- Email: [email protected]
3-D reconstruction of bluetongue virus tubules using cryoelectron microscopy
JOURNAL
OF STRUCTURAL
BIOLOGY
108,
35-48
(19%)
3-D Reconstruction of Bluetongue Virus Tubules Cryoeiectron Microscopy ELIZABETH
A. HEWAT,
TIMOTHY
F.
RICHARD H. WADE, AND POLLY
BOOTH,*
Laboratoire de Biologie Structurale, CEA and CNRS URA 1333, Virology and Environmental Microbiology, Mansfield Road, Oxford Health Sciences, School of Public Health, The University of Alabama Received
November
8, 1991,
and
Bluetongue virus (BTV) forms tubules in infected mammalian cells. These tubules are virally encoded entities which can be formed with only one protein, NSl. The NSl protein does not form a part of virus particles, and its function in viral infection is uncertain. Expression of the NSl gene in insect cells by recombinant baculovirus yields high amounts of NSl tubules (ca. 50% of cellular proteins) which are morphologically and immunologically similar to authentic BTV NSl and can be isolated to about 90% purity. The structure of these synthetic NSl tubules was investigated by cryoelectron microscopy. NSl tubules are on average 52.3 nm in diameter and up to 100 nm long. The structure of their helical surface lattice has been determined using computer image processing to a resolution of 40 A. The NSl protein is about 5.3 nm in diameter and forms a dimer-like structure, so that the tubules are composed of helically coiled ribbons of NSl “dimers,” with 21 or 22 dimers per turn. The surface lattice displays P2 symmetry and forms a one-start helix with a pitch of 9.1 nm. The NSl tubules exist in two slightly different pa-dependent conforma? 19% Academic Press, Inc. tional states.
INTRODUCTION Bluetongue virus (BTV) is the prototype virus of the Orbivirus genus within the Reoviridae. It is transmitted to vertebrates by Culicoides midges and causes disease in ruminants. The 10 segments of the BTV double-stranded RNA genome of BTV are located in the core of the virus particle. The core contains two major (VP3 and VP71 and three minor protein species (VPl, VP4, and VP6) and is surrounded by an outer capsid consisting of two major proteins, VP2 and VP5 (Huismans, 1979; Martins et al., 1973; Verwoerd et al., 1970, 1972). In addition, in BTVinfected cells three “nonstructural” proteins (NSl, NS2, and NS3), i.e., proteins which are not the capsid components of the mature virus particle, are also synthesized (Huismans, 1979). Their function in the
DBMSIDSV, CENG, 85X, OX1 3SR, United Kingdom; at Birmingham, University
in revised
form
December
Using ROY*‘+
38041, France; *NERC Institute of and iDepartment of Environmental Station, Birmingham, Alabama 35294
11. 1991
replication or morphogenesis of BTV is unclear; however, two virus-specified entities, tubules and inclusion bodies, are routinely observed in BTVinfected cells, and appear to be common to all Orbiviruses (Lecatsas, 1968). These morphological structures have been observed to be attached to the intermediate filament component of the cytoskeleton of infected cells (Eaton et al., 1988). When the NSl gene was expressed in insect cells by recombinant baculovirus, tubules were formed that were similar to those found in BTV-infected mammalian cells (Urakawa and Roy, 19881, confirming that the tubules can be made in cells from the NSl protein only. Similarly, it has been shown that the inclusion bodies can be formed in insect cells by the NS2 protein alone: this protein is phosphorylated and binds single-stranded RNA. The inner core proteins of BTV particles, VP3 and VP7, appear to be associated with the NSl tubules in infected mammalian cells (Hyatt and Eaton, 19881, and some virions and inner core particles also associate with the tubules (Eaton et al., 1988). The inclusion bodies composed of NS2, consist of reticulate fibrils, and are one of the sites of assembly of virus particles, which can be seen within them apparently in the process of formation by electron microscopy (Hyatt and Eaton: 1988; Roy et al., 1990). The NSl protein has been suggested as a diagnostic tool to distinguish BTV from other Orbiviruses (Ritter and Roy, 1988; Gould et al., 1988; Urakawa and Roy, 1988) since it is highly conserved at the nucleic acid and amino acid sequence levels, among different BTV serotypes, but shows little homology with the NSl of other Orbiviruses (Huismans and Cloete, 1987; Unger et al., 1988). Study of the detailed structure may give further clues as to the possible role of NSl and the function of the tubules in BTV infection and the nature of the protein-protein interactions involved in tubule structure formation.
All
1047-8477192 $3.00 Copyright i 1992 by Academic Press, Inc rights of reproduction in any form reserved.
36
HEWAT
In this study, we used cryo-electron microscopy (cryo-EM) to examine the structure of BTV NSl tubules in vitrified water. This technique avoids the artifacts obtained with conventional EM preparation techniques involving chemical treatments, dehydration, embedding, shadowing, or negative staining (Dubochet et al., 1985). Contrast in the unstained, vitrified specimens arises solely from the differences in electron scattering between the biological material and the solvent, and avoids the distortion and collapse of the specimen caused by airdrying, so allowing a three-dimensional (3-D) reconstruction to be made, a technique which is particularly useful for tubular structures (Mandelkow and Mandelkow, 1985). Contrast depends on the image defocus. This report decribes the determination of the low resolution three-dimensional structure of BTV NSl tubules, using reconstruction techniques with images obtained of frozen-hydrated specimens. Baculovirus-expressed BTV serotype 10 tubules (Urakawa and Roy, 1988) were used in these studies since these are easily obtained in large amounts, relatively free from other cellular or BTV proteins. Immunolabeling experiments to identify the tubular structures present are also described. MATERIALS Preparation
of NSl
ET AL.
FIG. 1. Polyacrylamide gel electrophoresis and immunoblotting analysis of NSl tubule preparation I. Lanes 3 and 4: Coomassie blue stain of separated NSl preparation (lane 3) and molecular weight standards (lane 4). Lanes 1 and 2: Immunoblots of lanes similar to lane 3, transferred to membranes and probed with anti-AcNPV virus particle antiserum (lane 1) and antiBTVlO-infected BHK cell antiserum (lane 2). NSl is indicated (arrows).
AND METHODS
Tubules
Spodoptera frugiperda (St9) insect cells grown in suspension culture were infected with the AcBTVlO-6 recombinant baculovirus as described by Urakawa and Roy (1988). This recombinant baculovirus contains the gene sequence coding for NSl under the control of the late promoter of the Autograph californica nuclear polyhedrosis virus (AcNPV) polyhedrin gene (Urakawa and Roy, 1988). After incubation at 28°C for 3 days, the cells were harvested, washed, and resuspended in lysis buffer (160 mM NaCl, 5 n&f MgClz, 10 n-&f Tris-HCl, pH 7.5, 0.5% w/v Nonidet p-401, vortexed, and allowed to equilibrate on ice for 5 min. The nuclei and cell debris were pelleted (15OOg for 5 min). The supernatant was loaded onto a 40% (w/v) sucrose pad in 0.15 M STE (150 mM NaCl, 1 mil4 EDTA, 10 m&f TrisHCl, pH 7.5) and centrifuged in a Beckman SW41 rotor (3 hr at 40 000 rpm). The pellet was resuspended in a small volume of STE and stored at 4°C (preparation I). An additional purification step involved layering preparation I from above onto sucrose gradients in STE buffer, and then centrifugation for 90 min at 24 K r-pm in a Beckman SW27 rotor, after the methods of Huismans and Els (1979), to yield preparation II, which was also resuspended in STE. Monolayers of BHK-21 cells were infected with U.S. BTVlO at a multiplicity of 5 plaque-forming units per cell and incubated at 37°C. Specimens were fixed at 36 or 40 hr postinfection.
Analysis of NSl Preparations and Immunoblotting
by SDS-PAGE
Tubule preparations purified from infected cells were analyzed by 10% polyacrylamide gel electrophoresis (PAGE) in the presence of SDS, and by immunoblotting, as previously described (Urakawa and Roy, 1988). For immunoblotting, separated polypeptides were transferred from the gel onto nitrocellulose membranes which were incubated in the presence of either rabbit anti-BTVlO serum or rabbit anti-AcNPV extracellular virus particle serum, both at dilutions of lOOO-fold. Controls included probing blots of uninfected Sf9 cell lysates, and testing of preimmune rabbit serum at the same dilution on identical blots of the NSl preparation. Bound antibodies were detected using a secondary antibody conjugated with alkaline phosphatase (Urakawa and Roy, 1988). Immunogold
EM
For immunogold-negative stain electron microscopy (EM), tubules were adsorbed onto a carbon filmed EM grid for 1 min, washed in STE buffer, and floated on 25-ml drops of 1:500 diluted rabbit anti-BTVlO-infected BHK cell antiserum (1 hr). After three further washes, antibodies were localized by incubating grids in goat anti-rabbit colloidal gold (lo-nm particle size; Bio Cell Res. Labs., Cardiff, UK), for 1 hr before a final wash and
FIG. 2. (a) Immunogold negative-stained EM localization of BTV NSl tubules with rabbit anti-BTVlO-infected BHK cell antiserum and 10 nm goat anti-rabbit colloidal gold conjugate. Small arrows indicate specifically bound gold particles on BTV NSl structures, while the baculovirus contaminant tubule (AcNPV) are unlabeled. (b) Control lysate of cells infected with wild-type acNPV treated like NS1 preparation I in a, and similarly immunogold labeled with antiBTV-10 antiserum.
