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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Nat Struct Mol Biol. Author manuscript; available in PMC 2010 October 1.
Published in final edited form as:
PMCID: PMC2920743
NIHMSID: NIHMS223152

Structural basis for receptor recognition by New World hemorrhagic fever arenaviruses

Abstract

New World (NW) hemorrhagic fever arenaviruses are rodent-borne agents that cause severe human disease. The GP1 subunit of the surface glycoprotein (GP) mediates cell attachment through transferrin receptor 1 (TfR1). We report the structure of Machupo virus (MACV) GP1 bound with human TfR1. Atomic details of the GP1:TfR1 interface clarify the importance of TfR1 residues implicated in NW arenavirus host specificity. Analysis of sequence variation among NW arenavirus GP1s and their host-species receptors in light of the molecular structure indicates determinants of viral zoonotic transmission. Infectivities of pseudoviruses in cells expressing mutated TfR1 confirm that contacts at the tip of the TfR1 apical domain determine the capacity of human TfR1 to mediate infection by particular NW arenaviruses. We propose that NW arenaviruses pathogenic to humans fortuitously acquired affinity for human TfR1 during adaptation to TfR1 of their natural hosts.

Keywords: arenavirus, transferrin receptor 1, hemorrhagic fever, pathogenic, host animal, rodent

Introduction

Arenaviruses have enveloped virions and single-stranded, bi-segmented, ambisense RNA genomes1. They fall into two groups, the “Old World” and “New World” (NW) arenaviruses; the latter are further divided into clades A, B, C, and a recombinant A/B clade24. Several NW clade B arenaviruses cause hemorrhagic fevers when transmitted from their host species to humans. These include the Machupo (MACV), Junín (JUNV), Guanarito (GTOV), and Sabiá (SABV) viruses, the agents of Bolivian, Argentine, Venezuelan, and Brazilian hemorrhagic fevers, respectively, all with high case fatality rates57. Chapare virus is a novel NW clade B hemorrhagic fever arenavirus recently isolated in Bolivia8.

The arenavirus envelope glycoprotein (GP) is the sole protein on the virion surface. During its maturation, GP is processed into three non-covalently associated subunits; the stable signal peptide (SSP), GP1, and GP2 (ref. 9). Unlike most signal peptides, SSP remains on the surface of virions and participates in maturation and pH-dependent membrane-fusion activity of the GP complex1013. The GP1 subunit interacts with cellular receptor(s), and GP2 mediates membrane fusion after virus particles are internalized into acidified endosomes1416.

The cellular receptor for the NW hemorrhagic fever arenaviruses MACV, JUNV, GTOV and SABV, is transferrin receptor 1 (TfR1)1719. TfR1 is a ubiquitously expressed, homodimeric, type II transmembrane glycoprotein that assists iron uptake into cells through endocytosis of iron-loaded transferrin20. The dimeric ectodomain of human TfR1 (residues 89–760) is a butterfly-shaped molecule with three subdomains: a “protease-like” domain, related in structure to certain Zn-metalloproteases, an “apical domain”, and a “helical domain”, which makes the principal dimer contacts21. Transferrin interacts with the protease-like and helical domains22, but does not interfere with the entry of MACV17. Mutational analysis implicates the TfR1 apical domain as the principal site of interaction with NW arenaviral GPs, and a short segment in the TfR1 apical domain (residues 208–212) is a critical determinant of virus host specificity18,23.

TfR1 orthologs from the natural host species of all clade B NW arenaviruses we have tested (MACV, JUNV, GTOV, AMAV, and TCRV) are functional cellular receptors for the corresponding virus, but human TfR1 (hTfR1) mediates entry only of the three that are human pathogens (Table 1)1719,23. Thus, there is a strong correlation between the capacity of hTfR1 to mediate infection and the capacity of a particular NW clade B arenavirus to cause human disease19,23.

