Structural studies of Chikungunya virus maturationMoh Lan Yapa,b, Thomas Klosea, Akane Urakamic, S. Saif Hasana, Wataru Akahatac, and Michael G. Rossmanna,1

aDepartment of Biological Sciences, Purdue University, West Lafayette, IN 47907; bDepartment of Biological Science, Faculty of Science, Universiti TunkuAbdul Rahman, 31900 Kampar, Perak, Malaysia; and cVLP Therapeutics, Gaithersburg, MD 20878

Edited by Robert M. Stroud, University of California, San Francisco, California, and approved November 10, 2017 (received for review July 25, 2017)

Cleavage of the alphavirus precursor glycoprotein p62 into theE2 and E3 glycoproteins before assembly with the nucleocapsid isthe key to producing fusion-competent mature spikes on alphavi-ruses. Here we present a cryo-EM, 6.8-Å resolution structure of an“immature” Chikungunya virus in which the cleavage site has beenmutated to inhibit proteolysis. The spikes in the immature virushave a larger radius and are less compact than in the mature virus.Furthermore, domains B on the E2 glycoproteins have less free-dom of movement in the immature virus, keeping the fusion loopsprotected under domain B. In addition, the nucleocapsid of theimmature virus is more compact than in the mature virus, protect-ing a conserved ribosome-binding site in the capsid protein fromexposure. These differences suggest that the posttranslationalprocessing of the spikes and nucleocapsid is necessary to produceinfectious virus.

alphavirus | Chikungunya virus | maturation | cryo-electron microscopy |conformational changes

Chikungunya virus (CHIKV) is a mosquito-borne virus, whichwas first reported in Tanzania in 1952 (1) and later emerged

as an epidemic in the French Reunion Island in 2005 (2). In thepast decade, CHIKV has spread to more than 40 countriesacross Africa, Asia, and Europe, causing over a million infectionsin the Americas alone since 2014 (3). Among the symptoms ofthe disease are rash, myalgia, high fever, and, typically, severearthritis (4).CHIKV is a member of the alphavirus genus in the Toga-

viridae family (5). Other closely related and well-studiedalphaviruses are Semliki Forest virus (SFV), Ross River virus(RRV), Sindbis virus (SINV), and Venezuelan Equine En-cephalitis virus (VEEV). Alphaviruses are spherical envelopedviruses with an ∼700-Å diameter and a T = 4 quasi-icosahedralsymmetry. The genome of alphaviruses is an ∼12-kb positive-sensed single-stranded RNA molecule encoding four non-structural proteins (nsP1–4), which are required for virus rep-lication, and five structural proteins (capsid protein C,glycoproteins E1, E2, E3, and 6K) (6). The structural proteinsare synthesized as a long polyprotein, which is then post-translationally cleaved into C, E1, 6K, and p62. A total of240 copies of the C protein associate with a newly synthesizedgenomic RNA molecule to form a nucleocapsid in the hostcell’s cytoplasm (7). The glycoproteins E1 and p62 interact toform heterodimers that subsequently trimerize into a viral spikein the endoplasmic reticulum (ER). The glycoprotein p62 isthen cleaved into E2 and E3 by cellular furin during its trans-portation from the acidic environment of the Golgi and earlyendosomes to the neutral pH environment of the cell surface,releasing E3 (Movie S1). Virus budding occurs at the cellmembrane where the nucleocapsid is enveloped by the glyco-proteins E1–E2 on the plasma lipid membrane. The protein 6Kfacilitates particle morphogenesis (8–10), but its position in theparticle remains to be verified.Alpha- and flaviviruses (11) have many similarities. Their

glycoprotein exteriors have icosahedral symmetry and surround alipid membrane that, in turn, surrounds their RNA genome,which is associated with the capsid protein. A major differencebetween alpha- (12) and flaviviruses (13) is the maturation

