Author response: Structural analyses at pseudo atomic resolution of Chikungunya virus and antibodies show mechanisms of neutralization (2013)

Full text Figures and data Side by side Abstract eLife digest Introduction Results and discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract A 5.3 Å resolution, cryo-electron microscopy (cryoEM) map of Chikungunya virus-like particles (VLPs) has been interpreted using the previously published crystal structure of the Chikungunya E1-E2 glycoprotein heterodimer. The heterodimer structure was divided into domains to obtain a good fit to the cryoEM density. Differences in the T = 4 quasi-equivalent heterodimer components show their adaptation to different environments. The spikes on the icosahedral 3-fold axes and those in general positions are significantly different, possibly representing different phases during initial generation of fusogenic E1 trimers. CryoEM maps of neutralizing Fab fragments complexed with VLPs have been interpreted using the crystal structures of the Fab fragments and the VLP structure. Based on these analyses the CHK-152 antibody was shown to stabilize the viral surface, hindering the exposure of the fusion-loop, likely neutralizing infection by blocking fusion. The CHK-9, m10 and m242 antibodies surround the receptor-attachment site, probably inhibiting infection by blocking cell attachment. https://doi.org/10.7554/eLife.00435.001 eLife digest The Chikungunya virus is carried by mosquitos and can cause a number of diseases in humans including encephalitis, which can be fatal in some cases, and severe arthritis. A recent mutation in the E1 protein of the virus has allowed it to efficiently reproduce in a different species of mosquitos, leading to a Chikungunya epidemic in Réunion Island in 2005 and the subsequent infection of millions of individuals in Africa and Asia. The virus also has the potential to spread to many areas of Europe and the Americas. Chikungunya virus has a single-stranded RNA genome that codes for four non-structural proteins and five structural proteins. Based on this knowledge it has been possible to develop virus-like particles that can be used to immunise non-human primates against Chikungunya infection by inducing antibody production. However, the development of vaccines for Chikungunya in humans will require a deeper understanding of how these antibodies produced by the vaccine interact with the virus and more detailed information about the structures of the virus and antibodies. Sun et al. have used two techniques – X-ray crystallography and electron cryo-microscopy – to determine the structure of Chikungunya virus-like particles, and to obtain new insights into the interactions of these particles with four related antibodies. Electron cryo-microscopy was used to figure out the structure of the particles at near atomic resolution, and X-ray crystallography was used to determine the atomic resolution structures of two of the four Fab antibodies that neutralize the Chikungunya virus. Electron cryo-microscopy was also used to probe the complex formed by the interactions between the virus-like particles and the antibodies. Sun et al. were able to identify the likely viral receptor site that is blocked by three of the antibodies when they neutralize the virus; the fourth antibody is thought to act by immobilizing one of the domains of protein E2, thereby hiding the “fusion loop” that allows the virus to enter and infect human tissue. It is hoped that these findings will contribute to efforts to combat the spread of the Chikungunya virus worldwide. https://doi.org/10.7554/eLife.00435.002 Introduction Chikungunya virus (CHIKV) is a mosquito-transmitted viral pathogen that causes fever, myalgia, rash, and severe arthritis in humans (Powers and Logue, 2007; Simon et al., 2008). The first reported human CHIKV infections occurred in East Africa in 1952 (Robinson, 1955). Prior to the 2005 epidemic on Réunion Island, CHIKV was not regarded as a highly prevalent virus. An adaptive mutation in the E1 protein (E1-A226V) that allowed CHIKV to replicate more efficiently in Aedes albopictus is considered to be the primary reason for its recent extensive spread, infecting