Table 1
Receptor usage by NW clade B arenaviruses1719,23

To examine the structural basis for hTfR1 recognition by NW clade B arenaviruses, we have determined the crystal structure of MACV GP1 complexed with the ectodomain of hTfR1. As anticipated, MACV GP1 binds TfR1 through an extensive network of contacts with the apical domain. The atomic details of the GP1:TfR1 interface clarify the importance of several receptor residues implicated in NW arenavirus host specificity. On the viral glycoprotein side of this interface, there is substantial amino-acid sequence variability among GP1 proteins that bind the human receptor. We correlate this variability with differences among the sequences of the host-species TfR1 orthologs, which can serve as efficient receptors for the corresponding virus18,23. The specificities of the viral glycoproteins reflect long-term adaptation of the viruses in their natural hosts24; our data suggest that the NW hemorrhagic fever arenaviruses independently and fortuitously acquired their ability to bind human TfR1 and hence to cause human disease.

Results

Structure determination

Recombinant MACV GP1 (residues 79–258) was expressed in insect cells with a His6-tag at the C-terminus and purified using nickel-affinity chromatography, then treated with elastase to remove the His6-tag and five N-terminal residues, and further purified by gel filtration. A stably-expressing Chinese hamster ovary (CHO) cell line was used to express a soluble form of the ectodomain of hTfR1 (residues 117–760), which was purified as described previously21. By SDS-PAGE analysis, the MACV GP1 and hTfR1 proteins migrated with molecular masses consistent with their full glycosylation (Supplementary Fig. 1). The proteins, mixed in equimolar ratio, crystallized in the space group C2221, with unit cell dimensions a=140 Å, b=173 Å, and c=104 Å. We determined the structure of the GP1:hTfR1 complex using molecular replacement, with the hTfR1 monomer (PDB ID: 1CX8)21 as a search model and refined it using data extending to 2.4 Å (but incomplete beyond 2.7 Å: Table 2). The final model of the GP1:hTfR1 complex contains residues 121–756 of the receptor, except for residues 316–319, which are disordered, and residues 86–241 of MACV GP1, with N-linked glycans at TfR1 positions 251 and 727, and at GP1 positions 95, 137, 166, and 178.

Table 2
Data collection and refinement statistics (molecular replacement)

Complex of MACV GP1 with human TfR1

MACV GP1 contacts the tip of the apical domain of TfR1, with 1,900 Å2 of total surface area buried at the interface (Fig. 1). The apical domain of TfR1 is an insertion between the first and second strands in the central β-sheet of the protease-like domain. It resembles a β-sandwich in which the two sheets are splayed apart, with a helix (αII-2) running along the open edge, a fold similar to domain 4 of aconitase21. Helix αII-2 and strand βII-2 in the apical domain form most of the contacts with MACV GP1 (Fig. 1b). Residues 208–212, built as an extended loop in previous structures of hTfR121,25, have a β-strand conformation in our structure of the complex, and form an extension of strand βII-2. This segment was poorly defined in previous structures of hTfR121,25, which were determined to lower resolution (3.1 Å and 2.9 Å), and re-analysis of the electron density in the unliganded hTfR1 structure (PDB ID 1CX8)21 in light of our higher-resolution model shows that residues 208–212 in unliganded hTfR1 adopt a very similar β-strand conformation (Supplementary Fig. 2a). Thus, GP1 binding induces no major main-chain distortions in the TfR1 apical domain. It does, however, increase order in the extended βII-2 strand, displace the backbone and change the orientation of Leu209, and switch the rotameric state of Tyr211 (Supplementary Fig. 2b).

Figure 1
Structure of the MACV GP1:human TfR1 complex. (a) The butterfly-shaped TfR1 dimer is shown oriented with the cell surface at the bottom. One TfR1 protomer is colored according to the TfR1 domains: the apical domain is green, the protease-like domain is ...

The core of MACV GP1 is a seven-stranded anti-parallel β-sheet (β1–7), with five interconnecting α-helical segments (αA to αE) (Fig. 2)26. Four N-linked glycans decorate surfaces of the viral glycoprotein not involved in receptor binding. We could interpret density for seven sugar residues on Asn178, three (including a fucosyl group) on Asn166, and a single N-acetyl glucosamine (NAG) at the remaining two positions (Asn95 and Asn137). Several extended loops protrude from the exposed, concave face of the MACV GP1 core to contact TfR1 (Fig. 2c). The loop between β-strands 3 and 4 (loop 3, residues 113–123) contacts the βII-2 strand in the apical domain of hTfR1; a second loop (loop 10, residues 220–234), which is disulfide-linked and contains a short α-helical segment, packs against the opposite side of theβII-2 strand. Loop 10 is longer in MACV (15 residues) than in the GPs of other NW arenaviruses; the other GP1s have only a short segment (5–6 residues, Fig. 2b). A third loop (loop 7, residues 165–174, between β-strands 5 and 6), containing the glycan at Asn166, also contacts TfR1 (Fig. 2c).

