Complete in vitro assembly of the reovirus outer capsid produces highly infectious particles suitable for genetic studies of the receptor-binding protein

Abstract

Mammalian reoviruses, prototype members of the Reoviridae family of nonenveloped double-stranded RNA viruses, use at least three proteins--sigma1, mu1, and sigma3--to enter host cells. sigma1, a major determinant of cell tropism, mediates viral attachment to cellular receptors. Studies of sigma1 functions in reovirus entry have been restricted by the lack of methodologies to produce infectious virions containing engineered mutations in viral proteins. To mitigate this problem, we produced virion-like particles by "recoating" genome-containing core particles that lacked sigma1, mu1, and sigma3 with recombinant forms of these proteins in vitro. Image reconstructions from cryoelectron micrographs of the recoated particles revealed that they closely resembled native virions in three-dimensional structure, including features attributable to sigma1. The recoated particles bound to and infected cultured cells in a sigma1-dependent manner and were approximately 1 million times as infectious as cores and 0.5 times as infectious as native virions. Experiments with recoated particles containing recombinant sigma1 from either of two different reovirus strains confirmed that differences in cell attachment and infectivity previously observed between those strains are determined by the sigma1 protein. Additional experiments showed that recoated particles containing sigma1 proteins with engineered mutations can be used to analyze the effects of such mutations on the roles of particle-bound sigma1 in infection. The results demonstrate a powerful new system for molecular genetic dissections of sigma1 with respect to its structure, assembly into particles, and roles in entry.

FIG. 1

Protein composition of r-cores+ς1. Purified virions, cores, r-cores, and two preparations of r-cores+ς1 (no. 1 and no. 2) (8 × 10¹⁰ particles each) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10% acrylamide) and Coomassie blue staining. Positions of viral proteins are indicated at left, and the position of ς1 is highlighted with arrows.

FIG. 2

Image reconstruction of r-cores+ς1. (a to c) Composite images comprising external (left half) and cutaway (right half) surface-shaded representations of virions (a), r-cores (b), and r-cores+ς1 (c) are shown. Features attributed to outer capsid proteins and the genome are labeled in panel a. (d) Difference map between r-cores+ς1 and r-cores. Only density features in the front half of the map are shown and are coded in red. (e) The difference map (red) superimposed upon a cutaway section of the r-core map. Features in the difference map at positions above and below the sectioning plane are not shown. (f) Magnified view of a portion of the image in panel e. One segment of the pentameric shutter that closes the λ2 turret is labeled (*). Scale bar, 200 Å.

FIG. 3

Capacity of r-cores+ς1 to bind to cells. (a) HA induced by purified virions, cores, r-cores, and r-cores+ς1 was determined by endpoint titration using human type A⁺ RBCs (American Red Cross), as described previously (20). Serial twofold virus dilutions were incubated with RBCs (0.4% [vol/vol]) in a 96-well round-bottomed plate at 4°C for 2 h, and HA was visually detected. The number of HA units induced by a virus preparation is given by the highest number of particles used (2 × 10¹⁰ particles) divided by the minimum number of particles required to induce HA. HA activity is expressed as the log₂(HA units). Averages ± standard deviations of three replicates are shown. (b) Attachment of purified virions, r-cores, and r-cores+ς1 to L cells at 4°C was determined by indirect immunofluorescence as described previously (18). Cells on coverslips were incubated with purified virus (5 × 10⁵ particles/cell) at 4°C for 1 h. Unbound virus was removed by washing with ice-cold phosphate-buffered saline. After the cells were fixed and permeabilized, bound virus was detected by using a 1:500 dilution of rabbit antireovirus serum (44) as primary antibody and a 1:100 dilution of fluorescein isothiocyanate-conjugated donkey anti-rabbit immunoglobulin G (Pierce) as secondary antibody.

FIG. 4

Infectious properties of r-cores+ς1 in L cells. (a) The infectivities of purified virions, cores, r-cores, and r-cores+ς1 were determined by plaque assay on L-cell monolayers and expressed as PFU per milliliter (26). After standardization against the number of virus particles per milliliter, the relative infectivity of each preparation was determined by dividing its PFU per milliliter by the PFU of cores per milliliter. Averages of three replicates are shown (standard deviation ≤ 0.10 log₁₀ units). (b) The capacity of anti-T1L ς1 antibody 5C6 to neutralize formation of plaques on L cells by purified virions, r-cores, and r-cores+ς1 was determined as described previously (12). Virus (100 PFU) was incubated with 5C6 (1 μg/ml) for 1 h at 37°C, and infectivity was measured by plaque assay. The extent of neutralization for each preparation (expressed as percent plaque survival) was the ratio of PFU obtained in the presence of antibody to the PFU obtained in its absence. Averages ± standard deviations of three replicates are shown. (c) Single-cycle growth curves of purified virions, r-cores+ς1, and chymotrypsin treatment mixtures containing ISVPs and proteinase-treated r-cores+ς1 (pr-core+ς1) (2 PFU/cell) were generated in L cells as described previously (13). Infectivity at a specified time (t) relative to time zero was expressed as log₁₀(PFU/ml)_t − log₁₀(PFU/ml)₀. Each data point represents the average of duplicate values.

FIG. 5

Capacity of r-cores+ς1 to mediate ς1-specific in vitro and in vivo properties. (a) HA of bovine RBCs (Colorado Serum Co.) induced by purified T1L virions, T3D virions, r-cores+ς1L, and r-cores+ς1D was determined as described for Fig. 3a. Averages ± standard deviations of three replicates are shown. (b) The effect of chymotrypsin treatment on infectivity of T1L virions (open squares), T3D virions (open circles), r-cores+ς1L (filled squares), r-cores+ς1D (filled circles), and r-cores+ς1D(T249I) (filled triangles) was determined as follows. Purified virus (1.5 × 10¹² particles/ml) was incubated with chymotrypsin (200 μg/ml) at 37°C. At specified times, an aliquot was removed onto ice and digestion was stopped with phenylmethylsulfonyl fluoride (2 mM). The infectivity of each aliquot was determined by plaque assay. Viral infectivity at a specified time (t) relative to time zero was expressed as described for Fig. 4c. Averages ± standard deviations of three independent experiments are shown. (c) Infectious yields produced in MEL and L cells by T1L virions, T3D virions, r-cores+ς1L, and r-cores+ς1D were determined as follows. Purified virus (5 PFU/cell) was incubated with 4 × 10⁶ cells for 1 h at room temperature. Unbound virus was removed by centrifugation (500 × g) and washing cells with phosphate-buffered saline. Cells were then resuspended in growth medium and incubated at 37°C for 24 h. Cell lysates were generated by freeze-thawing, and the amount of infectious virus in these lysates was determined by plaque assay. Growth yields were expressed as log₁₀(PFU/ml) at 24 h. Averages ± standard deviations of six trials from two independent experiments are shown.