TMPRSS2 isoform 1 cleaves and activates the influenza virus hemagglutinin.

<p>(A) Expression plasmids encoding FLUAV HA subtypes H1 (left) and H3 (right) and the indicated proteases or empty plasmid (pCAGGS) were transiently cotransfected into 293T cells. At 48 h post transfection the cells were treated with PBS or trypsin, and HA cleavage was determined by Western blotting. Similar results were obtained in three independent experiments. The HA₀ precursor (upper arrow) and the HA₁ (middle arrow) and HA₂ (lower arrow) subunits are indicated. (B) The indicated proteases were transiently expressed in 293T cells and the cells infected with FLUAV A/PR/8/34 (H1N1) at an MOI 0.01 (left) or FLUAV A/Panama/2007/99 (H3N2) at an MOI of 0.1 (right) and treated with either trypsin or PBS. At 48 h post infection, the virus release was measured by determination of infectious particles (ffu/ml) in the culture supernatant. The results of representative experiments performed with triplicate samples are shown. Error bars indicate standard deviations. Similar results were obtained in three independent experiments. ffu, focus forming units.</p

Expression of TMPRSS2 isoforms 1 and 2.

<p>(A) Sequence alignment of the N-termini of TMPRSS2 isoforms 1 and 2. Identical amino acids are marked with stars. Amino acids absent in isoform 2 are marked with ‘–‘. (B) Plasmids encoding TMPRSS2 isoform 1 and isoform 2, both equipped with an N-terminal myc tag, were transiently transfected into 293T cells. Empty plasmid (pCAGGS) served as a negative control. Protease expression in cell lysates was detected via Western blotting with anti-myc antibody. The β-actin expression served as a loading control. Black-filled arrowheads indicate the zymogen forms, while grey-filled arrowheads highlight cleavage products resulting from protease activation. (C) 293T cells were transfected as described for panel B but protease expression was determined using flow cytometry with an anti-TMPRSS2 antibody. The geometric mean channel fluorescence (GMCF) measured in a representative experiment performed with triplicate samples is shown. Error bars indicate standard deviations. Similar results were obtained in two independent experiments.</p

TMPRSS2 isoforms 1 and 2 colocalize with hemagglutinin.

<p>(A) COS-7 cells were transfected with plasmids encoding TMPRSS2 isoform 1 or isoform 2 or with empty plasmid which served as negative control. Subsequently, the cells were infected with FLUAV A/PR/8/34 (H1N1) at an MOI 0.5. At 24 h post infection, the cells were stained for FLUAV-HA (green) and TMPRSS2 isoforms (red) and images were taken at 63 x magnification. White squares show examples of colocalization of HA and TMPRSS2 (yellow signals) and were digitally magnified 2.5x from the original images. Similar results were obtained in three separate experiments. (B) Images obtained in (A) were analyzed with Fiji software, which allows calculation of the Pearson Correlation Coefficient (PCC), a measure for colocalization. The average PCC measured in three separate experiments is shown. For each experiment, 6–8 cells were analyzed. Error bars indicate standard error of the mean (SEM).</p

Expression of tmprss2 transcript variants in human organs and cell lines.

<p>RT-PCR analysis of the expression of tmprss2 transcript variants 1 and 2 in organ samples from adult men (left panel) or from cell lines of human origin (right panel). Expression of GAPDH was assessed in parallel. Plasmids encoding isoform 1 and 2 (left panel) or a mix of both plasmids (right panel) served as positive control. Similar results were obtained in two separate experiments.</p

TMPRSS2 isoform 1 cleaves and activates the SARS-coronavirus spike protein.

<p>(A) 293T cells were cotransfected with plasmid encoding SARS-S with a C-terminal V5 tag and plasmids encoding the indicated proteases. At 48 h post transfection, the cells were treated with PBS or trypsin, and SARS-S cleavage was analyzed by Western blotting with a V5-specific antibody. The expression of β-actin served as a loading control. The results are representative of three independent experiments with different plasmid preparations. Black-filled arrowhead, uncleaved SARS-S; gray-filled arrowhead, S2 subunit generated by trypsin digest; white-filled arrowheads, C-terminal cleavage fragments generated by TMPRSS2. (B) To analyze SARS-S-driven virus-cell fusion, 293T cells were transiently transfected with plasmids encoding the indicated proteases and ACE2. At 24 h post transfection, the cells were pretreated with medium supplemented with DMSO or 10 μM cathepsin B/L inhibitor MDL 28170, and transduced with pseudotypes bearing SARS-S. The luciferase activities in cell lysates were analyzed at 72 h post transduction. The results of a representative experiments performed with triplicate samples are shown. Error bars indicate standard deviations. Similar results were obtained in two separate experiments. (C) MERS-S-driven virus-cell fusion was analyzed as described for panel B but 293T cells transfected to express DPP4 were used as targets. The results of a representative experiment performed with triplicate samples are shown. Error bars indicate standard deviations. Similar results were obtained in three independent experiments. c.p.s., counts per second.</p

Differential transduction of fruit bat cells lines by different filovirus glycoproteins.