Figure 2
MACV GP1. (a) Domain organization of the MACV envelope glycoprotein (GP). GP is processed during its maturation into three non-covalently associated subunits: the stable signal peptide (SSP), GP1, and GP2. Sites of potential N-linked glycosylation are ...

MACV GP1:hTfR1 interface

We have analyzed the interface between MACV GP1 and the apical domain of TfR1 in terms of five interaction motifs, each anchored by a key residue on the receptor surface. Three of these five TfR1 residues – Tyr211, Asn348, and Val210 – have previously been implicated as determinants of NW arenavirus host specificity18,23.

Motif 1

Tyr211 in the apical domain of TfR1 is a critical determinant of MACV, JUNV, and GTOV cell entry18. This residue, conserved in all orthologs of TfR1 that support the entry of NW arenaviruses, is a central feature of the GP1:TfR1 interface (Fig. 3a). Tyr211 is found within the hTfR1βII-2 strand and fits snugly in a pocket on the viral glycoprotein. The hydroxyl group of TfR1 Tyr211 is hydrogen bonded with MACV GP1 Ser113, and its aromatic ring is surrounded by the side chains of MACV GP1 Arg111, Ile115, and Val117. The side chain of MACV GP1 Arg111 reaches past the aromatic ring to form a hydrogen bond with the main chain carbonyl of TfR1 Val210.

Figure 3
Five interaction motifs in the MACV GP1:human TfR1 interface. (a) Motif 1, centered on Tyr211 on the βII-2 strand in the receptor. (b) Motif 2, centered on TfR1 Asn348, which caps the C-terminus ofαII-2 helix. (c) Motif 3, centered on ...

Motif 2

Asn348 participates in a well-defined hydrogen-bonding network at the GP1-receptor interface (Fig. 3b). Asn348 caps the C-terminus of αII-2 with a hydrogen bond to the backbone carbonyl of Lys344. It participates in an extensive hydrogen-bonding network with MACV GP1 residues Asp114 and Ser116, and TfR1 residues Ser370 and Lys371. Mutation of TfR1 Asn348 to lysine interferes with the entry of MACV, GTOV, and in some contexts JUNV into cells expressing the altered receptor18.

Motif 3

Val210 participates in hydrophobic interactions with the side chains of Phe226 and Tyr228 on MACV GP1 (Fig. 3c). These MACV GP1 residues both lie within loop 10, the disulfide-linked GP1 insert unique to MACV. The hydroxyl group of MACV GP1 Tyr228 hydrogen bonds with the main-chain amide of TfR1 Val210, and in addition to a side chain contact with Val210, MACV GP1 Phe226 is in van der Waals contact with receptor side chains of Ile201 and Leu212. MACV GP1 Pro223, also in loop 10, is in van der Waals contact with the δ-carbon of TfR1 Leu212. Mutation of Val210 in hTfR1 to glycine allows the altered receptor to support entry of TCRV, a nonpathogenic NW arenavirus that is closely related to MACV and JUNV, but does not otherwise use hTfR123. We have not found a direct explanation for the effect of the Val210Gly mutation on hTfR1 usage by TCRV. TfR1 Val210 is well anchored within a β-strand; substitution with a glycine may nonetheless allow sufficient local flexibility to compensate for some other, unfavorable aspect of the TCRV:hTfR1 interface.

Motif 4

Lys344, in the TfR1 αII-2 helix, packs against MACV GP1 Met119 and participates in a hydrogen-bonding network that includes MACV GP1 Asp123 and Lys169, and TfR1 Glu294 (Fig. 3d). TfR1 Glu343, which precedes Lys344 in the αII-2 helix, accepts a hydrogen bond from the phenolic hydroxyl of MACV GP1 Tyr122. The Tyr122 ring also contacts TfR1 Ala293, Glu294, and Ala340, and Glu294 in turn has a salt bridge with GP1 Lys169. There is an additional hydrogen bond between the side chain of MACV GP1 Glu171 and the backbone amide of TfR1 Asn215. The contacts of motif 4 thus extend to the TfR1 loops between βII-2/βII-3 and βII-6/βII-7 at the base of the apical domain.

