Rotavirus Spike Structure and Polypeptide ... - Journal of Virology

Vol. 65, No. 8

JOURNAL OF VIROLOGY, Aug. 1991, p. 4334-4340

0022-538X/91/084334-07$02.00/0 Copyright C) 1991, American Society for Microbiology

Rotavirus Spike Structure and Polypeptide Composition INDUMATHY D. ANTHONY, STANLEY BULLIVANT, SHOBHNA DAYAL, A. RICHARD BELLAMY,* AND JOHN A. BERRIMANt Department of Cellular and Molecular Biology, University of Auckland, Auckland, New Zealand Received 29 November 1990/Accepted 10 May 1991

Negatively stained preparations of rotavirus imaged with a low dose of electrons provide sufficient contrast to reveal surface projections or spikes. The number of spikes found projecting from different particles indicates that not all 60 peripentonal sites are occupied. Treatment at pH 11.2 with 250 mM ammonium hydroxide specifically removes the spikes, yielding smooth double-shelled particles of the same diameter as that of the native virus. Protein analysis confirms that the released spikes are composed of polypeptide VP4 (or its two cleavage products VP5* and VP8*) and that the smooth particle retains the other major outer shell protein VP7. Spikeless particles can be decorated by a monoclonal antibody specific for the major immunodominant neutralizing domain of VP7, implying that removal of the spikes does not denature the VP7 that is retained on the surface of the smooth particle.

The outer shell of the double-layered rotavirus particle is constructed from two viral proteins. The most abundant of these is VP7, a 38-kDa glycoprotein which is the typespecific antigen (9, 12). VP7 is the translational product of genomic segment 9 (3, 17). The second protein, VP4 (88 kDa), is encoded by genomic segment 4 and is the viral hemagglutinin, a protein which also specifies cell tropism (9). Both VP7 and VP4 are assembled on the surface of the single-shelled rotavirus particle at some stage during the maturation of the virus in the lumen of the rough endoplasmic reticulum (20, 21). However, when trypsin is incorporated into the culture medium, VP4 is cleaved proteolytically to yield VP5* (60 kDa) and VP8* (28 kDa), an event which greatly enhances infectivity in vitro (4, 6, 7). The cleavage sites probably reside at Arg-241 and Arg-247, residues which are conserved in most strains of rotavirus for which cDNAs have been sequenced (2). The capsomeres of the inner shell have been shown by platinum shadowing to be arranged in a T = 13L configuration (19, 24). Prasad et al. (23) applied cryoelectron microscopy and image-averaging techniques to construct a model for the rotavirus particle with a resolution of 4.0 nm. Their work revealed that the outer shell is also arranged in a T = 13L symmetry and that there are 60 surface projections (spikes) approximately 4.5 nm long and 3.5 nm wide. They estimated from their model that each spike was one molecule of VP4. In a subsequent analysis (22), the spike was described as a thin structure extending to a height of 4.5 nm, with a further well-defined globular domain of approximately 5.5 nm in diameter, across which lies a bilobed structure 4.0 nm wide and 7.0 nm across. The estimated mass of the spike and the fact that two Fab fragments appeared to bind to each spike led these researchers to infer that the spike was a dimer of VP4. Yeager et al. (26) have also reconstructed images of rotavirus from cryoelectron micrographs and similarly describe the spike as having a complex bilobed morphology. The spikes were found to be shorter by the reconstruction method than could be measured on the micrograph, and they

suggested that morphological variability would be lost as a result of the averaging implicit in the reconstruction procedure. In discussing the oligomeric nature of the spike, they drew attention to the large disparity of stoichiometry between VP4 and VP7 from densitometer measurements of stained polyacrylamide gels (15) and suggested that purified viruses may well not all carry the full complement of 60 spikes. In a study of rotavirus by the freeze-etch method (la), it was found that while spikes on the surface of the intact virus could be clearly demonstrated, occupancy of the peripentonal sites was low. Here we present new evidence on the nature of the spikes by using negatively stained preparations examined with a low dose (26) of electrons (low-dose images). This method has enabled us to test many different extraction conditions for their effect on the virus. We have found that treatment of the intact virion with the weak base ammonium hydroxide at pH 11.2 removes the spikes, releases VP4 (or its cleavage products VP5* and VP8*) into the supernatant, and yields smooth particles that are otherwise intact. MATERIALS AND METHODS Cells and virus. The SAl1 strain of rotavirus was propagated in 1,585-cm2 roller bottle cultures of MA104 cells as described previously (25), using culture medium containing 25 pLg of trypsin (Hazleton Biologics Inc., Lenexa, Kans.) per ml. For the preparation of virus which contained the uncleaved form of VP4, this medium was removed 6 h postinfection and the monolayers were extensively washed and reincubated with medium lacking trypsin and containing 1% fetal calf serum. The infected cells were then harvested 40 h postinfection. Double-shelled virus was purified by banding on CsCl gradients that contained 10 mM Tris HCl (pH 7.5) and 10 mM CaCl2 (25) and stored at 4°C in CsCl prior to use. Trypsin cleavage of VP4 was achieved by first dialyzing the virus against TBS (140 mM NaCl, 0.7 mM Na2HPO4, 5 mM KCI, 20 mM Tris HCl [pH 7.5]), followed by digestion with 1-,ug/ml tolylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin (Sigma type XIII) for 30 min at 37°C. Aprotinin (100 ,uglml; Serva GmbH & Co., Heidelberg, Germany) was added to inhibit further trypsin digestion, the virus was rebanded on a preformed CsCl gradient, and then

* Corresponding author. t Present address: Laboratory of Molecular Biology, Medical Research Council, Cambridge CB2 2QH, England.

