Protein μ1-specific MAbs detect μ1 conformational change in vitro and within infected cells. (a) ³⁵S-labeled ISVPs and ISVP*s were immunoprecipitated with protein A-conjugated magnetic beads alone (none) or beads bound to μ1-specific MAb 10H2, 8H6, 4A3, or 10F6. Viral particles bound to antibody were quantitated by liquid scintillation counting. Error bars indicate standard deviations. (b to e) ISVPs were allowed to bind to CHX-pretreated Mv1Lu cells at 4°C, and cells were fixed at 0 or 2 h p.i. at 37°C. Cells were permeabilized and immunostained with MAb 10H2 (b), 8H6 (c), 4A3 (d), or 10F6 (e), followed by goat anti-mouse IgG conjugated to Alexa 488. Bars, 10 μm.

Anticore serum detects changes in particle structure in vitro and within infected cells. (a) ³⁵S-labeled ISVPs, ISVP*s, and cores were immunoprecipitated with protein A-conjugated magnetic beads alone (none) or beads bound to λ2-specific MAb 7F4 (α-λ2) or anticore serum (α-core). Viral particles bound to antibody were quantitated by liquid scintillation counting. Error bars indicate standard deviations. (b) ISVPs were allowed to adsorb to CHX-pretreated Mv1Lu cells at 4°C, and cells were fixed at 0 or 2 h p.i. at 37°C. Cells were permeabilized and immunostained with anticore serum, followed by goat anti-rabbit IgG conjugated to Alexa 594. (c) Cells were infected as described for panel b, fixed at 2 h p.i. at 37°C, permeabilized, and coimmunostained with anticore serum and 7F4, followed by goat anti-mouse IgG conjugated to Alexa 488 and goat anti-rabbit IgG conjugated to Alexa 594. Arrows in panel c insets indicate punctate spots detected by 7F4 but not anticore serum. Bars, 10 μm.

Kinetics of changes in particle structure and onset of viral protein synthesis within infected cells. ISVPs were allowed to adsorb to untreated or CHX-pretreated Mv1Lu cells in suspension at 4°C, and cells were fixed and permeabilized at different times p.i. at 37°C. CHX-treated cells were immunostained with μ1-specific MAb 10H2, 4A3, or 10F6 or with anticore serum (α-core), followed by goat anti-mouse or goat anti-rabbit IgG conjugated to Alexa 488. Both untreated and CHX-treated cells were immunostained with anti-μNS serum (α-μNS), followed by goat anti-rabbit IgG conjugated to Alexa 488. Cells were analyzed by flow cytometry. (a) Representative histograms. Ctrl. Ab, isotype-matched control antibody. Numbers above each histogram indicate the time in minutes p.i. (b) The geometric mean of each histogram was used as a measure of antibody binding, after subtraction of the value obtained with control antibody. Averages from two independent experiments are shown.

Particles resembling cores are generated in infected cells. (a to c) ISVPs were allowed to adsorb to CHX-pretreated Mv1Lu cells at 4°C, and cells were fixed at 2 h p.i. at 37°C. Cells were permeabilized and coimmunostained with anticore serum and μ1-specific MAb 10H2 (a), 4A3 (b), or σ1-specific MAb 5C6 (c), followed by goat anti-mouse IgG conjugated to Alexa 488 and goat anti-rabbit IgG conjugated to Alexa 594. The arrowhead in panel b indicates a punctate spot detected by both anticore serum and 4A3. (d) Intracellular localization of particles. CV-1 cells were infected as described above and fixed at 2 h p.i. Cells were permeabilized and coimmunostained with anticore serum and anti-LAMP-2 MAb H4B4 (α-LAMP-2), followed by goat anti-rabbit IgG conjugated to Alexa 488 and goat anti-mouse IgG conjugated to Alexa 594. Bars, 10 μm. (e and f) Biochemical evidence for particles resembling cores in infected cells. (e) ³⁵S-labeled ISVPs (∼5 × 10⁵ particles/cell) were allowed to adsorb to CHX-pretreated Mv1Lu cells (∼2 × 10⁶) in 60-mm dishes at 4°C, and cells were harvested and lysed in IP buffer at 0 or 2 h p.i. at 37°C. Cytoplasmic lysates were immunoprecipitated with λ2-specific MAb 7F4 (α-λ2) or anticore serum (α-core). Proteins within the immunoprecipitates were resolved by SDS-PAGE, and viral capsid proteins were detected by phosphorimaging. A reference lane for ISVPs is also shown. Positions of viral proteins are indicated to the left and right. (f) Bands in each lane in panel e corresponding to the μ1 proteolytic fragment δ and core proteins λ1, λ2, and λ3 were quantitated by densitometry as described previously (14). Protein μ1 content within each lane was computed as a ratio of band volumes [δ/(λ1 + λ2 + λ3)] and is shown as a percentage of that obtained for the ISVP reference lane. A similar quantitative analysis was also carried out with cytoplasmic lysates prior to immunoprecipitation (no IP). Averages and standard deviations for two (α-λ2, 0 h) or three lanes from two independent experiments are shown.