Motif 5

Leu209 of TfR1 is in van der Waals contact with MACV GP1 Phe98 and the β-carbon of MACV GP1 Ser97 (Fig. 3e). Two nearby TfR1 residues – Arg208 and Asp204 – have been implicated in the host specificity of NW arenaviruses. TfR1 Asp204 is replaced by a N-linked glycosylation site in the rodent host-species TfR1 orthologs (Supplementary Fig. 3), and removal of this glycan enhances the entry of MACV, JUNV, and GTOV on the altered receptor18. Arg208, at the tip of the apical domain, is the first residue in the TfR1 βII-2 strand. In addition to the Val210Gly mutation described above (motif 3), the Arg208Gly mutation when introduced into hTfR1 also allows TCRV to use the altered receptor23. This change probably increases flexibility or adaptability of the loop between βII-1 and βII-2 of hTfR1.

Conservation of the GP1:TfR1 interface

We have examined the amino-acid sequences and specificities of known NW arenavirus GP1s, in view of the interface analysis just described. The sequences of MACV, JUNV, GTOV and SABV GP1 diverge substantially – based on the alignment shown in Figure 2b, the pairwise identities of MACV GP1 residues 87–242 with the corresponding GP1 sequences of JUNV, GTOV and SABV are 46%, 27%, and 25%, respectively – but hTfR1 is a cell-entry receptor for all three viruses17,19. TCRV (41% identify with MACV GP1) can use hTfR1 when either Arg208 or Val210 in the hTfR1 βII-2 strand is mutated to glycine, suggesting that modest changes in its GP1 protein might also allow it to bind human TfR123. AMAV (29% GP1 sequence identity with MACV) can use hTfR1 when four mutations are made to the βII-1/βII-2 loop and βII-2 strand in the receptor23. Thus, all six GP1 receptor-binding domains appear to be complementary to the same surface. Location of conserved residues in GP1 justifies the further assumption that despite considerable sequence divergence, all NW arenavirus GP1s have the same framework structure.

MACV GP1 has two long-range disulfide bonds (one between cysteines 92 and 237, bounding the entire domain, and the other, between cysteines 135 and 164, bridging across the hydrophobic core) and two short-range disulfides (207 to 213 and 220 to 229, stabilizing local loops). The alignment in Figure 2b shows that the two long-range disulfide bonds are conserved, and that the short-range disulfides are unique to MACV. The alignment predicts that GTOV GP1 will have an additional long-range disulfide between 120 and 163 (MACV numbering) and that AMAV GP1 will have a long-range disulfide between 116 and 213 (MACV numbering). Both disulfide pairings are fully compatible with a conserved fold. SABV GP1 has a short insertion following residue 107 (MACV numbering) with an unpaired cysteine, which projects away from the TfR1 interface and probably bridges to a cysteine elsewhere in the viral glycoprotein.

GP1 residues contacting TfR1

Figure 4a shows the viral side of the GP1 interface, with GP1 residues grouped according to the five interaction motifs. In viral contributions to interaction motif 1, the hydrogen bond between MACV GP1 Ser113 and the phenolic hydroxyl group of TfR1 Tyr211 can also be made by the Thr at that position in SABV and by the Asp in JUNV and TCRV GP1 (Fig. 4b). GTOV and AMAV, however, have a hydrophobic residue at the equivalent of 113; a Glu nearby (replacing MACV GP1 Arg111) might accept the hydrogen bond instead. The residue at GP1 position 115, which lines the pocket occupied by TfR1 Tyr211, is Ile in MACV and JUNV, Leu in AMAV, and Val in TCRV, GTOV, and SABV. Residue 117, which contributes the other hydrophobic side chain lining the GP1 pocket for TfR1 Tyr211, is Val in MACV, JUNV, TCRV, and AMAV, Leu in SABV, and Pro in GTOV. MACV GP1 Arg111, which makes prominent contacts with the TfR1 βII-2 strand, is not conserved in any of the other viruses. In motif 2, TfR1 Asn348 has reciprocal hydrogen-bond interactions with the main-chain amide and carbonyl of MACV GP1 Ser116—the side chain of this residue does not participate, nor is it conserved in any of the GPs. Interaction motif 3 is unique to MACV as it involves the insert in loop 10. In motif 4, three of five MACV GP1 residues that contact TfR1 are conserved in the other clade B1 viruses TCRV and JUNV, and four of five are conserved between MACV and JUNV. Substantial variability is seen in this motif, however, when GTOV, AMAV, and SABV are compared with the clade B1 viruses. In motif 5, Phe98 is replaced by a more polar residue in the other viruses, as might be expected, since absence of loop 10 would leave this phenylalanine substantially more exposed.

