INTRODUCTION

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Mammalian orthoreoviruses (reoviruses) serve as useful models to study the viral and cellular determinants that enable nonenveloped viruses to enter cells and initiate infection. The mature reovirus virion comprises two concentric icosahedral capsids, which in turn surround the segmented double-stranded RNA genome. Outer-capsid proteins 1, 3, and µ1/µ1C play critical roles in virus entry. The first step in entry, binding to cell surface receptors, is mediated by the 1 trimer located at each fivefold axis in virions (24, 26, 49). Following receptor binding, reovirus virions are delivered into endocytic compartments, where they undergo partial uncoating. By this process the major outer-capsid proteins 3 and µ1/µ1C, present in 600 copies each, are proteolytically cleaved, yielding subvirion particles (16, 48) with similarities to the infectious subvirion particles (ISVPs) that can be generated by in vitro proteolysis (7, 30, 45). Notable features of these subvirion particles include loss of 3 and cleavage of µ1/µ1C within a defined region near its C terminus to generate particle-bound fragments µ1/ and  (40). Subsequent to the required cleavages of 3 and/or µ1/µ1C, the µ1/µ1C protein is thought to undergo a change in conformation analogous to those by the fusion proteins of enveloped viruses, giving it the capacity to perturb the integrity of the adjacent membrane bilayer (9, 14, 15, 27, 35, 39, 52). This interaction provides access to the cytoplasm for the resulting subvirion particle in which the particle-associated enzymes for transcription of the viral mRNAs are activated from their latent state in virions (8, 10, 14, 19, 22, 30, 45).

Although proteolysis of outer-capsid proteins is essential for productive infections (see below), the molecular basis for this requirement remains to be fully characterized. In nature, reoviruses infect via the enteric and respiratory tracts. Studies with proteinase inhibitors have shown that in the intestinal tract, proteolysis of outer-capsid proteins by pancreatic serine proteinases, generating ISVP-like subvirion particles, is required for at least some reovirus strains to adhere to M cells and infect intestinal target tissues (1, 6). In contrast, when reoviruses infect via the respiratory tract, where the concentration of extracellular proteinases is low, proteolysis more likely occurs after uptake of virions into the acidic endocytotic compartments of target cells. Studies with cultured cell lines have clearly demonstrated the need for intracellular proteolysis during infections with intact reovirus virions. In culture, infections with virions, but not ISVPs, can be blocked by treating cells with weak bases like NH₄⁺ (e.g., from NH₄Cl) that raise pH in acidic compartments in cells, including endosomes and lysosomes (3, 12, 50). Similar results are obtained with E-64, an inhibitor of papain family cysteine proteinases, including several that reside in mammalian lysosomes (3, 15). Treatment with pepstatin A, an inhibitor of aspartic proteinases including cathepsin D in mammalian lysosomes, however, has no effect on reovirus infections (32). Together, these data indicate that specific cellular proteinases participate in cleaving 3 and/or µ1/µ1C during entry into cells. Recent evidence indicates that the effects of E-64 and NH₄Cl on reovirus infections can be overcome by infecting cells either with ISVPs (3, 50) or with dpSVPs, distinct subvirion particles which are like ISVPs in lacking 3 but like virions in having very few µ1/µ1C molecules cleaved at the - junction (15). The latter finding suggests that cleavage of µ1/µ1C at the - junction during reovirus entry is dispensable for infection and that only cleavages of 3 are required. Many molecular details of reovirus entry, including which cleavages of 3 are needed to activate the membrane-disrupting potential of the underlying µ1/µ1C protein and which host proteinases effect these cleavages, remain to be determined.

To understand the molecular basis of reovirus entry in greater detail, we sought to develop a system in which the 3 protein could be altered (e.g., by site-directed mutagenesis) and assembled into virion-like particles, after which the consequences of these mutations on virion structure and entry into cells could be analyzed. Chang and Zweerink (16) provided the first evidence that 3 protein derived from reovirus-infected L cells can bind to ISVP-like subvirion particles generated during reovirus infection. Subsequent studies by Astell et al. (2) showed that the reassembled particles obtained in this manner are similar to native virions in density, appearance in electron micrographs, and insensitivity to transcriptase activation. Recent work in one of our laboratories showed that 3 generated by in vitro transcription-translation, using a 3-encoding S4 cDNA (31), can bind to purified ISVPs and remain bound through purification in a CsCl gradient (46). Moreover, the conformation of the 3 molecules bound to ISVPs appeared identical to that bound to virions, based on an identical pattern of 3 cleavage fragments following limited in vitro digestion with proteinase K (46).

