Receptor determinants of zoonotic transmission of New World hemorrhagic fever arenaviruses

Abstract

Transferrin receptor 1 (TfR1) is a cellular receptor for the New World hemorrhagic fever arenaviruses Machupo (MACV), Junín (JUNV), and Guanarito (GTOV). Each of these viruses is specifically adapted to a distinct rodent host species, but all cause human disease. Here we compare the ability of these viruses to use various mammalian transferrin receptor 1 (TfR1) orthologs, including those of the South American rodents that serve as reservoirs for MACV, JUNV, and GTOV (Calomys callosus, Calomys musculinus, and Zygodontomys brevicauda, respectively). Retroviruses pseudotyped with MACV and JUNV but not GTOV glycoproteins (GPs) efficiently used C. callosus TfR1, whereas only JUNV GP could use C. musculinus TfR1. All three viruses efficiently used Z. brevicauda TfR1. TfR1 orthologs from related rodents, including house mouse (Mus musculus) and rat (Rattus norvegicus), did not support entry of these viruses. In contrast, these viruses efficiently used human and domestic cat TfR1 orthologs. We further show that a local region of the human TfR1 apical domain, including tyrosine 211, determined the efficiency with which MACV, JUNV, and GTOV used various TfR1 orthologs. Our data show that these New World arenaviruses are specifically adapted to the TfR1 orthologs of their respective rodent hosts and identify key commonalities between these orthologs and human TfR1 necessary for efficient transmission of these viruses to humans.

Fig. 1.

New World arenavirus entry mediated by TfR1 orthologs. (A) CHO cells were transfected with plasmids encoding TfR1 orthologs from human (hTfR1), M. musculus (mTfR1), R. norvegicus (rTfR1), C. familiaris (cTfR1), F. domesticus (fTfR1), C. callosus (ccTfR1), or C. musculinus (cmTfR1) tissues. Cell surface expression was determined by flow cytometry using an anti-FLAG antibody recognizing a tag present at the C terminus of each of the TfR1 variants. (B) In parallel, transfected cells were incubated with 200 nM of the MACV GP1 truncation variant, GP1Δ, fused to the Fc domain of human IgG1 (GP1Δ-Fc), and TfR1 association was determined by flow cytometry. Mean fluorescence values were normalized to those of hTfR1. Error bars indicate the standard deviation of five experiments. (C) An aliquot of the cells used in A and B were transduced with MLVs expressing enhanced GFP and pseudotyped with the glycoproteins of MACV, JUNV, or GTOV. Forty-eight hours after transduction, cell entry was measured by flow cytometry. Mean fluorescence values were normalized to those of hTfR1-expressing cells. Error bars indicate the standard deviation of five experiments. (D) An experiment similar to that in A, except that CHO cells were transfected with plasmid encoding the Z. brevicauda TfR1 ortholog (zbTfR1), as well as those encoding hTfR1, mTfR1, ccTfR1, and cmTfR1. (E) An experiment similar to that in C in which cell entry efficiency was measured by using an aliquot of the cells used in D.

Fig. 2.

MACV, JUNV, and GTOV entry mediated by chimeras of human and mouse TfR1. (A) A representation of the TfR1 structural domains and human/mouse TfR1 chimeras. In the top bar, the protease-like, apical, and helical domains of human TfR1 are indicated as blue, red, and cyan, respectively. The N-terminal cytoplasmic domain and transmembrane domain are shown in white. Individual mouse/human chimeras are represented in gray, indicating murine sequence, and in white, indicating human sequence. A plus sign to the right of each chimera indicates efficient MACV GP1Δ-Fc association and MACV, JUNV, and GTOV GP-mediated entry, shown in C and D. (B) CHO cells were transfected with plasmids encoding hTfR1, mTfR1, and chimeras of these receptors. Cell surface expression was analyzed as in Fig. 1A. Mean fluorescence values were normalized to hTfR1. Error bars indicate the standard deviation of three experiments. (C) In parallel, cell surface binding of MACV GP1Δ-Fc was determined by flow cytometry, as in Fig. 1B. Mean fluorescence values were normalized to those of hTfR1-expressing cells. Error bars indicate the standard deviation of three experiments. (D) An aliquot of the cells used in B and C was transduced with MACV, JUNV, or GTOV pseudoviruses and analyzed as in Fig. 1C. Mean fluorescence values were normalized to hTfR1. Error bars indicate the standard deviation of three experiments.

