5 research outputs found

    AMAV and TCRV GPs bind and use the TfR1 orthologs of their respective host species.

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    <p>(A) HEK293T cells were transfected with plasmids encoding human TfR1 (hTfR1), <i>N. spinosus</i> TfR1 (NsTfR1), or <i>A. jamaicensis</i> TfR1 (AjTfR1). Cells were incubated 48 hr later with an α-flag antibody or Ig-fusion proteins comprising the truncated GP1 subunits <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000358#ppat.1000358-Radoshitzky1" target="_blank">[32]</a> of AMAV and TCRV (AMAV GP1Δ-Ig and TCRV GP1Δ-Ig, respectively). The association of these proteins with cells was measured by flow cytometry. The data shown are representative of two independent experiments, duplicated in each assay, with similar results. Mean fluorescence values for TfR1 ortholog expression were 435, 380, and 458 for human, Ns, and AjTfR1, respectively. Mean fluorescence values for AMAV GP1Δ-Ig binding to transfected cells were 1.5, 597, and 1.6, and those for TCRV GPΔ-Ig binding were 3.7, 425, and 553 for human, Ns, and AjTfR1, respectively. (B) HEK293T cells were transfected with plasmids encoding human, ZbTfR1, NsTfR1, AjTfR1 orthologs or vector alone. Cell surface expression of the TfR1 orthologs was determined as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000358#ppat-1000358-g003" target="_blank">Figure 3</a>. Aliquots of these cells were infected with AMAV, TCRV, or VSV pseudoviruses, and infection levels were assessed as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000358#ppat-1000358-g002" target="_blank">Figure 2</a>. (C) An experiment similar to the one performed in (B), except CHO cells were used for transfection and infected with MACV, JUNV, GTOV, or VSV pseudoviruses. Expression levels of the various TfR1 orthologs were normalized to that of human TfR1 (α-flag, left panels). Infection levels were normalized to that of mock-transfected cells.</p

    Modest mutations convert human TfR1 into an efficient receptor for AMAV and TCRV.

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    <p>(A) The structure of the human TfR1 dimer is shown, oriented with the cell membrane at the bottom. The apical, protease-like, and helical domains are indicated in green, red, and yellow, respectively, on one monomer. The other monomer is shown in cyan. In the right panel, the TfR1 apical domain is enlarged; a loop comprising residues 202-212, implicated as a site of interaction with the GPs of NW clade B arenaviruses, is shown. The side chains of residues D204, K205, R208, V210, and Y211 are colored yellow. The image was rendered using PyMol <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000358#ppat.1000358-DeLano1" target="_blank">[59]</a>. (B) Sequence alignment of residues 195 through 216 of human TfR1 with analogous sequences of the TfR1 orthologs of <i>Z. brevicauda</i> (ZbTfR1), <i>A. jamaicensis</i> (AjTfR1), and <i>N. spinosus</i> (NsTfR1). Variants of human TfR1 containing sequence from <i>Z. brevicauda</i> (zh1 and zh2), <i>A. jamaicensis</i> (ah2 through ah5), and <i>N. spinosus</i> (nh2, nh4, nh5, nh7, and nh8) TfR1 were generated based on this sequence alignment. Zb, Aj, and NsTfR1 sequences are shown in green, blue, and yellow, respectively. The right panel summarizes the entry data. ND = not determined. (C–E) HEK293T cells were transfected with plasmids encoding human, Zb, Aj, or NsTfR1 along with the zh variants (C), the ah variants (D), or the nh variants (E). The expression level of each TfR1 variant was assessed as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000358#ppat-1000358-g003" target="_blank">Figure 3</a>. In parallel, cells were infected with AMAV, TCRV, or VSV pseudoviruses. The expression levels of the various TfR1 orthologs were normalized to that of human TfR1 (α-flag, left panels), and infection levels were normalized to that of mock-transfected cells.</p

    Host-animal orthologs of TfR1 support the entry of infectious AMAV and TCRV.

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    <p>HEK293 cells were transfected with plasmids encoding human TfR1, AjTfR1, or NsTfR1, and infectious viruses added at 36 hr post-transfection. Incubation was continued for 12 hr, and the cells were washed, fixed, and stained as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000358#s4" target="_blank">Materials and Methods</a>.</p

    AMAV and TCRV pseudovirus entry does not depend on human TfR1.

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    <p>HEK293T cells were infected with GTOV, AMAV, TCRV, or MACV pseudoviruses expressing GFP in the presence or absence of the indicated concentrations of an α-human TfR1 (BD Pharmingen) or a control (α-HLA) antibody (BD Pharmingen). Infection levels were assessed 48 hr later by measuring GFP expression by flow cytometry. Mean fluorescence values were normalized to that of cells infected in the absence of antibody. GFP mean fluorescence values for virus entry in the absence of antibody were 144.0, 89.7, 92.3, and 115.0 for GTOV, AMAV, TCRV, and MACV, respectively.</p

    AMAV and TCRV pseudoviruses can use animal orthologs of TfR1.

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    <p>CHO cells ((A), left panel) were transfected with vector alone (mock) or plasmids encoding human, mouse, rat, feline, canine, <i>C. callosus</i> (Cc), <i>C. musculinus</i> (Cm), and <i>Z. brevicauda</i> (Zb) TfR1 orthologs. HEK293T cells ((B), right panel) were transfected with the same plasmids with the exception of the one encoding canine TfR1. Cell surface expression was determined 48 hr later by flow cytometry using an antibody directed against a flag tag present at the C-terminus of each ortholog. In parallel, aliquots of these cells were infected with AMAV, TCRV, or LCMV pseudoviruses. Infection levels were assessed as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000358#ppat-1000358-g002" target="_blank">Figure 2</a>. Expression levels of various TfR1 were normalized to that of human TfR1 (α-flag, top panels). Infection levels were normalized to that of mock-transfected cells.</p
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