consideration of the three dimensional structure of the adenovirus hexon from electron microscopy...

Micron, 1979, Vol.: 10. pp. 247-266. Pergamon Press Ltd. Printed in Great Britain

C O N S I D E R A T I O N O F T H E T H R E E D I M E N S I O N A L S T R U C T U R E

O F T H E A D E N O V I R U S H E X O N F R O M E L E C T R O N M I C R O S C O P Y

A N D C O M P U T E R M O D E L L I N G

M. V. NERMUT and W. J. PERKINS

National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, England

(First received 12 March 1979; in revised form 12 April 1979)

Abstraet--A three-dimensional model of the adenovirus hexon has been built up from electron microscopic observations and information obtained by computer modelling. It has been shown that the hexons (in upright position) are negatively stained from below and that the stain layer after drying is only 1-2nm high. Thus the top, i.e. the external part, of the hexon is triangular, the waist is hexagonal and the bottom is 'round' with a large axial hole. The top is twisted through 30 ° with respect to the waist. This morphological polarity is accompanied by differences in physico-chemical properties: the top appears to be negatively charged at neutral pH, whereas the bottom is rather hydrophobic. Because of the hexagonal region at the waist of the hexon the virus capsid becomes sealed allowing passage of small molecules only, presumably via a narrow central channel.

I N T R O D U C T I O N Electron microscopy provides an indication

of the shape and density of a biological specimen and for simple highly symmetrical structures the three-dimensional configuration may sometimes be inferred from the limited range of projections observed (Valentine et al., 1968). In the electron microscope the appearance of a given part of an object is related to thickness and hence electron scattering at that part and not on the surface contours that we encounter with the human eye. For more complex symmetrical structures, reconstruction may be possible (de Rosier and Klug, 1968; Crowther et aL, 1970) but this technique requires extensive computing facilities and the reconstruction at high resolution is restricted by the number of tilted images that can be obtained from a single specimen without extensive radiation damage. For simple structures, one approach has been to produce physical models for visual comparison with related electron micrographs (EMs) (Caspar, 1966) but these provide no density comparison for diverse projections. For more complex non-periodic structures, extensive and more precise manipulation of a model becomes necessary, together with a closer comparison

with micrographs of the specimen. A computer model can be easily and exactly manipulated or modified as required to provide a more precise classification of the observed images in respect of tilting axes or degrees of rotation about each axis. In addition to providing information on the contours of a structure at any orientation, density is also included. As background noise can affect the observed image, this too can be added to provide a more realistic simulation of the situation on the specimen grid.

The structure of interest in this study is the adenovirus hexon, the major component of the icosahedral virus capsid. This name was proposed by Ginsberg et al. (1966) to denote that it is surrounded by six neighbours, whereas the pentons--si tuated at the 12 vertices of the icosahedron--have five neighbours. There are 240 hexons in the capsid and four of them form the edge between two pentons. When treated with detergents the capsid disintegrates into the characteristic 'groups of nine hexons' (GONs) which originate from each of the 20 triangular facets of the icosahedron (for references see Horne, 1974 or Nermut, 1975).

Electron micrographs from negatively stained, freeze-dried or freeze-etched viruses or purified

247

248 M.V. Nermut and W. J. Perkins

hexons provided data for producing a model of the hexon (Nermut, 1975). However, the initial model was based on an assumption that the hexon was negatively stained from above because the measurement across both its lower portion in the horizontal position and across the triangular (Y-shape) image, was between 8.5 and 9.5nm, whereas both the upper end and the

round end-on image (O-shape) measured about 7.5-8.0nm. Therefore, it appeared logical at that time to assign the O-shape to the top and the Y-shape to the base o f the hexon (Fig. 1). Al though the O-shape image-- typical for the left-handed 'groups of nine hexons ' - -was satis- factorily explained by that model, it was more difficult to understand the larger measurements

Fig. I. The initial hexon model as proposed in 1975. The solid base is triangular (Y-shaped). the top contains a central cavity which gives rise to the O-shape when filled with stain. From Nermut (1975)

with permission.

3-D Structure of Adcnovirus. Hexon 249

from the Y pattern in the reversed orientation (=8.5nm). In addition to this, a variety of 'unorthodox' images have been observed by N. G. Wrigley and ourselves in this Institute when hexons were attached to hydrophilic or positively charged carbon films (see also Fig. 16). These images were often U, V or X-shaped and at first sight did not resemble the known hexon images. It was decided therefore to develop a model of the hexon with the aid of a computer, incorporating the available data and ideas of possible structure for comparison with related electron microscopic images. It was appreciated that the electron microscope images were of stained hexons and that for a direct comparison with the model images, the assumed effect of stain would also need to be incorporated into the model. However, as the stain pattern around a structure is dependent upon the shape and orientation of the structure, it was first necessary to produce an approximate basic model to which the simulation of stain could be applied. It appeared necessary to study the distribution of stain around hexons in the upright position as taken up in the groups of nine hexons and to ascertain whether they are stained from above or from below. Because some of our experience indicated that the orientation of the hexons on the film might be influenced by the properties of the supporting films, we studied the orientation of GONs on hydrophilic (or positively charged) and hydrophobic carbon films to determine their physico-chemical polarity. These observations have been incorporated into a new hexon model which seems to satisfy all the require- ments.