process. Flaviviruses are assembled as “immature” noninfectiousparticles in the ER of the host cell that are then proteolyticallymodified to produce infectious viruses on leaving the host cell.However, alphavirus components are proteolytically modifiedbefore assembly into mature viruses on the plasma membrane.In addition, a regular, icosahedral capsid shell is observed onlyin alphaviruses. During infection, a conserved sequence on theN-terminal regions of the capsid proteins binds to the host cell’s60S ribosomal subunits, initiating the dissociation of the nu-cleocapsid and the release of the RNA from the nucleocapsid(14). This ribosome-binding site (RBS) is buried during nu-cleocapsid assembly but is exposed at the end of the maturationprocess (15, 16).In alphaviruses, there are 20 trimeric spikes located on the

icosahedral threefold axes and another 60 trimeric spikes ingeneral positions that obey T = 4 quasi-symmetry (17–19).Glycoprotein E1 is involved in cell fusion (20), and glycoproteinE2 interacts with host receptors (21) whereas glycoproteinE3 facilitates E1-p62 heterodimerization and prevents the ex-posure of the E1 fusion loops from premature fusogenic acti-vation (22, 23). Cryo-EM studies have shown that E3 remainsassociated with the mature virus of SFV (24), RRV (18), andVEEV (25). However, SINV (26, 27) and CHIKV (28) releaseE3 after budding.

Significance

Chikungunya virus (CHIKV) belongs to the alphavirus family, themembers of which have enveloped icosahedral capsids. Thematuration process of alphaviruses involves proteolysis of someof the structural proteins before assembling with nucleocapsidsto produce mature virions. We mutated the proteolytic cleavagesite on E2 envelope protein, which is necessary in initiating thematuration process. Noninfectious virus-like particles (VLP)equivalent to “immature” fusion incompetent particles wereproduced to study the immature conformation of CHIKV. Wedescribe the 6.8-Å resolution electron microscopy structure of“immature” CHIK VLPs. Structural differences between the ma-ture and immature VLPs show that posttranslational processingof the envelope proteins and nucleocapsid is necessary to allowexposure of the fusion loop on glycoprotein E1 to produce aninfectious virus.

Author contributions: M.L.Y. and M.G.R. designed research; M.L.Y., T.K., A.U., and W.A.performed research; M.L.Y. and S.S.H. analyzed data; and M.L.Y. and M.G.R. wrotethe paper.

Conflict of interest statement: M.L.Y., T.K., S.S.H., and M.G.R. declare no competing fi-nancial interests. A.U. is an employee of VLP Therapeutics, and W.A. is an officer andshareholder of VLP Therapeutics.

This article is a PNAS Direct Submission.

Published under the PNAS license.

Data deposition: The final immature Chikungunya VLP electron density map was depos-ited in the Electron Microscopy Data Bank, https://www.emdatabank.org (accession codeEMD-8734), and structure coordinates have been deposited in the Protein Data Bank,www.rcsb.org/pdb (PDB ID code 5VU2).1To whom correspondence should be addressed. Email: mr@purdue.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1713166114/-/DCSupplemental.

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Here, we report the structure of immature CHIKV, which wasdetermined using virus-like particles (VLPs) with mutations atthe furin cleavage site on p62. The E3 remained associated withthe E2, mimicking the precursor p62 in its immature confor-mation. A crystal structure of the E1-p62 heterodimer [ProteinData Bank (PDB) ID code 3N40 (29)] was fitted into the cryo-EM electron density map of immature CHIKV VLPs to examinethe interactions of E1 and p62 with each other in the immaturevirus. A previous report showed that alphaviruses can be as-sembled in a partially mature, replication-competent state (25).Hence, the structure described here represents an intermediatestructure of CHIKV during the assembly and maturation pro-cess. We showed that there are significant conformational dif-ferences between the mature and immature viruses, including thenucleocapsid, the transmembrane helices, and the cellular at-tachment sites on E2. The presence of E3 in the immature virusstabilized domain B on E2, protecting the fusion peptide onE1 from becoming exposed and fusogenic.