Fig. 3.

TfR1 determinants of zoonotic transmission of New World hemorrhagic fever arenaviruses. (A) The structure of the human TfR1 dimer is shown oriented with the cellular membrane at the bottom. Protease-like, apical, and helical domains are indicated as blue, red, and cyan, respectively, on one monomer. The other monomer is shown in white. A loop composed of residues 208–212, critical for New World arenaviral entry, is indicated in green. (B) As in A, except that human TfR1 dimer is rotated 150° about the twofold axis of the dimer. (C) As in B, except that the apical domain is enlarged. Aspartic acid 204, corresponding to a potential glycosylation site in the Calomys species, is indicated in blue. Asparagine 348, necessary together with residues 208–212 to convert mTfR1 to an efficient MACV and GTOV receptor, is shown in yellow. Tyrosine 211, within the 208–212 loop, is also indicated. (D) An alignment of amino acid sequences from two proximal regions of the indicated TfR1 orthologs. Human TfR1 residues that convert mouse TfR1 to an efficient MACV, JUNV, and GTOV receptor are underlined. Tyrosine 211 and lysine 348 are shown in green and red, respectively. Potential glycosylation sites present in Calomys species and Z. brevicauda TfR1 orthologs are indicated in blue. Macaca mulatta (rhesus macaque) and Cricetulus griseus (Chinese hamster) TfR1 regions are shown with those of the TfR1 orthologs characterized here. Rhesus macaques can be used as a model for MACV and JUNV infection (46, 47, 52). Hamster CHO and BHK cell lines are refractory to entry mediated by MACV, JUNV, and GTOV GP (28, 41).

Fig. 4.

The arenavirus GP1-binding site of human TfR1. (A) CHO cells were transfected with plasmids encoding hTfR1, mTfR1, or human TfR1 variants N292E, N348K, or Y211T. Cell surface expression was analyzed as in Fig. 1. Mean fluorescence values were normalized to those of hTfR1-expressing cells. Error bars indicate the standard deviation of three experiments. (B) In parallel, cell surface binding of MACV GP1Δ-Fc was determined by flow cytometry. Mean fluorescence values were normalized to those of hTfR1-expressing cells. Error bars indicate the standard deviation of three to five experiments. (C) An aliquot of the cells used in A and B was transduced with MACV, JUNV, or GTOV pseudoviruses and analyzed as in Fig. 1C. Mean fluorescence values were normalized to hTfR1. Error bars indicate the standard deviation of three experiments.

Fig. 5.

Conversion of house mouse TfR1 to an efficient arenavirus receptor. (A) CHO cells were transfected with plasmids encoding hTfR1, mTfR1, and mTfR1 mutants (RLVYL, K348N, and RLVYL+K348N). Cell surface expression was analyzed as in Fig. 1. Mean fluorescence values were normalized to hTfR1. Error bars indicate the standard deviation of three experiments. (B) In parallel, cell surface binding of MACV GP1Δ-Fc was determined by flow cytometry. Mean fluorescence values were normalized to hTfR1. Error bars indicate the standard deviation of three to five experiments. (C) An aliquot of the cells used in A and B was transduced with MACV, JUNV, or GTOV pseudoviruses and analyzed as in Fig. 1C. Mean fluorescence values were normalized to hTfR1. Error bars indicate the standard deviation of three experiments.

Fig. 6.

Effect of C. callosus and C. musculinus asparagine 205 glycosylation on MACV, JUNV, and GTOV entry. (A) CHO cells were transfected with plasmids encoding hTfR1, C. callosus TfR1 (ccTfR1), C. musculinus TfR1 (cmTfR1), or N205A glycosylation mutants (ccTfR1 N205A or cmTfR1 N205A). Cell surface expression was analyzed as in Fig. 1. Mean fluorescence values were normalized to hTfR1. Error bars indicate the standard deviation of three experiments. (B) An aliquot of the cells used in A was transduced with MACV, JUNV, or GTOV pseudoviruses and analyzed as in Fig. 1C. Mean fluorescence values were normalized to hTfR1. Error bars indicate the standard deviation of three experiments.