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CsCl was removed by dialysis against TBS. Samples were prepared for microscopy at concentrations of about 2 mg of viral protein per ml. NH40H treatment of virus. Virus at 0.5 mg/ml in 4 M CsCl was dialyzed against a solution containing 25 mM NaCl, 1 mM Tris HCl (pH 7.5), and 1 mM CaCl2. Aprotinin was added to a final concentration of 10 ,ug/ml. NH40H (1.25 M) (prepared from a 25% NH3 solution [analytical grade]; BDH, Poole, England) was added to the virus preparation to give a final concentration of 250 mM NH40H, and the preparation was incubated at room temperature for 25 min. The treated virus (100 ,ul) was then overlaid onto a 20-,ul cushion of 30% sucrose prepared in TBS and contained in an airfuge tube (5 by 20 mm; Beckman Instruments, Inc., Palo Alto, Calif.). The mixture was then centrifuged at 22 lb/in2 for 90 s in a Beckman Airfuge with an A-100/30 rotor to pellet the smooth particles which were resuspended in TBS for electron microscopy. For protein analysis by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) (14), the supernatant (80 pul) was first neutralized with acetic acid, and the protein was concentrated by precipitation with 9 volumes of ethanol at -20°C for 1 h. Electron microscopy. Specimens were examined by using the low-dose facility of a Philips CM12 electron microscope operating at 80 kV. Suitable areas were recorded on Kodak SO-163 film at a magnification of x28,000 and a dose of about 10 electrons per 0.01 nm2. The dose was calibrated against the speed of the film, which was developed in full-strength D19 for 12 min. The magnification was verified with catalase crystal spacings and maintained by using a constant (eucentric) objective lens current. Cryoelectron microscopy. Films with perforations were made by dipping cleaned microscope slides into a 0.5% solution of collodion in acetone. The dried films were examined by phase-contrast microscopy to assess size and distribution of holes. Suitable films were floated onto water, and 400-mesh copper grids were applied to the surface. These grids were picked up, coated with platinum-carbon (2.0 nm) to improve electrical conductivity, and then coated with carbon (10.0 nm) using a freeze-fracture coating source. The plastic layer was dissolved away by rinsing individual grids in amyl acetate, and after drying the grids were examined once more. The very high contrast of the platinum layer made it possible to identify false holes or bounded depressions. A 5-pI sample of virus was applied to the grid, which was then blotted and plunged into liquid ethane (1). Without warming, the grid was transferred to liquid nitrogen and inserted into a cold stage (Gatan Inc., Warrendale, Pa.) designed to maintain the specimen in amorphous ice (5). To minimize contamination with ice in the microscope, a bladetype anticontaminator (10) was used in addition to the standard cold trap. Negative staining. Optimal results were obtained using freshly prepared 10% uranyl formate. One-milliliter aliquots of 1% uranyl acetate were dispensed into Eppendorf tubes. Then 0.1 ml of 1.0 M NaOH was added, and the tubes were sealed and shaken. The precipitate was sedimented by centrifugation, and the supernatant was discarded. The pellet was dissolved in 100 p1 of 5% formic acid by vigorous mixing and used immediately. A carbon-coated grid was floated on 5 p,l of virus on a Parafilm surface (American Can Co., Greenwich, Conn.). After about 1 min, the grid was sequentially washed and stained by transfer across 3 50-pd drops of TBS and 2 drops of stain before it was blotted dry with filter paper held to the side of the grid. Hemagglutination assays. Virus samples in TBS were

