Fig 1.
Generation of FAST protein-deficient recombinant pteropine orthoreovirus (PRV).
(A) Construction of FAST protein-deficient PRV strain MB (rsMB-ΔFAST) involved generation of S1 gene segment with replacement of the first codon of the FAST-p10 ORF (ATG) with ATT and the fifth codon (TGC) with TGA. (B, C) Syncytia are formed by wild-type rsMB, but not rsMB-ΔFAST. (B) Vero cells infected with rsMB or rsMB-ΔFAST were fixed, and viral antigen (sigmaC) was detected by indirect immunofluorescence assay with rabbit anti-sigmaC antibody followed by a CF488-conjugated secondary antibody. Cellular plasma membranes were visualized with anti-pan-cadherin antibody and CF594-conjugated secondary antibody (red). Phase contrast images (top) and fluorescence images (bottom) are shown. Scale bars are 200 μm (top) and 20 μm (bottom). (C) BSR cells infected with rsMB or rsMB-ΔFAST were fixed at 16 h post infection. Viral sigmaC (green) and FAST-p10 (red) antigens were visualized by indirect immunofluorescence assay with antibodies to sigmaC and FAST-p10 (residues 21–40), respectively. Scale bars are 200 μm. (D) Structural and non-structural viral protein expression in cells infected with rsMB or rsMB-ΔFAST. Whole-cell lysates of Vero cells infected with rsMB or rsMB-ΔFAST at a MOI of 0.1 PFU/cell and harvested at 24 h post infection were subjected to western blotting. Viral proteins were detected with anti-sigmaC, anti-p17, and anti-sigmaA antibodies. Plasmid vectors directing expression of hemagglutinin (HA)-sigmaC, HA-p17, and HA-sigmaA were transfected as positive controls for antibody detection, and β-actin staining was a control for protein loading.
Fig 2.
FAST protein enhances viral replication at an early phase of infection.
(A) Vero cells were infected with rsMB or rsMB-ΔFAST at a multiplicity-of-infection (MOI) of 0.01 plaque-forming units (PFU)/cell. Infectious virus titers in the supernatant and cell lysate were examined. Data are expressed as means ± SD (n = 3). (B) Time course of viral protein expression. Vero cells were infected with rsMB or rsMB-ΔFAST at a MOI of 0.01 PFU/cell. Whole-cell extracts were subjected to western blotting. Viral proteins were detected with antibodies to sigmaC, p17, and sigmaA. (C–E) Vero cells were infected with rsMB or rsMB-ΔFAST at a MOI of 0.01 PFU/cell and incubated for various intervals. (C) To determine the time course of syncytium formation, cells were fixed and viral sigmaC antigens were visualized by immunostaining. Numbers of cells involved per syncytium were plotted. Data are presented as a box plot (n = 11–30). (D, E) To determine the kinetics of viral replication, cells were disrupted at indicated times post infection by freeze-thawing. Copy numbers of the genomic RNA L1 gene (D) and infectious virus titers (E) were investigated by TaqMan quantitative PCR and plaque-formation assay, respectively. Arrows indicate the time points of primary increase of levels of viral genomes and infectious virus particles. NA, not available; ND, not detected. Data are expressed as means ± SD (n = 3). (F, G) Vero cells were infected with rsMB or rsMB-ΔFAST at a MOI of 0.001 or 10 PFU/cell. (F) Virus infectious titers were determined by the plaque assay. (G) Viral antigens in whole-cell extracts of Vero cells infected with rsMB or rsMB-ΔFAST at a MOI of 10 PFU/cell were detected with an anti-sigmaA antibody. An anti-β-actin antibody was used as a loading control. (H) Time course of virus spread in monolayers of Vero cells. Vero cells were infected with rsMB or rsMB-ΔFAST at a MOI of 0.01 PFU/cell. After virus adsorption at 37°C for 1 h, cells were overlaid with 0.8% agarose gel and incubated. At the indicated time points post infection, cells were fixed and viral antigens were detected by immunostaining with murine anti-PRV-MB serum followed by anti-mouse IgG-CF488. NA, not available. Scale bars are 200 μm. (I) Ratio between the viral genome copy number and the infectious virus titer in whole-cell lysates and purified virions. The genome copy number of positive-stranded L1 gene segments and the number of plaque-forming units of purified rsMB and rsMB-ΔFAST were calculated. Data are expressed as means ± SD (n = 3) and were statistically analyzed using the t-test.
Fig 3.
Cell–cell fusion activity correlates with virus propagation.