Figure 4
Conservation of the GP1:TfR1 interface. (a) Ribbon diagram of MACV GP1, as seen from the apical domain of TfR1 in the complex. GP1 residues that contact TfR1 are colored according to the interaction motifs shown in Figure 3, using the color scheme outlined ...

TfR1 residues contacting GP1

The NW hemorrhagic fever viruses vary in their capacity to infect cultured cells expressing the TfR1 orthologs from the natural hosts (Table 1)18,23. For example, MACV can use its host-species TfR1 ortholog (CcTfR1) as a receptor, but not that of the JUNV host species (CmTfR1)18. GTOV can use only the TfR1 ortholog of its host (ZbTfR1) and that of the closely related NW arenavirus AMAV (NsTfR1)18,23. The restricted receptor usage of each virus clearly reflects a relatively long period of virus-host co-adaptation24.

Figure 4c shows the receptor side of the GP1:TfR1 interface. TfR1 Tyr211, the central feature of motif 1 (Fig. 3a), is conserved in all the host-species receptors (Fig. 4d). TfR1 Asn348, which anchors the second interaction motif (Fig. 3b), is likewise conserved, except in CmTfR1 (JUNV host), which has a lysine at position 348. MACV cannot use CmTfR1 as a receptor, and the Asn348Lys substitution in the context of hTfR1 consistently disrupts the entry of MACV and GTOV into cells expressing this altered receptor18. The lysine at 348 would disrupt the hydrogen-bond network that includes the C-cap on TfR1 αII-2 and the main-chain hydrogen bonds with MACV GP1 Ser116 (Fig. 3b). Presumably JUNV, in adapting to the receptor of its native host, has evolved to accommodate Lys348 in binding to CmTfR1. In motif 3, the interaction unique to MACV, most of the receptors present a hydrophobic surface (valine or leucine at 210, and leucine at 212: Fig. 3c). The exceptions are Cm and AjTfR1. The former has a serine at 210 and a proline at 212; the latter, a glycine at 210. Loop 10 of MACV GP1 engages this hydrophobic surface, with MACV GP1 Phe226 inserted between the side chains of TfR1 residues 210 and 212. The details of the contact suggest that Cγ of proline at 212 would clash with the Phe226 side chain, consistent with the failure of MACV to efficiently infect cells expressing CmTfR118; a glycine would not interfere, and MACV indeed infects AjTfR1-expressing cells efficiently23.

The anchoring residue of motif 4, TfR1 Lys344, is conserved in all receptors except that of the MACV host (CcTfR1); its substitution by asparagine could lead to plausible rearrangements of the hydrogen-bond and salt-bridge network at the contact (Fig. 3d). Another difference in motif 4 is TfR1 residue 294, which is Asp in the host receptor for MACV (and also those for JUNV, GTOV, and AMAV), but Glu in hTfR1. An aspartic-acid side-chain at this position cannot reach the ε-amino group of GP1 Lys169 (conserved in the clade B1 viruses), but the salt bridge can be replaced by one from TfR1 residue 340, which is Glu rather than Ala in precisely those receptors that have the aspartic-acid substitution at 294.

The preceding analysis of contact motifs at the extensive GP1:apical-domain interface illustrates that the sometimes substantial amino-acid sequence differences among the various viruses and receptors are nonetheless consistent with an overall retention of local conformation within each motif, as key contacts have conserved characteristics. Moreover, the conservation of contacts among the viruses and their native host-species receptors at the base of the apical domain (motif 4), and the conserved packing of GP1 residues around TfR1 Tyr211, some 20 Å away near the tip of the apical domain (motif 1), together indicate that GP1 will dock against TfR1 in essentially the same orientation and position in any productive virus-receptor pairing. The general consistency of the analysis indicates that the interface between alternate pairings of GP1 proteins and receptors varies only in detail, and not in the overall mode in which one partner docks against the other.