millions of individuals in Africa and Asia (Tsetsarkin et al., 2007; Kumar et al., 2008; Santhosh et al., 2008). In some CHIKV-infected patients, severe damage in joint tissues can cause debilitating chronic arthritis. In the recent outbreaks, a change in pathogenesis was observed, with some cases progressing to fatal encephalitis. An autochthonous CHIKV outbreak in Italy in 2007 and the presence of the Aedes albopictus vector in many areas of Europe and the Americas have raised concern of further spread of the virus. Currently, there is no vaccine or antiviral agent approved for use in humans. CHIKV belongs to the alphavirus genus of the Togaviridae family (Kuhn, 2007). Alphaviruses are a group of positive-sense, single-stranded RNA, enveloped viruses transmitted by arthropods (Griffin, 2007). The alphavirus genome encodes four non-structural and five structural proteins. The non-structural proteins are required for virus replication, protein modification, and immune antagonism. The structural proteins (capsid-E3-E2-6K-E1) are synthesized as a polyprotein from a subgenomic promoter, and are cleaved post-translationally into separate proteins by an autoproteinase and signalase. The E1 glycoprotein participates in cell fusion (Lescar et al., 2001), whereas the E2 glycoprotein binds to cellular receptors (Smith et al., 1995; Zhang et al., 2005) and initiates clathrin-dependent endocytosis (Solignat et al., 2009). Virus core assembly is initiated by interactions between the genomic RNA and the nuclear capsid protein (NCP) (Tellinghuisen et al., 1999; Tellinghuisen and Kuhn, 2000; Linger et al., 2004) in the cytoplasm. The E3 protein is essential for the proper folding of p62, the precursor to E2, and the formation of the p62-E1 heterodimer (Mulvey and Brown, 1995; Carleton et al., 1997). Although E3 remains part of mature Semliki Forest virus (SFV) and Venezuelan equine encephalitis virus (VEEV), apparently, it is not a component of mature CHIKV (Simizu et al., 1984). The small 6 kDa protein, 6K, associates with the p62-E1 heterodimer and is transported to the plasma membrane prior to assembly. The 6K protein facilitates particle morphogenesis but is not stoichiometrically incorporated into virions (Gaedigk-Nitschko and Schlesinger, 1990, 1991). Alphaviruses are icosahedral particles that have T = 4 quasi-icosahedral symmetry (Paredes et al., 1993; Venien-Bryan and Fuller, 1994; Cheng et al., 1995; Zhang et al., 2002, 2011; Kostyuchenko et al., 2011). The ectodomain forms 80 spikes, each consisting of three copies of E1-E2 heterodimers. There are 20 icosahedral “i3” spikes, situated on icosahedral 3-fold axes, and 60 quasi-3-fold “q3” spikes in general positions containing a quasi-3-fold axis consistent with T = 4 symmetry (Figure 1A). Thus, each icosahedral asymmetric unit contains one complete q3 spike and one-third of an i3 spike. E1 of CHIKV is a 439 amino acid protein with an N-linked glycosylation site at residue Asn141. The ectodomain is formed by the 404 N-terminal residues, followed by a 30 residue transmembrane (TM) helix and a five amino acid cytoplasmic domain. The E1 ectodomain consists of three β-barrel domains (Lescar et al., 2001; Gibbons et al., 2004; Li et al., 2010; Voss et al., 2010). Domain I is spatially located between domains II and III with the fusion peptide lying at the distal end of domain II. E2 of CHIKV is 423 amino acids in length and has two N-linked glycans at positions Asn263 and Asn345. The E2 364 residue ectodomain is followed by a 26-residue TM helix and a 33-residue cytoplasmic domain. The E2 ectodomain consists of three distinct immunoglobulin-fold domains with domain A, the putative receptor-binding domain, lying between domains B and C (Li et al., 2010; Voss et al., 2010). In the mature virus, domain B covers the fusion loop in domain II of E1. The virus becomes fusogenic in acid pH (Wahlberg and Garoff, 1992) when sets of three E1 molecules combine to make a fusogenic trimer after expelling the three E2 proteins from the center of the spike. The three fusion loops are then exposed outwards for insertion into the host cell membrane (Gibbons et al., 2004). Similar large conformational changes occur in other enveloped