(A) Deduced amino acid sequences of pteropine orthoreovirus (PRV) strain MB (rsMB) FAST-p10 protein. Functional domains and peptide motifs are indicated [2]. (B) Vero cells were infected with the mutant PRV-MB strains, which harbored mutant FAST-p10 genes with single amino acid substitutions or C-terminal truncations. At 16 h post infection, the syncytium-forming activity of each mutant was determined by calculating the number of nuclei involved per syncytium Data are presented as a box plot (n = 8–14). (C) Propagation of FAST-p10 mutant viruses. Vero cells were infected with rsMB or FAST-p10 mutant viruses at a multiplicity-of-infection (MOI) of 0.01 plaque-forming units (PFU)/cell and incubated for 72 h. Infectious virus titers in cell lysates were determined. Data are expressed as means ± SD (n = 3). (D) Correlation of syncytium-forming activity and virus propagation, with a linear regression line. Mean values of fusion activity (B) and viral replication (C) were plotted on the x- and y-axis, respectively. (E–G) Lysophosphatidylcholine (LPC) impaired replication of rsMB, but not of the FAST-p10-deficient mutant rsMB-ΔFAST. (E) Vero cells were infected with rsMB or rsMB-ΔFAST at a MOI of 0.01 PFU/cell. At 2 h post infection, cell-culture medium was replaced with medium containing LPC (0–100 μM). Cells were fixed at 16 h post infection, and virus-infected foci were visualized by immunostaining for sigmaC. Fusion efficiency was determined by the number of nuclei involved per syncytium. Data are presented as a box plot and were statistically analyzed using the t-test (n = 14–26). (F–G) Vero cells were infected with (F) rsMB or (G) rsMB-ΔFAST at a MOI of 0.01 PFU/cell. At 2 h post infection, culture medium was replaced with medium containing LPC (0–100 μM). Infectious virus titers in cell lysates were determined by plaque-formation assay. Data are expressed as means ± SD (n = 3). * indicates p < 0.05 (Dunnett’s multiple comparison test). (H) Time course of viral protein expression. Vero cells were infected with rsMB or rsMB-ΔFAST at a MOI of 0.1 PFU/cell. Viral antigens in whole-cell extracts were detected with an anti-sigmaA antibody. An anti-β-actin antibody was used as a loading control.
Fig 4.
Cell–cell fusion by FAST proteins enhances replication of pteropine orthoreovirus (PRV).
(A) Enhancement of viral replication by recombinant PRV FAST-p10 protein. Vero cells were transfected with FAST-p10 expression vector at the indicated times either before or after infection with a FAST-p10-deficient PRV mutant (rsMB-ΔFAST). Infectious virus titers in cell lysates at 16 h post infection were determined. Data are expressed as means ± SD (n = 3) and were statistically analyzed using the t-test. (B) Whole-cell lysates of Vero cells transfected with FAST-p10 expression vector or empty vector 2 h before infection with rsMB-ΔFAST were prepared, and viral sigmaC protein was detected by western blotting. (C) Vero cells were transfected with FAST-p10 expression vector or empty vector (0.25–2.0 μg/well) 2 h before infection with wild-type PRV (rsMB) or rsMB-ΔFAST. Infectious virus titers in cell lysates at 16 h post infection were determined. Data are expressed as means ± SD (n = 3) and were statistically analyzed using the t-test. (D) Efficiency of syncytium formation by different members of the FAST protein family. Vero cells were transfected with recombinant expression vectors (0.5 μg/well) for PRV FAST-p10, avian orthoreovirus (ARV) FAST-p10, reptilian orthoreovirus (RRV) FAST-p14, or baboon orthoreovirus (BRV) FAST-p15, or with empty vector. At 16 h post infection, cells were fixed and numbers of nuclei per syncytium were counted (n = 5–33). * indicates p < 0.05 (Dunn’s multiple-comparison test). (E) Vero cells were transfected with expression vectors for PRV FAST-p10, ARV FAST-p10, RRV FAST-p14, or BRV FAST-p15, or with empty vector (0.1–2.0 μg/well) 2 h before infection with rsMB-ΔFAST. Infectious virus titers in cell lysates at 16 h post infection were determined. Data are expressed as means ± SD (n = 4). Viral titers in cells with 2.0 μg/well plasmid transfections were statistically compared with titers in mock-transfected samples. * indicates p < 0.05. (F) QT6 cells were transfected with 2 μg of PRV FAST-p10 expression vector or empty vector, followed by infection with ARV at a MOI of 0.001 PFU/cell. Infectious virus titers in cell lysates at 16 h post infection were determined and statistically analyzed using the t-test. Data are expressed as means ± SD (n = 3). (G) Time course of syncytium formation by PRV FAST-p10 and modified recombinant Sendai virus (SeV) F (Fc) proteins. Vero cells were transfected with expression vectors either for FAST-p10 or for SeV Fc and SeV HN (0.5 μg each/well). At indicated times post transfection, cells were fixed and numbers of nuclei involved per syncytium were counted. Data are presented as a box plot (n = 7–30). (H) Enhancement of viral replication by recombinant SeV Fc protein. Vero cells were transfected with expression vectors for SeV Fc and SeV HN proteins (1 μg each/well) 2 h before infection with rsMB-ΔFAST at a MOI of 0.001 PFU/cell. At 16 h post infection, infectious virus titers in the cell lysates were determined. Data are expressed as means ± SD (n = 3) and were statistically analyzed using the t-test.
