Cell-surface expression of CD26/DPP4 on bat cells.

<p>Cells of different bat cell lines were analyzed by flow cytometry after staining with goat anti-human DPP4/CD26 antibody (red line) or control antibody (black lines).</p

Six of ten tested bat cell lines are susceptible to MERS-CoV infection.

<p>(A and B) Ten different bat cell lines were exposed to MERS-CoV/EMC (A) or MERS-CoV/Jor (B) at an MOI of 1. Supernatants were harvested at days 0, 1, 3, and 5 after virus exposure, and virus yields were determined by plaque assay on Vero cells. Error bars indicate the standard deviation of triplicate samples. (C and D) Same experiment: immunofluorescence assay (IFA) images of bat cell lines exposed to MERS-CoV/EMC (C) or MERS-CoV/Jor. (D) 1 (D1) or 3 (D3) days after virus exposure and stained against MERS-CoV spike protein (green). (E) Same experiment: TEM images of bat cells infected with MERS-CoV/EMC at day 1 after virus exposure. Red arrows point at double-membrane vesicles (DMVs) typical of coronavirus infections.</p

Persistent MERS-CoV infection of bat cells induces downregulation of bat cell CD26/DPP4 expression.

<p>Bat cell lines susceptible to infection were infected with MERS-CoV/EMC (A) or MERS-CoV/Jor (B) at an MOI of 1. After 7 days, supernatants were harvested for virus yield analysis by plaque assay, and the cells were subcultured at a 1∶10 dilution in new flasks. Subsequently, the persistently infected cells were passaged at a 1∶10 dilution weekly. Error bars indicate the standard deviation of duplicate samples. (C and D) Same experiment: immunofluorescence assay (IFA) images of bat cells persistently infected with MERS-CoV/EMC (C) or MERS-CoV/Jor (D) at day +33 stained with anti-MERS-CoV spike protein antibody (green). (E) Same experiment: TEM images of bat cells persistently infected with MERS-CoV/EMC at day 56. (F) Flow cytometry data of CD26/DPP4 surface expression (red line: anti-human CD26-/DPP4 antibody; black line: control antibody) in persistently infected cells. (G) CD26/DPP4 expression in persistently infected EidNi/41.3 cells (day 63) as detected by western blot.</p

Expression of human CD26/DPP4 confers MERS-CoV susceptibility to otherwise resistant bat cells.

<p>(A) Viral yields from MERS-CoV-resistant PESU-B5L, R05T, R06E, and Tb1Lu bat cells. Cells were transfected with a plasmid expressing human CD26/DPP4 or empty control plasmid and exposed 48 h later to MERS-CoV/EMC at an MOI of 3. Supernatants were harvested at 24 h after virus exposure for quantification of virus yields by plaque assay. (B) Same experiment: representative immunofluorescence assay (IFA) images of cells stained with anti-MERS-CoV spike protein antibody (green, top) or anti-human CD26/DPP4 antibody (red, bottom). (C) Merged IFA images demonstrate colocalization of MERS-CoV spike protein and CD26/DPP4. (D). Viral yields from MERS-CoV-susceptible bat cells transfected with a plasmid expressing human CD26/DPP4 or empty control plasmid using procedures identical to resistant cells in (A) except that cells were exposed to virus 24 h after transfection. Error bars indicate the standard deviation of duplicate samples.</p

Origin of tested bat cell lines.

<p>Origin of tested bat cell lines.</p

Anti-human CD26/DPP4 antibody inhibits MERS-CoV infection of bat cells.

<p>RoNi/7.1 or Huh-7 cells (control) were treated with increasing concentrations (0, 1.25, 2.5, 5, 10, and 20 µg/ml) of anti-human CD26/DPP4 antibody or control antibody and then exposed to MERS-CoV/EMC at an MOI of 1. (A) After 24 h, viral yields in supernatants were determined by plaque assay. (B) Cellular infection was determined by immunofluorescence assay (IFA) with an anti-MERS-CoV spike protein antibody (green). (B left) The percentage of infected cells was analyzed by high content imaging. (B right) Representative IFA images. Error bars indicate the standard deviation of triplicate samples.</p

siRNA Screen Identifies Trafficking Host Factors that Modulate Alphavirus Infection

<div><p>Little is known about the repertoire of cellular factors involved in the replication of pathogenic alphaviruses. To uncover molecular regulators of alphavirus infection, and to identify candidate drug targets, we performed a high-content imaging-based siRNA screen. We revealed an actin-remodeling pathway involving Rac1, PIP5K1- α, and Arp3, as essential for infection by pathogenic alphaviruses. Infection causes cellular actin rearrangements into large bundles of actin filaments termed actin foci. Actin foci are generated late in infection concomitantly with alphavirus envelope (E2) expression and are dependent on the activities of Rac1 and Arp3. E2 associates with actin in alphavirus-infected cells and co-localizes with Rac1–PIP5K1-α along actin filaments in the context of actin foci. Finally, Rac1, Arp3, and actin polymerization inhibitors interfere with E2 trafficking from the trans-Golgi network to the cell surface, suggesting a plausible model in which transport of E2 to the cell surface is mediated via Rac1- and Arp3-dependent actin remodeling.</p></div

Rac1, Arp3 and formation of a Rac1:PIP5K1-α complex are important for VEEV infection.

