Structural Studies of Chikungunya Virus Maturation

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Structural studies of Chikungunya virus maturation

Moh 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 Tunku Abdul 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 the process. Flaviviruses are assembled as “immature” noninfectious E2 and E3 glycoproteins before assembly with the nucleocapsid is particles in the ER of the host cell that are then proteolytically the key to producing fusion-competent mature spikes on alphavi- modified to produce infectious viruses on leaving the host cell. ruses. Here we present a cryo-EM, 6.8-Å resolution structure of an However, alphavirus components are proteolytically modified “ ” immature Chikungunya virus in which the cleavage site has been before assembly into mature viruses on the plasma membrane. mutated to inhibit proteolysis. The spikes in the immature virus In addition, a regular, icosahedral capsid shell is observed only have a larger radius and are less compact than in the mature virus. in alphaviruses. During infection, a conserved sequence on the Furthermore, domains B on the E2 glycoproteins have less free- ’ dom of movement in the immature virus, keeping the fusion loops N-terminal regions of the capsid proteins binds to the host cell s protected under domain B. In addition, the nucleocapsid of the 60S ribosomal subunits, initiating the dissociation of the nu- immature virus is more compact than in the mature virus, protect- cleocapsid and the release of the RNA from the nucleocapsid ing a conserved ribosome-binding site in the capsid protein from (14). This ribosome-binding site (RBS) is buried during nu- exposure. These differences suggest that the posttranslational cleocapsid assembly but is exposed at the end of the maturation processing of the spikes and nucleocapsid is necessary to produce process (15, 16). infectious virus. In alphaviruses, there are 20 trimeric spikes located on the icosahedral threefold axes and another 60 trimeric spikes in alphavirus | Chikungunya virus | maturation | cryo-electron microscopy | general positions that obey T = 4 quasi-symmetry (17–19). conformational changes Glycoprotein E1 is involved in cell fusion (20), and glycoprotein E2 interacts with host receptors (21) whereas glycoprotein BIOPHYSICS AND

hikungunya virus (CHIKV) is a mosquito-borne virus, which E3 facilitates E1-p62 heterodimerization and prevents the ex- COMPUTATIONAL BIOLOGY Cwas first reported in Tanzania in 1952 (1) and later emerged posure of the E1 fusion loops from premature fusogenic acti- as an epidemic in the French Reunion Island in 2005 (2). In the vation (22, 23). Cryo-EM studies have shown that E3 remains past decade, CHIKV has spread to more than 40 countries associated with the mature virus of SFV (24), RRV (18), and across Africa, Asia, and Europe, causing over a million infections VEEV (25). However, SINV (26, 27) and CHIKV (28) release in the Americas alone since 2014 (3). Among the symptoms of E3 after budding. the disease are rash, myalgia, high fever, and, typically, severe arthritis (4). CHIKV is a member of the alphavirus genus in the Toga- Significance viridae family (5). Other closely related and well-studied alphaviruses are Semliki Forest virus (SFV), Ross River virus Chikungunya virus (CHIKV) belongs to the alphavirus family, the (RRV), Sindbis virus (SINV), and Venezuelan Equine En- members of which have enveloped icosahedral capsids. The cephalitis virus (VEEV). Alphaviruses are spherical enveloped maturation process of alphaviruses involves proteolysis of some viruses with an ∼700-Å diameter and a T = 4 quasi-icosahedral of the structural proteins before assembling with nucleocapsids symmetry.Thegenomeofalphavirusesisan∼12-kb positive- to produce mature virions. We mutated the proteolytic cleavage sensed single-stranded RNA molecule encoding four non- site on E2 envelope protein, which is necessary in initiating the structural proteins (nsP1–4), which are required for virus rep- maturation process. Noninfectious virus-like particles (VLP) “ ” lication, and five structural proteins (capsid protein C, equivalent to immature fusion incompetent particles were glycoproteins E1, E2, E3, and 6K) (6). The structural proteins produced to study the immature conformation of CHIKV. We describe the 6.8-Å resolution electron microscopy structure of are synthesized as a long polyprotein, which is then post- “ ” translationally cleaved into C, E1, 6K, and p62. A total of immature CHIK VLPs. Structural differences between the mature and immature VLPs show that posttranslational processing 240 copies of the C protein associate with a newly synthesized of the envelope proteins and nucleocapsid is necessary to allow genomic RNA molecule to form a nucleocapsid in the host exposure of the fusion loop on glycoprotein E1 to produce an cell’s cytoplasm (7). The glycoproteins E1 and p62 interact to infectious virus. form heterodimers that subsequently trimerize into a viral spike