METHODS

Preparation procedures for electron microscopy Hexons were obtained by disintegration of

adenovirus 5 (strain 75) with Na-deoxycholate and partial purification on a glycerol density gradient (Russell et aL, 1971). Fractions con- taining the hexons were dialysed against 5mM tris-HC1 buffer, pH 7.5, for several hours or overnight.

Negative staining of the hexons was carried out using one of the following procedures:

(i) A two stage procedure (see Nermut, 1973 for references) where hexons are first adsorbed to the supporting films, then washed with distilled water followed by negative staining.

(ii) A one stage procedure, in principle as described by Brenner and Horne (1959) where hexons are first mixed 1 : 1 with the stain then adsorbed onto the supporting films and dried.

Four percent silicotungstate (STA) pH 6.5 and 1 ~ uranyl acetate (UA) pH 4.4 were used as negative stains. Specimens were examined with the Philips EM-300 or 301 electron micro- scopes operating at 60 or 80kV, equipped with either a high resolution or a goniometer specimen stage. Their magnification was calibrated with negatively stained catalase (Wrigley, 1968). The grids were always oriented with the material up in the specimen holder, i.e. material down in the electron microscope (Finch and Klug, 1965). Some preparations were tilted around the X-axis in a JEM-100 C electron microscope (JEOL Co., U.K.).

Computer modelling procedures

The program provides user-interaction for building, manipulating and modifying a model by means of predetermined options selected by typing in simple instructions, e,g. RX 45 for rotate structure about the X axis by 45 ° (Perkins, Polihroniadis, Piper and Smart, 1976). The basis of the technique is to use a building unit that can be made up into sub-structures in three dimensions which can in turn be incorporated into the complete structure. A sphere was chosen as the basic building unit as this could be represented by a circle on the display screen for any orientation and stored as a triplet of X, Y, Z co-ordinates. Spheres can be brought into any desired position in three-dimensional space and others joined to them at appropriate angles. Density is given to a model by replacing each circle with a distribution of dots whose positions are randomly placed within the circle area to allow an increased number of dots for over- lapping spheres. Normally the front, side and top views of the model are displayed simul- taneously in three quadrants of a storage display oscilloscope, representing the three orthogonal planes (Fig. 2). For manipulating established structures, a particular view can be selected for display within the orthogonal axes then rotated about a specified axis by a given angle. Back- ground noise may be added to the display at any desired level by a random dot distribution over


L~ ~.

/ 5

Fig. 2. Notation for orthogonal axes as used in the computer. Z axis is normal to display (i.e. viewing screen or photograph). Clockwise rotation looking to the centre from the positive end of each axis is positive (arrows).

the whole display. The computer used was a Honeywell DDP 516 with 16 k words of 16 bit store and a 3.6M word disc.

The effect of negative staining of the hexon can be simulated in the computer model but as the stain profile depends upon the shape and orientation of a structure, this has to be established for each position of interest. As the images of the upright and reversed positions of the hexons can be readily determined from the micrographs of left-handed and right-handed GONs, this position was selected for simulation. A drawing is produced of one sub-unit of the hexon in this position together with the assumed stain profile around it, viewed from the front and side (for concrete example see Fig. 17). Horizontal sections throughout the stain profile are then drawn of the stain surrounding the three hexon sub-units. Each section is placed under a transparent conductive tablet and the outlines of each section and sub-section traced over sequentially with a conductive probe which transfers their X,Y co-ordinates into the computer (Perkins, Green and Burdett, 1976). A computer program then provides selected density levels for the stain and hexon areas of each section and these sections are combined to produce a variable density image of the stain surrounding the hexon for this particular orientation of the structure which can be compared directly with electron micrographs of this orientation (Perkins et al., 1979).

RESULTS

(a) Initial development of the computer model

Initial information for constructing the computer model of the hexon came from measurements on electron micrographs of hexons, combined with other observations and ideas about the possible structure. This information was considered at three levels, measurements being generally accepted as true data, observations as possibly true but needing confirmation and ideas as being not proven. At the first level, the hexon was shown to have three symmetrical polypeptides arranged at 120 '~, with an overall length of l lnm. The lengthwise images also showed a greater width dimension of 8.5-9.5nm at about one third of the length. At the second level, electron micrographs of hexons in the adenovirus capsid indicated that this wider section was nearer to the base. Measurements on left-handed (LH) and right-handed (RH) GONs also suggested that the end responsible for the triangular Y pattern was larger than the end responsible for the circular O pattern. At the third level was the assumption that because the Y was larger than the O, it should be associated with the larger base section. Although the possi- bility of a rotational twist within the sub-units had been suggested by Russell et al. (1973) and also anticipated by Nermut (1975), this was not incorporated into the initial model as at this stage it could not be determined with any certainty from the observed images. It was decided to incorporate this feature at a later stage and to study the effect of a twist on the model images.

