Source: https://jvi.asm.org/content/83/16/8259?ijkey=fc13c9e66574ad21b4754ad28ab12000d8ff1c32&keytype2=tf_ipsecsha
Timestamp: 2019-04-19 14:53:46+00:00

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New World arenaviruses, which cause severe hemorrhagic fever, rely upon their envelope glycoproteins for attachment and fusion into their host cell. Here we present the crystal structure of the Machupo virus GP1 attachment glycoprotein, which is responsible for high-affinity binding at the cell surface to the transferrin receptor. This first structure of an arenavirus glycoprotein shows that GP1 consists of a novel α/β fold. This provides a blueprint of the New World arenavirus attachment glycoproteins and reveals a new architecture of viral attachment, using a protein fold of unknown origins.
Pathogenic human and animal viruses constitute a growing and persistent threat to global health (25). Machupo virus (MACV), responsible for Bolivian hemorrhagic fever (HF), is an apt example, being zoonotic and highly virulent. MACV was first isolated in 1963 and, along with Junín virus (JUNV), Guanarito virus (GTOV), Sabia virus (SABV), and Chapare virus (CAPV), comprises the HF viruses within clade B of the New World arenavirus family (13, 21). Clinical features of MACV infection during initial disease onset generally include fever, malaise, and headaches, developing over 7 to 10 days into severe HF (13). The high fatality rate (∼20%) and potential for global spread of this rodent-borne virus by deliberate dissemination have resulted in its classification by the National Institute for Allergy and Infectious Diseases as a high-priority category A biothreat agent (6).
MACV is an ambisense RNA enveloped virus composed of a bisegmented genome. The L (large) segment encodes an RNA-dependent polymerase (L) and a zinc finger matrix protein (Z); the S (small) segment encodes the nucleoprotein (NP) and the viral glycoprotein precursor GPC (9). The L and NP proteins are coded in the conventional sense for a negative-sense RNA virus, while Z and GPC are transcribed in the opposite direction (Fig. 1). GPC is cleaved by the cellular proprotein convertase site 1 protease (39) to yield a stable complex composed of a 58-amino-acid signal peptide which is necessary for virus infectivity, a GP1 subunit which is involved in receptor attachment (199 amino acids), and a transmembrane-bound GP2 subunit (249 amino acids) which is putatively classified as a class I fusion protein (23, 38) (Fig. 1).
Schematic diagram of the ambisense, bisegmented arenavirus genome and details of the MACV GP1 sequence crystallized and ordered in the crystal structure. Both the L and S segments contain a central noncoding region (NCR). Arrows correspond to the coding directionality of the genes.
MACV GP1 maintains low sequence identity with the GP1s of other New World HF arenaviruses (47, 27, 31, and 30% for JUNV, SABV, GTOV, and CAPV, respectively). Nevertheless, recent studies have shown that the transferrin receptor (TfR1) is a common cellular receptor for the GP1s of MACV, JUNV, GTOV, and SABV (24, 35, 36). These studies are an important step toward defining the viral tropism, and this interaction provides a target for the development of antivirals and prophylactic vaccines to prevent New World arenavirus infection. Knowledge of the molecular determinants of arenavirus attachment and fusion is a prerequisite for the rational development of immunotherapeutic and antiviral reagents (analogous to the development of neuraminidase inhibitors for the treatment of influenza ). To this end, we have solved the structure of the MACV GP1.
The globular domain of MACV GP1 glycoprotein (MACV GP1) responsible for attachment to TfR1 (residues 87 to 257 from the complete mature GP1 which comprises residues 59 to 257; GenBank accession number AAS77647.1; cDNA synthesized by Codon Devices; Fig. 1) was cloned into the pHLsec vector containing the chicken RPTPσ signal sequence (5). This region was selected based on the disorder predictions of RONN (44) and consideration of potential disulfide bond patterns. MACV GP1 was expressed in HEK 293T cells transfected with 2 mg DNA/liter of cell culture in the presence of 5 μM kifunensine, which prevents glycosylation processing, resulting in protein bearing oligomannose-type glycans (12). MACV GP1 protein was purified from the cell supernatant by using immobilized metal affinity followed by size-exclusion chromatography (SEC) using a Superdex 200 10/30 column (Amersham) equilibrated in 150 mM NaCl and 10 mM Tris, pH 8.0 (Fig. 2A and B). Protein yields were ∼2.0 mg MACV GP1/liter of cell culture. The binding activity of MACV GP1 for TfR1 (GenBank NC_BC001188, residues 122 to 760 cloned into the pHLsec vector ) was confirmed by coexpression and purification (as described above) of a MACV GP1-TfR1 complex from GlcNAc transferase I (GnTI)-deficient HEK 293S cells (37) (Fig. 2C and D).