Results and DiscussionCryo-EM Structure of Immature CHIKV.The cryo-EM density map ofimmature CHIK VLPs attained a 6.8-Å resolution (Fig. 1A). Thevirions had a diameter of 660 Å and, like mature virions, haveT = 4 icosahedral symmetry. Central cross-sections of the re-construction showed that the immature virion (Fig. 1C) has anucleocapsid, enveloped by a plasma membrane and an out-ermost layer of glycoproteins. Unlike flaviruses, alphaviruses,

including CHIKV, have a well-ordered icosahedral nucleocapsidwithin the membrane envelope (Fig. 1B).Immature CHIKV virions, like mature CHIKV virions, have

spike-like features (Fig. 2A) on their surface. Intraspike contactsare formed between the three E2 molecules that form a spike.The glycoprotein E1 wraps around E2 and contributes to inter-spike interactions. Furthermore, E3 is located at the periphery ofthe E2 molecules (Fig. 2A). The trimeric immature spikes, al-though organized with T = 4 quasi-symmetry, are similar tomature CHIKV spikes, but are less compact with a hole alongtheir threefold axes, resulting in a bigger spike radius. The spikesare more densely packed on the surface of immature CHIKV,resulting in smaller holes along the icosahedral twofold (i2) andicosahedral fivefold (i5) symmetry axes and smaller separationbetween the spikes, in comparison with mature CHIKV (Fig.2B). Thus, the spikes undergo a structural rearrangement duringmaturation.

Glycoprotein Spikes. As described in the crystal structure of E1-p62 (29), E1 has three beta-sheet–rich domains, namely domainsI, II, and III. A fusion loop is located at the tip of domain II.E2 consists of three Ig-like domains (A, B, and C) and a longbeta-ribbon (domain D) connecting domain B to C. Domain Dinteracts extensively with E3. The E1 fusion loop is sandwichedbetween domains A and B of E2.The crystal structure of E1-p62 (PDB ID code 3N40) (29) was

fitted into the cryo-EM electron density map of immature

Fig. 1. Cryo-EM reconstruction of immature CHIK virus-like particle. (A) Three-dimensional cryo-EM map of immature CHIKV, viewed down anicosahedral twofold axis. An icosahedral asymmetric unit is marked by a black triangle. Icosahedral symmetry elements are shown as black-filledpentagon, triangles, and ellipse. Four unique subunits in an asymmetric unit are shown in white numbers. (B) Internal capsid protein shell of theimmature CHIKV. (C ) Central cross-sections of the immature CHIKV viewed down an icosahedral twofold axis. Components of the virus are shown indifferent colors as indicated in the figure. (D) Enlarged view of the region outlined by the black rectangle in C. Fitting of an E1-p62-C structure isshown in the cryo-EM map of immature CHIKV.

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CHIKV (Fig. 1D) using the EMfit program (30). Unlike themature CHIKV, the average electron density of domain B inE2 is higher than in the immature CHIKV (Table 1). This im-plies that domain B is more rigid in immature CHIKV. Thisresult supports the hypothesis that domain B of E2 is stabilizedby the presence of E3, in agreement with a previous study (29).The rigid domain B of E2 protects the fusion loop on E1 fromexposure and therefore inhibits cell fusion. However, domain Aof E2 is more flexible in the immature than in the matureCHIKV, as indicated by the poorer electron density (Table 1).This might be because the spikes in the immature CHIKV areless compact. Domain A of E2, which is situated close to thespike center, has fewer contacts with the neighboring moleculesthan in the mature virus. This domain is more stable and exposedin the mature conformation, which might be beneficial for host-cell binding.

Glycoproteins Transmembrane Helices and Capsid Protein. Thechymotrypsin-like capsid protein of alphaviruses consists of ahydrophobic pocket between the two β-barrel domains (31). Therather basic N-terminal residues 1 to ∼110 of the capsid protein aredisordered in the crystal structures of SINV (31, 32) and SFV (33).However, in the cryo-EM maps of VEEV and SINV, the ordered

part of the capsid protein starts at about residue 109 (VEEV) (25)and residue 97 (SINV) (19), presumably due to interactions withthe genomic RNA. This ordered part has a helical structure inVEEV. In the virus, the positively charged region of the capsidproteins is associated with the negatively charged RNA. The ex-ternal glycoproteins and the internal capsid protein shell are as-sociated with the E1 and E2 cytoplasmic tail binding to the capsidprotein (34). This interaction has been shown to be important invirus budding and fusion (35).The model of the E1 and E2 transmembrane (TM) domains