Although in vitro transcription-translation generates sufficient amounts of 3 for many types of analytical studies with recoated particles (46), that approach is not conducive to obtaining large numbers of virion-like particles. Here, we demonstrate that this limitation can be overcome by using 3 protein from lysates of insect cells that are infected with a recombinant baculovirus providing high levels of 3 expression. Using baculovirus-expressed 3 protein, we approximated stoichiometric recoating of large numbers of ISVPs and performed studies which indicate that these recoated ISVPs (rcISVPs) mimic both the appearance and the behavior of virions in several respects. We conclude from these studies that 3, and not the - cleavage of µ1/µ1C, is the primary determinant of structural and functional differences between virions and ISVPs. Since virion-like particles can be similarly reconstituted with 3 mutants (29), we can now explore the roles of 3 in reovirus entry by using an approach that we call recoating genetics. To demonstrate this approach, we used chimeric 3 proteins to localize the primary determinants of a strain-dependent difference in 3 cleavage rate to a C-terminal region of the ISVP-bound protein.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Cells. Spinner-adapted L cells were grown in Joklik's modified minimal essential medium (Irvine Scientific Co., Irvine, Calif.) supplemented to contain 2% fetal bovine serum, 2% neonatal bovine serum (HyClone Laboratories, Logan, Utah), 2 mM glutamine, 100 U of penicillin per ml, and 100 µg of streptomycin per ml (Irvine Scientific). Spodoptera frugiperda clone 21 (Sf21) insect cells (Invitrogen, Carlsbad, Calif.) were grown in TC-100 medium (Gibco BRL, Gaithersburg, Md.) supplemented to contain 10% heat-inactivated fetal bovine serum.

Recombinant baculovirus containing the reovirus T3D S4 gene. A cDNA copy of the reovirus type 3 Dearing (T3D) S4 gene (31) was subcloned into the EcoRI site of the transfer plasmid pEV/35K/ polybsmcr (34) under transcriptional control of the baculovirus polyhedrin promoter. Recombinant baculoviruses containing the polyhedrin-S4 construct (termed S4D-baculoviruses) were then obtained and amplified as described for the reovirus S3 gene (25). Expression of the T3D 3 protein upon infection of Sf21 cells with S4D-baculoviruses was confirmed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and immunoblotting.

Expression of 3 in insect cells. Sf21 cells (5 × 10⁶) were infected with S4D-baculovirus at 10 PFU/cell. Cells were harvested at 48 h postinfection, washed with phosphate-buffered saline (PBS: 137 mM NaCl, 8.1 mM Na₂HPO₄, 2.7 mM KCl, 1.5 mM KH₂PO₄ [pH 7.5]), and lysed with 800 µl of lysis buffer (20 mM Tris, 5 mM MgCl₂, 1% Triton X-100, 0.1 M NaCl, 5 µg of leupeptin per ml, 1 mM phenylmethylsulfonyl fluoride [pH 7.4]) by incubation on ice for 30 min. Immediately after incubation, the lysed cells were pelleted by centrifugation at 500 × g for 10 min at 4°C. The soluble (cytoplasmic) fraction was removed, and the pellet was resuspended in 800 µl of PBS adjusted to contain 500 mM NaCl. After centrifugation at 22,000 × g for 10 min, the new soluble fraction (nuclear lysate), which contained most of the 3 protein, was harvested.

Virions and ISVPs. Virions of reovirus type 1 Lang (T1L) or T3D were obtained by the standard protocol (24) and stored in virion buffer (150 mM NaCl, 10 mM MgCl₂, 10 mM Tris [pH 7.5]). To obtain T1L ISVPs, the same protocol was followed except that after the second freon extraction, virions were diluted in virion buffer and pelleted by centrifugation at 5°C in an SW28 rotor (Beckman Instruments, Palo Alto, Calif.) spun at 25,000 rpm for 2 h. The pelleted virions were resuspended in virion buffer at a concentration lower than 10¹³ particles/ml and treated with 200 µg of N-p-tosyl-L-lysine chloromethyl ketone (TLCK)-treated -chymotrypsin (CHT) (Sigma Chemical Co., St. Louis, Mo.) per ml for 50 min at 37°C. ISVPs were purified by centrifugation in a preformed CsCl gradient (24) and stored in virion buffer after dialysis. All particle concentrations were determined from A₂₆₀ (17).

rcISVPs. Purified T1L ISVPs were incubated at room temperature for 30 min with nuclear lysate derived from Sf21 cells infected with S4D-baculovirus. Stoichiometric recoating was routinely achieved by adding lysate from 5 × 10⁶ infected cells to 2.5 × 10¹² ISVPs. Virion buffer was added to decrease the NaCl concentration of the final mixture to 250 to 350 mM. Immediately after incubation, the sample was layered on a preformed CsCl density gradient (1.27 to 1.46 g/cm³) and subjected to centrifugation at 5°C in an SW41 rotor (Beckman) spun at 35,000 rpm for at least 4 h. The particle band was harvested and dialyzed into virion buffer. Particle concentration was determined from A₂₆₀ values, using the conversion factor corresponding to virions (17).

SDS-PAGE and immunoblotting. Samples to be analyzed by SDS-PAGE were diluted 2:1 with 3× sample buffer (375 mM Tris, 30% sucrose, 3% SDS, 6% -mercaptoethanol, 0.03% bromophenol blue [pH 8.0]) and boiled for 1 to 2 min. Ten percent acrylamide gels were used, and proteins were visualized by staining with Coomassie brilliant blue R-250 (Sigma). For immunoblots, protein samples were subjected to SDS-PAGE and transferred to nitrocellulose at 4°C overnight at 30 V in transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol [pH 8.3]). Mouse-derived, 3-specific monoclonal antibody 4F2 (53) was used at a 1/2,000 dilution of a 1-mg/ml stock. Detection of this antibody was achieved by using 1/3,000 alkaline phosphatase-coupled goat anti-mouse immunoglobulin (Bio-Rad Laboratories, Hercules, Calif.) with 300 µg of p-nitroblue tetrazolium chloride per ml and 150 µg of 5-bromo-4-chloro-3-indolyl phosphate p-toluidine per ml (Bio-Rad) in substrate buffer (100 mM Tris, 0.5 mM MgCl₂ [pH 9.5]).