Capacities of ISVP-like particles derived from recoated cores to mediate the lysis of erythrocytes and to undergo ISVP→ISVP* change in vitro. (a) Purified virions, r-cores(μ1-WT), or r-cores(μ1-HS) (8 × 10¹² particles/ml) were converted to their respective ISVPs or ISVP-like particles [pr-cores(μ1-WT) or pr-cores(μ1-HS)] by digestion with chymotrypsin for 15 min at 37°C. Viral capsid proteins were resolved by SDS-PAGE and visualized by Coomassie brilliant blue R-250 staining. Positions of viral proteins are indicated at the left and right. (b) Capacities of nonpurified pr-cores(μ1-WT) and pr-cores(μ1-HS) to mediate hemolysis, determined as previously described for ISVPs (13). Washed citrated bovine calf erythrocytes (3% [vol/vol]) were incubated with increasing concentrations of viral particles in virion buffer for 1 h at 37°C, and the extent of hemoglobin release into the supernatant was measured relative to that in controls containing only virion buffer and erythrocytes (0%) or water and erythrocytes (100%). Values from two independent experiments are shown. (c) Kinetics of hemolysis mediated by pr-cores(μ1-WT) and pr-cores(μ1-HS). Erythrocytes were incubated with viral particles (4 × 10¹² per ml) for different times at 37°C, and the extent of hemolysis was determined as described for panel b. Values from two independent experiments are shown. (d) Conformational status of protein μ1. Aliquots (10 μl) from the hemolysis reactions in panel c were incubated with trypsin (100 μg/ml) for 30 min at 4°C, digestion was stopped by the addition of soybean trypsin inhibitor (300 μg/ml), and viral capsid proteins were resolved by SDS-PAGE. Protein μ1 was visualized by immunoblotting with μ1-specific MAb 10F6. A portion of each immunoblot containing the region of interest is shown. Positions of viral proteins are indicated at the left. (e and f) Capacities of pr-cores±σ1(μ1-WT) and pr-cores±σ1(μ1-HS) to undergo conversion to ISVP*-like particles. pr-cores(μ1-WT) (e) and pr-cores(μ1-HS) (f) (4 × 10¹² per ml) were incubated with 300 mM NaCl (−) or CsCl (+) for 20 min at 37°C to inhibit or promote conversion to ISVP*-like particles, respectively (see the text for details). Viral particles were immunoprecipitated with protein A-conjugated magnetic beads alone (no Ab) or beads bound to 10H2 or 10F6. pr-cores+σ1(μ1-WT) (e) andpr-cores+σ1(μ1-HS) (f) were prepared and incubated with NaCl or CsCl as described above and immunoprecipitated with σ1-specific MAb 5C6 (α-σ1). Viral capsid proteins bound to beads were resolved by SDS-PAGE and visualized by Coomassie brilliant blue R-250 staining. Positions of viral proteins are indicated at the left and right. IgG H, IgG heavy chain; IgG L, IgG light chain; no IP, no immunoprecipitation (see the legend to Fig. 4). Asterisks indicate positions of protease bands.

Capacities of r-cores+σ1(μ1-WT), r-cores+σ1(μ1-HS), and their respective pr-cores+σ1 to synthesize viral proteins and produce viral progeny within cells. (a and b) pr-cores+σ1(μ1-WT) (a) or pr-cores+σ1(μ1-HS) (b) were allowed to adsorb to Mv1Lu cells in 60-mm dishes at 4°C, and cells were suspended, washed, and fixed at 0, 4, or 8 h p.i. at 37°C. Cells were permeabilized and immunostained with anti-μNS serum, followed by goat anti-mouse IgG conjugated to Alexa 488, and analyzed by flow cytometry. Protein μNS-negative (neg.) and μNS-positive (pos.) regions for each set of histograms were set so that 99.5% of cells fixed at 0 h were designated μNS negative. The tables (insets) indicate the percentages of μNS-negative and μNS-positive cells at each time point. (c) Infectivities of purified virions, r-cores+σ1(μ1-WT), r-cores+σ1(μ1-HS), r-cores(μ1-WT), and r-cores(μ1-HS) in L929 cells relative to their parent cores (infectivity set to 1), as determined by a plaque assay. Averages and standard deviations from three trials are shown.

