Article Figures & Data
Figures
- Fig. 1.
Schematic diagram of the amino acid sequences of SV5 HN proteins with truncated cytoplasmic tails. The cytoplasmic tail of HN is predicted to comprise the first 17 amino acids of the protein. The indicated nested set of HN proteins containing progressive N-terminal deletions was obtained by site-directed mutagenesis of the HN cDNA.
- Fig. 2.
Intracellular transport of HN cytoplasmic tail-truncated proteins. HN proteins were synthesized in CV-1 cells with the vaccinia virus T7 expression system. Cells were radiolabeled with35S-Promix for 15 min and then incubated in chase medium for the indicated times. Mock-transfected cells (lane M) were processed with no chase. HN was immunoprecipitated from cell lysates, and half of each immune complex was incubated with (+) and without (−) endo H. Polypeptides were analyzed by SDS-PAGE on 10% gels (A). R and S denote the migrations of endo H-resistant and endo H-sensitive HN proteins, respectively. (B) The radioactivities in the endo H-sensitive and -resistant HN species were quantitated with a BioImager.
- Fig. 3.
Expression of HN cytoplasmic tail-truncated proteins in recovered-virus-infected cells. CV-1 cells were mock infected or infected with the indicated recovered recombinant viruses and radiolabeled with 35S-Promix at 42 h p.i. SV5 HN and NP proteins were immunoprecipitated from cell lysates, and immune complexes were digested with peptide N-glycosidase F (PNGase). Polypeptides were then analyzed by SDS-PAGE on 8% gels. The positions of NP and unglycosylated HN are indicated.
- Fig. 4.
Growth curve analysis of HN cytoplasmic tail-truncated rSV5. MDBK cells were infected with the indicated viruses at an MOI of 1.0 PFU/cell, and the culture medium was harvested at the indicated times. Virus titers were determined by plaque assay on BHK-21F cells. Plaques (A) and growth curves (B) of wt rSV5, rSV5 HNΔ2-9, and rSV5 HNΔ2-13 are shown. Values plotted represent averages of results from two experiments.
- Fig. 5.
Density gradient purification of HN cytoplasmic tail-truncated SV5 virions. MDBK cells were infected with wt rSV5 (A), rSV5 HNΔ2-9 (B), and rSV5 HNΔ2-13 (C) at an MOI of 0.01 PFU/cell, and culture medium was harvested at 7 days p.i. Virus particles were pelleted by ultracentrifugation, resuspended, and centrifuged through sucrose gradients. Thirty-six equal fractions were taken from the top of the gradient, and fractions were assayed for NP protein by dot blotting. NP-containing fractions (fractions 20 to 29) were pooled, virus particles were pelleted by ultracentrifugation, and samples were further purified by centrifugation through a second sucrose gradient. Thirty-six fractions were collected and assayed for NP protein by dot blotting. The density and amount of NP (arbitrary units) for each fraction are shown.
- Fig. 6.
Polypeptide composition of HN cytoplasmic tail-truncated SV5 virions. wt rSV5, rSV5 HNΔ2-9, and rSV5 HNΔ2-13 were grown in MDBK cells, and virions were purified by centrifugation through two sequential sucrose gradients. Polypeptides from purified gradient fractions were fractionated by SDS-PAGE on 10% gels and visualized by silver staining. The positions of viral proteins, as well as cellular actin, are indicated.
- Fig. 7.
Localization of viral proteins in cells infected with HN cytoplasmic tail-truncated viruses. CV-1 cells grown on glass coverslips were infected with wt rSV5, rSV5 HNΔ2-9, and rSV5 HNΔ2-13. At 16 h p.i. cells were fixed with formaldehyde (and for M protein staining, they were permeabilized with 0.1% saponin) and bound with MAbs specific to the SV5 HN, M, or F protein and then with fluorescein isothiocyanate-conjugated secondary antibodies. Fluorescence was examined with a Zeiss LSM 400 confocal microscope with a 1-μm-thick optical section.
- Fig. 8.
Syncytium formation in cells infected with HN cytoplasmic tail-truncated viruses. CV-1 cells were mock infected or infected with wt rSV5, rSV5 HNΔ2-9, or rSV5 HNΔ2-13. At various times p.i., cells were fixed and stained with a modified Wright-Giemsa stain and representative fields were photographed with a Kodak DCS 420 digital camera.
Tables
- Table 1.
Surface expression of HN cytoplasmic tail-truncated HN proteinsa
Protein expressed Surface expression efficiency % of positive cells Relative mean fluorescence intensity HN 93.0 1.00 HNΔ2-3 94.1 0.75 HNΔ2-5 94.7 0.90 HNΔ2-7 92.6 0.94 HNΔ2-9 94.3 0.91 HNΔ2-11 94.0 0.84 HNΔ2-13 94.1 0.78 HNΔ2-15 93.8 0.73 HNΔ2-17 25.5 0.03 None (mock transfected cells) 0.01 ↵a HN proteins were synthesized in CV-1 cells with the vaccinia virus T7 expression system. Surface expression of HN was determined by flow cytometry at 16 h posttransfection with a cocktail of two HN-specific MAbs followed by a fluorescein isothiocyanate-conjugated secondary antibody. Values shown are averages of results from three experiments.
- Table 2.
Quantitation of virus releasea
Virus Approx. no. of cells infected (108) Total U of NP harvested U of NP harvested/106 cells Fold decrease in U/cell relative to that for wt rSV5 2.4 4,254 17.7 1.0 rSV5 HNΔ2-9 6.4 1,564 2.4 7.3 rSV5 HNΔ2-13 6.4 971 1.5 11.7 ↵a wt rSV5, rSV5 HNΔ2-9, and rSV5 HNΔ2-13 were grown in MDBK cells, and at 7 days p.i. the culture media were harvested and virions were purified by centrifugation through two sequential sucrose gradients. Thirty-six equal fractions were taken from the top of each gradient, and fractions 13 to 36 were assayed for NP protein by quantitative dot blotting. The total amount of NP protein harvested (arbitrary units [U]) was calculated by summing the amounts of NP protein detected in the gradient fractions.