Purification of MACV GP1 and MACV GP1-TfR1 complex. MACV GP1 and MACV GP1-TfR1 were expressed in HEK 293T (with 5 μM kifunensine) and GnTI-deficient HEK 293S cells, respectively. (A) SEC of MACV GP1 run on an S200 10/30 column. (B) A 4 to 12% gradient morpholineethanesulfonic acid-polyacrylamide gel electrophoresis assay of the resulting MACV GP1 from SEC run under reducing conditions (expected unglycosylated molecular mass, ∼22 kDa). The rightmost lane shows molecular mass markers. (C) SEC of MACV GP1-TfR1 complex run on an S200 10/30 column. Peak 1 corresponds to MACV GP1-TfR1 complex, and peak 2 corresponds to excess, unbound MACV GP1. (D) A 4 to 12% gradient morpholineethanesulfonic acid-polyacrylamide gel electrophoresis assay of the resulting protein from SEC run under reducing conditions. Lanes 1, 2, and 3 are consecutive fractions from peak 1, and lanes 4 and 5 are adjacent fractions from peak 2. The rightmost lane shows molecular mass markers. Note that peak 1 contains both GP1 (expected unglycosylated molecular mass, ∼22 kDa) and TfR1 (expected unglycosylated molecular mass, ∼71 kDa). Also, note that the apparent molecular mass difference observed between MACV GP1 in panel B and that in panel D is due to the different MACV GP1 glycoforms which result from expression in kifunensine-treated HEK 293T cells and GnTI-deficient HEK 293S cells.
Purified MACV GP1 (concentrated to 12.5 mg/ml) crystallized from sitting drops of 100 nl plus 100 nl (25% [wt/vol] polyethylene glycol 3350, 0.2 M NaCl, and 0.1 M bis-Tris, pH 5.5) equilibrated against 95-μl reservoirs for 78 days at room temperature (42). Crystals were cryoprotected by immersion in reservoir solution plus 25% (vol/vol) glycerol and cryocooled in a 100 K gaseous nitrogen stream. X-ray diffraction data were recorded at the European Synchrotron Radiation Facility (ESRF), Grenoble, France. For phase determination, a crystal was soaked for 21 h with ∼10 mM potassium tetrachloroplatinate (II) and diffraction data were collected to a resolution of 3.4 Å on BM-14 at λ = 0.886 Å (the L1 edge for platinum), λ = 1.072 Å (inflection of the platinum L3 edge), and λ = 1.180 Å (low-energy remote) and on beamline ID14 EH1 at λ = 0.9334 Å (“high-energy remote” between platinum L1 and L2 edges). High-resolution (1.7-Å) data were recorded from a native crystal on ID14 EH1. Images were indexed, integrated, and scaled using HKL2000 (32). Data collection and crystallographic statistics are presented in Tables 1 and 2.
Phase determination used the multiple-wavelength anomalous dispersion method. Heavy atom positions were identified using SHELXD (40) and refined using SHARP (20), solvent flattening was performed using SOLOMON (2) and DM (17), and the resulting low-resolution (4-Å) electron density map was used for initial model building of the single molecule in the asymmetric unit (solvent content, 50%). This initial model was placed into the high-resolution data using PHASER (30), and model building was completed automatically using ARP-wARP (33). Structure refinement iterated cycles of restrained refinement with TLS using REFMAC5 (31) and manual rebuilding using COOT (22). The final model was validated using MolProbity (19). Of the residues, 96.7% lie within favored regions of the Ramachandran plot and the remainder lie in additionally allowed regions (19).