viruses (Kielian and Rey, 2006) although little is known of the structural intermediates through which these changes take place. Figure 1 Download asset Open asset Structure of the CHIK VLPs. (A) Surface-shaded figure of ectodomain (left) and surface-shaded figure of nucleocapsid (right), colored according to the radial distance from the center of the virus. White triangles indicate one icosahedral asymmetric unit. (B) Cross-section of the virus showing density above 1.5 σ also colored according to the radial distance from the center of the virus. (C) Resolution of β-strands in the E1 domain III. https://doi.org/10.7554/eLife.00435.003 The nucleocapsid core consists of 30 hexamers around each 2-fold axis and 20 pentamers at each 5-fold vertex of the nuclear capsid (Figure 1A), and is separated from the ectodomains of E1 and E2 by a lipid membrane (Figure 1B). The N-terminal 118 amino acids of Sindbis (SINV) (Choi et al., 1991) and SFV (Choi et al., 1997) nuclear capsid contain positively charged residues that associate with the genomic RNA (Zhang et al., 2002). A similar positively charged N-terminal region exists in CHIKV NCP and functions to recognize the viral genome for packaging during NCP assembly (Perera et al., 2001; Hong et al., 2006). The carboxy-terminal region of the NCP has a chymotrypsin-like fold and acts as an auto-catalytic proteinase during processing of the structural polyprotein (Hahn and Strauss, 1990; Choi et al., 1991, 1997). In addition, there is a hydrophobic pocket in the C-terminal domain of the NCP that binds the 33 amino acid cytoplasmic tail of E2, effectively linking the proteins of the inner core with those on the surface of the virion (Lee et al., 1996; Skoging et al., 1996). This association of E2 with the nuclear capsid is essential for virus assembly (Owen and Kuhn, 1997). Many studies have analyzed the interaction of antibodies with alphaviruses (Meyer and Johnston, 1993; Griffin et al., 2001; Hunt et al., 2010). Most neutralizing antibodies bind to E2, as this glycoprotein is more exposed on the viral surface than E1. The mode of neutralization differs considerably among antibodies, with some blocking attachment to cells and others inhibiting the conformational changes required for fusion (Hernandez et al., 2008). The only published structural studies of alphavirus-antibody complexes were a cryoEM analysis of Ross River virus complexed with an antibody that blocked receptor attachment (Smith et al., 1995) and a cryoEM investigation of SINV complexed with an antibody that inhibited the conformational changes required to form fusogenic E1 trimers (Hernandez et al., 2008). Expression of the CHIKV structural C-E3-E2-6K-E1 polyprotein gives rise to virus-like particles (VLPs) that have been used to immunize and protect non-human primates against CHIKV infection (Akahata et al., 2010). Non-human primates immunized with these VLPs developed high-titer neutralizing antibodies that protected against viremia after high-dose challenges. Moreover, immunodeficient mice that were passively administered purified IgG from immunized non-human primates were protected against CHIKV challenge (Couderc et al., 2009). Previously, we published the structure of the CHIK VLPs at a resolution of 18 Å using cryoEM reconstruction of single particles (Akahata et al., 2010). Here, we report a 5.3 Å three-dimensional high-resolution reconstruction of the CHIK VLP. We also describe pseudo-atomic resolution structures of CHIK VLPs complexed with Fab fragments from four different neutralizing mouse monoclonal antibodies (MAb CHK-9, CHK-152, m10 and m242). These studies are the first structural investigations of CHIKV complexed with neutralizing MAbs, based on 15 Å resolution cryoEM reconstructions of the complexes, the 5.3 Å resolution structure of the VLPs and 3 Å resolution crystallographic studies of the Fab fragments. These structures suggest that MAb CHK-152 neutralizes infectivity by inhibiting fusion whereas the other MAbs probably neutralize by blocking attachment. Results and discussion CryoEM structure of CHIK VLPs The structure of CHIK VLPs was determined to a resolution of ∼5.3 Å. The trimeric appearance of individual spikes was recognizable