The above data and ideas were incorporated into a computer model by building up a series of sections along the Z axis (normal to the screen). Each section contained three sub-sections representing the sub-units at 0 °, 120 ° and 240 ~' respectively about the Z axis. These sections were made up of spheres, which also defined the thickness of the sections, and their shape and dimensions were obtained from electron micrographs of hexons in their horizontal position (parallel to the specimen grid). Several changes were made to the model before a reasonable resemblance to related electron microscope images was obtained. This stage of the model is shown in Fig. 3a. Comparison of the model images produced by rotation about the Z, Y and X axes (Fig. 3b) with electron micrographs showed a great deal of similarity between them. In particular it demonstrated that diverse images

3-D Structure of Adenovirus Hexon 251

RX 9 0 RY 9 0 + 9 0 + 4 5 0 - 4 5 - 9 0

Fig. 3. (a) Three orthogonal views of hexon model built up in a series of eight sections along the Z axis; density and background are included. RZ (rotated about Z) = 0 °, RY = 90 °, RX = 90 °. (b) Rotated from central position first about Z axis by 45 ° then about Y axis in steps of 45 ° to -4-45/90 ° and about X axis by ~45/90 ° for each step in Y. Figures indicate degrees of rotation.

U, V or X-shaped and parallel patterns could be obtained from the same structure.

In attempting to produce the X images and, in particular, similar ' tooth-shaped' images, the larger section of the model needed to be hollowed out which produced a hole at the bot tom layer. The opposite, narrower, end was also opened out to satisfy the X-images and this produced a Y-pattern. However this was now smaller than the 0 pattern produced at the other end and conflicted with one of the constraints for deter- mining the model. The basic computer model could not reveal different end-on views of O- and Y-patterns as the sum of the densities are the same from either end. The existence of two different O- and Y-images of the hexon in the upright position is indicative of morphological differences at the ends (i.e. that the specimen cannot be completely embedded in the negative stain) so that in these positions only the ends are visualized by negative staining. It was therefore necessary at this point to investigate more thoroughly the behaviour of negative stain around the hexons in these two positions.

(b) Distribution of neyative stain outside and inside the hexon in vertical orientation

The approach was to estimate the height of the dry negative stain around the hexons or GONs. Some of our observations of negatively stained adenovirus particles by means of stereo- electron microscopy showed that the dry stain can reach to between half and three-quarters of the height of the virions, particularly if they are close together. This would support the conclusion of Finch and Klug (1965) that small virus particles are usually stained from both sides. However it was decided to check whether this was also applicable for the single hexons.

A suspension of GONs and single hexons was adsorbed to normal (=hydrophobic) or positively charged carbon films (Dubochet et al., 1971). Negative staining was carried out using the two-stage procedure. Half of each grid was then shadowed with Pt-C at an angle of 45 °. Pictures were taken from both halves and from places with dense as well as thin population of hexons. Figure 4 shows that isolated hexons cast a distinct, long shadow whereas those which are


Fig. 4. Electron micrograph of hexons adsorbed to positively charged carbon films, negatively stained with STA and shadowed with Pt-C. Isolated hexons cast a distinct shadow whereas those in the middle (densely packed) cast either very short shadows or none at all, although they are not in contact with each

other. 200,000.

closely packed (see the central por t ion o f the mic rograph) cast only a very shor t shadow or none at all. A close inspection of the field revealed tha t those hexons are 5-10nm apart . This indicates tha t the stain has br idged this little gap between the hexons.

These results suggested tha t t i l t ing o f the specimen in the electron microscope could provide a more accurate est imate of the height o f the dry stain a round the hexons. Therefore, negatively s tained p repara t ions of hexons were t i l ted using a goniometer stage. In most favour- able cases, pictures were taken at + 4 5 °, + 3 0 °, 0 °, - 3 0 °, - 4 5 ° tilts and a t tent ion was pa id to changes in the original s t ructural features (e.g. O-pa t te rn or Y-pat tern) as well as to the cross-

section d imensions of the hexons. At the same time, in-scale drawings were carr ied out to help in unders tanding the observed features.