3-D
RECONSTRUCTION
OF BTV
37
TUBULES
A&l PV
38
HEWAT
negative staining in 2% many1 acetate. Controls included elimination of the primary antiserum, its replacement with preimmune rabbit serum, probing with an irrelevant antiserum (raised against purified BTV NS2 protein), and testing of preparations of uninfected cells. For postembedding, on-section immunogold labeling of NSl in both mammalian and insect cells, infected cultures were fixed in 0.1% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, dehydrated in ethanol series (50, 70, and 90% w/v) and embedded in LR white resin (London Resin Co., Basingstoke, UK) as described by Newman et al. (1983). Specimens were observed in a JEM 1OOC electron microscope at 100 kV. Preparation
and Observation
of Frozen-Hydrated
Specimens
Frozen-hydrated specimens were prepared on holey carbon films as described by Dubochet et al. (1985). A modified Zeiss cryoworking unit was employed since it allows manipulation of the specimen grid in gaseous rather than liquid nitrogen. The holey carbon films, supported on 400-mesh copper grids were glow-discharged in air immediately prior to use. Samples of the tubule suspension (4 pl) were applied to grids, which were blotted immediately with filter paper for l-6 set and then rapidly plunged into liquid ethane cooled by nitrogen gas at - 175°C. Specimens were observed at temperatures of - 165 to - 175°C in a Zeiss 1OC electron microscope equipped with a top entry cold stage and nontilting specimen holder. The microscope was operated at 100 kV and images were recorded at x 20 000 magnification with a dose of less than 10 e /Aa on Kodak SO163 electron image film which was developed in full-strength D19 for 12 min at room temperature. The magnification was calibrated using the 4.0-nm first layer line in computed Fourier transforms of microtubule images (Amos and Klug, 1974, Chretien, 1991). Conventional
Electron
Microscopy
Specimens were prepared for conventional electron microscopy using one of the following negative stains: 2% w/v uranyl acetate, pH 4.4,2% w/v phosphotungstic acid (PTA), pH 5.0,2% w/v PTA, pH 7.2, or 2% w/v sodium silicotungstate (SST), pH 7.2. These specimens were also observed in the Zeiss 1OC at room temperature at 100 kV under low-dose conditions. Heavy metal shadowing was carried out at room temperature in an Edwards vacuum evaporation unit. A suspension of NSl tubules (4 pl) was mixed with 1 M ammonium acetate (4 pl), and spread on freshly cleaved mica. The specimens were rotary shadowed at 8” from the surface normal with Pt/W and a carbon support film was then evaporated from above. Specimens were observed in a Zeiss 1OC as for the negatively stained specimens. Image
Processing
Preliminary examination of the micrographs was carried out using a CCD TV camera linked to a dedicated computer which produced the power spectrum of a 512 x 512 pixel image in 2 set (Tietz video and processing systems). With this system individual tubules were easily masked off with a black paper mask and assessed separately. Only images in which all helical diffraction data lay inside the first zero of the contrast transfer function were selected so no correction for the contrast transfer function was made. Selected micrographs were digitized for further analysis using a CCD TV camera coupled to a PC with an ADC card. A pixel of size of IO.5 p,rn on the micrograph was employed, which for a magnification of x 20 000, corresponds to a pixel size of 5.2 A at
ET AL. the specimen. Image analysis was performed on a Vaxstation 3200 using the Semper 6 Plus image analysis package and a modified version of the MRC helical reconstruction programs (DeRosier and Moore, 1970). Typically areas of 250 x 135 pixels, floated to 512 x 512, were Fourier transformed and no correction was required for unbending of the tubule images. The amplitude and phase information on each layer line was extracted and corrected for tilt of the helical axis away from the normal to the electron beam. Analysis was simplified by the fact that there was only one Bessel order present on each layer line so a complete low-resolution data set for reconstruction could be extracted from one image. The layer line data from near and far sides was averaged. Six different tubules were reconstructed separately using data out to 4NM - 1 from the meridian. Horizontal and cylindrical sections were calculated using the Fourier-Bessel transforms in the MRC programs and the surfaces of reconstructions were visualized using the Semper 6 Plus solid surface display facility. Filtered images were calculated for each side of the tubule by pseudooptical filtering of the layer line data (Baker and Caspar, 1983). RESULTS
Purity of Preparations of Structures
and Identification
When analyzed by SDS-PAGE and immunoblotting with antisera directed against either AcNPV virus particles or BTVlO-infected mammalian cells (Fig. 1) preparation I appeared to be approximately 70% NSl and was contaminated with several baculovirus proteins, of which a protein of about 40-kDa relative molecular mass was the most abundant (Fig. 1). The NSl protein in preparation I was also recognized in immunoblots using a monospecific antiserum raised against purified, baculovirusexpressed NSl (Urakawa and Roy, 1988; data not shown). Preparation I gave the better tubule morphology, as judged by negative-stained EM using uranyl acetate, although it was contaminated with empty baculovirus nucleocapsids (Fig. 2) which showed up on immunoblots using antiserum against AcNPV extracellular particles (Fig. 1). Bluetongue virus tubules in preparation I could be distinguished from the baculovirus nucleocapsid tubules by immunogold labeling with antiserum specific for BTVlOinfected mammalian cells (Fig. 2a) and by their general appearance in negative stain (Figs. 3a and 3b). The more striated appearance of the bluetongue virus tubules with their higher edge contrast with a repeating pattern of 9.1 nm was easily distinguished from the fine reticular “cross-weave” appearance of the surface of the empty AcNPV capsids (Fig. 3a). The rabbit antiserum raised against baculovirusexpressed BTVlO NSl also recognized the tubular structures formed in BTVl.O-infected BHK cells --.--
FIG. 3. EM images of negatively stained (2% uranyl acetate, pH 4.4) NSl tubules. (a) Preparation I, the different appearance of BTV NSl tubules and the contaminant baculovirus tubules (AcNPV) are evident. (b) Preparation II, additional purification leads to shorter tubules with little baculovirus contamination, with many short, unwound fragments (arrows) that can be seen unwinding from the ends of tubules or detached as circular forms or as short broken sections of filament.
3-D
RECONSTRUCTION
OF BTV
TUBULES
39
40
HEWAT
ET AL.
3-D
RECONSTRUCTION
FIG.
OF BTV
41
TUBULES
I.-Continued
(Fig. 4a). Both the BTV virions and the NSl tubules were observed in association with the intermediate filament component of the cytoskeleton of the BTVinfected mammalian cells. In all cases the controls confirmed the specificity of the antisera for the NSl tubular structures (Figs. 4a-4c) and the tubules reacting with the antiserum were identical in appearance to those previously isolated from BTV-infected mammalian cells (Huismans and Els, 1979). After the secondary centrifugation step using the method of Huismans and Els (19791, NSl tubules were produced that were over 90% pure as judged by SDS-PAGE (preparation II: data not shown). However, when preparation II was examined by negative-stained EM, the tubules were found to be shorter and their morphology less well preserved than those of preparation I (Fig. 3b). In addition, preparation II contained many short ribbons and, frequently, single strands unwinding from the ends of intact tubules were observed (Fig. 3b). Therefore, preparation I was used for the majority of the EM investigations. Dimers of NSl were not observed under any of the denaturing or native conditions tried. Tubules were found to be insoluble in the absence of
SDS at pH 7.5, 8.7, or 9.7. Heating of tubules at 60°C or freeze-thawing them, yielded disrupted aggregates of NSl that had a limited PAGE mobility in the absence of SDS at pH 8.7 or 9.7, showing a smear corresponding to high molecular weight NSl polymers, and a faint band of NSl monomers, while treatment with SDS or 2 M urea yielded monomers (data not shown). Treatment of tubules with the ionic detergent dodecyltrimethylammonium bromide (DTAB) at a concentration of 10 mM yielded highly distorted, unwound tubules that could be identified by immunogold labeling with antiserum directed against BTVlO-infected cells (Fig. 4~). The disrupted NSl tubules could thus be distinguished from the contaminating AcNPV tubules which were not affected by DTAB treatment (Fig. 4~). Cryoelectron
Microscopy
In cryo-EM images, frozen-hydrated NSl tubules are characterized by 9.1-nm repeat bands particularly visible along the “sides” of each tubule (Fig. 5). They are also distinguished by their tendency to form rafts in which the tubules are aligned parallel to each other with a separation of 2.0-2.5 nm. Some
FIG. 4. (a) Immunogold labeling of NSl tubules (arrows) in ultrathin plastic sections of BTVlO-infected BHK cells with rabbit antiserum raised against gel purified baculovirus-expressed NSl. T, tubular structures in section; VP, virus particles. (b) Control section incubated in anti-NS2 antiserum. The virus inclusion body (VIB) in this section is labeled but not the tubules (T) or the virus particles (VP). (c) Treatment of NSl tubules with 10 n&f DTAB: immunogold labeling demonstrates disruption of BTV NSl tubules (solid arrows); the baculovirus contaminant tubules (open arrow) are not disrupted by the detergent and are not labeled by immunogold.