Buoyant densities. Particles (2 × 10¹¹) were layered on a 10-ml 1.30- to 1.46-g/cm³ preformed CsCl gradient and subjected to centrifugation at 5°C in an SW41 rotor (Beckman) spun at 25,000 rpm for 12 to 16 h. Gradients were fractionated into 300-µl aliquots by collection from bottom to top, using a peristaltic pump (Rainin Instrument Co., Woburn, Mass.). Fractions containing virus particles were located by measuring A₂₆₀, and buoyant densities of the peak fractions were determined by measuring refractive indices with a digital refractometer (Bausch & Lomb, Rochester, N.Y.).

Electron microscopy and 3-D image reconstruction. Fresh rcISVPs samples were prepared for conventional transmission electron microscopy (TEM) as described previously (13). Samples of freshly generated rcISVPs were prepared for low-temperature, high-resolution scanning electron microscopy (cryo-SEM) and viewed as described previously (13). Images were acquired in digital format directly from the microscope by using Digital Micrograph (Gatan), and their brightness and contrast were optimized by using Photoshop (Adobe Systems, Mountain View, Calif.). For low-temperature, high-resolution TEM (cryo-TEM), purified rcISVPs were embedded in vitreous ice, and micrographs were recorded at a nominal magnification of ×38,000, using standard low-dose cryo-TEM procedures on a Philips CM200 microscope (5). Ninety-one particles were selected from three micrographs (defocus values of 2.1, 2.6, and 3.3 µm) and analyzed with image processing techniques for icosahedral particles (4, 23). The particle orientations were evenly distributed throughout the asymmetric unit, as evidenced by all inverse eigenvalues being <1.0 and at least 99% being <0.1 (23). The final three-dimensional (3-D) reconstruction was calculated at 33-Å resolution.

Plaque assays. Plaque assays to determine particle/PFU ratios of reovirus preparations were done as described previously (24). To determine infectious titers in other experiments, a modified procedure was used. Prior to addition of virus, the monolayers were washed to remove serum with PBS plus 2 mM MgCl₂. After a 1-h attachment period, the monolayers were covered with 2 ml of 1% Bacto Agar and serum-free medium 199 containing 10 µg of trypsin per ml (Sigma). Plaques were counted 2 days later.

Infectivity experiments. Single-step growth curves were determined as described elsewhere (15). Endpoint experiments with NH₄Cl were performed as for single-step growth curves except that only 0- and 24-h time point samples were harvested and 20 mM NH₄Cl was included in the culture medium. Endpoint experiments with E-64 (Sigma) and pepstatin A (Sigma) were performed as follows. L cells at 4 × 10⁵ cells/ml were dispensed in 2-dram vials and pretreated for 2 h at 37°C with 300 µM E-64 or 30 µM pepstatin A (each dissolved in dimethyl sulfoxide), or with dimethyl sulfoxide alone (for samples without inhibitor). After pretreatment, cells were chilled at 4°C for 15 min, the growth medium was removed, and virus particles were added at 3 PFU/cell. Adsorption proceeded for 1 h at 4°C, after which the inoculum was removed, and the original medium (with or without inhibitor) was added back to each vial. Vials were then transferred to a 37°C incubator, and 0- and 24-h time point samples were harvested by freezing.

Hemolysis and transcription. The capacity of reovirus particles to lyse erythrocytes was measured by a spectrophotometric assay for hemoglobin release (15, 39). The capacity of reovirus particles to mediate transcription of the viral mRNAs in vitro was measured by a radiometric assay for incorporation of [-³²P]GTP into acid-precipitable material (36).

Heat inactivation. Viral particles at a concentration of 4 × 10⁷ particles/ml in 500 µl of virion buffer were incubated at 52°C for 1 h or were kept on ice as a control. Heat treatment was ended by transferring the 52°C samples onto ice, and infectious titers were determined for all samples by plaque assay. The decrease in infectivity due to heat inactivation was expressed as the difference between the log₁₀ titers of each heat-treated sample and the corresponding control sample.

