Article Figures & Data
Figures
- Fig. 1.
In vitro assembly of baculovirus-expressed μ1 and ς3 proteins onto reovirus cores. (A) [35S]methionine/cysteine-labeled cytoplasmic extract prepared from 7.5 × 107 Tn High Five cells infected with a μ1- and ς3-expressing recombinant baculovirus was incubated with unlabeled reovirus cores (5 × 1012 particles). The resulting r-core particles were purified on two successive CsCl gradients. Both the extract (lane 1) and the purified r-cores (lane 2) were subjected to SDS-PAGE and phosphor imaging.35S-labeled cores (Core) and virions (Virion) were included as markers to indicate positions of the viral proteins. (B) Two independent preparations of purified r-cores (R-core 1 and R-core 2), generated as described above except by using unlabeled insect cell extract, were examined by SDS-PAGE and Coomassie blue staining. A lane of purified virions was included for comparison. (C) Purified cores, virions, and r-cores were examined by tricine-SDS-PAGE (44) on a 10 to 16% acrylamide gradient gel and Coomassie blue staining. Only those portions of the gel containing μ1 and its fragments are shown. Positions of molecular weight markers are indicated by Mr (103) at the left.
- Fig. 2.
Cryo-SEM of purified r-cores and pr-cores. Low-magnification (×150,000) images of cores (A), r-cores (B), and pr-cores (C) and high-magnification (×500,000) images of r-cores (D) and pr-cores (E) are shown. The particles in panels D and E are oriented near an axis of twofold symmetry. The scale bar in panel A applies to panels A to C, while that in panel E applies to panels D to E.
- Fig. 3.
Image reconstructions of r-cores from cryo-TEM images. The reconstruction of r-cores (C and F; 24 Å) is compared with previously reported reconstructions of cores (A and D; 32 Å) and virions (B and E; 28 Å) (15). (A to C) Surface-shaded views down a twofold axis of symmetry for each particle. (D to F) Same views as in panels A to C except that the reconstructions were radially cropped to remove all densities below 321 Å, thereby isolating features attributable to outer-capsid proteins μ1, ς3, ς1, and λ2 in the display.
- Fig. 4.
Close-up surface-shaded views of the λ2 turret in r-cores. The reconstructions of r-cores (C and F), cores (A and D), and virions (B and E) are shown in close-up for a representative λ2 turret. Asterisks indicate one of the five petal-like elements that form the central λ2 flower in virions and r-cores and then move outward and upward during conversion of virions to cores. Triangles in panels B and E indicate the drop-like density in virions attributed to ς1 that is absent in the r-core reconstruction. (A to C) Top views. (D to F) Views generated by tilting images by 45° about the horizontal axis. Images were sectioned so as to permit a clear view of the λ2 turret.
- Fig. 5.
Proteolytic processing of r-cores to pr-cores by CHT. Viral proteins were visualized by SDS-PAGE and Coomassie blue staining. (A) Purified virions or r-cores were treated with CHT for 1 h, and the resulting ISVPs or pr-cores were purified by centrifugation through CsCl density gradients. Viral proteins were resolved on a 5 to 20% acrylamide gradient gel (39). Positions of molecular weight markers are indicated by Mr (103) at the right. A lane of purified cores was included for comparison. (B) Purified virions were treated with CHT for specified times. A marker lane of untreated virions was included for comparison (M). (C) Same as panel B except that purified r-cores were used instead of virions.
- Fig. 6.
Capacity of pr-cores to induce lysis of RBCs in vitro. Bovine calf RBCs (final concentration, 3% [vol/vol]; Colorado Serum Co., Denver, Colo.) were incubated with purified virions, r-cores, cores, or CHT treatment mixtures containing ISVPs or pr-cores (3 × 1012 particles/ml) at 37°C for 40 min in the presence of CsCl (200 mM). Reactions were terminated by removal onto ice, and RBCs were pelleted by centrifugation at 300 × g for 5 min. The extent of hemolysis in each reaction was determined by measuring A415 of the supernatant and was expressed as a percentage (hemolysis by distilled water = 100%). Each bar represents the mean ± SD from three trials.
- Fig. 7.
Capacity of ς1-specific and core-specific antibodies to neutralize infectivity of cores and r-cores. Purified virions, cores, r-cores, or CHT treatment mixtures containing ISVPs or pr-cores were incubated with a ς1-specific MAb (5C6), a core-specific antiserum (anticore), or phosphate-buffered saline. Virus-antibody mixtures were used to infect L-cell monolayers, and infectious titers were measured by plaque assay. Neutralization of infectivity was expressed as the percentage of plaques remaining after antibody treatment. Each bar represents the mean plaque survival ± SD derived from three independent experiments for 5C6 treatment. Overlaid bars represent values from two experiments with the core-specific antiserum.