42
HEWAT
ET AL.
tubules have an abrupt change in diameter or “dislocation” along their length (Fig. 5). They range in diameter from 48.5 to 56.5 nm and in length from a few tens to 1000 nm. The average tubule diameter calculated from 50 measurements is 52.3 nm. The tubule length is typically five or six times the diameter. A histogram of tubule diameter is shown in Fig. 6. Tubules are seen either perpendicular to their axis or end-on. This is due to surface effects and constraints placed on their orientation by their presence in a thin film of buffer immediately prior to freezing. Empty nuclear polyhedrosis virus (AcNPV) capsids found in the same preparation have a much smoother appearance and their fine cross-weave surface lattice is occasionally visible. Electron
Microscopy
of Negatively
Stained Tubules
The 9.1-nm repeat is clearly visible with all of the four negative stains used (Figs. 3 and 7). In preparation I the NSl tubules also had a tendency to be aligned in bundles which, in contrast to the cryoelectron microscopy images, were not necessarily coplanar. In the more highly purified, more dilute preparation II the tubules are seen in isolation. The highest resolution images were obtained with sodium silicotungstate stain (Fig. 8~). Indexation Filtered
FIG. 5. Cryoelectron microscope images of BTV NSl tubules at a defocus of 3 pm. Note the parallel alignment of tubules (a) and the step in diameter of a tubule (b, small arrow). Short end-on tubules are also visible.
of Diffraction Images
Patterns
and
Computed diffraction patterns were obtained from images of frozen-hydrated and from sodium silicotungstate-stained NSl tubules (Fig. 8). All of these diffraction patterns fall into two categories as exemplified by Fig. 8a (type A) and Fig. 8b (type B). There is a clear difference in the position of the layer lines except for the layer line close to the meridian at 9.1 nm. The layer lines visible in type A diffraction patterns are indexed as 1 = 0, 1,4,5, and 6 and in type B diffraction patterns as 1 = 0,2,5,7, and 9 to a very good approximation. No intermediate values were ever seen. In preparation I, observed by cryo-EM, 49 tubules were of type A and 23 of type B out of a total of 72 analyzed. However, in 2% PTA, pH 7.2, or SST, pH 7.2, all tubules were of type B and in 2% PTA, pH 5.0, or uranyl acetate, pH 4.4, all tubules were of type A. Hence, we conclude that the transformation from type A to type B is pH dependent. Because the preparations observed by cryo-EM were nominally pH 7.5 and both types A and B were observed, it is probable that the presence of other ionic species (as in the negative stain) modifies the transition pH.
3-D
46
49
50
51
52
53
54
RECONSTRUCTION
55
56
57
nm
diameter FIG. 6. Histogram of frozen-hydrated The values of the diameters for the and 22 are indicated.
NSl tubule reconstructions
diameters. for U = 21
Some of the images obtained using sodium silicotungstate staining diffract to 3.0-nm resolution (Fig. 8c), which is better than that obtained in cryo-EM images which contain some information to 3.5 nm. Unfortunately, this higher resolution information cannot be exploited, because it is evident from the one-sided filtered images that both near and far sides of the collapsed tubules interact with each other. This causes distortion of the lattice, rendering the filtered images impossible to interpret. Filtered cryo-EM images of type A tubules were useful in assigning layer lines to one or another side of the helix. Only the combination shown in Fig. 8
FIG.
7.
Comparison
of electron
microscope
images
of negatively
OF BTV
43
TUBULES
gives a reasonable one-sided filtered image. The filtered image is shown both including and excluding the 1 = 0, n = 0 layer line, i.e., the equator, (Figs. 9a and 9b, respectively). The apparent horizontal dimension of the unit cell is smaller at the edges of the tubule than in the center as expected for the projection of a cylinder. The indexation regarding n is based on three criteria; first, the parity of related reflections (after correction for tilt away from the normal to the electron beam direction); second, the requirement that all reconstructed density falls within the average radial density envelope defined by the n = 0, I = 0 layer line (Egelman et al., 1989); and third, end-on views of short NSl tubules in ice were used as confirmation of the n indexation of similar diameter tubules since the subunits are visible in some favorable cases (Fig. 10). The selection rule for the type A tubules is 1 = 5n + (5U - l)m,
where U is an integer. Of the six images of type A tubules used for 3-D reconstruction, one had U = 20, two U = 21, and three had U = 22. From the observed distribution in tubule diameter, U = 21 and 22 are the most common values (Fig. 6). The number of units (dimers)/ turn = U - 0.2. Indexation of the diffraction patterns from type B is not as clear as this type was less well preserved under the conditions of observation by cryo-EM: in
stained
BTV
NSl
tubules.
(a) 2% SST,
pH 7.2; (b) 2% PTA,
pH 5.0.
44
HEWAT
ET AL.
FIG. 9. One-sided filtered images of a type A NSl tubule in ice including (a) and excluding (b) the I = 0, n = 0 layer line.
types A and B have the opposite hand. In view of the observation that type A can be converted into type B without unwinding, by changing the pH, they almost certainly have the same hand.
FIG 8. Computed diffraction patterns from NSl tubules images (a) type A in ice, (b) type B in ice, and (c) type B stained with SST, pH 7.2. The indexing shown is for U = 22.
general, the diffraction patterns are not very symmetrical. In addition, it is not evident from the amplitude distribution on layer lines I = 5 and 1 = 9 which has the higher n value. It was not possible to make satisfactory reconstructions of type B tubules. However, since type B tubules transform into type A tubules as a function of pH, it follows that both types have like-handed helices. Hence, the indexation shown in Fig. 8b is the most probable. The alternative assignment of diffraction peaks implies that
FIG. 10. End-on view of a short NSl tubule in ice.
3-D
The selection
The number Shadowed
RECONSTRUCTION
OF BTV
45
TUBULES
rule for type B is
1 = 7n + (7U - 2lm. of units/turn = U - 2/7. tubules
Images of NSl PtW-shadowed tubules only show the 9.1-nm repeat along the axis (data not shown). Although the handedness was not evident in all of these images due to distortion of the tubules on drying, the computed diffraction patterns showed the n = 1 helix to be most probably left-handed.
0 0
A-’
0.025 N-22 .
0.125,
L-l
,180
3-D Reconstructions The data from the analysis of six of the type A tubules proved to be suitable for reconstruction. An example of the layer line data for a U = 22 tubule is shown after correction for a 4.5” tilt and averaging over near and far sides (Fig. 11). It is notable that for each layer line, the phase flips between two values which differ by approximately IT. This is characteristic of a surface lattice which has a twofold axis perpendicular to the helix axis (Toyoshima and Unwin, 1990, Hutchinson et al., 1990). The layer line data in Fig. 11 was used to reconstruct the helix shown in Fig. 12. The reconstruction shows a solid surface representation of the front surface only (Fig. 12a), a horizontal section (Fig. 12b), and a half cylindrical section of the helix viewed from the outside (Fig. 12~). The surface lattice has a clear P2 symmetry; this has not been enforced on the data. The largest sphere which can be accommodated in the lattice shown has a diameter of 5.5 nm. This is close to the 5.3-nm diameter expected for a globular 64-kDa protein assuming a partial specific volume of 1.24 A” per dalton, so we assign this to the NSl monomer seen at low resolution. The structure can be described as a ribbon of what appears to be NSl dimers wound into a left-handed helix. The selection rules for the six reconstructed tubules are I = 5n + (5 x 20 - l)m for one, 1 = 5n + 15 x 21 - l)m for two, and 1 = 5n + (5 x 22 -. 1)m for three of the tubules. So they have 19.8, 20.8, and 21.8 dimersiturn, respectively. Each reconstruction gives essentially the same surface lattice with only slight differences in the density at the intermolecular contacts. While we could not make a satisfactory reconstruction of the type B tubules, we can determine some information about their surface lattice. Compared with type A tubules, the type B lattice is slightly skewed, and the number of dimersiturn changes; for example, from 21.8 to 21.7 units per turn (Fig. 12~). There is also a small change in distribution of intensity on layer lines 1 = 3 and 1 = 5 which become 1 = 5 and 1 = 9, respectively (Figs. 8a
0
A-’
0.025 N--21
L-4
io/ -j&f&i0
0.125
.
.a-
0
-
0.025 N-l
0.125
*
.
*.
L-5
180
.
A-’ - 180
.
. -0 -OB
N-23
p-
il:‘.’