immediately, along with the inclined positions of the three E1 molecules surrounding each spike. Further inspection of the E1 and E2 molecules showed β-barrel structures surrounding empty cavities, thus yielding a good indication of the positions of each domain. Although resolution of individual β-strands was possible only in some instances (Figure 1C), identification of individual side chains was not possible. The E3 glycoprotein, which is observed in some alphavirus structures (Zhang et al., 2011), was not apparent in the CHIK VLP cryoEM density map. Densities for the E2 B-domain were weak in all four E1-E2 heterodimers, consistent with the crystal structures of the CHIKV E1-E2 heterodimer (Voss et al., 2010) and the SINV trimeric spike (Li et al., 2010). Within the CHIK VLP structure, the four quasi-equivalent chymotrypsin-like nucleocapsid proteins were organized as pentamers around the 5-fold vertices and hexamers around the 2-fold icosahedral symmetry axes (Figure 1A). The cryoEM density height of the nucleocapsid proteins was roughly 5% less than that of the glycoproteins. The glycoproteins and the nuclear capsid were separated by a ∼45 Å-wide lipid membrane. The external and internal lipid leaflets had a density of about 5 σ, whereas the protein density of the glycoproteins and the NCPs had a density of about 7 σ. The two lipid leaflets were separated by a ∼15 Å wide region, where the density was less than 1.5 σ, representing the loosely packed aliphatic chains of the lipid molecules (Figure 1B). The membrane was traversed by 60 × 4 pairs of α helices representing the E1 and E2 carboxy-terminal regions (Figure 1B). The quality of the membrane density in the icosahedrally averaged map demonstrates that the symmetry of the glycoproteins and nuclear capsid impose icosahedral symmetry on the flexible membrane by confining it to a limited space. Refinement and verification of the structure A number of procedures have been developed for the refinement of atomic parameters when the resolution of the structural data is insufficient to follow the polypeptide chains with certainty or to recognize the identity of amino acids (Chapman, 1995; Volkmann and Hanein, 1999; Tama et al., 2004; Fabiola and Chapman, 2005; Goddard et al., 2007; Topf et al., 2008; Trabuco et al., 2008; Lasker et al., 2010; Schröder et al., 2010; Zhu et al., 2010). These procedures increase the number of refinable parameters beyond those necessary to position and orient a rigid known structure into a relatively low-resolution cryoEM density map, thus reducing the ratio of observed data points (e.g. structure amplitudes) to refinable parameters. However severe restraints are placed on the geometry of the structure (thus providing additional observational data) while improving agreement between observed and calculated data in real or reciprocal space. Verification in reciprocal space can be achieved by tests on data that had been excluded in the refinement process, for instance by calculating Rfree (Brünger, 1992). Verification in real space can be established by observing anticipated structural features that had not been considered in the restrained refinement process. In the analysis of the CHIKV VLPs described here, the initial interpretation was performed by fitting the structure of the CHIKV E1-E2 heterodimer (Voss et al., 2010) into the cryoEM map of the CHIKV VLPs assuming strict T = 4 symmetry between quasi-equivalent structures. Subsequently, the number of refinable parameters was increased by breaking the structure into domains, based on the assumption that the domains likely are more rigid than the hinges in the polypeptide chain that hold the domains together. In addition, constraints imposed by the assumption of strict T = 4 symmetry were abandoned, thus further multiplying the number of refinable parameters by four. Verification came from the improved correlation of the domain-fitted structure with the amino acid sequences, the improved correlation between density heights of quasi-equivalent parts of the structure with each other, and from the emergence of features such as the position of the N-linked carbohydrate moieties. Fitting of the CHIKV E1-E2 crystal structure