Fig 5.
Cell–cell fusion has heterogeneous effects on replication of different viruses.
(A–D) Effects of cell–cell fusion on replication of non-fusogenic mammalian orthoreovirus (MRV) and group A rotavirus (RVA). (A, B) Vero cells were transfected with pteropine orthoreovirus (PRV) FAST-p10 expression plasmid vectors or empty vector (1 μg/well). (C, D) BSR cells were transfected with Sendai virus (SeV) modified recombinant F (Fc) and HN expression plasmid vectors or empty vector (1 μg each/well). At 2 h post transfection, cells were infected with MRV strain T1L (A, C) or RVA strain SA11 (B, D) at a multiplicity-of-infection (MOI) of 0.001 plaque-forming units (PFU)/cell. Infectious virus titers in cell lysates at 16 h post infection were determined by plaque-formation assay. Data are expressed as means ± SD (n = 3) and were statistically analyzed using the t-test. (E–G) Effects of cell–cell fusion by PRV FAST-p10 on replication of (E) infectious bursal disease virus (IBDV) in DF1 cells, (F) encephalomyocarditis virus (EMCV) in Vero cells, and (G) vaccinia virus (VV) in BSR cells. Monolayers of DF1, Vero, or BSR cells were transfected with FAST-p10 expression plasmid vectors or empty vector 4 h (E) or 2 h (F, G) before viral infection at a MOI of 0.001 PFU/cell (E–F) or 0.001 TCID50/cell (G). At 14 h post infection (IBDV), 12 h post infection (EMCV), and 20 h post infection (VV), infectious virus titers were determined. Data are expressed as means ± SD (n = 3) and were statistically analyzed using the t-test. p < 0.05 was considered statistically significant.
Fig 6.
FAST protein is a determinant of pteropine orthoreovirus (PRV) pathogenicity.
(A, B) C3H mice (male, 3-week-old, n = 10/group) were intranasally infected with 4 × 105 plaque-forming units (PFU) of wild-type (rsMB) or FAST protein-deficient (rsMB-ΔFAST) PRV. The mice were monitored for (A) bodyweight changes and (B) survival rate for 14 days following virus inoculation. Survival curves were statistically compared with that in the control group using the log rank test. (C) Replication of PRV in mouse lungs. C3H mice (male, 3-week-old, n = 5) were intranasally infected with 4 × 105 PFU of rsMB or rsMB-ΔFAST. Animals were euthanized at 5 days post infection. Infectious virus titers in lung homogenates were determined by plaque-formation assay. Virus infectious titers were statistically compared between the two groups using the t-test. (D) Histopathological examination of C3H mice infected with PRV. Lungs of C3H mice 7 days after infection with rsMB or rsMB-ΔFAST were subjected to histopathological examination by hematoxylin–eosin (HE) staining and immunohistochemistry (IHC) with anti-sigmaC serum. Arrowheads indicate sigmaC-positive cells in IHC. Original magnifications: 200× (HE) and 400× (IHC). Scale bars indicate 100 μm (HE) or 20 μm (IHC).
Fig 7.
Use of FAST-p10-deficient virus as live attenuated vaccine.
(A) Schedule of immunization and challenge infection. Male, 3-week-old C3H mice (n = 6/group) were intranasally infected with 4 × 105 plaque-forming units (PFU) of FAST-p10-deficient pteropine orthoreovirus (PRV) mutant (rsMB-ΔFAST) either once (on day 1) or twice (on day 1 and day 7). Control mice were intranasally inoculated with phosphate-buffered saline (PBS). On day 14, mice were intranasally infected with a lethal dose of wild-type PRV (rsMB, 4 × 105 PFU). Animals were monitored daily for 14 days after challenge infection. (B) Survival curves of non-infected mice and mice inoculated with one dose (×1) or two doses (×2) of rsMB-ΔFAST, or with PBS, and challenged with rsMB. Survival curves were statistically compared with that in the control group using the log rank test. (C) Bodyweight changes in mice inoculated with one dose (×1) or two doses (×2) of rsMB-ΔFAST, or with PBS, and challenged with rsMB. Black arrows indicate immunization on day 0 or day 7; white arrows indicate challenge infection on day 14.