Recognition of human TfR1

Among the TfR1 sequences we have compared, Arg208 is a unique feature of the human receptor. The corresponding position is occupied by glycine in all NW arenavirus host-species receptors examined here, with the exception of the MACV host (CcTfR1), in which it is occupied by an asparagine (Fig. 4d). The Arg208Gly mutation converts hTfR1 into a receptor for TCRV23. Because most of the viruses are probably best adapted to the absence of a large side chain at the 208 position in TfR1, we asked whether an Arg208Gly change in hTfR1 also affects the infectivity of MACV, JUNV, and GTOV. We generated fusion proteins comprising the truncated GP1 subunits of MACV, JUNV, or GTOV fused to the Fc portion of human IgG (GP1Δ-Ig)17 and tested them for binding to CHO cells transfected with wt-hTfR1 or hTfR1-R208G. MACV GP1Δ-Ig bound CHO cells expressing either wt-hTfR1 or hTfR1-R208G, but bound the latter more strongly (Fig. 5a). JUNV and GTOV GP1Δ-Ig bound only cells expressing hTfR1-R208G. Lack of detectable binding by JUNV and GTOV GP1Δ-Ig to cells expressing wt-hTfR1 probably results from the low affinity of these proteins for the wild-type receptor. Consistent with these data, the R208G mutation modestly enhanced infectious entry of MACV pseudovirus, but led to substantial increases in infection by JUNV and GTOV pseudoviruses (Fig. 5b). The tip of the TfR1 apical domain (motif 5) is thus a determinant of entry by both pathogenic and nonpathogenic NW clade B arenaviruses. The rodent receptors all have a simple, four-residue β-turn in the βII-1/βII-2 loop, while human TfR1 has a five-residue turn. The pathogenic viruses (MACV, JUNV, and GTOV) have presumably adapted in their natural hosts to the shorter turn, which places a glycine at the 209 position instead of leucine. The substitution eliminates the hydrophobic contact we see between hTfR1 Leu209 and MACV GP1 Phe98 (Fig. 3e). The alternative interactions that we have suggested might be present in different virus-host pairs vary in character and position, and we therefore cannot readily predict from GP1 sequences alone how many amino-acid changes might be needed to make any particular nonpathogenic NW clade B arenavirus infectious in humans without compromising ability to propagate in its natural host.

Figure 5
Importance of the tip of the apical domain in determining the role of human TfR1 as a receptor for pathogenic NW arenaviruses. (a) CHO cells were transfected with vector alone (Mock) or plasmids encoding hTfR1 (Human), or hTfR1 with the Arg208Gly mutation ...

Discussion

The structure we have determined shows that MACV GP1 contacts the apical domain of TfR1. Although present in all mammalian TfR1s, the apical domain does not have a known physiological role: neither transferrin nor the hereditary hemochromatosis associated protein, HFE, interact with it22,25. As NW arenavirus GPs engage TfR1 in a manner that does not seem to interfere with binding of its known physiological ligands17, antibodies that target the TfR1 apical domain might in principle be useful as broadly-acting therapeutic agents for acute treatment of NW hemorrhagic fevers.

Our structure, when compared with the structure of unliganded MACV GP1 (ref. 26), shows that no significant conformational change occurs upon receptor binding (Cα r.m.s.d.=0.55). Receptor binding is therefore unlikely to prime GP for pH-induced membrane fusion before virions are internalized into endosomes, as seen, for example, with the avian leukosis virus27,28. The events during NW arenavirus cell entry may be more similar to those during entry of influenza viruses into cells, in which HA attachment to a cell surface receptor (sialic acid) serves primarily to traffic virions to acidified endosomes rather than to initiate a series of conformational rearrangements14.