in the endoplasmic reticulum (ER). The glycoprotein p62 is Author contributions: M.L.Y. and M.G.R. designed research; M.L.Y., T.K., A.U., and W.A. then cleaved into E2 and E3 by cellular furin during its trans- performed research; M.L.Y. and S.S.H. analyzed data; and M.L.Y. and M.G.R. wrote portation from the acidic environment of the Golgi and early the paper. endosomes to the neutral pH environment of the cell surface, Conflict of interest statement: M.L.Y., T.K., S.S.H., and M.G.R. declare no competing fi- releasing E3 (Movie S1). Virus budding occurs at the cell nancial interests. A.U. is an employee of VLP Therapeutics, and W.A. is an officer and membrane where the nucleocapsid is enveloped by the glyco- shareholder of VLP Therapeutics. proteins E1–E2 on the plasma lipid membrane. The protein 6K This article is a PNAS Direct Submission. facilitates particle morphogenesis (8–10),butitspositioninthe Published under the PNAS license. particle remains to be verified. Data deposition: The final immature Chikungunya VLP electron density map was deposited in the Electron Microscopy Data Bank, https://www.emdatabank.org (accession code Alpha- and flaviviruses (11) have many similarities. Their EMD-8734), and structure coordinates have been deposited in the Protein Data Bank, glycoprotein exteriors have icosahedral symmetry and surround a www.rcsb.org/pdb (PDB ID code 5VU2). lipid membrane that, in turn, surrounds their RNA genome, 1To whom correspondence should be addressed. Email: [email protected]. which is associated with the capsid protein. A major difference This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. between alpha- (12) and flaviviruses (13) is the maturation 1073/pnas.1713166114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1713166114 PNAS Early Edition | 1of5 Downloaded by guest on September 27, 2021 Here, we report the structure of immature CHIKV, which was including CHIKV, have a well-ordered icosahedral nucleocapsid determined using virus-like particles (VLPs) with mutations at within the membrane envelope (Fig. 1B). the furin cleavage site on p62. The E3 remained associated with Immature CHIKV virions, like mature CHIKV virions, have the E2, mimicking the precursor p62 in its immature confor- spike-like features (Fig. 2A) on their surface. Intraspike contacts mation. A crystal structure of the E1-p62 heterodimer [Protein are formed between the three E2 molecules that form a spike. Data Bank (PDB) ID code 3N40 (29)] was fitted into the cryo- The glycoprotein E1 wraps around E2 and contributes to inter- EM electron density map of immature CHIKV VLPs to examine spike interactions. Furthermore, E3 is located at the periphery of the interactions of E1 and p62 with each other in the immature the E2 molecules (Fig. 2A). The trimeric immature spikes, al- virus. A previous report showed that alphaviruses can be as- though organized with T = 4 quasi-symmetry, are similar to sembled in a partially mature, replication-competent state (25). mature CHIKV spikes, but are less compact with a hole along Hence, the structure described here represents an intermediate their threefold axes, resulting in a bigger spike radius. The spikes structure of CHIKV during the assembly and maturation pro- are more densely packed on the surface of immature CHIKV, cess. We showed that there are significant conformational dif- resulting in smaller holes along the icosahedral twofold (i2) and ferences between the mature and immature viruses, including the icosahedral fivefold (i5) symmetry axes and smaller separation nucleocapsid, the transmembrane helices, and the cellular at- between the spikes, in comparison with mature CHIKV (Fig. tachment sites on E2. The presence of E3 in the immature virus 2B). Thus, the spikes undergo a structural rearrangement during stabilized domain B on E2, protecting the fusion peptide on maturation. E1 from becoming exposed and fusogenic. Glycoprotein Spikes. As described in the crystal structure of E1- Results and Discussion p62 (29), E1 has three beta-sheet–rich domains, namely domains Cryo-EM Structure of Immature CHIKV. The cryo-EM density map of I, II, and III. A fusion loop is located at the tip of domain II. immature CHIK VLPs attained a 6.8-Å resolution (Fig. 1A). The E2 consists of three Ig-like domains (A, B, and C) and a long virions had a diameter of 660 Å and, like mature virions, have beta-ribbon (domain D) connecting domain B to C. Domain D T = 4 icosahedral symmetry. Central cross-sections of the re- interacts extensively with E3. The E1 fusion loop is sandwiched construction showed that the immature virion (Fig. 1C) has a between domains A and B of E2. nucleocapsid, enveloped by a plasma membrane and an out- The crystal structure of E1-p62 (PDB ID code 3N40) (29) was ermost layer of glycoproteins. Unlike flaviruses, alphaviruses, 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 an icosahedral twofold axis. An icosahedral asymmetric unit is marked by a black triangle. Icosahedral symmetry elements are shown as black-filled pentagon, triangles, and ellipse. Four unique subunits in an asymmetric unit are shown in white numbers. (B) Internal capsid protein shell of the immature CHIKV. (C) Central cross-sections of the immature CHIKV viewed down an icosahedral twofold axis. Components of the virus are shown in different 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 is showninthecryo-EMmapofimmatureCHIKV.