The 1.7-Å resolution structure of MACV GP1 is shown in Fig. 3. Although the MACV GP1 used for crystallization included residues 87 to 257 (Fig. 1), we do not see electron density for the last 20 C-terminal residues (the residues preceding the point of GP1/GP2 cleavage). MACV GP1 appears to be monomeric in the crystal. Structural database comparison (27, 45) suggests that MACV GP1 is composed of a fold that has not been previously observed. The N and C termini extend in the same direction, and the secondary structure consists of seven antiparallel β-strands forming a left-handed sheet (designated 1 to 7); three α-helices, one of which is preceded by a 310 helix; and two additional 310 helices (Fig. 3A and B; see also Fig. S1 in the supplemental material). When looking onto the edge of the β-sheet, the overall fold appears to be similar to the shape of a positive meniscus lens where the three large helices protect the convex side of the β-sheet whereas the concave side of the β-sheet is largely uncovered (Fig. 3B and D). The secondary structure is stabilized by four disulfide bonds which are present around the plane of the lens (Fig. 3C). Two of these disulfide bonds appear to be conserved across the New World HF arenaviruses, a third is also found in the GP1 of JUNV, while the fourth stabilizes a MACV-specific insertion (Fig. 3C to E). The presence of an additional conserved disulfide bond between MACV GP1 and JUNV GP1 reflects the close relationship between these two viruses with respect to other New World arenaviruses (8, 11, 14); thus, for the portion of the GP1 that we have analyzed, the sequence identity is 40% (Fig. 3E), and we would therefore expect the two structures to agree with approximately 1.2-Å root mean square deviation over matching Cα atoms (16).
Structure of MACV GP1. (A) Cartoon diagram of MACV GP1 colored as a rainbow with the N terminus shown in blue and the C terminus in red. The N and C termini are marked with blue and red spheres, respectively. (B) View of MACV GP1 rotated by 90° from that in panel A. (C) Cα trace of MACV GP1 colored gray with carbohydrate moieties (GlcNAc) observed at N-linked glycosylation sites (nitrogen atoms colored blue, oxygen atoms colored red, and carbon atoms colored yellow) and disulfide bonds (carbon atoms colored gray and sulfur atoms colored gold) shown as sticks. Disulfide bond pairs are numbered in magenta according to panel E. (D) View of MACV GP1 rotated by 90° from that in panel C. (E) Sequence alignment of residues observed in the MACV GP1 crystal structure with the GP1s of JUNV, GTOV, SABV, and CAPV (determined by ClustalW , plotted by ESPRIPT , and adjusted by hand). Secondary structure elements are shown with an arrow (β-strand, β1 to -7), and helices are shown as spirals, with α-helices shown in bold (α-helix, α1 to -3; 310 helix, η1 to -3). Residues which are highlighted red are fully conserved, residues which are colored red are partially conserved, and residues which are black are not conserved. Residues which are solvent accessible (as determined by ESPRIPT ) are highlighted by bars below the sequence and are colored light blue (partially accessible) or dark blue (fully accessible). Conserved amino acids which are solvent exposed are annotated below the alignment with yellow spheres. Amino acids which correspond to predicted N-linked glycosylation sites are marked with blue boxes and annotated beneath the alignment as yellow (conserved across all sequences) and green (not conserved) spheres. The bottom row of the alignment marks the disulfide bond pairs as marked in panel C.
Four N-linked glycosylation sites lie on the perimeter of the plane of the β-sheet (Fig. 3C and D). Electron density was observed for at least one N-acetylglucosamine (GlcNAc) residue at each of these sites (Fig. 4). B-factors for glycan atoms (Table 2) are not significantly greater than those of other surface atoms. Deglycosylation of MACV GP1 with endo F1 resulted in precipitation (data not shown), suggesting that, as reported for some other systems (34), glycans solubilize the protein. Protein-carbohydrate interactions were observed for several glycans. For example, GlcNAc at Asn95 forms a classic stacking interaction between the side chain of Phe98 and the pyranose ring (Fig. 4A) (7, 34, 43) (the rest of the glycan projects across a protein surface which bears further solvent-exposed aromatic residues). The glycan at Asn178 lies at the center of a cavity on the protein surface, braced by a network of hydrogen bonds (Fig. 4C), including one to Glu184 which may stabilize the loop following the β6 strand. Oligomannose structures are often observed on glycoproteins from enveloped viruses, for example, human immunodeficiency virus type 1, dengue virus, and Ebola virus (3, 18, 29, 41), where they can influence viral tropism and stimulate the host immune response (28). While glycosylation processing of complex-type structures is influenced by tissue-specific processing, oligomannose-type glycans are frequently highly conserved between recombinant material and infectious virions. For example, the oligomannose-type glycans of human immunodeficiency virus type 1 gp120 are also present in recombinant gp120 monomers expressed in CHO cells (46). However, analysis of N-linked glycosylation by matrix-assisted laser desorption ionization-time of flight mass spectrometry (Fig. 4D; see also Table S1 in the supplemental material) demonstrated that recombinant MACV GP1 contains instead highly branched, extensively heterogeneous, complex-type glycans when expressed in the absence of any glycosidase inhibitors.
