10 research outputs found

    Endogenous IFITM1 restricts both direct and ADE-mediated DENV infection of K562 cells.

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    <p>(A) K562 cells were stably transduced to express shRNA targeting IFITM1, 2, or 3 or non-targeting control shRNA (scrambled). IFITM protein expression was measured by Western blot, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034508#pone-0034508-g001" target="_blank">Fig. 1A</a>. (B) shRNA-transduced K562 cells characterized in panel A were infected with the indicated pseudoviruses as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034508#pone-0034508-g001" target="_blank">Fig. 1C</a>. (C) shRNA-transduced K562 cells characterized in panel A were infected with infectious DENV2 NGC strain as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034508#pone-0034508-g001" target="_blank">Fig. 1D</a>. Error bars indicate standard error. Single and double asterisks indicate statistically significant (<i>P</i><0.05 and <i>P</i><0.005, respectively) differences between cells expressing IFITM1 and control shRNA for corresponding infection conditions. Experiment is representative of three with similar results.</p

    IFITM proteins restrict direct and ADE-mediated infection with similar efficiencies.

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    <p>(A) K562 cells stably transduced to express human IFITM1, 2, or 3 or with control vector were infected as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034508#pone-0034508-g001" target="_blank">Fig. 1D</a>, except that an MOI of 3.0 was used for non-opsonized DENV, and an MOI of 1.0 was used for DENV pre-opsonized with the antibodies 2H2 or 4G2. Different MOIs were used to achieve comparable infection levels between direct and ADE-mediated infection. (B) K562 cells stably transduced to express control shRNA or shRNA targeting IFITM1 were infected as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034508#pone-0034508-g002" target="_blank">Fig. 2C</a>, except that an MOI of 1.6 was used for non-opsonized DENV, and an MOI of 1.0 was used for DENV pre-opsonized with the indicated antibodies. Standard error bars are shown. For each transduction condition, differences between direct infection and ADE-mediated infection were not statistically significant.</p

    IFITM proteins restrict both direct and ADE-mediated DENV infection of K562 cells.

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    <p>(A) K562 cells were treated with the indicated concentrations of human interferon-α or mock treated, and lysed for Western blot analysis 48 h later, using an antibody that recognizes IFITM1 alone, or one that recognizes both IFITM2 and IFITM3. (B) K562 cells were stably transduced to express human IFITM1, 2, or 3 or with the control vector pQCXIP. IFITM protein expression was measured by Western blot using an anti-c-myc antibody. (C) Stably transduced K562 cells characterized in panel B were infected with an MLV-based retrovirus expressing EGFP and pseudotyped with MACV or IAV H5 entry proteins, as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034508#pone.0034508-Huang1" target="_blank">[9]</a>. Infection was determined by flow cytometry, and normalized to K562 cells transduced with vector alone. (D) K562 cells characterized in panel B were infected with infectious DENV2 NGC strain (labeled “DENV2” for virus only) at the indicated MOI, or the same amount of infectious DENV2 pre-opsonized with enhancing titers of antibodies against DENV structural proteins prM or E (“+2H2” and “+4G2” respectively) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034508#pone.0034508-Brass1" target="_blank">[8]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034508#pone.0034508-Diamond1" target="_blank">[21]</a>. Cells were washed after 1.5 h and incubated for ∼24 h. Intracellular staining of DENV antigen was performed with a DyLight-649-conjugated antibody against prM and infection was determined by flow cytometry. Experiment is representative of three with similar results. (E) An experiment similar to that shown in panel D was performed except that infection was assayed by measuring viral loads in the supernatant by plaque assays using BHK cells. (F) An experiment similar to that in panel D was performed, except that J774A.1 murine macrophage cells were stably transduced to express murine orthologs of IFITM1, 2, or 3 or with the control vector pQCXIP. Stably transduced cells were infected with infectious DENV2 NGC strain at ∼MOI 5 and incubated for ∼2 days before harvesting for flow cytometry. Error bars indicate standard error. Single and double asterisks indicate statistically significant (<i>P</i><0.05 and <i>P</i><0.005, respectively) differences between IFITM protein expressing and control cells for corresponding infection conditions.</p

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

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    <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.

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    <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.

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    <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

    hTIM1-mediated EBOV and MARV entry is dependent on the presence of the intracellular receptor NPC1.

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    <p>CHO cells lacking NPC1 (NPC1<sup>−/−</sup>) and the same cells stably expressing mouse NPC1 (NPC<sup>−/−</sup>mNPC1) were mock transduced or transduced to express hTIM1. (A) Cells were assessed for hTIM1 expression levels using anti-hTIM1 antibody as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003232#ppat-1003232-g001" target="_blank">Figure 1A</a>. (B) Cells were infected for 0.5–1 h with EBOV or MARV pseudoviruses, which were concentrated 3–5 fold by ultracentrifugation, or with unconcentrated TCRV or H7N1 pseudoviruses as positive and negative controls for TIM1 use. Infection levels for all viruses were assessed 2 days after infection by measuring GFP expression. Regardless of hTIM1 expression, NPC1<sup>−/−</sup> cells showed no GFP positive cells with either EBOV or MARV infection when inspected by fluorescence microscopy. Figure depicts representative results of one of three experiments performed in duplicates.</p

    hTIM1 increases the infection of diverse replication-competent viruses.

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    <p>293T cells transduced with hTIM1 or hACE2 (mock) were infected for 1–6 h with infectious TCRV (A), RRV (B), SINV (C), H1N1 (D), or DENV (E) at increasing titers. Cells were trypsinized the following day (D and E) or 2 days later (A, B and C), stained either with immune ascitic fluids (A, B and C) or with anti-FLUAV (D) or anti-DENV (E) antibodies. Figures show a representative of the two duplicated experiments (A) or mean ± SD of two independent, duplicated experiments (B–E). LD50: Lethal dose 50, as determined in suckling mice at ATCC.</p

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

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    <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.

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    <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
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