2of5 | www.pnas.org/cgi/doi/10.1073/pnas.1713166114 Yap et al. Downloaded by guest on September 27, 2021 part of the capsid protein starts at about residue 109 (VEEV) (25) and residue 97 (SINV) (19), presumably due to interactions with the genomic RNA. This ordered part has a helical structure in VEEV. In the virus, the positively charged region of the capsid proteins is associated with the negatively charged RNA. The ex- ternal glycoproteins and the internal capsid protein shell are associated with the E1 and E2 cytoplasmic tail binding to the capsid protein (34). This interaction has been shown to be important in virus budding and fusion (35). The model of the E1 and E2 transmembrane (TM) domains and capsid protein as observed in mature CHIKV (28) was fitted into the cryo-EM map of immature CHIKV (Fig. 1D). There are a number of differences between the structure of immature and mature CHIKV. These include a small change in orientation and location of the TM helices (Fig. 3A and Fig. S1), resulting in a more compact nucleocapsid in the immature CHIKV (Fig. 2B). The additionally ordered amino terminal region of the capsid protein in the virus is a part of the RBS (Fig. 3B). This fragment consists of the residues 98–112 (KPGRRERMCMKIEND) of the capsid protein, which are the conserved RBS in alphaviruses (14). The life cycle of alphaviruses can be described as starting with the mature virus in which the B domain of E2 is only loosely associated with the underlying domain II of the E1 glycoprotein. Thus, after virus recognition of a cell, the virus is enclosed into the low pH environment of an endosome. This causes the formation of trimeric fusogenic spikes resulting in the fusion of the viral and cellular membranes. As a result, the nucleocapsid is BIOPHYSICS AND exposed to the cell’s cytoplasm containing ribosomes at low pH. COMPUTATIONAL BIOLOGY The RBS on the icosahedral capsid of the mature virus is then available to bind to ribosomes. The association of ribosomes with the nucleocapsid causes the viral capsid to disintegrate (14) while guiding the genome to a ribosome. The replicated genome Fig. 2. Structural characteristics of the immature CHIKV. (A) A trimeric spike then associates with newly synthesized capsid proteins to make of the immature CHIKV. The E2 molecules (red) form interactions within a new nucleocapsids. These are transported to the plasma mem- spike whereas the E1 molecules (yellow) wrap around E2 molecules and form – interactions between spikes. The E3 molecules (green) are located at the brane where they will associate with mature E1 E2 to form new periphery of the E2 molecules. (B) Central cross-section of the immature mature particles that bud out from the membrane. In some (Left) and mature (Right) CHIKV. The icosahedral symmetry axes are in- cases, the E3 glycoprotein, although cleaved from E2, will re- dicated by white arrows. Components of the viruses are shown in different main on the particle. colors as indicated in the figure. The immature virus has smaller holes In the present case, the cleavage site has been mutated around the i2 and i5 symmetry axes compared to the mature virus. The di- resulting in no cleavage of p62 but producing the assembly of ameter of the nucleocapsid in the immature virus is smaller than in the immature particles. The diameter of the nucleocapsids in these mature virus. immature particles is about 20 Å smaller than in the mature particles, making the particles more compact and less likely to CHIKV (Fig. 1D) using the EMfit program (30). Unlike the expose the RBS. When these immature particles bind to a cell mature CHIKV, the average electron density of domain B in surface the (E1p62)3, trimeric spikes cannot fuse with the cell E2 is higher than in the immature CHIKV (Table 1). This im- membrane because the E3 glycoprotein stops the exposure of the plies that domain B is more rigid in immature CHIKV. This fusion loop. Observations of the immature particles indicate that result supports the hypothesis that domain B of E2 is stabilized furin cleavage of glycoprotein spikes followed by their associa- by the presence of E3, in agreement with a previous study (29). tion with the preformed nucleocapsid is required to produce The rigid domain B of E2 protects the fusion loop on E1 from fusion- and replication-competent particles. exposure and therefore inhibits cell fusion. However, domain A of E2 is more flexible in the immature than in the mature Table 1. Average density height of the densities at the atomic CHIKV, as indicated by the poorer electron density (Table 1). positions (sumf) on fitting of the atomic structure of CHIKV This might be because the spikes in the immature CHIKV are heterodimer E1-p62 into the immature cryo-EM density map less compact. Domain A of E2, which is situated close to the spike center, has fewer contacts with the neighboring molecules Protein Protein domain T = 4 fitting Independent molecule fitting than in the mature virus. This domain is more stable and exposed E1 I 14.2 15.3 in the mature conformation, which might be beneficial for host- II 14.2 16.0 cell binding. III 15.6 16.0 E2 A 11.1 13.2 Glycoproteins Transmembrane Helices and Capsid Protein. The chymotrypsin-like capsid protein of alphaviruses consists of a B 10.0 11.9 hydrophobic pocket between the two β-barrel domains (31). The C 16.3 18.8 rather basic N-terminal residues 1 to ∼110 of the capsid protein are D 13.9 14.1 disordered in the crystal structures of SINV (31, 32) and SFV (33). E3 12.9 13.1 Average 13.5 14.8 However, in the cryo-EM maps of VEEV and SINV, the ordered