and capsid protein as observed in mature CHIKV (28) was fittedinto the cryo-EM map of immature CHIKV (Fig. 1D). There area number of differences between the structure of immature andmature CHIKV. These include a small change in orientationand location of the TM helices (Fig. 3A and Fig. S1), resulting ina more compact nucleocapsid in the immature CHIKV (Fig.2B). The additionally ordered amino terminal region of thecapsid protein in the virus is a part of the RBS (Fig. 3B). Thisfragment consists of the residues 98–112 (KPGRRERMCMKIEND)of the capsid protein, which are the conserved RBS inalphaviruses (14).The life cycle of alphaviruses can be described as starting with

the mature virus in which the B domain of E2 is only looselyassociated with the underlying domain II of the E1 glycoprotein.Thus, after virus recognition of a cell, the virus is enclosed intothe low pH environment of an endosome. This causes the for-mation of trimeric fusogenic spikes resulting in the fusion of theviral and cellular membranes. As a result, the nucleocapsid isexposed to the cell’s cytoplasm containing ribosomes at low pH.The RBS on the icosahedral capsid of the mature virus is thenavailable to bind to ribosomes. The association of ribosomes withthe nucleocapsid causes the viral capsid to disintegrate (14)while guiding the genome to a ribosome. The replicated genomethen associates with newly synthesized capsid proteins to makenew nucleocapsids. These are transported to the plasma mem-brane where they will associate with mature E1–E2 to form newmature particles that bud out from the membrane. In somecases, the E3 glycoprotein, although cleaved from E2, will re-main on the particle.In the present case, the cleavage site has been mutated

resulting in no cleavage of p62 but producing the assembly ofimmature particles. The diameter of the nucleocapsids in theseimmature particles is about 20 Å smaller than in the matureparticles, making the particles more compact and less likely toexpose the RBS. When these immature particles bind to a cellsurface the (E1p62)3, trimeric spikes cannot fuse with the cellmembrane because the E3 glycoprotein stops the exposure of thefusion loop. Observations of the immature particles indicate thatfurin cleavage of glycoprotein spikes followed by their associa-tion with the preformed nucleocapsid is required to producefusion- and replication-competent particles.

Fig. 2. Structural characteristics of the immature CHIKV. (A) A trimeric spikeof the immature CHIKV. The E2 molecules (red) form interactions within aspike whereas the E1 molecules (yellow) wrap around E2 molecules and forminteractions between spikes. The E3 molecules (green) are located at theperiphery of the E2 molecules. (B) Central cross-section of the immature(Left) and mature (Right) CHIKV. The icosahedral symmetry axes are in-dicated by white arrows. Components of the viruses are shown in differentcolors as indicated in the figure. The immature virus has smaller holesaround the i2 and i5 symmetry axes compared to the mature virus. The di-ameter of the nucleocapsid in the immature virus is smaller than in themature virus.

Table 1. Average density height of the densities at the atomicpositions (sumf) on fitting of the atomic structure of CHIKVheterodimer E1-p62 into the immature cryo-EM density map

Protein Protein domain T = 4 fitting Independent molecule fitting

E1 I 14.2 15.3II 14.2 16.0III 15.6 16.0

E2 A 11.1 13.2B 10.0 11.9C 16.3 18.8D 13.9 14.1

E3 12.9 13.1Average 13.5 14.8

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Conclusion. During the maturation process of alphaviruses,cleavage of p62 into E2 and E3 exposes the fusion loop onE1 and arranges the glycoprotein spikes into a mature con-formation. Association of the mature spikes with the pre-assembled nucleocapsid expands the nucleocapsid to a lesscompact form. This step exposes the RBS on the capsid pro-tein, thus priming the nucleocapsid to be disassembled uponrelease into the host-cell cytoplasm during the next infectioncycle. The events described here for VLPs would correspondto the release of the genome into a host cell after virus entryand may be similar to the mechanism of genome release inmany other viruses.