0.025
A ’
0
FIG. 11. Layer line data from a type A NSl tubule corrected for a 4.5- tilt of the tubule away from the normal to the electron heam and averaged over near and far sides.
and 8b). This must reflect protein density distribution.
a change
in the radial
DISCUSSION
Cryoelectron microscopy and 3-D reconstruction of the bluetongue NSl tubules show that they consist of a ribbon of dimers wound into a left-handed helix about 52 nm in diameter. There are usually 21- or 22-dimer-like units per turn and the pitch is 9.1 nm. The quantification of tubule diameter as a function of dimer units per turn is somewhat
46
HEWAT
ET AL.
a
IOnm
FIG. 12. Reconstruction of a type A NSl tubule with U = 22. Visualization of the front surface of the tubule only (a), section perpendicular to the tubule axis (b), and a half-cylindrical section at a radius of 25 nm viewed from the outside (c) are shown. The P2 symmetry of the surface lattice is indicated. The unit cell of the surface lattice of the type B tubule is shown dotted. The skew angle between the type A and type B tubules is increased by a factor of two for clarity.
masked by the spread in values due to distortion of the tubules and errors in measurements. It would appear that the dimeric form of NSl is unstable outside the context of the dimer ribbon, and that the protein-protein interactions between adjacent dimer units must be of a similar nature to the intradimer bonding. Thus, there is one of the four protein-protein interactions that each NSl molecule makes within the tubule that is closer than the other three interactions, and hence appears dimeric. The fact that under certain conditions the tubules unwind at their ends into dimer ribbons, and breakage of these into “circular” forms was also observed, suggests that the bonding between the filaments in the direction of the tubule axis is weaker than the bonding within the dimer filament. A possible model for the unwinding of the dimer filament is shown in Fig. 13. The Orbivirus tubules clearly differ in structure from those associated with retrovirus infections (Dales, 1963) and tubules associated with rotavirus infection which consist of a structural capsid protein of the virus particle (Holmes et al., 1975; Kimura and Murakami, 1977). In previous EM studies, the expressed NSl tubules were also contaminated with baculovirus nu-
cleocapsid tubules which superficially have a similar appearance and this has led to confusion in the interpretation of the morphological changes observed (e.g., Marshall et al., 1990). The AcNPV tubules are formed from the 40-kDa nucleocapsid protein, and represent an immature, empty form of the nucleocapsid without the genomic nucleic acid or the cap-like ends (data not shown). The structure of AcNPV tubules consists of rings of protein subunits which are stacked one on top of another with a slight rotation between each layer to form a cylinder (unpublished data). In this study, baculovirusexpressed BTV NSl tubule preparations were purified to about 90% NSl (estimated by SDS-PAGE);
FIG. 13. of the dimer
Model showing ribbon.
the
proposed
unwinding
mechanism
3-D
RECONSTRUCTION
however, they still contained a significant amount of baculovirus tubules. We unequivocally identified these two different tubular structures using both immunological and structural criteria. Tubules composed of BTV NSl were distinguished from the AcNPV tubules by immunogold labeling, and both had a distinctive appearance by both cryo-EM and negative-stained EM. In addition, the diffraction patterns allowed the two different structures to be readily distinguished. The empty AcNPV capsids have identical diffraction patterns to those shown in previous electron microscope studies (Beaton and Filshie, 1976). We have shown that the expressed NSl tubules are apparently immunologically identical to those made in BTV-infected mammalian cells and have an identical appearance to the previously published negative-stained electron micrographs of authentic BTV NSl tubules purified from infected BHK cells (Huismans and Els, 1979). The cryo-EM images of frozen-hydrated NSl tubules gave computed diffraction patterns which fell into two distinct groups (types A and B). The difference between type A and type B involves a skew in the surface lattice with respect to the tubule axis, with the angle for the type B tubule being about 3” greater than that of the type A tubule (see Fig. 12~). A typical sample was composed of 68% type A, and 32% type B, out of 72 tubules examined. All tubules which gave a clear diffraction pattern could be assigned as type A or B to a very good approximation. However, after negative staining using solutions of different pH, the conformation of the BTV tubules was found to be dependent on the pH of the stain used: At pH 7.2, using either PTA or SST, 100% of the tubules were type B, whereas at pH 4.4 or 5.0 (PTA or uranyl acetate), 100% of the tubules oberved were of type A. This result suggests that the lattice undergoes a transition during the drying process in the presence of increasing concentrations of ionic species (buffer and heavy metal salts): this transition thus appears to be influenced by the initial pH of the solution. The fact that the type B NSl tubule at higher pH cpH 7.2), has a 3” greater angle of skew with respect C;Othe tubule axis than the type A at lower pH (below pH 5.0) suggests that the diameter of the tubule decreases with pH. This change in shape of the lattice is equivalent to a change in the number of dimers per turn of 21.8 at low pH to 21.7 at high pH. There is also probably a small change in the radial density distribution of the dimer. Future studies will include examination of these pH effects in the frozen-hydrated state to determine whether this change can occur under native conditions. The data presented in this initial 3-D reconstruction of the BTV NSl tubules will be of use in further studies employing genetic engineering to determine
OF BTV
47
TUBULES
the effects of mutations in the NSl gene on the formation of oligomeric tubular structures. Further studies to compare the tubular NSl structures from other Orbiviruses will be possible, and the cryo-EM technique will be available in analyzing the results of attempts to generate tubules containing NSl from different viruses, or tubules containing mutant chimerit species of the NSl protein. The function of NSl remains unknown. It may be involved as a molecular chaperone to prevent the core particles from assembling before the correct minor core proteins (VPl, VP4, and VP61 and/or genome have been incorporated. It has been demonstrated (French and Roy, 1990) that expressed BTV VP3 and VP7 can self-assemble to form a core like particle in the absence of other BTV proteins. Further clues as to the function of these NSl tubules in BTV infection must await generation of mutant and recombinant BTV strains with defective NSl. It is a pleasure to thank Christian Closse, Samantha Griffiths, and Katie Monstyrskaya for expert assistance, and Annie Barge of the EMBL Grenoble for assistance with the heavy metal shadowing. We also thank Stephen Fuller for supplying the helical reconstruction programs employed. The financial contributions of EMBO and NERC are gratefully acknowledged. REFERENCES Amos,
L. A., and
Klug,
A. (19741
J. Cell
Sci.
Baker, T. S., and Caspar, D. L. D. (1983) Proceedings of the 41st Annual Meeting cisco Press, San Francisco, CA. Beaton. 161.
C. D., and
Filshie,
B. K. (1976).
14, 523-549.
in Bailey, G. W. (Ed.), of EMSA, San FranJ. Gen.
Chretien D. (1991) Apports de la cryomicroscopie l’etude de microtubules assembles in vitro. Joseph-Fourier, Grenoble. Dales,
S. (1963)
Proc.
Natl.
Acad.
Sci.
D. d., and Moore,
Dubochet, Trends
J., Adrian, M., Lepault, J., and McDowall, Biochem. Sci. 6, 143-146. A. D.. and
White,
Egelman, E. H., Wu S.-S., Amrein, / 19891 J. V~rol. 63, 2233-2243. i;i-ench. (would, Res.
‘I or and
Hii>
1’
A. K., Pritchard, 11. 97-107.
Holmes, 11975)
1990.
,i
1,. 1.. and
H. $1979)
Huismans,
H.. and
Cloete,
Huismans,
H., and
Els,
Hutchinson, J. Struct.
G., Tichelaar, W.. Weiss, Biol. 103, 75-88.
Hyatt, Kimura,
A. D.. and
Virology
Eaton,
T., and Murakami,
H. J. (1979)
B. T. 11988) T. (1977)
B
R. F..
.52, 355-369. A. W. (1985) Virology
163,
A,, and Murti,
tij,
Ta\aiia,
92, 385 M. (1987
Biol.
Portner,
I‘rr~,i
I. H.. Ruck, B. J., Bishop, J. Virol. 16, 937-943.
Huismans,
J. Mol.
J. R. (1988)
M.,
151-
50, 268-275.
DeRosier,
Eaton, B. T.. Hyatt, 527 -537
31,
electronique Thesis, Universite
USA
P. B. (1970)
Viral.
i 5Rti
:.%6
&l. D. * i988i and
G
Davidson,
i-l/ (A,> G. P.
396
t Virology Vtrology
158, 373-380. 92, 397-405.
H., and Leonard, J. Gen. Infect.
K. (19901
Viral.
69, 8055815.
Immun.
17, 157-160.
48
HEWAT
Lecatsas, G. (1968) Onderstepoort J. Vet. Res. 35, 139-150. Mandelkow, E.-M., and Mandelkow, E. (1985) J. Mol. Biol. 181, 123-135. Marshall, J. J. A., Fayard, B., and Roy, P. (1990) J. Gen. Viral. 71, 1839-1844. Martins, S. A., Pett, 12, 194-198. Newman, G. R., Jasani, J. 15, 543-555. Ritter, D. G., and Roy,
D. M.,
and
Zweerink,
B., and Williams, P. (1988)
Virus
H. J. (1973) E. D. (1983) Res.
11, 3347.
J. Viral. Histochem.
ET AL. Roy, P., Adachi, A., Urakawa, T., Booth, T. F., and Thomas, C. P. (1990) J. Viral. 64, l-8. Toyoshima, C., and Unwin, N. (1990) J. CelZBiol. 111,2623-2635. Unger, R. E., Chuang, R. Y., Chuang, L. F., Osburn, B. I., and Doi, R. H. (1988) Virology 167, 296-298. Urakawa, T., and Roy, P. (1988) J. Viral. 62, 3919-3927. Verwoerd, D. W., Els, H. J., de Villiers, E. M., and Huismans, H. (1972) J. Viral. 10, 783-794. Verwoerd, D. W., Louw, rol. 5, l-7.