Firs t a p repara t ion o f single hexons, negatively s tained with UA, was tilted. A t 0 '~ tilt most hexons show a dist inct central hole o f abou t 2 .0-2.5nm in d iameter which is less than after STA staining (Fig. 5a). After t i l t ing to + 3 0 ° a round the X-axis (JEM-100C microscope) , this central da rk spot moves ei ther in the direct ion of the tilt or agains t it (Fig. 5b). This unexpected behaviour can be explained if we accept that the spot is caused by stain si tuated at different levels above the suppor t ing film as shown in the sketch of Fig. 6. I t is concluded tha t in this par t icular p repa ra t ion the stain dr ied in an internal cavity

Legend for Page 253 Fig. 5. Hexons on glow discharged (in air) carbon films, negatively stained with UA. (a) Zero tilt, (b) + 30 ° (JEM 100-C). Axis of tilt is normal to the double arrow. Central dot moved downwards in some xonshe (boxed), in others upwards (encircled) as a result of tilting. Note hexon with hexagonal profile

(triangular marker). ~ 600,000.

3-D StructUre .of Adenovirus H~xon 253


I I

I I !

I

I !

I

I I

I I I i , I I

Fig. 6. Diagram of tilting of a truncated cone by 30 ° with a layer of stain at the bottom about 1.5nm high. The two black dots could be pockets of stain. The diameter

of the circle is reduced by 20 ~ upon tilting (right).

o f the hexon situated either close to the carbon film or close to the top o f the hexon. Abou t 45 o f the hexons on the micrograph of Fig. 5a are oriented with the central hole down, about 30 are oriented with the central hole up, the rest being difficult to decide. The appearance o f the central hole in the upside down position is probably specific for UA, since after negative staining with STA hexons reveal either a clear- cut hole or a triangular shape without any hole (Nermut, 1975), exceptionally with a tiny central

dot (Fig. 7). Evidently UA stays (and dries) in the cavity even when the hexons are attached by the other end. However, observations on single hexons do not allow any conclusions about the top and base o f the hexon. This is possible in the case of G O N s (see below).

At the same time, the cross-section dimensions o f hexons at 0 ° tilt were compared with those tilted by 30 ° . The average diameter of the untilted hexons (measured in the direction o f the following tilt) was 10.0nm and after tilting only 8.2nm (=82~o). Measurements f rom a drawing such as in Fig. 6 showed that this low reduction in diameter is only possible if the layer o f stain a round the hexon is about lnm high. I f the stain layer were 5.0nm high the diameter would decrease by about 50~,~ and if the whole hexon was embedded in the stain its image would probably disappear after a 30 ° tilt. Similar reasoning indicates that the height of the dark spot in some of the hexons is about 6-8nm. Sometimes two spots were observed after tilting which could mean that both ends o f the central channel contained stain.

In an attempt to localize the central hole, preparations o f GONs, negatively stained with STA, were tilted in the electron microscope. Left-handed G O N s showed a clear-cut central hole at 0 ° and this was also observed after tilting by 30 ° (Fig. 8) or even 45L This demonstrates that the central hole is located near the supporting

i

Fig. 7. Groups of nine hexons (GONs) in left-handed (a) or right-handed (b) orientation. Note central holes in left-handed and 3 subunits in right-handed GONs. Negative staining with STA. Markham's

rotation technique was used in both cases. ~ 1,000,000 approx.

3-D Structure of Adenovirus HeXon 255

Fig. 8. Left-handed groups of nine hexons (a) tilted by +30 ° (Philips 301). (b) Note the presence of central holes after tilting. Negative staining with STA. x 500,000.

film. The drawing in Fig. 6 shows clearly that any stain located near the top of the hexon would be projected outside the area of the base for the above tilts. The drawing also shows how the diameter of the hexon is influenced by the

height of stain attached to the outside of the hexon. I t is quite clear that the original image would disappear if the hexon was fully enveloped in the stain, i.e. up to the top or, say, three- quarters of the height. The original diameter of


the hexons (8.7nm) was reduced to 7.2nm which is about 80~ . This reduction accounts for a height of lnm only. Certainly, one has to take into account that the measured hexons or GONs were not close to each other and attached mainly to hydrophobic carbon films. This certainly can influence the final result of negative staining. Similar observations were obtained with RH GONs by Franklin et al. (1972) who tilted R H GONs by 45 ° (see Fig. 7 in their paper).

On the basis of the above findings, that most hexons or GONs are stained from below, it is concluded that the large central hole is close to the base of the hexon, i.e. that side which points towards the virus interior. Consequently, the Y-shape image is assigned to the top of the hexon. Figure 9 shows a diagrammatic representation of negatively stained GONs in a cross-section, both LH and RH. These results have shown that the stain, though covering the hexons with a high layer, drains out during drying and remains attached to the carbon film and the lower portion of the macromolecule. This also indicates that the central channel, anticipated formerly (Nermut, 1975), is free for water or small molecules.