into the cryoEM density map In contrast to the interpretation of the cryoEM density of VEEV (Zhang et al., 2011), which had a resolution of 4.8 Å, we were able to use the crystallographically determined structure of the glycoproteins to aid in the analysis. The crystal structure of CHIKV E1-E2 (Voss et al., 2010) was fitted into the 5.3 Å cryoEM map using the program EMfit to maximize the average value of the density (sumf) at all atomic positions (Table 1A), to minimize the steric clash between symmetry related structures, and to minimize the percentage of atoms in low density (Rossmann et al., 2001). Initially, the E1-E2 heterodimer crystal structure was fitted as a rigid body generating the four quasi-equivalent structures at positions #1, #2, #3, and #4 (Figure 2A) while refining the best positions of the quasi-symmetry axes (see Materials and Methods), and using the icosahedral symmetry to generate the structure in neighboring icosahedral asymmetric units. The resultant locations of the four quasi-equivalent CHIKV heterodimers were used as starting positions for refinement of individual domains. The average height of the density at all fitted atoms (sumf) improved by ∼12% for the independently fitted domains. The value of sumf was at least 38% lower for the B domains than for any of the other domains. This is consistent with the disorder of the B domain in the crystal structure of the SINV spike (Li et al., 2010) and the large temperature factors for the atoms in the B domain in the crystal structure of the CHIKV heterodimer (Voss et al., 2010). The height of the average value of the cryoEM density taken over all atomic positions within a molecular domain was higher at the quasi-equivalent position #4 than at the other three quasi-equivalent positions (Table 1A). This might be due to crowding around the 5-fold symmetry axes, which reduces the variability in the heterodimer position. Table 1 Quality of fitting the atomic structural fragments into the cryoEM density https://doi.org/10.7554/eLife.00435.004 A) Average height of the densities at the atomic positions (sumf) (Rossmann et al., 2001) on fitting the E1E2 heterodimer CHIKV structure into the cryoEM map at four quasi equivalent positionsAll atomsT = 4 fittingIndependent domain fitting#1#2#3#4Average#1#2#3#4AverageE1*I16.115.817.017.716.618.217.018.519.918.5II15.717.218.816.717.018.018.619.018.518.6III15.714.215.218.015.718.715.918.220.718.3E2*A15.518.019.017.517.418.817.819.919.418.8B10.09.49.69.69.810.911.311.712.411.7C15.315.419.019.116.821.019.920.222.420.5D13.316.415.816.115.416.417.216.016.816.5Average14.915.817.016.816.117.817.318.219.018.1Main chain atoms onlyT = 4 fittingIndependent domain fitting#1#2#3#4Average#1#2#3#4AverageE1*I20.518.421.522.920.823.221.622.626.323.4II17.020.923.020.220.321.723.323.522.822.8III18.915.317.522.318.522.317.922.626.622.4E2*A16.521.122.219.419.822.020.323.422.622.1B10.79.49.910.110.010.811.213.013.412.1C18.719.527.426.923.127.727.127.430.828.3D13.418.718.518.517.319.419.819.119.019.3Average16.517.620.020.018.521.020.221.723.121.5B) Average height of the densities at the atomic positions (sumf) (Rossmann et al., 2001) on fitting the CHIKV homology model of the capsid protein into the cryoEM map at the four quasi equivalent positionsAll atomsT = 4 fittingIndependent domain fitting#1#2#3#4Average#1#2#3#4AverageD1†14.913.313.916.114.616.114.615.417.315.8D2†17.017.418.118.117.618.319.117.419.018.6Average16.015.416.017.216.117.417.116.518.217.3Main chain atoms onlyT = 4 fittingIndependent domain fitting#1#2#3#4Average#1#2#3#4AverageD1†18.516.517.818.917.920.416.919.222.719.8D2†21.321.521.423.221.922.824.220.425.323.2Average19.919.019.621.119.921.821.019.924.121.6C) Average height of the densities at the atomic positions (sumf) (Rossmann et al., 2001) on fitting the CHIKV model of the transmembrane protein into the cryoEM map at the four quasi equivalent positionsAll atomsT = 4 fittingIndependent domain fitting#1#2#3#4Average#1#2#3#4AverageE1‡13.714.613.218.615.013.914.414.717.515.1E2‡14.712.811.817.514.215.114.113.718.915.5Average14.413.512.317.914.614.514.214.218.215.3Main chain atoms onlyT = 4 fittingIndependent domain