H., and
Oellermann,
R. A. (1970)
J. Vi-
OF STRUCTURAL
BIOLOGY
108,
35-48
(19%)
3-D Reconstruction of Bluetongue Virus Tubules Cryoeiectron Microscopy ELIZABETH
A. HEWAT,
TIMOTHY
F.
RICHARD H. WADE, AND POLLY
BOOTH,*
Laboratoire de Biologie Structurale, CEA and CNRS URA 1333, Virology and Environmental Microbiology, Mansfield Road, Oxford Health Sciences, School of Public Health, The University of Alabama Received
November
8, 1991,
and
Bluetongue virus (BTV) forms tubules in infected mammalian cells. These tubules are virally encoded entities which can be formed with only one protein, NSl. The NSl protein does not form a part of virus particles, and its function in viral infection is uncertain. Expression of the NSl gene in insect cells by recombinant baculovirus yields high amounts of NSl tubules (ca. 50% of cellular proteins) which are morphologically and immunologically similar to authentic BTV NSl and can be isolated to about 90% purity. The structure of these synthetic NSl tubules was investigated by cryoelectron microscopy. NSl tubules are on average 52.3 nm in diameter and up to 100 nm long. The structure of their helical surface lattice has been determined using computer image processing to a resolution of 40 A. The NSl protein is about 5.3 nm in diameter and forms a dimer-like structure, so that the tubules are composed of helically coiled ribbons of NSl “dimers,” with 21 or 22 dimers per turn. The surface lattice displays P2 symmetry and forms a one-start helix with a pitch of 9.1 nm. The NSl tubules exist in two slightly different pa-dependent conforma? 19% Academic Press, Inc. tional states.
INTRODUCTION Bluetongue virus (BTV) is the prototype virus of the Orbivirus genus within the Reoviridae. It is transmitted to vertebrates by Culicoides midges and causes disease in ruminants. The 10 segments of the BTV double-stranded RNA genome of BTV are located in the core of the virus particle. The core contains two major (VP3 and VP71 and three minor protein species (VPl, VP4, and VP6) and is surrounded by an outer capsid consisting of two major proteins, VP2 and VP5 (Huismans, 1979; Martins et al., 1973; Verwoerd et al., 1970, 1972). In addition, in BTVinfected cells three “nonstructural” proteins (NSl, NS2, and NS3), i.e., proteins which are not the capsid components of the mature virus particle, are also synthesized (Huismans, 1979). Their function in the
DBMSIDSV, CENG, 85X, OX1 3SR, United Kingdom; at Birmingham, University
in revised
form
December
Using ROY*‘+
38041, France; *NERC Institute of and iDepartment of Environmental Station, Birmingham, Alabama 35294
11. 1991
replication or morphogenesis of BTV is unclear; however, two virus-specified entities, tubules and inclusion bodies, are routinely observed in BTVinfected cells, and appear to be common to all Orbiviruses (Lecatsas, 1968). These morphological structures have been observed to be attached to the intermediate filament component of the cytoskeleton of infected cells (Eaton et al., 1988). When the NSl gene was expressed in insect cells by recombinant baculovirus, tubules were formed that were similar to those found in BTV-infected mammalian cells (Urakawa and Roy, 19881, confirming that the tubules can be made in cells from the NSl protein only. Similarly, it has been shown that the inclusion bodies can be formed in insect cells by the NS2 protein alone: this protein is phosphorylated and binds single-stranded RNA. The inner core proteins of BTV particles, VP3 and VP7, appear to be associated with the NSl tubules in infected mammalian cells (Hyatt and Eaton, 19881, and some virions and inner core particles also associate with the tubules (Eaton et al., 1988). The inclusion bodies composed of NS2, consist of reticulate fibrils, and are one of the sites of assembly of virus particles, which can be seen within them apparently in the process of formation by electron microscopy (Hyatt and Eaton: 1988; Roy et al., 1990). The NSl protein has been suggested as a diagnostic tool to distinguish BTV from other Orbiviruses (Ritter and Roy, 1988; Gould et al., 1988; Urakawa and Roy, 1988) since it is highly conserved at the nucleic acid and amino acid sequence levels, among different BTV serotypes, but shows little homology with the NSl of other Orbiviruses (Huismans and Cloete, 1987; Unger et al., 1988). Study of the detailed structure may give further clues as to the possible role of NSl and the function of the tubules in BTV infection and the nature of the protein-protein interactions involved in tubule structure formation.
All
1047-8477192 $3.00 Copyright i 1992 by Academic Press, Inc rights of reproduction in any form reserved.
36
HEWAT
In this study, we used cryo-electron microscopy (cryo-EM) to examine the structure of BTV NSl tubules in vitrified water. This technique avoids the artifacts obtained with conventional EM preparation techniques involving chemical treatments, dehydration, embedding, shadowing, or negative staining (Dubochet et al., 1985). Contrast in the unstained, vitrified specimens arises solely from the differences in electron scattering between the biological material and the solvent, and avoids the distortion and collapse of the specimen caused by airdrying, so allowing a three-dimensional (3-D) reconstruction to be made, a technique which is particularly useful for tubular structures (Mandelkow and Mandelkow, 1985). Contrast depends on the image defocus. This report decribes the determination of the low resolution three-dimensional structure of BTV NSl tubules, using reconstruction techniques with images obtained of frozen-hydrated specimens. Baculovirus-expressed BTV serotype 10 tubules (Urakawa and Roy, 1988) were used in these studies since these are easily obtained in large amounts, relatively free from other cellular or BTV proteins. Immunolabeling experiments to identify the tubular structures present are also described. MATERIALS Preparation
of NSl
ET AL.
FIG. 1. Polyacrylamide gel electrophoresis and immunoblotting analysis of NSl tubule preparation I. Lanes 3 and 4: Coomassie blue stain of separated NSl preparation (lane 3) and molecular weight standards (lane 4). Lanes 1 and 2: Immunoblots of lanes similar to lane 3, transferred to membranes and probed with anti-AcNPV virus particle antiserum (lane 1) and antiBTVlO-infected BHK cell antiserum (lane 2). NSl is indicated (arrows).
AND METHODS
Tubules
Spodoptera frugiperda (St9) insect cells grown in suspension culture were infected with the AcBTVlO-6 recombinant baculovirus as described by Urakawa and Roy (1988). This recombinant baculovirus contains the gene sequence coding for NSl under the control of the late promoter of the Autograph californica nuclear polyhedrosis virus (AcNPV) polyhedrin gene (Urakawa and Roy, 1988). After incubation at 28°C for 3 days, the cells were harvested, washed, and resuspended in lysis buffer (160 mM NaCl, 5 n&f MgClz, 10 n-&f Tris-HCl, pH 7.5, 0.5% w/v Nonidet p-401, vortexed, and allowed to equilibrate on ice for 5 min. The nuclei and cell debris were pelleted (15OOg for 5 min). The supernatant was loaded onto a 40% (w/v) sucrose pad in 0.15 M STE (150 mM NaCl, 1 mil4 EDTA, 10 m&f TrisHCl, pH 7.5) and centrifuged in a Beckman SW41 rotor (3 hr at 40 000 rpm). The pellet was resuspended in a small volume of STE and stored at 4°C (preparation I). An additional purification step involved layering preparation I from above onto sucrose gradients in STE buffer, and then centrifugation for 90 min at 24 K r-pm in a Beckman SW27 rotor, after the methods of Huismans and Els (1979), to yield preparation II, which was also resuspended in STE. Monolayers of BHK-21 cells were infected with U.S. BTVlO at a multiplicity of 5 plaque-forming units per cell and incubated at 37°C. Specimens were fixed at 36 or 40 hr postinfection.
Analysis of NSl Preparations and Immunoblotting
by SDS-PAGE
Tubule preparations purified from infected cells were analyzed by 10% polyacrylamide gel electrophoresis (PAGE) in the presence of SDS, and by immunoblotting, as previously described (Urakawa and Roy, 1988). For immunoblotting, separated polypeptides were transferred from the gel onto nitrocellulose membranes which were incubated in the presence of either rabbit anti-BTVlO serum or rabbit anti-AcNPV extracellular virus particle serum, both at dilutions of lOOO-fold. Controls included probing blots of uninfected Sf9 cell lysates, and testing of preimmune rabbit serum at the same dilution on identical blots of the NSl preparation. Bound antibodies were detected using a secondary antibody conjugated with alkaline phosphatase (Urakawa and Roy, 1988). Immunogold
EM
For immunogold-negative stain electron microscopy (EM), tubules were adsorbed onto a carbon filmed EM grid for 1 min, washed in STE buffer, and floated on 25-ml drops of 1:500 diluted rabbit anti-BTVlO-infected BHK cell antiserum (1 hr). After three further washes, antibodies were localized by incubating grids in goat anti-rabbit colloidal gold (lo-nm particle size; Bio Cell Res. Labs., Cardiff, UK), for 1 hr before a final wash and
FIG. 2. (a) Immunogold negative-stained EM localization of BTV NSl tubules with rabbit anti-BTVlO-infected BHK cell antiserum and 10 nm goat anti-rabbit colloidal gold conjugate. Small arrows indicate specifically bound gold particles on BTV NSl structures, while the baculovirus contaminant tubule (AcNPV) are unlabeled. (b) Control lysate of cells infected with wild-type acNPV treated like NS1 preparation I in a, and similarly immunogold labeled with antiBTV-10 antiserum.