From this point of view the results obtained by means of one stage negative staining are of interest. This procedure (the original negative staining technique described by Brenner and Horne, 1959) allows the stain to infiltrate the hexon from both sides and often results in denser staining than the two stage procedure. Obviously the stain surrounds the structures to higher levels and possibly stains both ends. This

seems to be well demonstrated in Fig. 10 where the R H GONs have clear-cut central holes as do the LH GONs. If the hexon is stained from both sides, the large dense hole would override the fine Y-pattern in both orientations. It seems

Fig. 10. Electron micrograph of GONs mixed 1 : 1 with 4~o STA and allowed to dry on hydrophobic carbon films. Both the right-handed (R) and left-handed (L)

GONs display a clear-cut central hole. "~ 300,000.

I.~..I 3 0 1.4-1 j,.~.._ . .~ . j " - - - - - ~ I " ~ - - -

Fig. 9. Diagram of a cross-section through negatively stained GONs in a left-handed (left) and a right- handed orientation (right) and their electron microscope images (below). The shapes of dry negative

stain are approximate, Dimensions are in/~ units.


justified to conclude that the central channel, though free, is probably very narrow and does not allow stain from the top cavity to drain out during the drying period, or the stain might be retained in the channel by capillary attraction.

(c) Observations on the polarity of hexons

In order to complete the image of the hexon and particularly to understand its function in the virus capsid, it appeared necessary to determine whether the morphological polarity is accompanied by a functional polarity, e.g. by differences in the surface properties. If so it should be possible to find conditions when the GONs are attached predominantly by their bases or by their tops; in other words they would appear as left-handed or as right-handed. The existence of handedness (Pereira and Wrigley, 1974) proved very useful in this type of study.

First, we adsorbed GONs onto normal carbon films which are predominantly hydrophobic and onto carbon films made hydrophilic by glow- discharging in the presence of amylamine (Dubochet et al., 1971). Such films are positively charged. The results, summarized in Table 1, indicate that the base is hydrophobic whereas the top is negatively charged (at pH 7.0).

Next, the GONs were dialysed against Na- citrate buffer, pH 4.4, and again adsorbed onto normal as well as positively charged films. At this pH, close to the isoelectric point of the hexon (Shortridge and Biddle, 1970), the surface charge is rather neutralized or slightly reversed, and the results reflect this situation. Most of the GONs on normal carbon films are found to be LH whereas on positively charged film both LH and RH in approximately equal amounts can be

found. These results indicate that hydrophobic interaction between the base of the hexon and the carbon film is responsible for the predominantly LH orientation, whereas a statistical 50:50 orientation is observed on positively charged films (although non-charged patches can be partly responsible for this result).

(d) Further development of the hexon model

The tilting experiments confirmed that the bottom (hydrophobic) portion of the hexon could be responsible for the O-shaped image as suggested in section (b) of the Results since negative staining provides information only about the end portion of the hexon, approximately 1.5nm high. We thus amended the first of the eight horizontal sections (each 1.4nm high), as seen in Fig. 1 l a, so that the cross- section dimension corresponded to 7.5-8.0nm as measured on the LH GONs. Similarly, the top two sections (7th and 8th) were shaped so as to match the Y-pattern and to give 8.5-9.0nm across the triangle. The central channel opens up towards both ends, which produces the X-pattern but is very narrow in the middle portion of the hexon as indicated by the negative staining experiments (section b).

The third and largest section was shaped to provide a hexagonal profile of the hexon at this level, as this can occasionally be observed in the micrographs (see Fig. 5a, b and Pettersson et al. (1967). Also, the width of the middle portion, when measured from horizontally-oriented hexons, is often over 8.5nm, usually 9.5nm. On the other hand, the width of the hexon as derived from the length of the icosahedron edge is 8.5nm (see Nermut, 1975), This discrepancy

Table 1. Orientation of groups of nine hexons on carbon films

LH ~ RH ~o Total

pH 7.0-7.4 Normal carbon 1204 92 115 8 1319

film Positively charged 178 6 1587 94 17655

carbon film

pH 3.8--4.4 Normal carbon 211 83 75 17 286

film Positively charged 492 36 557 64 1049

carbon film

LH, left-handed groups of nine hexons RH, right-handed groups of nine hexons

258 M.V. Nermut and W..1. Perkins

b

an angle of 60 ° with the edge of the icosahedron. Consequently, it deviates from the height of the triangular facet by 30 ° to the right in the RH orientation or to the left in the LH orientation. This is best demonstrated when RH GONs are averaged by means of the photographic averag- ing technique (Markham et al., 1963) and reversed to become LH. The large triangle in Fig. 12 represents one of the capsid facets. The angle formed by the height and the azimuth of one of the hexons is 30 °. Several GONs were processed in this way and the angle found was 30 ° + 5 °. It is therefore concluded that the top of the hexon is rotated by 30 ° in respect to the

Fig. 11. Final computer model. (a) One hexon subunit in orthogonal views (bottom). Sections of the hexon (top and centre) built from spheres. Numbers I-8 refer to layers (1 ~ bottom at -Z, 8 top at I Z). (b) Orth- ogonal views of the hexon. Spheres filled with densities, defocussed, with background included. RZ (rotated about

