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 ma- ture 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 depos- ited 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.