Construction of T1L and T3D 3 chimeras. DNA clones of the T1L and T3D S4 genes, inserted into pBluescript KS⁺ (Stratagene, La Jolla, Calif.) at the EcoRI site, were cleaved with BsaI and StyI (both within the S4 gene) or with StyI and EcoRV (the latter within the vector beyond the 3' end of the S4 plus strand) to obtain middle (S4 nucleotides 552 to 791, corresponding to 3 amino acids 186 to 265) or C-terminal (S4 nucleotides 792 to 1095, corresponding to 3 amino acids 266 to 365) fragments of S4, respectively. These fragments were exchanged between the T1L and T3D S4 constructs to generate chimeras as shown in Fig. 10A. The chimeric S4 genes were subcloned into the pEV/35K/polybsmcr plasmid (34), and the resulting constructs were used to express each 3 protein in insect cells by using a novel baculovirus system (28). Protein was labeled at 40 h postinfection with 25 µCi of [³⁵S]methionine/cysteine per ml, and 8 h later cells were harvested as described above.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Expression of recombinant 3 protein in insect cells. To obtain high levels of 3 expression for stoichiometric recoating of ISVPs, we constructed a recombinant baculovirus containing a cDNA copy of the S4 gene from reovirus T3D under control of the baculovirus polyhedrin promoter. At 48 h after infection with the recombinant baculovirus, insect cells were harvested and lysed. This treatment, followed by centrifugation at 500 × g, rendered a soluble supernatant (cytoplasmic fraction) and a pellet containing nuclei and insoluble material. SDS-PAGE and immunoblot analyses of these two fractions (Fig. 1 and data not shown) confirmed high levels of 3 expression and revealed that most 3 was in the pellet. This distribution could indicate either that 3 was insoluble or that it had been localized to the nucleus in insect cells as previously observed for 3 expressed in HeLa cells from an S4 cDNA (56) and in L cells after reovirus T3D infection (43). To distinguish between these possibilities, we resuspended the pellet and disrupted the nuclei with a series of buffers containing increasing amounts of salt. After centrifugation at 22,000 × g, SDS-PAGE of each new supernatant (nuclear lysate) and pellet fraction demonstrated recovery of 3 in the supernatant, with more 3 protein recovered at higher salt concentrations (data not shown). These findings suggest that 3 was recovered in the initial pellet because of its localization to the nucleus in insect cells. In subsequent experiments, buffer containing 0.5 M NaCl was used to release 3 from the pellet (as shown in Fig. 1) since higher concentrations did not improve recovery and were a concern for binding experiments.

View larger version (41K): [in this window] [in a new window]   FIG. 1.   Expression of 3 in insect cells by using recombinant S4D-baculovirus. (A) Cytoplasmic (cyt) and nuclear lysate (nuc) fractions from Sf21 cells infected with LacZ-baculovirus (lanes 1 and 2) or S4D-baculovirus (lanes 3 and 4) were subjected to SDS-PAGE and Coomassie blue staining. A sample of reovirus T3D virions (vir; lane 5) was included to give a size marker for 3. The positions of -galactosidase (-gal) and 3 proteins are indicated by arrows. Molecular weight markers (sizes in kilodaltons) are indicated at the left. (B) The lysate fractions analyzed in panel A were subjected to SDS-PAGE followed by immunoblot analysis using 3-specific monoclonal antibody 4F2 (53). The origin and significance of the high-molecular-weight band in lane 5 of panel B remain unconfirmed; however, this band is likely to represent a dimeric form of 3 that resists disruption in preparation for electrophoresis. This form of 3 may arise during particle preparation and/or storage since it is seen only with purified particles (also see Fig. 2B, lanes 1 and 3).

Stoichiometric recoating of ISVPs with recombinant 3. To determine if baculovirus-expressed T3D 3 protein can bind to ISVPs, purified ISVPs of reovirus T1L (generated from virions by in vitro CHT digestion and chosen over T3D ISVPs for their greater stability) were incubated with nuclear lysate from 3-expressing insect cells. After incubation, the particles were subjected to centrifugation in a preformed CsCl gradient to separate soluble 3 from any that was particle associated. SDS-PAGE and immunoblot analyses revealed that 3 had bound to the particles and remained bound through centrifugation (Fig. 2). Different ratios of 3-containing lysate and ISVPs led to different extents of 3 binding (data not shown). With excess lysate, however, the amount of bound 3 appeared to approximate that present in virions (Fig. 2), suggesting that nearly all potential binding sites for 3 had been occupied. To quantify the extent of recoating, SDS-PAGE followed by Coomassie blue staining and densitometry was performed with both native virions and ISVPs that appeared to have been fully recoated with 3. For each sample, the intensity of the 3 band was expressed relative to that of the -protein band. Comparison of 3/ ratios indicated that the 3 content of the recoated particles was equivalent to that of virions (Table 1), consistent with stoichiometric recoating.

View larger version (38K): [in this window] [in a new window]   FIG. 2.   SDS-PAGE and immunoblot analysis of rcISVPs. T1L ISVPs (2.5 × 1012) were incubated with the nuclear fraction from 5 × 106 Sf21 cells infected with S4D-baculovirus. rcISVPs were purified by centrifugation through a CsCl density gradient and dialyzed extensively against virion buffer. Virions (lane 1), ISVPs (lane 2), and rcISVPs (lane 3) (5 × 1010 each) were subjected to SDS-PAGE and Coomassie blue staining (A) or immunoblotting with the 3 monoclonal antibody 4F2 (53) (B).

View this table: [in this window] [in a new window]   TABLE 1.   Amounts of 3 protein in rcISVPs

The 3 coats of virions and rcISVPs appear structurally identical, as determined by electron microscopy and 3-D image reconstruction. rcISVPs were analyzed by conventional TEM after negative staining to compare them to native virions and ISVPs. A homogeneous population of particles very similar in morphology to virions was observed (Fig. 3A). Measured diameters of the particles ranged from 72 to 76 nm (data not shown), which is consistent with the diameter of native virions previously determined by negative-stain TEM (7, 24). Like virions (24), rcISVPs displayed a smooth perimeter with characteristic flattened edges that arise at fivefold axes where the µl-3 lattice is interrupted by 2 (21, 37).