Z) - 0 , RY ~90" (right), RX ,, 90' (bottom).

can be solved by accepting a hexagonal profile with a corner-to-corner distance of 9.8nm and an edge-to-edge distance of 8.5nm. In this case four hexons on the edge of the icosahedron would measure 34nm as derived previously (Nermut, 1975). The orientation of these hexons determines the orientation of the hexons at the triangular facet. The triangular top can be in register with the corners of the hexagonal 3rd section or it can be rotated to the right or to the left. It has been shown in a previous paper (Nermut, 1975) that the triangular hexons in the RH GONs have a constant azimuth which forms

Fig. 12. Diagram showing how the azimuth of the triangular top of the hexons forms an angle of 30 with

the height of the triangular facet of the capsid.

lower portion of the hexon (3rd hexagonal section). A diagram of one triangular facet of the capsid with the triangular top superimposed over the hexagonal lower portion is given in Fig. 13. The shapes and dimensions of the remaining layers were defined from geometric considerations and observations of hexons in the horizontal plane. The sections of the proposed twisted model are shown in Fig. l l a together with the orthogonal views of one polypeptide. Sections 4-8 were rotated about the Z-axis with respect to section 3 in 6 ° steps to 30 ° clockwise and sections 2-1 from - 6 ° to - 12 °. The hexon in orthogonal views is shown in Fig. i lb.

3-D Structure of Adenovirus HeXon 259

Fig. 13. Diagram of one triangular facet of the icosahedrai capsid. P, pentons. Dark shaded area, group of nine hexons with triangular top and hexagonal lower portion.

(e) Comparison of model images with electron micrograph images of the hexon

Model images. To observe the diversity of patterns of the computer model images and to assess their trends for sequential rotations, the basic model was rotated stepwise about the Y and X axes respectively for Z axis positions of 0 ° and 45 °. When in association with other hexons in the capsid, the hexon has a stable orientation with the longest dimension normal to the plane of the triangular facet, so this was made the reference position (0 °, 0 °, 0 ° in X, Y, Z). Figure 14a shows the images obtained for rotations from Z 45 °, in steps of 15 °, first in Y to _+ 15/30 ° then about X for each step in Y to _ 15/30 °. Figure 14b shows orientations of the proposed model for rotations about the axes from Z at 0 °, then Y in steps of 45 ° to _ 90 ° and about X of ___ 45/90 ° for each step in Y.

Electron microscope images. For comparison of the model images with the electron microscope images, the latter were sorted into three main categories: category A, hexons seen end-on, i.e. attached normal to the supporting film, category C, hexons oriented lengthwise, i.e. tilted by 90 ° to the previous ones, and category B, hexons in an intermediate orientation between A and C. Figure 15a shows diagramatically the hexon images observed and categorized according to the previous definition, and Fig. 15b shows similar patterns obtained by orientation of the model. Figure 16 shows selected micrographs corresponding to A, B and C categories. Cate-

gory A comprises the O- and Y-patterns, but also a few deviating to a certain extent, which could be described as eccentric O, or those with two spots. Comparison with Fig. 14a reveals that those hexons are attached to the carbon film, either normally or tilted by about + 15 °. Those in category B include changing patterns from U- to V-shape (BI to B2) and from a common tooth shape (B3) to an X-pattern (B4). Figure 14b again shows that they are fixed in an inclined position, i.e. at an angle between 30 and 60 ° . Controlled tilting in the computer allows us to identify the ends of the V, X or tooth-shaped images as shown in Fig. 16b. For example, the denser end of the X-shape image corresponds to the lower portion of the hexon, the thinner V-like arms are the tops. This might prove useful in studies of antibody binding sites, if the hexons become immobilized in an inclined position. Such pictures have indeed been observed by N. G. Wrigley (personal communication). Cate- gory C contains the horizontal views of the hexon; in some of them two subunits side by side can be distinguished, one being often more electron dense, others are of equal density and trapezoid in shape.

The proportion of these three categories varies from preparation to preparation and seems to be influenced mainly by the properties of the supporting carbon films. A very high proportion of category A is usually found on normal (i.e. hydrophobic) carbon films; for example, out of 1000 hexons, 7 0 ~ were in category A, 20~o in


+30

+15

+90

+45

0 0

-15 -45

-30 -90

-30 -15 0 +15 +30 -90 -45 0 +45 +90

Fig. 14. (a) Computer model from Fig. 11 rotated about Z axis by 45, then about Y axis by ~_ 15/30 and about X axis by 1::15/30 '~ for each step in Y. (b) As (a) but for Z at 0 and in steps of :4:45/90.

Figures indicate degrees of rotation.