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diluted in microtitration dishes (Behring Institute, Behringwerke, Marburg, Germany), yielding diluted samples of 100-,ul volume. An equal volume of a freshly prepared 0.5% suspension of human group 0 erythrocytes (suspended in the same buffer) was added to each well, and the assay was read after 3 h at room temperature. RESULTS Rotavirus spike morphology. Images of rotavirus in ice show low-contrast spikes extending from the surface of the intact virions (Fig. la) as described previously (23, 27). Low-dose uranyl formate-stained images (Fig. lb) also reveal these structures, which were radiation sensitive and which were lost using conventional imaging methods. Figure lb reveals that different numbers of spikes project as a corona from individual particles. The number of spikes is so low for some virions that the variation cannot be due to chance orientation but rather must be due to a reduced occupancy of the 60 sites in the capsid. Another feature of the negatively stained spikes is the presence of apparent Y and looped conformations (indicated by arrowheads in Fig. lb). However, while initially this could be taken as supportive evidence that the spike is dimeric, this may be due to images of neighboring spikes being superimposed. Virus that had not been exposed to trypsin showed the same wide variability in numbers of spikes (Fig. 2a), and these were more frequently seen as single straight structures. The absence of Y and looped conformations in undigested preparations could be interpreted as indicating the dependence of these structures on prior trypsin treatment, but it might merely be the consequence of variability in staining conditions between preparations. Removal of spikes by treatment at elevated pH. A wide range of experimental conditions were investigated for their ability to remove spikes from the intact virion as observed by low-dose electron microscopy and gel electrophoresis. As anticipated, the spikes were resistant to sonication, isopycnic banding on CsCl gradients, and other methods traditionally used in virus purification. Digestion with a wide range of proteolytic enzymes, including chymotrypsin, thermolysin, papain, trypsin, subtilisin, pepsin, and V8 protease, was found to be ineffective. Treatment with heat and exposure to urea, methods which successfully release reovirus spikes (8), also were ineffectual. The nonionic detergents Triton X-100, Nonidet P-40, and ,-octyl glucoside also failed to disrupt or solubilize the viral proteins. While investigating the effects of pH, the best method which reproducibly removed the spikes yet yielded otherwise intact particles was found to be treatment with dilute ammonium hydroxide. Higher concentrations of this weak base were found to release an increasing proportion of spikes, with a concentration of 250 mM being optimal (Fig. 2b) at pH 11.2. Factors other than pH must be involved in this process as NaOH-phosphate and NaOH-glycine buffers caused release at around pH 13 but also disrupted the virus. The material in the background of Fig. 2b is taken to be released spike protein. When separated by centrifugation, the resuspended pellet brought to pH 7.4 (Fig. 2c) gave images of intact particles with a good circular outline and evidence for good structural integrity. In these viruses, it can be seen that the stain-filled channels running through the viral coat are more highly contrasted where the spikes are absent. The supernatant derived from the NH40H treatment (Fig. 2d) was free of intact particles and viral capsomeres but

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FIG. 1. Rotavirus (isolated in the presence of trypsin) imaged by cryoelectron microscopy in amorphous ice and by low-dose negative staining with uranyl formate. (a) An image of the unstained preparation, underfocused by 3 ,um, shows the randomly oriented intact particles in the thin layer (about 100 nm thick) of ice. Spikes can be seen projecting from the surface with an indication of a terminal domain. (b) In a stained preparation imaged closer to focus, the projections exhibit looped and bilobed conformations (indicated by arrowheads) but these may be the result of overlap between pairs or groups of spikes. Bar, 100 nm.

contained material exhibiting globular and short fibrous structures. Although this material could not be identified by microscopy, SDS-PAGE (Fig. 3) showed that the released material (lanes 4 and 8) was almost exclusively VP4 (or VP5* and VP8* in trypsin-treated virus). The spike protein was almost completely released by alkaline treatment while the major external protein VP7 was retained (Fig. 3, lanes 3 and 7). Minor amounts of VP6 present on the gel must derive from a small fraction of particles that are either disrupted during centrifugation or inefficiently sedimented, because this protein was also present in the controls (Fig. 3, lanes 2 and 6). To confirm that the proteins of the virus have not been denatured by the ammonium hydroxide treatment, the

treated particles were incubated with a monoclonal antibody that recognizes the serotype-specific epitope present on region A of the single large immunodominant neutralizing domain of VP7 (18). Cryoelectron microscopy (Fig. 4) revealed that the monoclonal antibody bound to the smooth particle, implying that alkaline treatment had not grossly denatured the VP7 protein which remained on the surface of the smooth particle. Hemagglutination. In view of the assignment of VP4 as the viral hemagglutinin (11), the hemagglutinating ability (HA) of virus treated to either cleave or remove VP4 was determined by using human erythrocytes. Table 1 shows that virus prepared in the presence of trypsin exhibited a consistently lower hemagglutination titer (approximately two- to fourfold

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FIG. 2. Effect of ammonium hydroxide treatment on rotavirus as revealed by negative staining and low-dose microscopy. (a) The uncleaved (VP4) preparation of virus (cf. with Fig. lb) shows many single spike projections. (b) Virus preparation following addition of ammonium hydroxide and incubation at pH 11.2. Note the complete release of the spikes from the virus surface. The material in the background may be released spike protein which appears aggregated when stained at this high pH. (c) Particles separated by centrifugation and brought to neutral pH have a smooth circular profile and show very clear stain-filled channels radiating through the capsid. The background is free of released protein. (d) The supernatant brought to neutral pH yields images (white arrowheads) of short (20- to 40-nm) linear molecules which may be the released spike protein. Small globular material may be either denatured protein from the spike or the inner capsid VP6. Bar, 100 nm.

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