The structure of MACV GP1 bound to human TfR1 reveals that NW clade B arenaviruses contact TfR1 on a surface with substantial sequence variability among viruses. This sequence variability, which presumably reflects the independent evolution of individual viruses in their various host-species24, is best illustrated by the insert in MACV loop 10, which forms extensive receptor contacts yet is much shorter and lacks these contacts in the other viruses (Fig. 3b). Nonetheless, a number of similarities among the GP1 receptor-binding domains strongly support the assumption of a shared fold within members of the family. Conservation of key receptor residues in several of the interaction motifs indicates conservation of local features of the interface, as well as of the overall docking of the two partners. The structure of the complex may therefore prove useful in predicting whether a novel NW arenavirus is likely to use TfR1. For example, extending the analysis shown in Figure 4 to the recently isolated NW hemorrhagic fever arenavirus Chapare suggests that this virus will indeed use TfR1 as its receptor (Supplementary Fig. 4)8.

The loop between the TfR1 βII-1 and βII-2 strands appears to be a key determinant of whether any particular GP1 can bind with adequate affinity to a TfR1 ortholog18,23. That loop is longer by one residue in human TfR1 than in its ortholog in any of the NW arenavirus native hosts, except for that of TCRV. Moreover, at the end of that loop, Arg208 in the human receptor becomes glycine in the host-species receptors (with the exception of the MACV host, where it is an Asn), and the Arg208Gly mutation enhances infectivity of JUNV and GTOV and allows infection by TCRV. The large arginine residue thus presents a barrier to use of human TfR1 by both nonpathogenic23 and pathogenic NW clade B arenaviruses (Fig. 5). The NW clade B arenaviruses appear to be adapted best to their host-species receptors. We suggest that those infecting humans accidently acquired the capacity to bind human TfR1 during co-evolution with the receptor in their natural hosts. The structure of MACV GP1 bound to hTfR1 thus clarifies the complex structural basis for the zoonotic transmission of this important class of emerging human pathogens.

Methods

Expression, purification, and crystallization of MACV GP1 and human TfR1

We expressed MACV GP1 (residues 79–258) with the addition of a His6-tag at the C-terminus in Sf9 insect cells and purified the protein by nickel affinity chromatography. We subjected the protein to limited proteolysis with elastase and further purified it by size exclusion chromatography. We purified the soluble ectodomain of hTfR1 as previously described21. We concentrated both proteins separately in buffer containing 25 mM Tris, pH 7.5, 150 mM NaCl, and mixed them in equimolar ratio to a final concentration of 10 mg ml−1. Crystals grew in 1.6 M NaKPO4, pH 5.5, 150 mM NaCl. We flash froze crystals in mother liquor containing 20% (v/v) glycerol for data collection.

Data collection and structure determination

The MACV GP1:hTfR1 complex crystallized in the C2221 space group, with unit cell dimensions a= 140 Å, b= 173 Å, and c= 104 Å. We collected X-ray diffraction data at 100 K° at NE-CAT beamline ID-24C at the Advanced Photon Source (Argonne National Laboratory). Because of anisotropic diffraction, we integrated the data using an ellipsoidal mask with resolution cutoffs of 2.4 Å along the a and b axes, and 2.7 Å along the c axis29. We processed the data using HKL2000 (HKL Research, Inc.), and determined the structure of the complex by molecular replacement with PHASER30, using the hTfR1 monomer (PDB ID: 1CX8)21 as a search model. We built the model into density with COOT31. Iterative model building and refinement with PHENIX32 yielded final Rfree of 24% and Rwork of 20% (Table 2); 96.8% of all residues were in the most favored region of Ramachandran space, and 99.9% (782/783 residues), in allowed regions. We calculated buried surface areas and simulated-annealing omit maps with CNS33 and made figures with PyMol34.

Sequence alignment

We used ClustalW35 to align the NW arenaviral GP1 and TfR1 sequences and adjusted the positions of gaps in the sequence alignments by referring to the three-dimensional structures. We selected the following GP sequences: AMAV (strain BeAn 70563, GenBank accession no. AF512834), Chapare virus (810419, NC_010562), GTOV (INH-95551, NC_005077); JUNV (MC2, D10072); MACV (Carvallo, NC_005078); SABV (SPH114202, NC_006317). The sequence of TCRV GP (a gift from Stefan Kunz, University of Lausanne, Switzerland) is that of a laboratory adapted strain, which has a deletion spanning residues 121–132, and substitutions at three residues (I134A, G418S and E458R) compared to GenBank NC_004293. We used the following TfR1 sequences: human TfR1 (NM_003234), C. callosus TfR1 (EU164540), C. musculinus TfR1 (EU164541), Z. brevicauda TfR1 (EU340259), A. jamaicensis TfR1 (FJ154605), N. spinosus TfR1 (FJ154604). We made figures and did pairwise sequence identity calculations using Jalview36,37.