B and only 10~ in C. On the other hand, on hydrophilic carbon films, up to 4 0 ~ were in category B and only 30 ~ in category A.

(f) Simulation of stain for the upright position of the proposed model

The stained images obtained for the normal (LH) and reversed (RH) positions of hexons when in groups of nine are given in Fig. 17. As only one or two sections are involved for staining in either position, the effect of rotating sections for the twisted version has little effect upon the stained images. These pictures fully confirm our previous conclusion that the bottom of the hexon is O-shaped and the top Y-shaped. At this stage we decided to build the hexon model from wooden sections and its twisted configuration is shown in several views in Fig. 18.

DISCUSSION

The tilting experiments with negatively stained hexons or GONs demonstrated that those hexons or GONs which are not closely packed are stained from below. This is rather surprising, taking into account the height of the hexon in relation to the height of the capillary layer of stain left on a hydrophilic grid before drying which has been estimated to be several hundred times higher than the hexon (Nermut, 1977). On the other hand, the height of stain left after draining on hydrophobic grids is determined by the height of the structure, since most stain was

quickly removed from such grids with the filter paper. Presumably the results of negative staining on different types of film depend to a great extent on the size and particularly the surface properties of the structure being studied. In certain cases capillary attraction in narrow cavities or channels has also to be taken into account. Finch and Klug (1965) reported that only about 1 ~ particles of rabbit papilloma virus are stained from below, most of them being stained from both sides. This does not seem to be the case with the hexon in a vertical orientation. This finding of course greatly influenced our concept of the location of the two characteristic features: the O- and Y-patterns. Having assigned the O-shape to the bottom end of the hexon we had to reduce the cross-section of the first section to about 7.5-8.0nm and support for our solution of this problem has been mentioned in the Results section.

Negatively stained hexons in a vertical orientation provide information only about the top or bottom parts of the hexon. Our dimensions for the middle portion were derived from measurements of stained images and development of the model to produce similar images. For example, the dimensions and shape of the 3rd section of the final computer model (Fig. 11) have been derived (i) from measurements of stained horizontal hexons which most probably give lower figures than of the hexon structure itself, (ii) by calculating the cross-section of our hexon from the edge of intact virions (Nermut, 1975),

3-D Structu.re of Adenovirus Hexon 261

a

A

B

12

2 3 T 4

3 4 5

2 UYJiililiJJJii a

Fig. 15. (a) Diagrammatic representation of electron micrscope images, categorized into: A-hexons oriented vertically, B-hexons in an intermediate orientation and C-hexons oriented horizontally. Com- pare with Fig. 16a, b, c. (b) Similar patterns obtained from orientations of the computer model. Symbols

refer to categories from (a). A1, bottom section of hexon; A4, top section of hexon.

(iii) from occasionally observed hexagonal pro- files (Pettersson et al., 1967 and Fig. 5).

The hexagonal shape of the waist of the hexon suggests that the hexons are in close contact in the capsid and their orientation is determined by the edge-to-edge distance (8.5nm) (Fig. 13). The corner-to-corner orientation would increase

the length of the virus edge. On the other hand, the orientation of the top (triangular shape) is not determined by the dimensions. I f there was no twist along the hexon the top triangles would point towards the corners of the lower hexagons. As the azimuth of the RH GONs is constantly to the right by 30 ° (Crowther and Franklin,


(a)

\

NORMAL

Outer FRONT VIEW

Section Levels

r 8

6

5

3

2 Stain 1

Inner SIDE VIEW

Fig. 17. Computer simulation of negative staining of the final model (see Fig. 11 ) in an upright orientation. (a) Diagram of stain simulation around one hexon subunit for normal and reversed orientation. (b) Stain profile of bottom layer (section 1) of hexon seen from above. (c) Computer image of negatively stained hexon in normal position, i.e. as in left-handed GONs. (d) Same as (c) for reversed position,

i.e. as in right-handed GONs.

1971; Nermut, 1975), the top portions of the hexon must be rotated by 30 ° in respect to the middle portion (to the left in the capsid). The twist below the 3rd section in our model is purely arbitrary in that it follows the same rate of twist as above; we have no reliable information in this respect. Twisted subunits are certainly in closer contact than the straight ones and this makes the structure rather tough and

stable. Separation of the single subunits in a native (unfolded) state has not been achieved yet (see Horwitz et al., 1970 and Philipson and Pettersson, 1973 for references).

The hexagonal shape of the lower portion of the hexon, suggested in the model, has now been demonstrated by X-ray crystallography. (Burnett et al., 1978; Burnett, personal communication). This implies that the hexons are closely packed


A

D Fig. 18. Physical (wooden) model of the hexon in different orientations. A, front view to B; D, reversed

end-on view of C.

instead of interacting with each other via corner- edge as suggested in our previous paper (Nermut, 1975). This means that the capsid is sealed against the penetration of large molecules such as DNase or ATP. The central channel, if free, would allow passage of water or ions only.