View larger version (109K): [in this window] [in a new window]   FIG. 3.   Conventional TEM and cryo-SEM of rcISVPs. (A) Population of negatively stained rcISVPs viewed by conventional TEM at a magnification of ×100,000. Bar, 50 nm. (B) Population of chromium-coated rcISVPs viewed by high-resolution cryo-SEM at a magnification of ×100,000. Bar, 50 nm. (C and D) Single chromium-coated rcISVPs viewed by cryo-SEM at a magnification of ×300,000. Particles oriented near a fivefold axis (C) and near a twofold axis (D) are shown. Bar, 25 nm.

View larger version (75K): [in this window] [in a new window]   FIG. 4.   3-D image reconstruction of rcISVPs obtained from cryo-TEM images (E and F; 33-Å resolution) compared with previously reported reconstructions of T1L virions (A and B; 28-Å resolution) and ISVPs (C and D; 29-Å resolution) (21). (A, C, and E) Surface-shaded views down a twofold axis of symmetry for each particle. (B, D, and F) Same views as in panels A, C, and E except that the reconstructions were radially cropped to remove all densities below 41 nm, thereby isolating features attributable to 3 in the display. Bar, 30 nm.

Regeneration of ISVPs from rcISVPs. In vitro treatment of virions with alkaline proteinases such as CHT or trypsin can remove 3 from the particle surface and cleave µ1/µ1C at the - junction, thus generating ISVPs (7, 30, 45). Several observations suggest that this process is analogous to the early proteolytic steps during reovirus infection (16, 48). To determine if rcISVPs can be similarly processed by alkaline proteinases, we digested purified rcISVPs under conditions of CHT treatment designed to generate ISVPs (200 µg of CHT per ml for 20 min at 37°C). SDS-PAGE of the reaction sample revealed that 3 was completely removed from rcISVPs (Fig. 5) but the remaining structural proteins were cleaved little if at all. Treatments with trypsin gave identical results (data not shown). The buoyant density of CHT-treated particles was measured at 1.375 g/cm³ (±0.001, n = 3), consistent with the value of 1.38 g/cm³ characteristic of ISVPs. Hence, our results show that ISVPs can be regenerated from rcISVPs by in vitro treatment with alkaline proteinases and that addition and removal of 3 has little if any adverse effect on the other capsid proteins.

View larger version (78K): [in this window] [in a new window]   FIG. 5.   Regeneration of ISVPs from rcISVPs. rcISVPs (7.5 × 1010) were treated with CHT at 200 µg/ml for 20 min at 37°C in a final volume of 20 µl. The reaction was stopped by adding 1 mM phenylmethylsulfonyl fluoride and transferring the sample to 4°C. After disruption in sample buffer, the CHT-treated sample was analyzed by SDS-PAGE and Coomassie blue staining (lane 4), as was a sample containing the same number of untreated rcISVPs (lane 3). Equivalent numbers of purified virions (lane 1) and the purified ISVPs from which the rcISVPs were generated (lane 2) were also run on the gel for comparison.

rcISVPs are fully infectious. Structural similarities between rcISVPs and virions suggest that rcISVPs will provide a useful tool for analyzing 3 structure in virions. However, meaningful studies of the function and fate of virion-bound 3 in vivo depend on the extent to which rcISVPs remain infectious. The relative infectivity of rcISVPs was determined in plaque assays in which L cells were infected with purified virions, rcISVPs, or the ISVPs that had been used to generate the rcISVP preparations. Similar determinations were made with CHT digests of purified rcISVPs designed to regenerate ISVPs. Infectivity was correlated with particle concentration to obtain the relative infectivity of each preparation expressed as a particle/PFU ratio. We found the mean relative infectivities of virions and rcISVPs to be very similar: 74 particles/PFU for T1L virions and 70 particles/PFU for three preparations of rcISVPs. The mean relative infectivities of the corresponding ISVPs were also very similar: 69 particles/PFU for T1L ISVPs and 82 particles/PFU for CHT digests of two preparations of rcISVPs. These results indicate that neither addition of 3 to generate rcISVPs nor its subsequent removal to regenerate ISVPs has a substantial effect on infectivity.

Virions and rcISVPs exhibit identical replication kinetics. There is a characteristic difference in the kinetics of viral growth when cells are infected with reovirus virions or ISVPs. The lag phase extends for 8 to 10 h postadsorption with virions but for only 4 to 6 h with ISVPs (3, 15, 18, 50). The absence of 3 and cleavage of µ1/µ1C at the - junction represent two major structural differences between ISVPs and virions that could be related to the different kinetics. Since rcISVPs contain 3 as do virions, but cleaved µ1/µ1C as do ISVPs, we used them to determine whether either of these structural features affects the lag phase in single-cycle growth curves. Virions, rcISVPs, and ISVPs generated from each by in vitro CHT treatment were adsorbed to L cells at 4°C, and samples were harvested periodically over a 24-h period at 37°C for measurement of infectious titers. Infections with virions and rcISVPs demonstrated nearly identical growth kinetics, with the lag phase ending near 10 h postadsorption (Fig. 6). Infections with ISVPs derived from virions or rcISVPs also exhibited indistinguishable kinetics, but the lag phase ended at about 4 h postadsorption (Fig. 6). Hence, the presence or absence of 3, and not the - cleavage of µ1/µ1C, appears to be the primary determinant of growth kinetics for infections with the different particle types.