3-D
RECONSTRUCTION
OF BTV
37
TUBULES
A&l PV
38
HEWAT
negative staining in 2% many1 acetate. Controls included elimination of the primary antiserum, its replacement with preimmune rabbit serum, probing with an irrelevant antiserum (raised against purified BTV NS2 protein), and testing of preparations of uninfected cells. For postembedding, on-section immunogold labeling of NSl in both mammalian and insect cells, infected cultures were fixed in 0.1% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, dehydrated in ethanol series (50, 70, and 90% w/v) and embedded in LR white resin (London Resin Co., Basingstoke, UK) as described by Newman et al. (1983). Specimens were observed in a JEM 1OOC electron microscope at 100 kV. Preparation
and Observation
of Frozen-Hydrated
Specimens
Frozen-hydrated specimens were prepared on holey carbon films as described by Dubochet et al. (1985). A modified Zeiss cryoworking unit was employed since it allows manipulation of the specimen grid in gaseous rather than liquid nitrogen. The holey carbon films, supported on 400-mesh copper grids were glow-discharged in air immediately prior to use. Samples of the tubule suspension (4 pl) were applied to grids, which were blotted immediately with filter paper for l-6 set and then rapidly plunged into liquid ethane cooled by nitrogen gas at - 175°C. Specimens were observed at temperatures of - 165 to - 175°C in a Zeiss 1OC electron microscope equipped with a top entry cold stage and nontilting specimen holder. The microscope was operated at 100 kV and images were recorded at x 20 000 magnification with a dose of less than 10 e /Aa on Kodak SO163 electron image film which was developed in full-strength D19 for 12 min at room temperature. The magnification was calibrated using the 4.0-nm first layer line in computed Fourier transforms of microtubule images (Amos and Klug, 1974, Chretien, 1991). Conventional
Electron
Microscopy
Specimens were prepared for conventional electron microscopy using one of the following negative stains: 2% w/v uranyl acetate, pH 4.4,2% w/v phosphotungstic acid (PTA), pH 5.0,2% w/v PTA, pH 7.2, or 2% w/v sodium silicotungstate (SST), pH 7.2. These specimens were also observed in the Zeiss 1OC at room temperature at 100 kV under low-dose conditions. Heavy metal shadowing was carried out at room temperature in an Edwards vacuum evaporation unit. A suspension of NSl tubules (4 pl) was mixed with 1 M ammonium acetate (4 pl), and spread on freshly cleaved mica. The specimens were rotary shadowed at 8” from the surface normal with Pt/W and a carbon support film was then evaporated from above. Specimens were observed in a Zeiss 1OC as for the negatively stained specimens. Image
Processing
Preliminary examination of the micrographs was carried out using a CCD TV camera linked to a dedicated computer which produced the power spectrum of a 512 x 512 pixel image in 2 set (Tietz video and processing systems). With this system individual tubules were easily masked off with a black paper mask and assessed separately. Only images in which all helical diffraction data lay inside the first zero of the contrast transfer function were selected so no correction for the contrast transfer function was made. Selected micrographs were digitized for further analysis using a CCD TV camera coupled to a PC with an ADC card. A pixel of size of IO.5 p,rn on the micrograph was employed, which for a magnification of x 20 000, corresponds to a pixel size of 5.2 A at
ET AL. the specimen. Image analysis was performed on a Vaxstation 3200 using the Semper 6 Plus image analysis package and a modified version of the MRC helical reconstruction programs (DeRosier and Moore, 1970). Typically areas of 250 x 135 pixels, floated to 512 x 512, were Fourier transformed and no correction was required for unbending of the tubule images. The amplitude and phase information on each layer line was extracted and corrected for tilt of the helical axis away from the normal to the electron beam. Analysis was simplified by the fact that there was only one Bessel order present on each layer line so a complete low-resolution data set for reconstruction could be extracted from one image. The layer line data from near and far sides was averaged. Six different tubules were reconstructed separately using data out to 4NM - 1 from the meridian. Horizontal and cylindrical sections were calculated using the Fourier-Bessel transforms in the MRC programs and the surfaces of reconstructions were visualized using the Semper 6 Plus solid surface display facility. Filtered images were calculated for each side of the tubule by pseudooptical filtering of the layer line data (Baker and Caspar, 1983). RESULTS
Purity of Preparations of Structures
and Identification
When analyzed by SDS-PAGE and immunoblotting with antisera directed against either AcNPV virus particles or BTVlO-infected mammalian cells (Fig. 1) preparation I appeared to be approximately 70% NSl and was contaminated with several baculovirus proteins, of which a protein of about 40-kDa relative molecular mass was the most abundant (Fig. 1). The NSl protein in preparation I was also recognized in immunoblots using a monospecific antiserum raised against purified, baculovirusexpressed NSl (Urakawa and Roy, 1988; data not shown). Preparation I gave the better tubule morphology, as judged by negative-stained EM using uranyl acetate, although it was contaminated with empty baculovirus nucleocapsids (Fig. 2) which showed up on immunoblots using antiserum against AcNPV extracellular particles (Fig. 1). Bluetongue virus tubules in preparation I could be distinguished from the baculovirus nucleocapsid tubules by immunogold labeling with antiserum specific for BTVlOinfected mammalian cells (Fig. 2a) and by their general appearance in negative stain (Figs. 3a and 3b). The more striated appearance of the bluetongue virus tubules with their higher edge contrast with a repeating pattern of 9.1 nm was easily distinguished from the fine reticular “cross-weave” appearance of the surface of the empty AcNPV capsids (Fig. 3a). The rabbit antiserum raised against baculovirusexpressed BTVlO NSl also recognized the tubular structures formed in BTVl.O-infected BHK cells --.--
FIG. 3. EM images of negatively stained (2% uranyl acetate, pH 4.4) NSl tubules. (a) Preparation I, the different appearance of BTV NSl tubules and the contaminant baculovirus tubules (AcNPV) are evident. (b) Preparation II, additional purification leads to shorter tubules with little baculovirus contamination, with many short, unwound fragments (arrows) that can be seen unwinding from the ends of tubules or detached as circular forms or as short broken sections of filament.
3-D
RECONSTRUCTION
OF BTV
TUBULES
39
40
HEWAT
ET AL.
3-D
RECONSTRUCTION
FIG.
OF BTV
41
TUBULES
I.-Continued
(Fig. 4a). Both the BTV virions and the NSl tubules were observed in association with the intermediate filament component of the cytoskeleton of the BTVinfected mammalian cells. In all cases the controls confirmed the specificity of the antisera for the NSl tubular structures (Figs. 4a-4c) and the tubules reacting with the antiserum were identical in appearance to those previously isolated from BTV-infected mammalian cells (Huismans and Els, 1979). After the secondary centrifugation step using the method of Huismans and Els (19791, NSl tubules were produced that were over 90% pure as judged by SDS-PAGE (preparation II: data not shown). However, when preparation II was examined by negative-stained EM, the tubules were found to be shorter and their morphology less well preserved than those of preparation I (Fig. 3b). In addition, preparation II contained many short ribbons and, frequently, single strands unwinding from the ends of intact tubules were observed (Fig. 3b). Therefore, preparation I was used for the majority of the EM investigations. Dimers of NSl were not observed under any of the denaturing or native conditions tried. Tubules were found to be insoluble in the absence of
SDS at pH 7.5, 8.7, or 9.7. Heating of tubules at 60°C or freeze-thawing them, yielded disrupted aggregates of NSl that had a limited PAGE mobility in the absence of SDS at pH 8.7 or 9.7, showing a smear corresponding to high molecular weight NSl polymers, and a faint band of NSl monomers, while treatment with SDS or 2 M urea yielded monomers (data not shown). Treatment of tubules with the ionic detergent dodecyltrimethylammonium bromide (DTAB) at a concentration of 10 mM yielded highly distorted, unwound tubules that could be identified by immunogold labeling with antiserum directed against BTVlO-infected cells (Fig. 4~). The disrupted NSl tubules could thus be distinguished from the contaminating AcNPV tubules which were not affected by DTAB treatment (Fig. 4~). Cryoelectron
Microscopy
In cryo-EM images, frozen-hydrated NSl tubules are characterized by 9.1-nm repeat bands particularly visible along the “sides” of each tubule (Fig. 5). They are also distinguished by their tendency to form rafts in which the tubules are aligned parallel to each other with a separation of 2.0-2.5 nm. Some
FIG. 4. (a) Immunogold labeling of NSl tubules (arrows) in ultrathin plastic sections of BTVlO-infected BHK cells with rabbit antiserum raised against gel purified baculovirus-expressed NSl. T, tubular structures in section; VP, virus particles. (b) Control section incubated in anti-NS2 antiserum. The virus inclusion body (VIB) in this section is labeled but not the tubules (T) or the virus particles (VP). (c) Treatment of NSl tubules with 10 n&f DTAB: immunogold labeling demonstrates disruption of BTV NSl tubules (solid arrows); the baculovirus contaminant tubules (open arrow) are not disrupted by the detergent and are not labeled by immunogold.
