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

<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.

<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.

<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.

<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.

<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

TIM1-mediated pseudovirus internalization does not always coincide with TIM1-mediated entry enhancement.

<p>(A) Some pseudoviruses that do not use hTIM1 for productive entry are still efficiently internalized by hTIM1. Mock- or hTIM1-transduced 293T cells were infected for 2 h with purified, RT-activity normalized MLVgag-GFP pseudovirions. Uninternalized virions were detached by acid-stripping and extensive trypsinization, after which internalized virions were detected by flow cytometry. Data shown are representative of two independent experiments performed in duplicates. (B) hTIM1-mediated pseudovirus internalization is blocked by PS-containing liposomes. Mock- or hTIM1-transduced 293T cells were preincubated for 20 min at room temperature with medium alone (none), or with liposomes consisting of either 50% PS and 50% PC (PS/PC) or PC alone (PC). MLVgag-GFP pseudovirions, prepared as in A, were then added for a 2 h infection at 37°C, after which bound virions were detached and internalized virions were detected as in A. Figure shows mean+SD of two independent, duplicated experiments.</p

hTIM1-mediated entry of pseudoviruses and VLPs is PS dependent.

<p>(A) A hTIM1 variant deficient in PS binding (AA-hTIM1) does not support viral entry. 293T cells were transduced with hACE2 (mock), hTIM1 or AA-hTIM1, and infected 2 days later with the indicated pseudoviruses or WNV VLPs. Entry was determined by GFP-expression measured by flow cytometry. Figure depicts representative results from one of three independent, duplicated experiments. M.f.i.: mean fluorescent intensity. (B) In parallel with the infection in A, expression levels of hTIM1 and AA-hTIM1 were assessed as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003232#ppat-1003232-g001" target="_blank">Figure 1A</a>. (C) hTIM1-mediated virus entry enhancement is efficiently blocked by PS-containing liposomes. 293T cells transduced with hACE2 (mock) or hTIM1 were preincubated for 20 min at room temperature with medium alone (none) or with liposomes consisting of either 50% PS and 50% PC (PS/PC) or PC alone (PC). Pseudoviruses or WNV VLPs were then added for a 30 min infection at 37°C, after which unbound liposomes and viruses were washed away and cells supplemented with fresh medium. Entry was quantified as in A and normalized to those of untreated hTIM1-expressing cells. Figure shows mean+SD of three independent, duplicated experiments.</p

hTIM1 promotes infection mediated by a range of viral entry proteins.

<p>(A) Human 293T cells do not express TIM1. 293T and control cells were stained with anti-hTIM1 antibody (red) or with mFc (black). (B) Murine 3T3 cells do not express TIM1. 3T3 and control cells were stained with anti-mTIM1-PE antibody (red). Unstained cells (black) and cells stained with anti-mIFNγ-PE (blue) served as negative controls. (C) Exogenous hTIM1 increases the entry of various pseudoviruses in 293T cells. 293T cells were transfected with plasmids expressing hTIM1 or, as a negative control, hACE2. 48 h later cells were infected with MLV pseudoviruses or WNV VLPs, both carrying the GFP reporter gene. The following day, GFP expression was quantified by flow cytometry. Fold changes in entry were calculated by dividing mean fluorescence intensity observed in hTIM1-expressing 293T cells by those in hACE2-expressing 293T cells. Figure shows mean+SD from three independent, duplicated experiments. (D) hTIM1 usage by pseudoviruses was similarly assessed in 3T3 cells transduced with hTIM1 or hACE2. (E) Entry inhibition by an anti-hTIM1 antibody parallels hTIM1 use by pseudoviruses. 293T cells transduced with hACE2 (mock) or hTIM1 were preincubated for 30 min at room temperature with medium alone (none), the anti-hTIM1 antibody 3D1 or mIgG, and infected with pseudoviruses overnight in the presence of the respective blocking agents. Infection levels were normalized to those of untreated hTIM1-expressing cells. Figure shows mean+SD from two independent, duplicated experiments. (F) Human Huh7 cells express high levels of TIM1. Huh7 cells and control cells were stained for TIM1 expression as in A. (G) An anti-hTIM1 antibody inhibits entry of various pseudoviruses in Huh7 cells. Huh7 cells were incubated with antibodies and infected as in E. Infection levels were assessed 48 h later as in C and normalized to those of untreated cells. Figure shows mean+SD from three independent, duplicated experiments.</p

Other PS-binding receptors similarly promote entry of hTIM1-using pseudoviruses.

<p>(A) Exogenous hAxl usage in 293T cells. 293T cells were transfected with hAxl or hACE2, infected with pseudoviruses or WNV VLPs, both carrying a GFP reporter gene, and infection levels were assessed the following day by measuring GFP expression by flow cytometry. Fold changes in entry were calculated by dividing mean fluorescence intensity observed in hAxl-expressing 293T by those of hACE2-expressing 293T cells. Figure shows mean+SD from three independent, duplicated experiments. Note that the entry of WNV VLPs is only little increased by hAxl. (B) Exogenous hTIM3 and hTIM4 use in 293T cells. 293T cells were transfected with plasmids expressing hTIM3, hTIM4 or hACE2, infected with the indicated pseudoviruses or WNV VLPs and infection levels were assessed as in A. Shown are mean+SD from three independent experiments. Note that, unlike hTIM1 and hTIM4, hTIM3 only weakly enhanced infection of TCRV pseudoviruses and WNV VLPs. (C) Both hTIM3 and hTim4 are expressed efficiently. The same cells as in B were stained with anti-myc tag antibody. Figure shows representative results of one of three independent experiments. (D) PS receptors contribute to EBOV VLP entry in macrophages. Mouse peritoneal macrophages plated the previous day were preincubated for 30 min at room temperature with medium alone (none) or with liposomes consisting of 50% PS and 50% PC (PS/PC) or PC alone (PC). VP40-Bla VLPs bearing the entry proteins of EBOV, LASV, or no GP (negative control) were then added for a 2 h infection at 37°C, after which cells were detached, washed, loaded with the Bla substrate CCF2-AM, washed again and analyzed by flow cytometry. Figure is representative of two experiments performed in duplicates.</p

A model of viral PS receptor usage.

<p>Human TIM proteins (TIMs) and other PS-binding receptors efficiently, but nonspecifically, promote internalization of various enveloped viruses. The TAM receptors Axl, Mer and Tyro (TAMs) similarly bind and internalize many viruses, albeit indirectly via the PS-binding bridging proteins Gas6 and/or Protein S (Prot S) in serum <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003232#ppat.1003232-Morizono1" target="_blank">[35]</a>. For most viruses PS-dependent internalization results in a moderate to strong increase of productive infection. Note that the degree of enhancement will depend on the cellular background in which experiments are performed. However, in the case of other viruses, PS receptor-mediated internalization may lead to a compartment that is not productive for infection. In addition, there is a third class of viruses, which is not efficiently bound and/or internalized by PS receptors. The indicated viruses were categorized based on hTIM1 usage and internalization results obtained with pseudoviruses and VLPs (black) or replication-competent viruses (blue). Note, however, that the consequences or efficiency of internalization can vary depending on the PS receptors and their expression levels.</p