View larger version (20K): [in this window] [in a new window]   FIG. 6.   Single-step growth curve after infection with rcISVPs. T1L virions (vir), rcISVPs (rcI), or ISVPs generated by CHT digestion of each (vir+CHT or rcI+CHT, respectively) were used to infect L cells at 3 PFU/cell. After 1 h at 4°C, unbound virus was removed, and infected cells were added to 2-dram vials containing growth medium. Infected cells were incubated at 37°C and then harvested at different times postadsorption for determination of infectious titers by plaque assay. The change in log10 titer over time is expressed relative to the corresponding 0-h sample. Each point represents the average of two independent infections.

Infections with rcISVPs are blocked by NH₄Cl and E-64 but not pepstatin A. Cells treated with inhibitors of acid-dependent lysosomal proteinases, such as the weak base NH₄Cl and cysteine proteinase inhibitor E-64, resist infections with reovirus virions at least in part because cleavages of 3 and/or µ1/µ1C are blocked (3, 15, 50). These agents, however, have no effect on infections with ISVPs (3, 15, 50). The requirement for proteolysis with virions, but not ISVPs, is thought to partially explain the different kinetics of growth after infections with these particles. Since most µ1/µ1C molecules are already cleaved at the - junction in rcISVPs, it was important to determine whether infections with these particles are also dependent on cleavages by the acid-dependent cysteine proteinases. Virions, rcISVPs, and ISVPs generated from each by in vitro CHT treatment were adsorbed to L cells at 4°C. Infections then proceeded at 37°C in the presence or absence of 20 mM NH₄Cl or 300 µM E-64, and infectious titers were determined after 24 h. As expected, infections with virions were blocked by NH₄Cl or E-64 whereas infections with virion-derived ISVPs were not blocked by either compound (Fig. 7A and B). Similarly, infections with rcISVPs were blocked, but infections with rcISVP-derived ISVPs were not blocked, by either compound (Fig. 7A and B). Thus, virions and rcISVPs, despite prior cleavage of µ1/µ1C at the - junction in the latter, are similarly dependent on the processing of 3 by acid-dependent cysteine proteinases for productive infection.

View larger version (20K): [in this window] [in a new window]   FIG. 7.   Viral growth in the presence of NH4Cl, E-64, or pepstatin A after infection with rcISVPs. T1L virions, rcISVP, or their corresponding ISVPs (vir + CHT or rcI + CHT, respectively) were used to infect L cells at 3 PFU/cell. After attachment for 1 h at 4°C, infected cells were incubated at 37°C for 0 or 24 h in the absence (; open bars) or presence (+; shaded bars) of 20 mM NH4Cl (A), 300 µM E-64 (B), or 30 µM pepstatin A (C). Infectious titers were determined by plaque assay. The change in log10 titer after 24 h is expressed relative to the corresponding 0-h sample. Each bar represents the average ± standard deviation of three independent infections.

rcISVPs cannot undergo in vitro activation to mediate hemolysis or transcription of the viral mRNAs. In vitro, ISVPs but not virions can be activated to interact with lipid bilayers (9, 15, 27, 35, 39, 52) and to transcribe the viral mRNAs (8, 10, 15, 19, 22, 30, 45). Productive infection in vivo depends on analogous activities. Available data (see above) suggest that virions require proteolysis for infection in order to generate subvirion particles that can undergo activation to mediate these activities. dpSVPs, which lack 3 but have most molecules of µ1/µ1C uncleaved at the - junction as in virions, can also be activated to interact with lipid bilayers and to mediate transcription in vitro in the absence of further proteolysis (15). Thus, the - cleavage appears to be dispensable for these activities. Because rcISVPs, in contrast to other particles, have a full 3 coat and most µ1/µ1C molecules cleaved at the - junction, we used them to determine whether the presence of 3 influences the activation of particles to mediate hemolysis and transcription.

View larger version (16K): [in this window] [in a new window]   FIG. 8.   Capacity of rcISVPs to be activated to mediate hemolysis or transcription. Each bar represents the mean ± standard deviation from three trials. (A) A 3% solution of calf erythrocytes was incubated with 5 × 1010 T1L virions (vir), rcISVPs (rcI), or ISVPs generated by CHT digestion of each (vir + CHT or rcI + CHT, respectively) at 37°C for 30 min in the presence of 200 mM CsCl as an accelerant (8, 15). The extent of hemolysis was expressed as a percentage of that obtained by hypotonic lysis. (B) T1L virions, rcISVPs, or their respective ISVPs (5 × 1010 each) were incubated with a transcription mixture including 2 mM ribonucleoside triphosphate, 1.13 µCi of [-32P]GTP, and 200 mM CsCl. The incorporation of 32P into viral transcripts was measured after precipitation with trichloroacetic acid (36).