<p>Vesicular stomatitis virus (VSV)-based pseudotypes (VSVpp) harboring the indicated filovirus glycoproteins or the glycoprotein of VSV (VSV-G; positive control), or no glycoprotein (pCAGGS; negative control) were used to inoculate human (HEK-293T), non-human primate (Vero) and fruit bat cell lines (RoNi/7, HypNi/1.1, EidNi/41, EpoNi/22.1). Mock-treated cells that received only fresh culture medium served as additional negative controls (mock). The VSVpp decorated with the respective GPs or VSV-G were either infectivity-normalized (A) or applied undiluted (B) to the target cells. At 18 h post inoculation, the activity of virus-encoded firefly luciferase (given in counts per second; cps) as an indicator for transduction efficiency was quantified. The results of a single representative experiment carried out with quadruplicate samples is shown. Similar results were obtained in three independent experiments carried out with separate pseudotype preparations. Error bars indicate standard deviations (SD).</p

Filovirus glycoproteins and distribution of fruit bats believed to serve as natural reservoir.

<p>(A) Phylogenetic tree based on the amino acid (aa) sequences of the glycoproteins (GP) of different filoviruses was generated using MEGA 6 (version 6.06). Viruses from which GPs were examined in the present study are written in bold. Viruses were named as follows: Filovirus species (abbreviation)/Country where the sample specimen originates from (abbreviation)/year of sampling/isolate-specific name (if available). In addition to GPs from viruses that have caused infection in humans and non-human primates, GP sequences of one Reston virus from a pig (diamond), as well as four Marburg virus-related and two Ravn virus-related GP sequences from bats (circles) were included. Construction of the tree was performed by the neighbor joining method with 1,000 bootstrap replications, using the MEGA 6 software (version 6.06). Small numbers at the nodes and the scale bar indicate bootstrap values and the number of aa substitutions per site, respectively. GenBank accession numbers for all GPs are given in brackets after the virus name. (B) Schematic drawing of the wildtype (wt) Ebola virus (EBOV) GP and EBOV-GP mutants lacking the mucin-like domain and the furin cleavage site. (Abbreviations: SP = signal peptide; RBD = receptor binding domain; FP = internal fusion peptide; HR1/2 = heptad repeat 1/2; TD = transmembrane domain). (C) Maps of the distribution of the four different fruit bat species from which the cell lines used in this study originated: <i>Rousettus aegyptiacus</i> (RoNi/7), <i>Hypsignathus monstrosus</i> (HypNi/1.1), <i>Eidolon helvum</i> (EidNi/41), <i>and Epomops buettikoferi</i> (EpoNi/22.1). The data on the bat distribution have been obtained from <a href="http://www.iucnredlist.org/" target="_blank">www.iucnredlist.org</a> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149651#pone.0149651.ref056" target="_blank">56</a>].</p

Information on the fruit bat cell lines used in this study.

<p>Information on the fruit bat cell lines used in this study.</p

Glycoprotein-mediated entry into human and fruit bat cells relies on the same host cell factors.

<p>Equal volumes of vesicular stomatitis virus (VSV)-based pseudotypes (VSVpp) harboring the indicated glycoproteins were used to inoculate human (HEK-293T) and fruit bat (EpoNi/22.1, EidNi/41) cell lines pre-incubated with the indicated inhibitors for 3 h. Cells treated with solvent (water or dimethyl sulfoxide) alone served as controls (vehicle control, VC). At 18 h post inoculation, the activity of virus-encoded firefly luciferase as an indicator for transduction efficiency was quantified and normalized against the values of the respective VC (x-fold changes). The results of a representative experiment carried out with quadruplicate samples are shown and were confirmed in an independent experiment, conducted with a separate pseudotype batch. The following inhibitor concentrations were used: Mannan (final concentration: 25 μg/ml), ammonium chloride (NH₄⁺; 50 mM), bafilomycin A1 (50 nM), tetrandrine (2 μM), E-64d (50 μM), MDL28170 (50 μM), camostat mesylate (100 μM), U18666A (20 μM). Error bars indicate SD. An unpaired student’s t-test was used to test statistical significance (* = p < 0.05, ** = p < 0.01, *** = p < 0.001).</p

Furin cleavage and mucin-like domain are dispensable for EBOV-GP-driven transduction of fruit bat cells.

<p>Equal volumes of vesicular stomatitis virus (VSV)-based pseudotypes (VSVpp) harboring the indicated glycoproteins were inoculated onto human (HEK-293T), primate (Vero) and fruit bat cell lines (RoNi/7, HypNi/1.1, EidNi/41, EpoNi/22.1). EBOV1976-GP(∆Cleav) cannot be cleaved by furin while EBOV1976-GP(∆MLD) does not contain the mucin-like domain. VSVpp that did not harbor a viral glycoprotein at all served as negative controls (pCAGGS). At 18 h post inoculation, the activity of virus-encoded firefly luciferase in cell lysates was quantified as an indicator for transduction efficiency. Transduction mediated by the tested GPs is shown relative to transduction mediated by wt EBOV1976-GP, which was set as 1. The average of three independent experiments with separate pseudotype preparations is shown. Error bars indicate standard error of the mean. A paired student’s t-test was performed to test statistical significance (* = p < 0.05).</p