Induction of μ1 conformational change and generation of particles resembling cores within cells infected with pr-cores+σ1(μ1-WT) or pr-cores+σ1(μ1-HS). (a to c) pr-cores+σ1(μ1-WT) or pr-cores+σ1(μ1-HS) were allowed to adsorb to CHX-pretreated Mv1Lu cells at 4°C, and cells were fixed at 0 or 2 h p.i. at 37°C. Cells were permeabilized and immunostained with μ1-specific MAb 10H2 (a) or 10F6 (b) or with anticore serum (α-core) (c), followed by goat anti-mouse IgG or goat anti-rabbit IgG conjugated to Alexa 488. Bars, 10 μm. (d and e) Suspension cultures of CHX-pretreated Mv1Lu cells were infected and immunostained as described above and analyzed by flow cytometry. The geometric mean of each histogram was used as a measure of antibody binding, after subtraction of the value obtained with control antibody. Averages and standard deviations from three independent experiments are shown.

Localization of viral particles, the δ region of μ1, and LAMP-1-positive vacuoles in cells infected with pr-core+σ1(μ1-WT) or pr-core+σ1(μ1-HS). pr-cores+σ1(μ1-WT) (a and c) or pr-cores+σ1(μ1-HS) (b and d) were allowed to adsorb to CHX-pretreated Mv1Lu cells at 4°C, and cells were fixed at 2 h p.i. at 37°C. (a and b) Cells were permeabilized and immunostained with λ2-specific MAb 7F4 (α-λ2), followed by goat anti-mouse IgG conjugated to Alexa 594 and then by μ1-specific MAb 10H2 directly conjugated to Alexa 488. The arrow in the panel a inset indicates a punctate spot detected by 7F4 but not by 10H2. The arrowhead indicates a punctate spot detected by both 7F4 and 10H2. (c and d) Cells were permeabilized and coimmunostained with a LAMP-1-specific polyclonal antiserum (α-LAMP-1) and 10H2, followed by goat anti-mouse IgG conjugated to Alexa 488 and goat anti-rabbit IgG conjugated to Alexa 594. Arrowheads in panel d indicate punctate spots that were detected by 10H2 and that colocalized with LAMP-1-positive structures. Images were captured by spinning-disk confocal microscopy and processed as described in Materials and Methods. Bars, 20 μm.

Capacities of ISVPs, pr-cores+σ1(μ1-WT), and pr-cores+σ1(μ1-HS) to potentiate α-sarcin (αs) intoxication of L929 cells. ISVPs, pr-cores+σ1(μ1-WT), or pr-cores+σ1(μ1-HS) were allowed to adsorb to L929 cells in 24-well plates at 4°C, and cells were washed and incubated in methionine-free growth medium containing [³⁵S]methionine-cysteine and with or without α-sarcin (50 μg/ml) for different times at 37°C. Proteins were then precipitated with trichloroacetic acid, and acid-precipitable radioactivity was measured by scintillation counting. Averages and standard deviations from three trials are shown.

Updated model for reovirus entry. Whether productive infection by ISVPs can be initiated by penetration at the plasma membrane remains unresolved and is not illustrated here. Wild-type ISVPs and pr-cores+σ1(μ1-WT) are converted to ISVP*-like particles following cell adhesion and/or uptake. This step is characterized by conformational changes in μ1 and λ1, release of σ1 from viral particles, and derepression of core transcriptases. ISVP*-like particles initiate membrane penetration by inserting viral protein sequences into a cellular lipid bilayer. During or following membrane penetration, protein μ1 is removed from ISVP*s by the action of an unknown cellular factor(s), yielding particles that resemble cores. The δ fragment of released μ1 localizes to the cytoplasm and nucleus. Particles within the cytoplasm initiate viral mRNA synthesis. Unlike their μ1-WT-containing counterparts, pr-cores+σ1(μ1-HS) fail to be converted to ISVP*-like particles, to penetrate the cytoplasm, and to undergo the release of μ1. These mutant ISVP-like particles accumulate within late endosomes or lysosomes (LE/LY). EE, early endosomes.