rcISVPs and virions are similarly thermostable. Reovirus virions are more resistant to heat inactivation than ISVPs (44). This property may enable virions to survive a wider range of conditions outside host organisms, thereby improving transmission of the virus between hosts (41). rcISVPs were analyzed to distinguish the roles of 3 and cleavage of µ1/µ1C at the - junction in determining the thermostability of particles. Virions, rcISVPs and ISVPs derived from each by in vitro CHT treatment were incubated at either 52 or 4°C in virion buffer for 1 h, and samples were then assayed for infectivity (Fig. 9). After 1 h at 52°C, ISVP samples exhibited a large decrease in titer, to <0.01% of that of the 4°C controls. In contrast, the titers of virions and rcISVPs decreased only to ~10% of control levels. Thus, virions and rcISVPs exhibited comparable greater thermostability than their respective ISVPs. The CHT present in the ISVP samples was not responsible for the loss of infectivity observed with heating since inactivation experiments performed with purified T1L ISVPs, with or without added CHT, gave very similar results (reference 44 and data not shown). These results demonstrate that 3 is the primary determinant of the different thermostabilities of virions and ISVPs and that cleavage of µ1/µ1C at the - junction has little or no effect on this property. These conclusions are consistent with a previous analysis showing that the S4 gene (which encodes 3) determines strain-specific differences in the thermostability of reovirus virions (20).

View larger version (16K): [in this window] [in a new window]   FIG. 9.   Sensitivity of rcISVPs to heat inactivation. T1L virions (vir), rcISVPs (rcI), or ISVPs generated by CHT digestion of each (vir + CHT or rcI + CHT, respectively) were subjected to heat inactivation at 52°C for 1 h. Corresponding samples were kept at 4°C as controls. Infectious titers were determined by plaque assay, and the change in log10 titer with heat treatment is expressed relative to the corresponding 4°C sample. Each bar represents the mean ± standard deviation from three trials.

Analysis of 3 cleavage rate by recoating genetics using chimeric 3 proteins bound to ISVPs. Proteolysis of reovirus outer-capsid proteins by luminal alkaline proteinases in the mammalian small intestine is required for at least some reovirus strains to adhere to M cells and infect intestinal target tissues (1, 6). During the course of experiments to dissect the cascade of cleavages that occur during alkaline proteolysis of 3, we treated purified T1L and T3D virions in vitro with endoproteinase Lys-C (EKC), a serine alkaline proteinase, and observed a difference in the rate at which full-length 3 protein was cleaved in these two strains, the 3 protein in T1L virions being cleaved faster than that in T3D virions (29). A genetic analysis using T1L × T3D reassortants indicated that the determinants of this difference reside within the two 3 proteins themselves (29). To localize these determinants within 3, we took advantage of our method for recoating ISVPs with baculovirus-expressed 3 protein. Eight distinct types of [³⁵S]methionine/cysteine-labeled 3 protein were separately expressed and used for recoating T1L ISVPs: T1L 3, T3D 3, and six chimeric proteins generated by exchanging amino acids 1 to 185, 186 to 265, and/or 266 to 365 between the T1L and T3D 3 proteins (Fig. 10A). The resulting particles were purified from CsCl gradients and subjected to EKC cleavage, followed by visualization and quantitation of full-length 3 protein by SDS-PAGE and phosphorimaging (Fig. 10B). The extent of 3 cleavage was assessed by comparing the intensities of the full-length 3 bands from EKC-treated and untreated samples of each particle preparation (Fig. 10C). The results demonstrate that the C-terminal 100 amino acids of 3 contain the primary determinants of the different rates of EKC cleavage of T1L and T3D 3: all 3 proteins with the C-terminal region from T1L exhibited a higher rate of cleavage than those with the C-terminal region from T3D (Fig. 10C). Interestingly, the N-terminal and middle regions of 3 made secondary contributions to cleavage rate, but in the directions opposite those expected from behaviors of the parental proteins: chimeric 3 proteins containing the N-terminal and/or middle regions from T3D exhibited an increased rate of cleavage, whereas chimeric 3 proteins containing one or both of those regions from T1L exhibited a decreased rate of cleavage. These findings suggest that different regions of 3 can contribute to the overall rate of cleavage but indicate a more prominent role for C-terminal sequences. The latter conclusion concurs with that from another recent study, which showed that a mutation near the C terminus of 3 affects the proteinase sensitivity of the virion-bound protein in a reovirus mutant selected during persistent infection (55).