- Fig. 8.
Capacity of r-cores to replicate in the absence and presence of inhibitors of viral infectivity. Purified virions, cores, r-cores, or CHT treatment mixtures containing ISVPs or pr-cores were used to infect L-cell monolayers at an MOI of 0.01 PFU/cell in the absence of inhibitor (solid bars) or presence of 20 mM NH4Cl (hatched bars) or 300 μM E-64 (gray bars) in the growth medium. Infectious titers at times 0 and 24 h were measured by plaque assay. Each bar represents the mean [log10(PFU/ml)t = 24 h − log10(PFU/ml)t = 0] ± SD derived from three independent experiments for no inhibitor and NH4Cl treatments. Overlaid bars represent values from two experiments for E-64 treatment.
- Fig. 9.
Generation of pr-cores(μ1-581D) and measurement of their capacity to induce lysis of RBCs and to initiate infection. (A) Purified r-cores(μ1-581D) or r-cores(μ1-581Y) were incubated with TLCK-treated CHT or TPCK-treated TRY to generate pr-cores(μ1-581D) or pr-cores(μ1-581Y), respectively. Viral proteins were resolved by SDS-PAGE and visualized by Coomassie blue staining. A marker lane of untreated r-cores(μ1-581D) or r-cores(μ1-581Y) was loaded onto each gel for comparison (M). (B) Purified r-cores(μ1-581D) or CHT treatment mixtures containing pr-cores(μ1-581D) or pr-cores(μ1-581Y) (2 × 1012 particles/ml) were incubated with bovine calf RBCs at 37°C for 30 min in the presence of CsCl, and the extent of hemolysis induced by each virus preparation was measured as described for Fig. 6. Each bar represents the mean ± SD from three trials except in the case of r-cores(μ1-581D) where two trials were performed, and the average of both is shown. The extent of hemolysis was lower than that in Fig. 6 because a lower concentration of particles was used. (C) The capacity of purified r-cores(μ1-581D) or CHT treatment mixtures containing pr-cores(μ1-581D) to replicate in L cells over a 24 h-period in the absence (solid bars) or presence (hatched bars) of 20 mM NH4Cl was determined from three independent experiments as described for Fig. 8. An MOI of 0.01 PFU/cell was used.
Tables
- Table 1.
Quantitation of μ1C and ς3 proteins in r-coresa
Particle type Prepn λ/μ1Cb ς3/μ1Cb Virion 1 0.67 ± 0.02 0.78 ± 0.03 2 0.59 ± 0.03 0.77 ± 0.06 r-core 1 0.67 ± 0.03 0.76 ± 0.02 2c 0.68 ± 0.03 0.81 ± 0.03 3 0.65 ± 0.03 0.72 ± 0.06 r-core(μ1-581D) 1 0.60 ± 0.06 0.73 ± 0.06 a See text for descriptions of r-cores.
b Volume-based intensities of the λ, μ1C, and ς3 protein bands were determined by densitometry of Coomassie blue-stained SDS-polyacrylamide gels on which the proteins from purified particles were separated. Band ratios were calculated from three different lanes for each particle preparation. Mean ± SD of the three values is reported.
c Same as r-core preparation 2 in Fig. 1B and preparation B-2 in Table 2.
- Table 2.
Infectivity of purified r-coresa
Particle type Prepnb P/PFUc Relative P/PFUd Core A 1.1 × 109 1.0 B 5.0 × 108 1.0 r-core A-1 2.1 × 106 530 A-2 2.3 × 106 480 B-1 1.4 × 106 360 B-2e 1.1 × 106 450 r-core(μ1-581D) B-1 1.9 × 106 260 Virion 1 150 7.3 × 106 2 110 9.7 × 106 a See text for descriptions of r-cores.
b Each core preparation is designated with a capital letter. Each r-core preparation is designated with a letter, representing the core preparation from which it was derived, followed by the preparation number.
c Particle (P) per PFU values for each preparation were obtained by dividing P per milliliter by PFU per milliliter and are presented as an average of three determinations (SD < 0.10 log10 unit for all values). Concentrations of virion and core preparations were estimated by A260 (13, 51). Concentrations of r-core preparations were determined by densitometry of Coomassie blue-stained SDS-polyacrylamide gels compared with virion standards. Viral infectivity was measured by plaque assay (18).
d Defined as the ratio of P/PFU value of a preparation of cores to P/PFU value of a matching core or r-core preparation. Relative P/PFU values of virion preparations were calculated by using the PFU-per-milliliter value of core preparation A.
e Same as r-core preparation 2 in Fig. 1B and Table 1.
