themonocistronic genome include C preM andE (Figure 4 [Virus DB VBRC genome browser]) As with thealphaviruses the flavivirus polyproteins are inserted into the


100 A

(a) (b)



Stopcodon

cEarly

P123

P123 RdRp nsP4

Late

Late

CP E3 E2 6k E1

Subgenomic RNA



DLP



3998400

3998400

5998400

5998400 c




2 Review



(a)

(b)


5998400





3998400-OH

Signal peptidase

Golgi protease

NS3 protease






1

2

3

4

5

k

h

1 4 9 16 25

1 2 3

3 7 13 21

4 5


2

T







(a) (b)

(c) (d) (e) (f) (g) (h)








(a) (b)


(A) (B)



















350

290

230

(A)

(a) (b) (c)

350

225

100

(A)

(d) (e) (f)

(A)202

160

(g) (h) (i)






(a)

(b) (c) (d)

(e) (f) (g)
















100

90

80

70

60

50

40

30

20

10

04 15 24 37


EmptyFull

Full

empt

y pa

rtic

les (

o

f tot

al)

(a)

1000

900

800

700

600

500

400

300

200

100

0015 30 45 60

Time (min)

EmptyFull

Full

empt

y pa

rtic

les (

o

f tot

al)

(b)






(a)

(b)


(a) (b) (c)



Surfa

ceC

entr

al cr

oss-

sect

ion


IClass II III IV

30 A130 A180 A

240 A260 A280 A


















Disclaimer






Acknowledgments


References










































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


100 A

(a) (b)



Stopcodon

cEarly

P123

P123 RdRp nsP4

Late

Late

CP E3 E2 6k E1

Subgenomic RNA



DLP



3998400

3998400

5998400

5998400 c




2 Review



(a)

(b)


5998400





3998400-OH

Signal peptidase

Golgi protease

NS3 protease






1

2

3

4

5

k

h

1 4 9 16 25

1 2 3

3 7 13 21

4 5


2

T







(a) (b)

(c) (d) (e) (f) (g) (h)








(a) (b)


(A) (B)



















350

290

230

(A)

(a) (b) (c)

350

225

100

(A)

(d) (e) (f)

(A)202

160

(g) (h) (i)






(a)

(b) (c) (d)

(e) (f) (g)
















100

90

80

70

60

50

40

30

20

10

04 15 24 37


EmptyFull

Full

empt

y pa

rtic

les (

o

f tot

al)

(a)

1000

900

800

700

600

500

400

300

200

100

0015 30 45 60

Time (min)

EmptyFull

Full

empt

y pa

rtic

les (

o

f tot

al)

(b)






(a)

(b)


(a) (b) (c)



Surfa

ceC

entr

al cr

oss-

sect

ion


IClass II III IV

30 A130 A180 A

240 A260 A280 A


















Disclaimer






Acknowledgments


References










































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a)

(b)


5998400





3998400-OH

Signal peptidase

Golgi protease

NS3 protease






1

2

3

4

5

k

h

1 4 9 16 25

1 2 3

3 7 13 21

4 5


2

T







(a) (b)

(c) (d) (e) (f) (g) (h)








(a) (b)


(A) (B)



















350

290

230

(A)

(a) (b) (c)

350

225

100

(A)

(d) (e) (f)

(A)202

160

(g) (h) (i)






(a)

(b) (c) (d)

(e) (f) (g)
















100

90

80

70

60

50

40

30

20

10

04 15 24 37


EmptyFull

Full

empt

y pa

rtic

les (

o

f tot

al)

(a)

1000

900

800

700

600

500

400

300

200

100

0015 30 45 60

Time (min)

EmptyFull

Full

empt

y pa

rtic

les (

o

f tot

al)

(b)






(a)

(b)


(a) (b) (c)



Surfa

ceC

entr

al cr

oss-

sect

ion


IClass II III IV

30 A130 A180 A

240 A260 A280 A


















Disclaimer






Acknowledgments


References










































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


1

2

3

4

5

k

h

1 4 9 16 25

1 2 3

3 7 13 21

4 5


2

T







(a) (b)

(c) (d) (e) (f) (g) (h)








(a) (b)


(A) (B)



















350

290

230

(A)

(a) (b) (c)

350

225

100

(A)

(d) (e) (f)

(A)202

160

(g) (h) (i)






(a)

(b) (c) (d)

(e) (f) (g)
















100

90

80

70

60

50

40

30

20

10

04 15 24 37


EmptyFull

Full

empt

y pa

rtic

les (

o

f tot

al)

(a)

1000

900

800

700

600

500

400

300

200

100

0015 30 45 60

Time (min)

EmptyFull

Full

empt

y pa

rtic

les (

o

f tot

al)

(b)






(a)

(b)


(a) (b) (c)



Surfa

ceC

entr

al cr

oss-

sect

ion


IClass II III IV

30 A130 A180 A

240 A260 A280 A


















Disclaimer






Acknowledgments


References










































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b)

(c) (d) (e) (f) (g) (h)








(a) (b)


(A) (B)



















350

290

230

(A)

(a) (b) (c)

350

225

100

(A)

(d) (e) (f)

(A)202

160

(g) (h) (i)






(a)

(b) (c) (d)

(e) (f) (g)
















100

90

80

70

60

50

40

30

20

10

04 15 24 37


EmptyFull

Full

empt

y pa

rtic

les (

o

f tot

al)

(a)

1000

900

800

700

600

500

400

300

200

100

0015 30 45 60

Time (min)

EmptyFull

Full

empt

y pa

rtic

les (

o

f tot

al)

(b)






(a)

(b)


(a) (b) (c)



Surfa

ceC

entr

al cr

oss-

sect

ion


IClass II III IV

30 A130 A180 A

240 A260 A280 A


















Disclaimer






Acknowledgments


References










































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




(a) (b)


(A) (B)



















350

290

230

(A)

(a) (b) (c)

350

225

100

(A)

(d) (e) (f)

(A)202

160

(g) (h) (i)






(a)

(b) (c) (d)

(e) (f) (g)
















100

90

80

70

60

50

40

30

20

10

04 15 24 37


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Full

empt

y pa

rtic

les (

o

f tot

al)

(a)

1000

900

800

700

600

500

400

300

200

100

0015 30 45 60

Time (min)

EmptyFull

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IClass II III IV

30 A130 A180 A

240 A260 A280 A


















Disclaimer






Acknowledgments


References










































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology














350

290

230

(A)

(a) (b) (c)

350

225

100

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160

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(b) (c) (d)

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100

90

80

70

60

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40

30

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04 15 24 37


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Full

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1000

900

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700

600

500

400

300

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IClass II III IV

30 A130 A180 A

240 A260 A280 A


















Disclaimer






Acknowledgments


References










































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a)

(b) (c) (d)

(e) (f) (g)
















100

90

80

70

60

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1000

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IClass II III IV

30 A130 A180 A

240 A260 A280 A


















Disclaimer






Acknowledgments


References










































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology













100

90

80

70

60

50

40

30

20

10

04 15 24 37


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Full

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IClass II III IV

30 A130 A180 A

240 A260 A280 A


















Disclaimer






Acknowledgments


References










































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a)

(b)


(a) (b) (c)



Surfa

ceC

entr

al cr

oss-

sect

ion


IClass II III IV

30 A130 A180 A

240 A260 A280 A


















Disclaimer






Acknowledgments


References










































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Surfa

ceC

entr

al cr

oss-

sect

ion


IClass II III IV

30 A130 A180 A

240 A260 A280 A


















Disclaimer






Acknowledgments


References










































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












Disclaimer






Acknowledgments


References










































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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