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 infection causes actin rearrangements into actin foci that are Rac1- and Arp3-dependent and that co-localize with Rac1, PIP5K1-α, and E2.

<p>(<b>A</b>) Representative confocal images of mock-, VEEV-, CHIKV-, or RVFV-infected HeLa cells (MOIs = 0.5, 5, or 3, respectively). Cells were fixed and stained with virus-specific antibodies (VEEV and CHIKV E2, RVFV nucleoprotein; shown in green) and phalloidin (red) 18 h (VEEV) or 24 h (CHIKV, RVFV) after infection. Nucleus staining is shown in blue. Representative actin foci are indicated by asterisks. (<b>B</b>) High-content quantitative image-based analysis was used to measure infection rates of VEEV, CHIKV, and RVFV (left panel), and the number of actin foci per cell (number of actin foci/total cell number, right panel). Analysis is based on single Z sections. ***, <i>p</i> < 0.0001, Student's <i>t</i> test (between the sample and mock). (<b>C</b>) VEEV-infected HeLa cells (MOI = 0.5) were fixed in formalin at the indicated time points, stained, and analyzed as in (<b>B</b>). (<b>B</b>–<b>C</b>) Values represent the mean ± SD, n ≥12. (<b>D</b>) Representative confocal images of VEEV-infected HeLa cells (MOI = 0.5) pretreated with the Rac1 inhibitor EHT1864 or Arp3 inhibitor CK548. Cells were fixed 18 h after virus addition and stained with VEEV E2-specific antibody (green), phalloidin (red), and a nuclear stain (blue). (<b>E</b>) High-content quantitative image-based analysis was used to measure infection rates of VEEV and the number of actin foci per cell. (<b>F</b>) Confocal images of VEEV-infected HeLa cells (MOI = 5). Co-localization of hemagglutinin (HA)-tagged PIP5K1-α (top panel) or Rac1 (bottom panel) (blue), actin (red), and VEEV E2 (green), at a single z section is shown (left panel). Insets: zoom on actin filaments indicated by white arrows. Single channel intensities were measured along lines crossing different actin clusters (right panel). VEEV was added to HeLa cells that were reverse-transfected with a plasmid encoding HA-tagged PIP5K1-α or tetracycline-induced T-Rex HeLa cells that expressed Rac1 fused to eGFP. Cells were fixed 20 h later, permeabilized, and stained with VEEV E2-specific antibody, phalloidin, and an antibody against HA.</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

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

Actin, Rac1, and Arp3 inhibitors block E2 transport to cell surface.

<p>(<b>A</b>) Representative confocal images of primary human astrocytes treated with DMSO, EHT1864, or CK548 and subsequently infected with VEEV (MOI = 0.005) for 18 h. Cells were stained with VEEV E2 (green)- and TGN46-specific antibodies (red), and a nuclear stain (blue). Representative cells showing co-localization of E2 and TGN46 are indicated with white arrows. (<b>B</b> and <b>C</b>) Upper panels: Geometrical mean fluorescent intensity of cell-surface E2 staining in HeLa cells infected with VEEV TC-83 (MOI = 10) and treated with EHT1864, CK548, cytochalasin D, latrunculin A, or nocodazole as measured by flow cytometry. HeLa cells were infected with VEEV TC-83 for 5 h and subsequently treated with increasing concentrations of the inhibitors or DMSO (control). Five (<b>B</b>) or six (<b>C</b>) h later cells were dissociated and stained against VEEV E2 and with a 7-amino-actinomycin D viability dye. Bottom panel: Immunoblot of total E2 expression in whole cell lysates of HeLa cells described in (<b>B</b>) and (<b>C</b>). GAPDH was used as a loading control. Densitometric analysis of western blots was performed with ImageJ. (<b>D</b>) Model for trafficking of alphavirus E2 from the TGN to the cell surface. (1) Biogenesis of vacuoles (CPV-II) containing E1/E2 at the TGN is dependent on ADP-ribosylation factor 1 (Arf1) and Rac1. (2) CPV-II containing E1/E2 traffic to the cell surface via actin by a Rac1- and Arp3-dependent mechanism. Rac1 and PIP5K1-α are also localized to these actin filaments. (3) Actin tunneling nanotubes mediate alphavirion spread to neighboring cells.</p

Sources of Human-sequence Reagents.

<p>Sources of Human-sequence Reagents.</p

siRNA screen identifies host regulators of alphavirus infection.

<p>(<b>A</b>) Schematic representation of the siRNA screen and the high-content quantitative image-based analysis of relative VEEV infection rates. HeLa cells were transfected with siRNAs against 140 host trafficking factors and inoculated with VEEV (multiplicity of infection [MOI] = 0.5) for 20 h. Cells were fixed and immunostained for cell surface VEEV envelope glycoprotein (E2) expression. Infection rates were determined using an Opera confocal imager and normalized to infection rates observed using non-targeting control siRNA. Representative images of cells treated with control (Cont) or Rac1 siRNA are shown. VEEV E2 staining is shown in green, and nucleus staining is shown in blue. (<b>B</b>) High-content quantitative image-based analysis was used to measure relative infection rates (normalized to control siRNA-treated cells) of VEEV (top panel) in HeLa cells pretreated with the indicated siRNAs. Cells were infected for 20 h (VEEV, MOI = 0.5), fixed, and stained with antibodies against E2. Values represent the mean ± SD, n = 3. Protein levels of Rac1, Arp3, and actin (loading control) following siRNA treatments were determined by immunoblotting (bottom left 2 panels). mRNA levels of PIP5K1-α (PIP5K1A) following siRNA treatments were determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR, bottom right panel). (<b>C</b>) VEEV titer following treatment of HeLa cells with siRNAs against Rac1, Arp3, PIP5K1A (PIP5K1-α), or control siRNA. Cells were inoculated with VEEV as in (<b>B</b>), and virus-containing media were analyzed by plaque assay. **, <i>p</i> <0.01, Student's <i>t</i> test (between the sample and control siRNA).</p