Analysis of N-linked glycans on MACV GP1. Enlarged view of the GlcNAc residues at Asn95 (A), Asn137 and Asn166 (B), and Asn178 (C). MACV GP1 is shown as a cartoon representation with asparagines and GlcNAc structures shown as sticks. Carbon atoms of the GlcNAc moieties are shown in yellow, and those of asparagine side chains are gray. Dashed lines correspond to hydrogen bonds between GlcNAc moieties and amino acids. A maximum likelihood weighted 2Fo-Fc electron density map was calculated using the final refined model and is displayed around the GlcNAc residues contoured at 1σ. (D) Mass spectrometric analyses of MACV GP1. Matrix-assisted laser desorption ionization-time of flight mass spectrometry of desialylated N-linked glycans ([M + Na]+ ions) released from MACV GP1 expressed in HEK 293T cells. Symbols used for the structural formulae are as follows: ⋄, Gal; ⧫, GalNAc; ▪, GlcNAc; ○, Man; ⟐, Fuc. The linkage position is shown by the angle of the lines linking the sugar residues (vertical line, 2-link; forward slash, 3-link; horizontal line, 4-link; backward slash, 6-link). Full annotation of the spectra, together with the anomericity indicated by full lines for β bonds and broken lines for α bonds, is provided in Table S1 in the supplemental material.
Analyses of N-linked glycosylation sites across our GP1 segment from all New World HF arenaviruses (MACV, JUNV, GTOV, SABV, and CAPV) reveal a total of 10 potential sites which, when mapped onto the structure of MACV GP1, decorate solvent-accessible loops on the perimeter of the β-sheet (Fig. 5A). Two sites are completely conserved, an additional site is conserved between MACV and JUNV, and the majority of glycans cluster at a specific side of the sheet (Fig. 5A). In contrast, solvent-accessible residues completely conserved across the New World HF arenaviruses are distributed in an almost complementary pattern across the surface (Fig. 5B). We suggest that the area containing the carbohydrate cluster is likely to be structurally variable among New World HF arenaviruses and that surfaces involved in receptor binding and interaction with GP2 are likely to lie outside this region, perhaps focused on the regions of the surface containing the conserved residues (Fig. 5B).
Conservation of amino acid sequence and N-linked glycosylation sites across New World HF arenaviruses mapped onto the structure of MACV GP1 (gray cartoon). (A) N-linked glycosylation sites from MACV GP1, JUNV GP1, GTOV GP1, SABV GP1, and CAPV GP1 mapped as spheres on the structure of MACV GP1. Spheres colored yellow are conserved across all viruses, and spheres colored green are not conserved. Residues that are clearly visible are labeled according to residue number. (B) Conserved amino acids which are solvent exposed (calculated by ESPRIPT ) (Fig. 3E) from MACV GP1, JUNV GP1, GTOV GP1, SABV GP1, and CAPV GP1 mapped as spheres on the structure of MACV GP1. Spheres colored orange correspond to residues conserved across all New World HF arenaviruses.
The crystal structure of the MACV GP1 reported herein represents the first structure of a New World arenavirus GP1. In agreement with previous phylogenetic studies (8, 11, 14), our structure-based analysis of conserved disulfide bonds and predicted N-linked glycosylation suggests that MACV GP1 is likely to have the greatest structural similarity to the JUNV GP1. More generally, given the conserved tropism of both pathogenic and nonpathogenic New World clade B arenaviruses for TfR1-expressing cells (1), we propose that this new protein fold defines the architecture of New World arenavirus attachment glycoproteins. The origins of this protein fold are unknown; it bears no relation to the host ligand of TfR1, transferrin. However, we note that the GPC gene is antisense, if we consider the Arenaviridae to be aberrant negative-sense RNA viruses. We suggest that either the antisense protein GP1 may have been recruited from the host (although the structure of GP1 does not resemble any seen so far in a host protein) or it may have originated de novo relatively recently, which might explain the large divergence in sequence between GP1s from different viral isolates (10).
Coordinates and structure factors have been deposited with the Protein Data Bank (PDB code 2WFO).
We thank W. Lu for technical assistance with tissue culture, K. Harlos for assistance with soaking experiments and data collection, Lukasz Jaroszewski for help with MACV GP1 fold assignment, and the staff of ID14 EH2 and BM14 at the ESRF.
This work was funded by the UK Medical Research Council, the Wellcome Trust, and the European Commission as SPINE2 Complexes (FP6-RTD-031220). S.C.G. was a Nuffield Medical Fellow, and E.Y.J. is a Cancer Research UK Principal Research Fellow.
↵▿ Published ahead of print on 3 June 2009.
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