Article Figures & Data
Figures
- FIG. 1.
(A). Hydropathy plot of G3L protein. The numbers at the bottom of the figure are the amino acid residues. (B) Alignment of the deduced amino acid sequences for vaccinia G3L and G3L orthologues in other poxviruses. VACV, vaccinia virus (WR strain); CPXV, cowpox virus (Brighton Red strain); VARV, variola virus (INDIA-1967/isolate IND3); MYXV, myxoma virus; LSDV, lumpy skin disease virus; YMTV, Yaba-like monkey tumor virus; FWPV, fowlpox virus; AMV, Amsacta moorei entomopoxvirus; MSV, Melanoplus sanguinipes entomopoxvirus. The boxed sequences in gray are conserved amino acid sequences. (C) Expression of G3L protein in infected cells and purified IMV virions. BSC40 cells were infected with vT7LacOI at an MOI of 5 PFU per cell and harvested at the indicated times postinfection; the lysates were then separated by SDS-12.5% PAGE and transferred to nitrocellulose for immunoblotting with the anti-G3L antiserum. M, mock-infected cells; V, purified IMV particles. When araC treatment was required, the drug (40 μg/ml) was added to the cells immediately after infection. The arrow shows the position of G3L protein.
- FIG. 2.
Membrane and core proteins after NP-40-DTT extraction of vaccinia IMV. Sucrose-purified vaccinia IMVs were incubated with buffer containing 1% NP-40 with or without 50 mM DTT. After centrifugation, the supernatant (S) and pellet (P) were analyzed on immunoblots with antibodies to G3L, H3L, or core proteins as indicated in Materials and Methods.
- FIG. 3.
(A) Schematic diagram of viG3L virus. The G3L and J2R (tk) loci in the viG3L recombinant virus are indicated. The J2R locus contains T7 RNA polymerase and the lacI repressor gene as described previously (42). The inducible G3L (shaded box)/Gpt selectable marker (black box) gene cassette is also shown. The arrows indicate the transcription direction. Abbreviations: T7 Pol, bacteriophage T7 RNA polymerase; LacO, E. coli lac operator; LacI, E. coli lac repressor gene; Gpt, E. coli xanthine guanine phosphoribosyltransferase gene; p7.5 and p11, viral promoters; T7, promoter for T7 RNA polymerase. (B) Expression of G3L in viG3L-infected cells. BSC40 cells were infected with viG3L at an MOI of 5 PFU per cell, incubated in culture medium with or without 50 μM IPTG, then harvested at the indicated times, and subjected to SDS-PAGE and immunoblot analyses with the anti-G3L antiserum.
- FIG. 4.
(A) ViG3L virus does not form plaques in BSC40 cells when the expression of G3L protein is repressed. BSC40 cells were infected with viG3L and incubated in medium with or without IPTG for 3 days, fixed, stained with crystal violet, and photographed. (B) One-step growth curve analysis of viG3L. BSC40 cells were infected with vT7LacOI or viG3L at an MOI of 5 PFU per cell and then incubated in the presence (viG3L+) or absence (viG3L− and vT7LacOI) of 50 μM IPTG for 0, 8, 12, 16, 24, or 48 h p.i. Virus titers in the lysates were determined by plaque formation on BSC40 cells. These experiments were performed three times.
- FIG. 5.
Electron micrographs of vaccinia virion morphogenesis in cells infected by viG3L. BSC40 cells were infected with viG3L at an MOI of 5 PFU per cell in the presence or absence of IPTG and then fixed at 12 h (A) or 24 h (B) p.i. for electron microscopy. Photographs were taken at magnifications of ×3,000 (top rows in panels A and B) and ×9,300 (lower rows in panels A and B). The arrows in panel A represent typical viral intermediate structures such as crescents and IV.
- FIG. 6.
Actin tail formation in association with CEV in infected cells. HeLa cells were infected with viG3L at an MOI of 5 PFU per cell, cultured in medium with (+IPTG) or without (−IPTG) 50 μM IPTG for 17 h p.i., fixed with 4% paraformaldehyde, permeabilized, and stained for 1 h at room temperature with anti-B5R MAb (1:2,500) (green) and Alexa Fluor 647-phalloidin (Molecular Probes) (red) and then for 30 min with Alexa Fluor 488-conjugated goat anti-mouse IgG (1:1,000) (Molecular Probes). DNA was visualized by staining with DAPI (Molecular Probes) (blue); the cells were then washed, and images were collected on an LSM510 META confocal laser scanning microscope (Carl Zeiss, Germany) using a ×63 objective lens. The white arrows indicate representative CEV with actin tails on their tips.
- FIG. 7.
Structural analyses of G3L+ and G3L− IMV by electron microscopy and SDS-PAGE. (A) Purified G3L+ and G3L− IMV on 25 to 40% sucrose gradients. (B) Electron micrographs of negatively stained G3L+ and G3L− IMV. Virions were deposited on grids, washed with water, and stained with 7% uranyl acetate in 50% ethanol for 30 s. (C) SDS-PAGE of sucrose gradient-purified wild-type (WR) vaccinia, G3L+, or G3L− IMV. The numbers of particles were determined from the optical density at 260 nm, and equal amounts of the three types of virions were analyzed by SDS-PAGE with Coomassie blue staining. (D) Immunoblot of G3L+ and G3L− IMV. Equivalent amounts of G3L+ and G3L− IMV were separated on SDS-PAGE and analyzed by immunoblots with antibodies recognizing the indicated envelope proteins.
- FIG. 8.
G3L is required for virion penetration to release cores into the cytosol. HeLa cells were infected for 1 h at 4°C with equivalent amounts of G3L+ and G3L− virions (determined from the optical density at 260 nm) and then the cells were washed extensively and either immediately fixed (A) or incubated for a further 2 h at 37°C in the presence of cycloheximide before fixation (B). Cells were stained with anti-L1R MAb, followed by Cy5-conjugated goat anti-mouse IgG antibody (red) or with rabbit anti-A4L antiserum, followed by FITC-conjugated goat anti-rabbit IgG antibody (green). DNA was visualized by staining with DAPI. Optical sections were obtained by confocal microscopy and are displayed as maximum-intensity projections as described previously (5). The numbers of fluorescent-staining particles were counted from multiple photos and averaged.
- FIG. 9.
G3L is required for cell fusion. Freshly confluent BSC-1 cells were infected with viG3L virus at an MOI of 5 PFU per cell and incubated at 37°C for 21 h in medium with or without IPTG. Cells were washed three times with PBS (pH 7.2) and treated with PBS (pH 7.2) or PBS (pH 4.7) for 3 min at room temperature, and washed again, and the PBS was then replaced with normal medium. These cells were incubated for another 3 h and photographed with a Nikon inverted microscope.