42
HEWAT
ET AL.
tubules have an abrupt change in diameter or “dislocation” along their length (Fig. 5). They range in diameter from 48.5 to 56.5 nm and in length from a few tens to 1000 nm. The average tubule diameter calculated from 50 measurements is 52.3 nm. The tubule length is typically five or six times the diameter. A histogram of tubule diameter is shown in Fig. 6. Tubules are seen either perpendicular to their axis or end-on. This is due to surface effects and constraints placed on their orientation by their presence in a thin film of buffer immediately prior to freezing. Empty nuclear polyhedrosis virus (AcNPV) capsids found in the same preparation have a much smoother appearance and their fine cross-weave surface lattice is occasionally visible. Electron
Microscopy
of Negatively
Stained Tubules
The 9.1-nm repeat is clearly visible with all of the four negative stains used (Figs. 3 and 7). In preparation I the NSl tubules also had a tendency to be aligned in bundles which, in contrast to the cryoelectron microscopy images, were not necessarily coplanar. In the more highly purified, more dilute preparation II the tubules are seen in isolation. The highest resolution images were obtained with sodium silicotungstate stain (Fig. 8~). Indexation Filtered
FIG. 5. Cryoelectron microscope images of BTV NSl tubules at a defocus of 3 pm. Note the parallel alignment of tubules (a) and the step in diameter of a tubule (b, small arrow). Short end-on tubules are also visible.
of Diffraction Images
Patterns
and
Computed diffraction patterns were obtained from images of frozen-hydrated and from sodium silicotungstate-stained NSl tubules (Fig. 8). All of these diffraction patterns fall into two categories as exemplified by Fig. 8a (type A) and Fig. 8b (type B). There is a clear difference in the position of the layer lines except for the layer line close to the meridian at 9.1 nm. The layer lines visible in type A diffraction patterns are indexed as 1 = 0, 1,4,5, and 6 and in type B diffraction patterns as 1 = 0,2,5,7, and 9 to a very good approximation. No intermediate values were ever seen. In preparation I, observed by cryo-EM, 49 tubules were of type A and 23 of type B out of a total of 72 analyzed. However, in 2% PTA, pH 7.2, or SST, pH 7.2, all tubules were of type B and in 2% PTA, pH 5.0, or uranyl acetate, pH 4.4, all tubules were of type A. Hence, we conclude that the transformation from type A to type B is pH dependent. Because the preparations observed by cryo-EM were nominally pH 7.5 and both types A and B were observed, it is probable that the presence of other ionic species (as in the negative stain) modifies the transition pH.
3-D
46
49
50
51
52
53
54
RECONSTRUCTION
55
56
57
nm
diameter FIG. 6. Histogram of frozen-hydrated The values of the diameters for the and 22 are indicated.
NSl tubule reconstructions
diameters. for U = 21
Some of the images obtained using sodium silicotungstate staining diffract to 3.0-nm resolution (Fig. 8c), which is better than that obtained in cryo-EM images which contain some information to 3.5 nm. Unfortunately, this higher resolution information cannot be exploited, because it is evident from the one-sided filtered images that both near and far sides of the collapsed tubules interact with each other. This causes distortion of the lattice, rendering the filtered images impossible to interpret. Filtered cryo-EM images of type A tubules were useful in assigning layer lines to one or another side of the helix. Only the combination shown in Fig. 8
FIG.
7.
Comparison
of electron
microscope
images
of negatively
OF BTV
43
TUBULES
gives a reasonable one-sided filtered image. The filtered image is shown both including and excluding the 1 = 0, n = 0 layer line, i.e., the equator, (Figs. 9a and 9b, respectively). The apparent horizontal dimension of the unit cell is smaller at the edges of the tubule than in the center as expected for the projection of a cylinder. The indexation regarding n is based on three criteria; first, the parity of related reflections (after correction for tilt away from the normal to the electron beam direction); second, the requirement that all reconstructed density falls within the average radial density envelope defined by the n = 0, I = 0 layer line (Egelman et al., 1989); and third, end-on views of short NSl tubules in ice were used as confirmation of the n indexation of similar diameter tubules since the subunits are visible in some favorable cases (Fig. 10). The selection rule for the type A tubules is 1 = 5n + (5U - l)m,
where U is an integer. Of the six images of type A tubules used for 3-D reconstruction, one had U = 20, two U = 21, and three had U = 22. From the observed distribution in tubule diameter, U = 21 and 22 are the most common values (Fig. 6). The number of units (dimers)/ turn = U - 0.2. Indexation of the diffraction patterns from type B is not as clear as this type was less well preserved under the conditions of observation by cryo-EM: in
stained
BTV
NSl
tubules.
(a) 2% SST,
pH 7.2; (b) 2% PTA,
pH 5.0.
44
HEWAT
ET AL.
FIG. 9. One-sided filtered images of a type A NSl tubule in ice including (a) and excluding (b) the I = 0, n = 0 layer line.
types A and B have the opposite hand. In view of the observation that type A can be converted into type B without unwinding, by changing the pH, they almost certainly have the same hand.
FIG 8. Computed diffraction patterns from NSl tubules images (a) type A in ice, (b) type B in ice, and (c) type B stained with SST, pH 7.2. The indexing shown is for U = 22.
general, the diffraction patterns are not very symmetrical. In addition, it is not evident from the amplitude distribution on layer lines I = 5 and 1 = 9 which has the higher n value. It was not possible to make satisfactory reconstructions of type B tubules. However, since type B tubules transform into type A tubules as a function of pH, it follows that both types have like-handed helices. Hence, the indexation shown in Fig. 8b is the most probable. The alternative assignment of diffraction peaks implies that
FIG. 10. End-on view of a short NSl tubule in ice.
3-D
The selection
The number Shadowed
RECONSTRUCTION
OF BTV
45
TUBULES
rule for type B is
1 = 7n + (7U - 2lm. of units/turn = U - 2/7. tubules
Images of NSl PtW-shadowed tubules only show the 9.1-nm repeat along the axis (data not shown). Although the handedness was not evident in all of these images due to distortion of the tubules on drying, the computed diffraction patterns showed the n = 1 helix to be most probably left-handed.
0 0
A-’
0.025 N-22 .
0.125,
L-l
,180
3-D Reconstructions The data from the analysis of six of the type A tubules proved to be suitable for reconstruction. An example of the layer line data for a U = 22 tubule is shown after correction for a 4.5” tilt and averaging over near and far sides (Fig. 11). It is notable that for each layer line, the phase flips between two values which differ by approximately IT. This is characteristic of a surface lattice which has a twofold axis perpendicular to the helix axis (Toyoshima and Unwin, 1990, Hutchinson et al., 1990). The layer line data in Fig. 11 was used to reconstruct the helix shown in Fig. 12. The reconstruction shows a solid surface representation of the front surface only (Fig. 12a), a horizontal section (Fig. 12b), and a half cylindrical section of the helix viewed from the outside (Fig. 12~). The surface lattice has a clear P2 symmetry; this has not been enforced on the data. The largest sphere which can be accommodated in the lattice shown has a diameter of 5.5 nm. This is close to the 5.3-nm diameter expected for a globular 64-kDa protein assuming a partial specific volume of 1.24 A” per dalton, so we assign this to the NSl monomer seen at low resolution. The structure can be described as a ribbon of what appears to be NSl dimers wound into a left-handed helix. The selection rules for the six reconstructed tubules are I = 5n + (5 x 20 - l)m for one, 1 = 5n + 15 x 21 - l)m for two, and 1 = 5n + (5 x 22 -. 1)m for three of the tubules. So they have 19.8, 20.8, and 21.8 dimersiturn, respectively. Each reconstruction gives essentially the same surface lattice with only slight differences in the density at the intermolecular contacts. While we could not make a satisfactory reconstruction of the type B tubules, we can determine some information about their surface lattice. Compared with type A tubules, the type B lattice is slightly skewed, and the number of dimersiturn changes; for example, from 21.8 to 21.7 units per turn (Fig. 12~). There is also a small change in distribution of intensity on layer lines 1 = 3 and 1 = 5 which become 1 = 5 and 1 = 9, respectively (Figs. 8a
0
A-’
0.025 N--21
L-4
io/ -j&f&i0
0.125
.
.a-
0
-
0.025 N-l
0.125
*
.
*.
L-5
180
.
A-’ - 180
.
. -0 -OB
N-23
p-
il:‘.’