<p>(<b>A</b>) High-content quantitative image-based analysis of relative VEEV infection rates in HeLa cells pretreated with increasing concentrations of two Rac1 inhibitors (EHT1864 or NSC23766), two Arp3 inhibitors (CK548 or CK869), or dimethyl sulfoxide (DMSO). Cells were inoculated with compounds 1 h prior to VEEV addition. Cells were fixed and stained with virus-specific antibodies 20 h later. Results are normalized to DMSO-treated samples. (<b>B</b>) Representative confocal images of (<b>A</b>). VEEV E2 staining is shown in green and nucleus/cytoplasm staining is shown in red. (<b>C</b>) Primary human astrocytes were treated with increasing concentrations of EHT1864, NSC23766, or CK548, and subsequently inoculated with VEEV (MOI = 0.005). Cells were fixed 19 h later, stained, and analyzed as in (<b>A</b>). (<b>D</b>) Representative confocal images of (<b>C</b>). VEEV E2 staining is shown in green and nucleus staining is shown in blue. (<b>E</b>) Flp-In T-REx 293 cells with tetracycline-inducible expression of wild-type Rac1, constitutively active Rac1 (G12V) or dominant-negative Rac1 (T17N) were generated, and analyzed for protein expression by immunoblotting (actin was used as a loading control). (<b>F</b>) High-content quantitative image-based analysis of VEEV or RVFV infection rates in Flp-In T-REx 293 cells pre-induced to express chloramphenicol acetyltransferase (CAT), wild-type Rac1, or variants thereof. Cells were fixed 18 h (VEEV) or 24 h (RVFV) after virus inoculation and stained with virus-specific antibodies. (<b>G</b>) Immunoblot of tetracycline-induced expression of wild-type Rac1, or Rac1 K186E in Flp-In T Rex 293 cells as in (<b>E</b>). (<b>H</b>) High-content quantitative image-based analysis of VEEV or RVFV infection rates in Flp-In T-REx 293 cells pre-induced to express CAT, wild-type Rac1, or Rac1 K186E. Cells were infected and stained as in (<b>F</b>).</p

Alphavirus E2 co-localizes with actin filaments and associates with actin.

<p>(<b>A-B</b>) Representative STED images of HeLa cells or primary human astrocytes infected with VEEV or with CHIKV (MOI = 5). Cells were fixed, permeabilized, and stained with E2-specific antibodies (green) and phalloidin (red). Scale bar: 10 μm. (<b>C</b>) Electron-microscopic images of VEEV-infected HeLa cells (MOI = 5). CPV-II structures and thin filaments, which probably correspond to actin, are indicated by filled and open arrows, respectively. An asterisk indicates CPV-I structures. (<b>D</b>) Western blot analysis of input lysates and immunoprecipitates (IP) of mock-, VEEV-, or RVFV-infected HeLa cells under different lysis conditions. Cells were infected for 8 h (MOI = 1), lysed, and VEEV E2-, RVFV Gn-, or actin-binding proteins were immunoprecipitated using specific antibodies and immunoblotted with antibodies against VEEV E2, RVFV Gn, or actin. (*) indicates a non-specific band.</p

Actin polymerization plays a role at a late stage of alphavirus infection.

<p>(<b>A</b> and <b>B</b>) High-content quantitative image-based analysis of relative VEEV and VEEV TC-83 infection rates in time-of-addition experiments. (<b>A</b>) VEEV-infected HeLa cells (MOI = 0.5) were treated with increasing concentrations of latrunculin A at the indicated time points prior to (-1 h) or after (+1–7 h) virus addition. Cells were fixed 20 h after addition of virus and stained for high-content quantitative image-based analysis with virus-specific antibodies. (<b>B</b>) VEEV TC-83 (MOI = 1)-infected HeLa cells were treated with cytochalasin D as in (<b>A</b>). Cells were fixed 12 h after addition of virus, stained, and analyzed as in (<b>A</b>). (<b>C</b>) HeLa cells were infected with VEEV (MOI = 0.5) for 3 h and then treated with increasing concentrations of cytochalasin B, cytochalasin D, latrunculin A, or nocodazole. Cells were fixed in formalin 17 h later, stained, and analyzed as in (<b>A</b>). (<b>A-C</b>) Results are normalized to DMSO-treated samples. (<b>D</b>) HeLa cells were infected as in (<b>C</b>) for 3 h and then treated with increasing concentrations of cytochalasin D or latrunculin A. After 17 h, virus titer in the supernatants was determined by plaque assay. Values represent the mean ± SD, n = 2. (<b>E</b>) Primary human astrocytes were infected with VEEV TC-83 (MOI = 0.005) for 5 h and then treated with increasing concentrations of inhibitors. After 6 h, virus titer in the supernatants was determined by plaque assay. (<b>F</b>) Aliquots of the cells treated in (<b>A</b>) were lysed and analyzed for E2 expression by immunoblotting (GAPDH was used as a loading control). Densitometric analysis of western blots was performed with ImageJ. (<b>G</b>) VEEV copy number (intracellular vRNA) in HeLa cells following treatment with inhibitors was determined by qRT-PCR. HeLa cells were inoculated with VEEV TC-83 (MOI = 2) and 5 h later treated with the indicated inhibitors. Cells were lysed and analyzed for virus copy number 11 h after virus addition. (<b>A-C, E, G</b>) Values represent the mean ± SD, n = 3. *, <i>p</i> < 0.05; **, <i>p</i> < 0.01; ***, <i>p</i> < 0.001; n.s., not significant, Student's <i>t</i> test (between the sample and DMSO-treated cells).</p