fitting#1#2#3#4Average#1#2#3#4AverageE1‡16.217.616.024.118.515.717.316.723.918.4E2‡16.715.013.620.716.517.716.715.823.718.5Average16.516.014.422.017.516.717.016.223.818.4 * Domain definition: E1 I (residues 1-36, 132-168, 273-293), II (residues 37-131, 169-272), III (residues 294-393). E2 A (residues 16-134), B (residues 173-231), C (residues 269-342), D (residues 7-15, 135-172, 232-268). † Domain definition: D1(residues 119 to 183), D2(residues 184 to 267). ‡ Domain definition: E1 (residues 394-439), E2(residues 343-423). Figure 2 Download asset Open asset of between amino acid residues in (A) of the E1 and E2 according to T = 4 icosahedral The show the in which the four were The indicate the of generating of the icosahedral asymmetric units. symmetry are shown as triangles and are shown as triangles and (B) between the E1 and E2 (C) between (B) and (C) show the number of between the The quasi T = 4 related positions #1, #2, and #4 are by their icosahedral symmetry identification A, C and The center of the “i3” icosahedral spike is by a and the center of the “q3” quasi-3-fold spike is by an the individual domains were fitted (Figure the geometry between the end of one domain and the amino end of the domain were The distance between these prior to (Table not Å the flexible domain that the quality of fit was of the between domains in the four quasi-equivalent positions (Table not for the of the B domain in that the length of the domain of is about Å, this a of about 6 Å for atoms from the to a of about three between structures. Figure 3 Download asset Open asset of the atomic structure into the cryoEM density. (A) of the E1E2 (B) of the capsid The quasi equivalent to the icosahedral 5-fold axis was for Table 2 in Å between and C of fitted domains equivalent to II to I to II to I to III 15 to A to to to to to Table 3 change between domains in different heterodimers change in E1 between domain I and domain II change in E1 between domain I and domain III change in E2 between domain A and domain B change in E2 between domain A and domain C fitting of the domains, the distance between equivalent atoms when between E1-E2 heterodimers from Å to Å (Table This showed that the structures of the four quasi-equivalent parts of the model were more to each other than to the crystal structure of the CHIKV heterodimer (Voss et al., 2010). such is to occur by this to the of the used to the cryoEM map. The change of the E1-E2 heterodimers in the VLPs to the crystal structure is the of the different quasi-equivalent environments. In contrast to the for the related VEEV (Zhang et al., 2011), the presence of than the between atoms between the four quasi-equivalent structural components of the CHIKV VLPs of the quasi equivalent density not the map. Table 4 in Å between quasi equivalent atoms The quality of the side chain was determined by their densities in the four quasi-equivalent The density with each residue was taken as the average density of all the atoms in that A correlation (see Materials and for that the of cryoEM density along each polypeptide chain was the average density for each residue all atoms in the side a correlation also of the side chain These correlation (Table from to which was significantly than the for the initial T = 4 rigid body there was a correlation of the average crystallographic temperature factors for each amino acid residue of the CHIKV heterodimer crystal structure (Voss et al., 2010) with the height of the cryoEM density at each amino acid position (Table Table 5 between amino acid and cryoEM density A) between cryoEM densities in the four quasi equivalent positions of the E1 and E2 atomsT = 4 fittingIndependent domain chain atoms onlyT = 4 fittingIndependent domain between cryoEM densities of the four quasi equivalent capsid atomsT = 4 fittingIndependent domain chain atoms onlyT = 4 fittingIndependent domain between cryoEM densities of the four quasi equivalent TM and atomsT = 4 fittingIndependent domain chain atoms onlyT = 4 fittingIndependent domain The N-linked carbohydrate The fitting of the domains of the CHIKV heterodimer into the cryoEM map was further by the densities with N-linked after to all densities at points within Å of fitted The densities to the on E1 and E2