View larger version (23K): [in this window] [in a new window]   FIG. 10.   Analysis of 3 cleavage rate by recoating genetics using chimeric 3 proteins bound to ISVPs. (A) T1L and T3D 3 proteins, as well as six 3 chimeras generated by exchanging N-terminal, middle, and C-terminal regions (junctions indicated by amino acid number) of the 3 proteins of reoviruses T1L and T3D, were expressed in insect cells (see Materials and Methods). Each type of 3 protein is designated by three letters indicating the origin (L or D) of the three regions. (B) [35S]methionine/cysteine-labeled 3 proteins of the eight types shown in panel A were separately bound to T1L ISVPs, which were then purified in a CsCl gradient and dialyzed into virion buffer. The purified particles were subsequently treated with EKC (20 µg/ml) for 1 h at 37°C, and the reaction was stopped using 1 mM TLCK (Sigma). Equivalent numbers of EKC-treated (+) and untreated () particles were analyzed by SDS-PAGE and phosphorimaging. The bands corresponding to full-length 3 are shown from two representative gels on which all eight types of 3-containing particles were analyzed. (C) The 3 bands were quantitated by phosphorimaging, and the amount of full-length 3 remaining in each EKC-treated sample was expressed as a percentage of that in the corresponding untreated sample. The filled and open bars indicate measurements from two pairs of samples for each particle preparation.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Assembly of 3 into virions in vivo and in vitro. In reovirus-infected cells, mature virions undergo a regulated process of assembly that remains poorly characterized. One observation, however, is that outer-capsid assembly is blocked and core-like particles accumulate when cells are infected at nonpermissive temperatures with virus containing a temperature-sensitive lesion in the S4 gene (tsG453) (38, 47). This finding suggests that the µ1 protein cannot be assembled into particles in the absence of functional 3. In fact, µ1 and 3 are known to form complexes in solution (33), and this interaction modifies the conformation of 3 such that its proteinase sensitivity is increased to the level seen in virions (46). In addition, the µ1 protein in µ1-3 complexes becomes susceptible to cleavage near its N terminus, yielding fragments µ1N and µ1C through what is thought to be an autocatalytic mechanism (42, 51). Together, these findings suggest that during assembly of virions in infected cells, µ1-3 complexes are formed first, which modifies the conformation of both µ1 and 3 and allows these complexes to bind to nascent particles (38, 47).

Binding to 3 is not blocked by cleavages of µ1 at the µ1N-µ1C and - junctions in ISVPs. As noted above, the µ1 molecules in virions have undergone several changes in structure relative to soluble µ1, including conformational changes and a cleavage near their N termini to generate the µ1N and µ1C fragments by which most µ1 molecules are represented in virions. Moreover, when ISVPs are subsequently generated from virions by proteolysis, the µ1/µ1C molecules undergo an additional cleavage near their C termini to generate fragments µ1/ and  (40). It was conceivable that one or more of these changes might have modified µ1 such that, in its ISVP-bound form, it was no longer competent to bind soluble 3 protein. However, because soluble 3 can bind to µ1/ and/or  in ISVPs, as directly shown in this study and previously (2, 46), the sites in µ1 that mediate 3 binding cannot have been irreversibly modified as a consequence of any of the changes in µ1 that accompany complex formation, µ1N-µ1C cleavage, particle binding, or - cleavage. In partial support of this conclusion, a 3-D reconstruction of dpSVPs, in which very few µ1/µ1C molecules have been cleaved at the - junction (15), failed to demonstrate any clear structural differences from ISVPs (54), suggesting that the - cleavage in ISVPs has little effect on µ1 conformation.

Effect of 3 on functional properties of reovirus particles in vitro and in vivo. After addition of 3 to ISVPs, to generate rcISVPs, these particles behave like virions in all of the functional properties tested in this study (summarized in Table 2). Thus, 3 is a key determinant of particle behavior for each of these properties. An important structural distinction between virions and rcISVPs is that the latter contain most molecules of µ1/µ1C cleaved at the - junction, like ISVPs. As a result, our findings with rcISVPs also indicate that cleavage of µ1/µ1C at the - junction is not an important determinant of the differences in behavior between virions and ISVPs studied here. Previous experiments with dpSVPs, a distinct type of subvirion particle that lacks 3 but contains most molecules of µ1/µ1C still uncleaved at the - junction, provided support for this conclusion by showing that dpSVPs behave like ISVPs in a similar set of tested properties (15) (summarized in Table 2), despite the low level of - cleavage in dpSVPs. The analyses of rcISVPs here and of dpSVPs in the previous study (15) are thus consistent and complementary in demonstrating the key role of 3 and the negligible role of - cleavage in determining the different particle behaviors.

rcISVPs as reagents for mutational studies of 3. The lack of an effective method for engineering mutations into the genomes of double-stranded RNA viruses in the family Reoviridae has hindered studies of various aspects of their biochemistry and biology. Our new method for recoating ISVPs, however, represents a simple yet effective system that can overcome this limitation in a partial way for the reovirus 3 protein. By engineering selected mutations into an S4 cDNA clone, expressing mutant forms of recombinant 3 protein, and using these mutant 3 molecules to generate rcISVPs (29), we can now perform detailed characterizations of the effects of these mutations, both in vitro and in vivo, on the properties of 3 relating to reovirus entry. A notable advantage of this system is that it does not rely on viral replication for recovery of infectious virions containing the mutant 3 proteins. Thus, mutations that may have lethal effects on infection, and would be difficult or even impossible to amplify by using a more classical reverse-genetic approach, can be readily studied with our approach. The genetic analysis of 3 cleavage rate included in this study provides a concrete demonstration of the utility of the recoating approach as a genetic tool and also demonstrates how chimeric proteins constructed to have different regions derived from different virus strains can be used in localizing the sequence determinants of strain-dependent differences in the behaviors of particle-bound proteins.