Materials and MethodsProduction and Purification of Immature CHIK VLP. The coding sequence forthe CHIKV strain 37997 structural proteins, C-E3-E2-6K-E1, was synthesized bythe Blue Heron Company and cloned into a pUC119-derived vector under thecontrol of a human cytomegalovirus early immediate promoter. The ex-pression plasmid for the furin cleavage-resistant CHIKV VLP was generatedby mutating amino acids 61–64 in E3 to Ser-Gly-Gly-Gly-Gly-Ser, usingthe QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technol-ogies). The VLPs were produced in FreeStyle 293-F cells (Thermo Fisher

Scientific). The cells were transfected with VLP-expressing plasmids bypolyethylenimine reagent (Polysciences). Four days after transfection, thecell culture supernatant was harvested and clarified by centrifugation andfiltration through a 0.45-μm polyethersulfone (PES) membrane. The VLPssecreted in the culture supernatant were collected by using OptiPrepDensity Gradient Medium (Sigma-Aldrich), as described previously (36),and further purified by Hiprep 16/60 Sephacryl S-500 HR column (GEHealthcare Life Sciences). The eluates containing purified VLPs wereconcentrated by Amicon Ultra-15 centrifugal filter units (EMD Millipore)and filtered with a 0.20-μm PES membrane.

Electron Microscopy and 3D Reconstruction. Aliquots of a 2.5-μL sample at3 mg/mL concentration were loaded on glow-discharged C-Flat grids (CF-2/2–4C). These grids were blotted for 5 s and flash-frozen in liquid ethaneusing a Gatan CP3 plunge freezer. The grids were viewed using the FEITitan Krios electron microscope operated at 300 kV. Images were recordedwith a Gatan K2 Summit detector calibrated to have a magnification of38,461, yielding a pixel size of 0.65 Å. A total dose of 36 e−/Å2 and anexposure time of 7.6 s were used to collect 38 movie frames. Fully auto-mated data collection was implemented using Leginon (37). The MotionCorrsoftware (38) was used to correct the beam-induced motion. A total of5,325 images were collected, and 76,806 particles were boxed using theEMAN2 package (39). Contrast transfer function parameters were esti-mated using CTFFIND3 (40). The 2D classification was performed usingRELION (41), and the 3D reconstruction was performed using the JSPRsoftware (42). The final electron density map was reconstructed using72,944 particles and was estimated to have a resolution of 6.8 Å based onthe gold-standard Fourier shell correlation (FSC) criterion of 0.143 (43)(Fig. 4). The map was deposited in the Electron Microscopy Data Bank(www.emdatabank.org) with Electron Microscopy Data Bank accessioncode EMD-8734, and structure coordinates have been deposited with thePDB (PDB ID code 5VU2).

Data Analysis and Figure Preparation. The crystal structure of E1-p62 (PDB IDcode 3N40) and models of the E1 and E2 TM domains and capsid protein (PDBID code 3J2W) were fit into the cryo-EMmap using the EMfit program (30) tomaximize the average density height (sumf value) at all atomic positions.The model was fit as a rigid body using the T = 4 quasi-symmetry and alsowas fit independently as a rigid body into four unique positions in anasymmetric unit. All figures were prepared using Chimera (44).

ACKNOWLEDGMENTS. We thank Sheryl Kelly and Yingyuan Sun for help inthe preparation of this manuscript and technical support, respectively, andthe Purdue Cryo-EM Facility for instrument access and technical support. Thework was funded by NIH Grant AI095366 (to M.G.R.). Part of this work wassupported by VLP Therapeutics.

Fig. 3. Comparisons of the E1-E2-C structure in the immature and mature CHIKV. (A) Superposition of the E1-p62-C structure in the immature conformation(magenta) to E1-E2-C structure in the mature conformation (cyan). The E3 molecule in p62 is in blue. The capsid protein has a similar orientation in bothimmature and mature conformations. However, the TM helices have a different orientation and location in the immature form compared with the matureform. (B) Density of the capsid protein. The ordered structure of the capsid protein consists of residues 113–261. Additional density seen only in the cryo-EMreconstruction of the virus (outlined with black dashes) belongs to the N-terminal region of the capsid protein. This region is the RBS (residues 98–112). Thehydrophobic pocket of the capsid protein is indicated by a red star.

Fig. 4. Gold-standard FSC curve for refinement. The resolution corre-sponding to the 0.143 FSC cutoff is 6.8 Å.

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