Further, the adenovirus capsid represents a unique case of a hexagonally-organized capsid made by macromolecules with a three-fold sym- metry. The number of structure units is in fact 720 instead of 1440 derived for a morphological subunit with six structure units (Horne, 1974). This makes a total of 780 structure units in the virion supposing that the penton is made of five polypeptides.

We have shown that the structural polarity of the hexon is accompanied by a physico-chemical polarity, i.e. differences in surface properties between the top and the base. The iso-electric point of the hexon has been estimated by Short- ridge and Biddle (1970) to be close to 4.8, so that

at a neutral pH the hexon is negatively charged. This has been confirmed in this laboratory by cellulose acetate electrophoresis (unpublished results). In addition, the present experiments showed that the negative charge is confined to the top of the hexon and this has been confirmed by experiments with cationized ferritin which binds to the virion surface (unpublished results). Recent chemical studies by J6rnvall et al. (1978) have shown the presence of acidic groups at the C-terminal end of the polypeptide and only a small number of hydrophobic amino acids. These preliminary results indicate that the surface of hexons is predominantly hydrophilic. Pre- sumably the hydrophobic amino acids are situated at the base judging from the preferential LH orientation on hydrophobic carbon films. More- over the hexons are in close contact with the virus core which has been shown to be a compact isometric (probably icosahedral) body (Nermut et al., 1975; Brown et al., 1975; Nermut, 1978)


with a surface protein shell made mainly by virus protein V (Everitt et al., 1975).

The computer modelling procedure, by providing accurate manipulation (tilting and rotation) and display of the model, allowed hypotheses about the structure to be tested and made it possible to identify and specify most of the observed images. It has been shown, for example, that on hydrophilic carbon films the hexons become immobilized in various positions thus presenting images such as V, X or tooth- shaped ones. On the other hand, most hexons look round on hydrophobic films. This might be one possible explanation for the diversity of images observed, and opinions expressed, by previous workers. Some of them described the hexons as 'round bodies with a hole' (Home et al., 1959; Valentine and Pereira, 1965; Petter- sson et aL, 1967), others as 'hollow polygons' (Horne, 1962; Wilcox et al., 1963; Norrby and Ankerst, 1969; Home et al., 1975). Franklin et al. (1972) described the hexon as a polygon with 3 cigar-shaped subunits and an axial hole. Their conclusions were based both on negative staining (and tilting) and X-ray crystallography. Computer modelling also enabled us to assess the feasibility of the suggested models and the correctness of the main features of the final model. This has ben facilitated in particular by simulating the effects of negative staining on the structure. It also proved useful in identifying the ends of tilted hexons (in X-shapes) which would help to localize the antibody binding sites on the hexon.

Although some structural features have been resolved with a resolution of about 1.0-1.5nm (Y image), others are much less defined, e.g. the shape of the central portion. A high resolution X-ray model by Bumett et al. (1978 and personal communication), provides more detailed information on the shape and configuration of this part of the hexon.

On the other hand, determination of the top and the base of the hexons and their mutual orientation in the capsid, can only be derived from direct observations of the virions by electron microscopy. Most recently Berger et aL (1978) compared their preliminary hexon model derived from X-ray crystallography with our previous model (Nermut, 1975) and found that its diameter is smaller than that of the X-ray model. The radius of gyration of our model was found to be only 4.1nm, whereas the X-ray model has Rg = 4.9nm. This is understandable

because, in general, measurements on negatively stained structures provide smaller dimensions than they really are. Our present model is certainly closer to the preliminary model described by the above authors both in respect of dimensions and shape. In earlier work, Lundquist (cited by Franklin et al., 1972) derived the following dimensions from his X-ray studies for the hexon of the adenovirus type 2: height of l l . l n m and width of 8.8 + 0.6nm.

The techniques described for the computer simulation of stain around models can be used for studying the behaviour of stain around known structures subjected to controlled stain experiments. The crystallographic models can thus be effectively stained in the computer, as for our model, and compared directly with the EM images to provide a precise model of the hexon and a clearer insight into the interpreta- tion of stained EM images.

Acknowledgements--The authors wish to thank Drs. W. C. Russell and E. G. Gray for comments on the manuscript, Dr. N. G. Wrigley for kind permission to use one of his electron micrographs (Fig. 16b), to Mr. K. Ibe (JEOL Co., U.K., Colindale) for tilting hexon preparations in JEM-100C electron microscope (Fig. 5) and Miss L. D. Williams and Miss M. Swies for technical assistance.

Note addedinproof: Dr. Nermut acknowledges discussions with Dr. R. M. Burnett during the final preparation of this paper. The major structural features of his high resolution model appeared in J. Supramol. Struct. Suppl. 3, p. 92, 1979, after this paper was submitted.

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consideration of the three dimensional structure of the adenovirus hexon from electron microscopy...

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