Yap et al. PNAS Early Edition | 3of5 Downloaded by guest on September 27, 2021 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 both immature and mature conformations. However, the TM helices have a different orientation and location in the immature form compared with the mature form. (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-EM reconstruction 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). The hydrophobic pocket of the capsid protein is indicated by a red star.

Conclusion. During the maturation process of alphaviruses, Scientific). The cells were transfected with VLP-expressing plasmids by cleavage of p62 into E2 and E3 exposes the fusion loop on polyethylenimine reagent (Polysciences). Four days after transfection, the cell culture supernatant was harvested and clarified by centrifugation and E1 and arranges the glycoprotein spikes into a mature con- μ formation. Association of the mature spikes with the pre- filtration through a 0.45- m polyethersulfone (PES) membrane. The VLPs secreted in the culture supernatant were collected by using OptiPrep assembled nucleocapsid expands the nucleocapsid to a less Density Gradient Medium (Sigma-Aldrich), as described previously (36), compact form. This step exposes the RBS on the capsid pro- and further purified by Hiprep 16/60 Sephacryl S-500 HR column (GE tein, thus priming the nucleocapsid to be disassembled upon Healthcare Life Sciences). The eluates containing purified VLPs were release into the host-cell cytoplasm during the next infection concentrated by Amicon Ultra-15 centrifugal filter units (EMD Millipore) cycle. The events described here for VLPs would correspond and filtered with a 0.20-μm PES membrane. to the release of the genome into a host cell after virus entry Electron Microscopy and 3D Reconstruction. Aliquots of a 2.5-μLsampleat and may be similar to the mechanism of genome release in 3 mg/mL concentration were loaded on glow-discharged C-Flat grids (CF-2/ many other viruses. 2–4C). These grids were blotted for 5 s and flash-frozen in liquid ethane using a Gatan CP3 plunge freezer. The grids were viewed using the FEI Materials and Methods Titan Krios electron microscope operated at 300 kV. Images were recorded Production and Purification of Immature CHIK VLP. The coding sequence for with a Gatan K2 Summit detector calibrated to have a magnification of − the CHIKV strain 37997 structural proteins, C-E3-E2-6K-E1, was synthesized by 38,461, yielding a pixel size of 0.65 Å. A total dose of 36 e /Å2 and an the Blue Heron Company and cloned into a pUC119-derived vector under the exposure time of 7.6 s were used to collect 38 movie frames. Fully auto- control of a human cytomegalovirus early immediate promoter. The ex- mated data collection was implemented using Leginon (37). The MotionCorr pression plasmid for the furin cleavage-resistant CHIKV VLP was generated software (38) was used to correct the beam-induced motion. A total of by mutating amino acids 61–64 in E3 to Ser-Gly-Gly-Gly-Gly-Ser, using 5,325 images were collected, and 76,806 particles were boxed using the the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technol- EMAN2 package (39). Contrast transfer function parameters were esti- ogies). The VLPs were produced in FreeStyle 293-F cells (Thermo Fisher mated using CTFFIND3 (40). The 2D classification was performed using RELION (41), and the 3D reconstruction was performed using the JSPR software (42). The final electron density map was reconstructed using 72,944 particles and was estimated to have a resolution of 6.8 Å based on the 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 accession code EMD-8734, and structure coordinates have been deposited with the PDB(PDBIDcode5VU2).

Data Analysis and Figure Preparation. The crystal structure of E1-p62 (PDB ID code 3N40) and models of the E1 and E2 TM domains and capsid protein (PDB ID code 3J2W) were fit into the cryo-EM map using the EMfit program (30) to maximize 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 also was fit independently as a rigid body into four unique positions in an asymmetric unit. All figures were prepared using Chimera (44).

ACKNOWLEDGMENTS. We thank Sheryl Kelly and Yingyuan Sun for help in the preparation of this manuscript and technical support, respectively, and the Purdue Cryo-EM Facility for instrument access and technical support. The Fig. 4. Gold-standard FSC curve for refinement. The resolution corre- work was funded by NIH Grant AI095366 (to M.G.R.). Part of this work was sponding to the 0.143 FSC cutoff is 6.8 Å. supported by VLP Therapeutics.

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