0.025
A ’
0
FIG. 11. Layer line data from a type A NSl tubule corrected for a 4.5- tilt of the tubule away from the normal to the electron heam and averaged over near and far sides.
and 8b). This must reflect protein density distribution.
a change
in the radial
DISCUSSION
Cryoelectron microscopy and 3-D reconstruction of the bluetongue NSl tubules show that they consist of a ribbon of dimers wound into a left-handed helix about 52 nm in diameter. There are usually 21- or 22-dimer-like units per turn and the pitch is 9.1 nm. The quantification of tubule diameter as a function of dimer units per turn is somewhat
46
HEWAT
ET AL.
a
IOnm
FIG. 12. Reconstruction of a type A NSl tubule with U = 22. Visualization of the front surface of the tubule only (a), section perpendicular to the tubule axis (b), and a half-cylindrical section at a radius of 25 nm viewed from the outside (c) are shown. The P2 symmetry of the surface lattice is indicated. The unit cell of the surface lattice of the type B tubule is shown dotted. The skew angle between the type A and type B tubules is increased by a factor of two for clarity.
masked by the spread in values due to distortion of the tubules and errors in measurements. It would appear that the dimeric form of NSl is unstable outside the context of the dimer ribbon, and that the protein-protein interactions between adjacent dimer units must be of a similar nature to the intradimer bonding. Thus, there is one of the four protein-protein interactions that each NSl molecule makes within the tubule that is closer than the other three interactions, and hence appears dimeric. The fact that under certain conditions the tubules unwind at their ends into dimer ribbons, and breakage of these into “circular” forms was also observed, suggests that the bonding between the filaments in the direction of the tubule axis is weaker than the bonding within the dimer filament. A possible model for the unwinding of the dimer filament is shown in Fig. 13. The Orbivirus tubules clearly differ in structure from those associated with retrovirus infections (Dales, 1963) and tubules associated with rotavirus infection which consist of a structural capsid protein of the virus particle (Holmes et al., 1975; Kimura and Murakami, 1977). In previous EM studies, the expressed NSl tubules were also contaminated with baculovirus nu-
cleocapsid tubules which superficially have a similar appearance and this has led to confusion in the interpretation of the morphological changes observed (e.g., Marshall et al., 1990). The AcNPV tubules are formed from the 40-kDa nucleocapsid protein, and represent an immature, empty form of the nucleocapsid without the genomic nucleic acid or the cap-like ends (data not shown). The structure of AcNPV tubules consists of rings of protein subunits which are stacked one on top of another with a slight rotation between each layer to form a cylinder (unpublished data). In this study, baculovirusexpressed BTV NSl tubule preparations were purified to about 90% NSl (estimated by SDS-PAGE);
FIG. 13. of the dimer
Model showing ribbon.
the
proposed
unwinding
mechanism
3-D
RECONSTRUCTION
however, they still contained a significant amount of baculovirus tubules. We unequivocally identified these two different tubular structures using both immunological and structural criteria. Tubules composed of BTV NSl were distinguished from the AcNPV tubules by immunogold labeling, and both had a distinctive appearance by both cryo-EM and negative-stained EM. In addition, the diffraction patterns allowed the two different structures to be readily distinguished. The empty AcNPV capsids have identical diffraction patterns to those shown in previous electron microscope studies (Beaton and Filshie, 1976). We have shown that the expressed NSl tubules are apparently immunologically identical to those made in BTV-infected mammalian cells and have an identical appearance to the previously published negative-stained electron micrographs of authentic BTV NSl tubules purified from infected BHK cells (Huismans and Els, 1979). The cryo-EM images of frozen-hydrated NSl tubules gave computed diffraction patterns which fell into two distinct groups (types A and B). The difference between type A and type B involves a skew in the surface lattice with respect to the tubule axis, with the angle for the type B tubule being about 3” greater than that of the type A tubule (see Fig. 12~). A typical sample was composed of 68% type A, and 32% type B, out of 72 tubules examined. All tubules which gave a clear diffraction pattern could be assigned as type A or B to a very good approximation. However, after negative staining using solutions of different pH, the conformation of the BTV tubules was found to be dependent on the pH of the stain used: At pH 7.2, using either PTA or SST, 100% of the tubules were type B, whereas at pH 4.4 or 5.0 (PTA or uranyl acetate), 100% of the tubules oberved were of type A. This result suggests that the lattice undergoes a transition during the drying process in the presence of increasing concentrations of ionic species (buffer and heavy metal salts): this transition thus appears to be influenced by the initial pH of the solution. The fact that the type B NSl tubule at higher pH cpH 7.2), has a 3” greater angle of skew with respect C;Othe tubule axis than the type A at lower pH (below pH 5.0) suggests that the diameter of the tubule decreases with pH. This change in shape of the lattice is equivalent to a change in the number of dimers per turn of 21.8 at low pH to 21.7 at high pH. There is also probably a small change in the radial density distribution of the dimer. Future studies will include examination of these pH effects in the frozen-hydrated state to determine whether this change can occur under native conditions. The data presented in this initial 3-D reconstruction of the BTV NSl tubules will be of use in further studies employing genetic engineering to determine
OF BTV
47
TUBULES
the effects of mutations in the NSl gene on the formation of oligomeric tubular structures. Further studies to compare the tubular NSl structures from other Orbiviruses will be possible, and the cryo-EM technique will be available in analyzing the results of attempts to generate tubules containing NSl from different viruses, or tubules containing mutant chimerit species of the NSl protein. The function of NSl remains unknown. It may be involved as a molecular chaperone to prevent the core particles from assembling before the correct minor core proteins (VPl, VP4, and VP61 and/or genome have been incorporated. It has been demonstrated (French and Roy, 1990) that expressed BTV VP3 and VP7 can self-assemble to form a core like particle in the absence of other BTV proteins. Further clues as to the function of these NSl tubules in BTV infection must await generation of mutant and recombinant BTV strains with defective NSl. It is a pleasure to thank Christian Closse, Samantha Griffiths, and Katie Monstyrskaya for expert assistance, and Annie Barge of the EMBL Grenoble for assistance with the heavy metal shadowing. We also thank Stephen Fuller for supplying the helical reconstruction programs employed. The financial contributions of EMBO and NERC are gratefully acknowledged. REFERENCES Amos,
L. A., and
Klug,
A. (19741
J. Cell
Sci.
Baker, T. S., and Caspar, D. L. D. (1983) Proceedings of the 41st Annual Meeting cisco Press, San Francisco, CA. Beaton. 161.
C. D., and
Filshie,
B. K. (1976).
14, 523-549.
in Bailey, G. W. (Ed.), of EMSA, San FranJ. Gen.
Chretien D. (1991) Apports de la cryomicroscopie l’etude de microtubules assembles in vitro. Joseph-Fourier, Grenoble. Dales,
S. (1963)
Proc.
Natl.
Acad.
Sci.
D. d., and Moore,
Dubochet, Trends
J., Adrian, M., Lepault, J., and McDowall, Biochem. Sci. 6, 143-146. A. D.. and
White,
Egelman, E. H., Wu S.-S., Amrein, / 19891 J. V~rol. 63, 2233-2243. i;i-ench. (would, Res.
‘I or and
Hii>
1’
A. K., Pritchard, 11. 97-107.
Holmes, 11975)
1990.
,i
1,. 1.. and
H. $1979)
Huismans,
H.. and
Cloete,
Huismans,
H., and
Els,
Hutchinson, J. Struct.
G., Tichelaar, W.. Weiss, Biol. 103, 75-88.
Hyatt, Kimura,
A. D.. and
Virology
Eaton,
T., and Murakami,
H. J. (1979)
B. T. 11988) T. (1977)
B
R. F..
.52, 355-369. A. W. (1985) Virology
163,
A,, and Murti,
tij,
Ta\aiia,
92, 385 M. (1987
Biol.
Portner,
I‘rr~,i
I. H.. Ruck, B. J., Bishop, J. Virol. 16, 937-943.
Huismans,
J. Mol.
J. R. (1988)
M.,
151-
50, 268-275.
DeRosier,
Eaton, B. T.. Hyatt, 527 -537
31,
electronique Thesis, Universite
USA
P. B. (1970)
Viral.
i 5Rti
:.%6
&l. D. * i988i and
G
Davidson,
i-l/ (A,> G. P.
396
t Virology Vtrology
158, 373-380. 92, 397-405.
H., and Leonard, J. Gen. Infect.
K. (19901
Viral.
69, 8055815.
Immun.
17, 157-160.
48
HEWAT
Lecatsas, G. (1968) Onderstepoort J. Vet. Res. 35, 139-150. Mandelkow, E.-M., and Mandelkow, E. (1985) J. Mol. Biol. 181, 123-135. Marshall, J. J. A., Fayard, B., and Roy, P. (1990) J. Gen. Viral. 71, 1839-1844. Martins, S. A., Pett, 12, 194-198. Newman, G. R., Jasani, J. 15, 543-555. Ritter, D. G., and Roy,
D. M.,
and
Zweerink,
B., and Williams, P. (1988)
Virus
H. J. (1973) E. D. (1983) Res.
11, 3347.
J. Viral. Histochem.
ET AL. Roy, P., Adachi, A., Urakawa, T., Booth, T. F., and Thomas, C. P. (1990) J. Viral. 64, l-8. Toyoshima, C., and Unwin, N. (1990) J. CelZBiol. 111,2623-2635. Unger, R. E., Chuang, R. Y., Chuang, L. F., Osburn, B. I., and Doi, R. H. (1988) Virology 167, 296-298. Urakawa, T., and Roy, P. (1988) J. Viral. 62, 3919-3927. Verwoerd, D. W., Els, H. J., de Villiers, E. M., and Huismans, H. (1972) J. Viral. 10, 783-794. Verwoerd, D. W., Louw, rol. 5, l-7.
H., and
Oellermann,
R. A. (1970)
J. Vi-