Cells and plasmids

We maintained HEK293T cells (human embryonic kidney, ATCC, CRL-11268) in Dulbecco’s modified Eagle’s medium, and CHO cells (Chinese hamster ovary epithelial, ATCC, CCL-61) in F12 medium, in the presence of 10% fetal bovine serum. Plasmids encoding hTfR1 and hTfR1 with the R208G mutation, with the addition of a C-terminal flag tag, have been described previously23, as have MACV (Carvallo strain), JUNV (MC2), and GTOV (INH-95551) GP-expressor plasmids17.

GP1Δ-Ig binding assays

We generated MACV GP1Δ-Ig, JUNV GP1Δ-Ig, and GTOV GP1Δ-Ig by PCR amplification of MACV GP1 residues 87–258, JUNV GP1 residues 87–241, and GTOV GP1 residues 79–228. We cloned these fragments into a previously described pcDM8-based plasmid containing the CD5 signal sequence and the Fc region of human IgG138. The SARS-CoV RBD Ig-fusion protein has been described previously38. For Ig-fusion protein production, we transfected HEK293T cells with the appropriate plasmid and harvested the supernatant at 48 h post-transfection. For binding assays, we transfected CHO cells with hTfR1 or hTfR1 containing the R208G mutation. After 48 h, we detached the cells with 1 mM EDTA/phosphate buffered saline (PBS) and incubated them in 100 μl of PBS-2% goat serum containing 0.5 μg of an anti-flag M2 monoclonal antibody (Sigma), or supernatant containing 0.5 μg of the indicated Ig-fusion protein. We then stained the cells with anti-mouse IgG, or with anti-human IgG (Fc-specific) antibodies conjugated with phycoerythrin (Jackson Immuno Laboratories).

Pseudovirus infection

We generated recombinant retroviruses pseudotyped with arenaviral GPs by transfecting HEK293T cells in a 1:1:1 ratio of plasmids encoding the respective arenaviral GP, the MLV gag and pol genes, and the pQCXIX retroviral vector (BD Biosciences) expressing GFP, as previously described17. We harvested cell supernatants 24 h post-transfection and filtered them through a 0.45 μm filter. We transfected CHO cells with plasmids expressing wt-hTfR1 or hTfR1-R208G, and replated the cells at 24 h for flow cytometry and pseudovirus infection. At 48 h post-transfection, we assessed TfR1 expression levels by flow cytometry using an anti-flag M2 antibody. In parallel, the cells were infected with the pseudoviruses for 1 h. Forty-eight hours after transfection, we detached the cells with trypsin and measured the GFP expression levels by flow cytometry. We normalized GFP expression levels to that of mock-transfected cells.

Supplementary Material

Acknowledgments

We thank Marina Babyonyshev for help with protein production, Simon Jenni for advice and instruction on methods of structure determination, and the staff at NE-CAT (APS, Argonne National Laboratory) for assistance with X-ray data collection. The work was supported by NIH Grants CA13202 (to S.C.H.) and R01 AI074879 (to H.C.). S.C.H. is an investigator in the Howard Hughes Medical Institute. J.A. is a Howard Hughes Medical Institute Gilliam fellow.

Abbreviations

NW arenavirus
New World arenavirus
MACV
Machupo virus
JUNV
Junín virus
GTOV
Guanarito virus
SABV
Sabiá virus
TfR1
transferrin receptor 1
TCRV
Tacaribe Virus
AMAV
Amapari virus
GFP
green fluorescent protein

Footnotes

Accession codes

The coordinates for the structure of MACV GP1 bound to hTfR1 have been deposited in the Protein Data Bank with accession code 3KAS.

Author contributions

J.A. designed and performed the experiments, analyzed the data, and wrote the paper. K.D.C. assisted with data collection, molecular replacement, re-interpretation of the unliganded TfR1 structures, and edited the paper. M.F. and H.C. assisted with data analysis and interpretation and edited the paper. S.C.H. helped design experiments, advised on model building and interpretation, and participated in writing and editing the paper.

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