Dependence on acidic endosomes for virus penetration. (A) Evaluation of treatment with increasing NH₄Cl concentrations on acidic intracellular compartments by AO vital staining. AO stains in red acidic organelles and nuclei in green. (B) Effects of pretreatment with different concentrations of NH₄Cl (0, 25, or 50 mM) on virus infection analyzed by plaque assay and by determination of the percentage of infected cells in immunofluorescence studies using I1, 2H12, and 5C4 antibodies to detect VSV, SVDV, and FMDV, respectively. Appropriate secondary antibodies coupled to Alexa Fluor 488 were used for detection of virus-specific antibodies. (C) Effects of pretreatment with concanamycin A on SVDV infection studied by plaque assay. (D) Colocalization assay of SVDV with the early endosomes marker EEA1. Cells were infected with SVDV (MOI of 100) for 1 h on ice and then transferred to 37°C 15 or 45 min, fixed, and processed for immunofluorescence using a mouse MAb to stain EEA1 (red) and a rabbit polyclonal antiserum to detect SVDV VP1 (green). Colocalization is shown in yellow (circles). Anti-mouse IgG secondary antibodies labeled with Alexa Fluor 594 and anti-rabbit IgG Alexa Fluor 488-coupled secondary antibodies were used to detect primary antibodies. (E) Quantification of colocalization between EEA1 and SVDV. (F) Colocalization assays of SVDV with TF and microtubules. Cells were infected as in panel D in combination with TF for 1 h on ice, transferred to 37°C 15 min, fixed, and processed for immunofluorescence using a rabbit polyclonal antiserum against SVDV VP1 and a mouse MAb against tubulin. Anti-mouse Alexa Fluor 555-labeled and anti-rabbit Alexa Fluor 647-labeled secondary antibodies were used. Alexa Fluor 488-labeled TF is shown in green, tubulin is shown in red, and SVDV is shown in blue. Colocalization between blue and green appears in aqua (circles) in the merged image. Arrowheads point to SVDV loaded endosomes in close association with microtubules. Statistically significant differences between control and drug treatments are indicated by one asterisk (P < 0.05) or two asterisks (P < 0.005).

Dynamin and cell signaling requirements for virus infection. The effects of dynasore (A), Bis (B), OV (C), genistein (D), PP2 (E), and wortmannin (F) on VSV, SVDV, and FMDV infection were studied by plaque assay. The following treatments induced measurable reported effects: dynasore, inhibition of TF internalization (49); wortmannin, endosomal vacuolation (46); and OV, tyrosine phosphorylation (73). Statistically significant differences between control and drug treatments are indicated by one asterisk (P < 0.05) or two asterisks (P < 0.005).

Role of clathrin for SVDV endocytosis in IBRS-2 cells. (A) Hypertonic medium inhibits TF endocytosis. Cells pretreated 30 min with sucrose were incubated 15 min with Alexa Fluor 488-labeled TF maintaining the sucrose, acid washed to eliminate extracellular TF, and fixed. (B) Cpmz inhibits TF endocytosis. Cells pretreated 30 min with chlorpromazine were incubated as for panel A with fluorescent TF. (C) Sucrose and Cpmz inhibit internalization of SVDV. Confocal sections of IBRS-2 cells were pretreated with sucrose or Cpmz and incubated with SVDV (MOI of 100) for 25 min at 37°C. SVDV was detected by immunofluorescence staining with rabbit polyclonal antiserum against VP1 and a secondary antibody labeled with Alexa Fluor 488. Nuclei were stained with To-Pro-3. (D) Effect of sucrose and Cpmz treatment on SVDV infection determined by plaque assay. (E) TEM of SVDV-infected cells. IBRS-2 cells were incubated with SVDV (MOI of 300) for 1 h on ice prior to incubation for 5 min at 37°C, fixed, and processed for electron microscopy. White bars (A to C), 20 μm; black bar (E), 100 nm. Statistically significant differences between control and drug treatments are indicated by one asterisk (P < 0.05) or two asterisks (P < 0.005).

Differential cytoskeleton requirements for intracellular sorting of viruses. (A) Effects of nocodazole on VSV, SVDV, and FMDV infection studied by plaque assay. (B) Evaluation of nocodazole treatment on microtubule and Golgi integrity. Cells treated or not with nocodazole were fixed and processed for immunofluorescence using rabbit polyclonal antiserum against tubulin and MAb 25H8 against cis-Golgi protein gp74. Anti-rabbit Alexa Fluor 488-coupled and anti-mouse Alexa Fluor 594-coupled secondary antibodies were used. Bar, 20 μm. (C) Effects of Cyt D on virus infection studied by plaque assay. (D) Evaluation of Cyt D treatment on actin cytoskeleton integrity. Cells treated or not with Cyt D were fixed and processed for immunofluorescence using Alexa Fluor 488-labeled phalloidin to stain the cellular actin. Bar, 20 μm. Statistically significant differences between control and drug treatments are indicated by one asterisk (P < 0.05) or two asterisks (P < 0.005).

Differential requirements for cholesterol in virus infection. (A) Effects of MβCD and αCD on cellular cholesterol content of IBRS-2 cells studied by filipin staining. Bar, 20 μm. (B) Effect of cholesterol depletion by MβCD on virus infection evaluated by plaque assay and the percentage of infected cells in immunofluorescence studies using I1, 2H12, and 5C4 antibodies to detect VSV, SVDV, and FMDV, respectively, and appropriate secondary antibodies coupled to Alexa Fluor 488. (C) Effects of treatment with αCD on virus infection. (D) VSV, SVDV, and FMDV infections do not induce caveolin-1 phosphorylation on Tyr14. IBRS-2 cells were infected (MOI of 100) for 1 h on ice and then incubated for 15, 30, or 60 min at 37°C; lysed; and processed for Western blotting using MAb anti-Tyr¹⁴ phosphorylated caveolin-1, and polyclonal serum to caveolin-1 to detect total caveolin-1. An extract of IBRS-2 cells treated with 1 mM OV 60 min was included as a control of MAb anti-Tyr¹⁴ phosphorylated caveolin-1. Results obtained with β-actin are also shown as a control for protein loading. Statistically significant differences between control and drug treatments are indicated by one asterisk (P < 0.05) or two asterisks (P < 0.005).