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Crystallographic Study of an Adenovirus-Induced Protein Crystal in KB cells: a structural model
Alain Lifchitz, Alexandre Rimsky, Gérard Torpier, Pierre Boulanger
To cite this version:
Alain Lifchitz, Alexandre Rimsky, Gérard Torpier, Pierre Boulanger. Crystallographic Study of an
Adenovirus-Induced Protein Crystal in KB cells: a structural model. Acta Crystallographica Section
B, Structural Science, Crystal Engineering and Materials, International Union of Crystallography,
1975, 31, pp.441-445. �10.1107/S0567740875002981�. �hal-00015153�
Aeta Cryst. (1975). B31, 441
Crystallographic Study of an Adenovirus-Induced Protein Crystal in KB Cells:
a Structural Model
441
BY A. LIFCHITZ AND A. RIMSKY
Laboratoire de Minkralogie et de Cristallographie associO au CNRS, Universitk de Paris VI, Tour 16, 4 Place Jussieu, 75230 Paris Cedex 05, France
AND G. TORPIER AND P. A. BOULANGER
UnitO de Recherches n ° 102 de Virologic Mol~culaire de I ' I N S E R M , 2 Place de Verdun, 59045 Lille Cedex, France (Received 19 June 1974; accepted 18 September 1974)
The physical parameters of a protein crystal induced by adenovirus type 5 in KB cells were determined from images of positively contrasted, sectioned intranuclear crystals. Fourier transforms of digitized images showed that the net appearing on transverse sections of the crystals, thus far considered as hexagonal, was in fact pseudohexagonal, rectangular. The three-dimensional characteristics of the crys- tal unit cell were: ao=630, bo = 1200, Co=900 A; ~= ~'= 90, //= 95-5 °. The space group was P21. The asymmetric unit of the crystal was found to consist of a tubule centred by a densely stained tubular filament; its calculated molecular weight was 130 x 106 Daltons. A three-dimensional model was recon- structed from the crystallographic data.
Introduction
Intranuclear proteinic crystalline inclusions have been observed in cells infected with adenovirus type 5 (Leuchtenberger & Boyer, 1957; Morgan, Godman, Rose, Howe & Huang, 1957; Morgan, Godman, Breitenfeld & Rose, 1960) and type 2 (Weber & Stich, 1969; Henry, Stifkin, Merkow & Pardo, 1971; Henry
& Atchison, 1971). In previous studies we have de- scribed the electron microscopic and biochemical characteristics of these crystalline bodies (Torpier &
Petitprez, 1968; Boulanger, Torpier & Biserte, 1970):
they consist of parallel tubules arranged on a crystal- line lattice. From the ultrastructural data and from the results of biochemical and immunological studies on infected KB cell extracts and of immunofluorescent staining (Torpier & Boulanger, 1971) we have proposed a hypothetical model for this crystal (Boulanger, Tor- pier & Biserte, 1970), assuming that it consists mainly of adenovirus capsid material, viz. hexon, penton and fibre capsomers, synthesized in considerable excess by the infected cell (Schlesinger, 1969). This hypothesis was first supported by Henry et al. (1971), then dis- proved by other workers, on the basis of immuno- fluorescent staining of crystal with an anti-adenovirus core hyperimmune serum, suggesting that crystals were related to adenovirus internal arginine-rich protein(s) (Henry & Atchison, 1971; Marusyk, Norrby & Maru- syk, 1972).
It is the aim of the present study to present some ad- ditional information on the structure of adenovirus type 5 induced protein crystals in KB cell nuclei, with the aid of crystallographic data obtained from electron micrographs of longitudinally and transversely sec-
tioned crystals (DeRosier & Klug, 1968; Crowther, Amos, Finch, DeRosier & Klug, 1970; Lake, 1972).
Such a crystallographic analysis of adenovirion crystals in KB cell nuclei has recently permitted the determina- tion of the mode of arrangement and association of the virions within the crystal packings (Boulanger, Torpier
& Rimsky, 1974).
Materials and methods
KB cells were grown as monolayers in 1 litre bottles (3x 107 to 4 x 107 cells per bottle) in Eagle's basal medium supplemented with 10 % calf serum. The cells were infected with adenovirus type 5 at a multiplicity of 100 infecting cell units per cell (ICU, Warocquier, M6nard & Samaille, 1966), and harvested at 40 hr post- infection. The cell pellet was fixed in 2.5 % glutaralde- hyde in 0-1M cacodylate buffer, pH 7.2, then post-fixed with osmium tetroxide and embedded in Araldite.
Sections were stained in uranyl acetate + lead hydroxide.
Electron micrographs of transverse and longitudinal sections of adenovirus-induced protein crystals were densitometered, using a C A L I F E automatic scanning microdensitometer in the Laboratoire Central de l'Armement (Arcueil). Computations were carried out at the Centre Interdisciplinaire R6gional de Calcul Electronique (Orsay) on an IBM 370-165 computer with a 2100 K bytes core memory, using programs written in Fortran IV.
The Fourier transforms were performed on the digitized electron microscopic plates, by the Fast Fourier Transform algorithm (Cooley & Tukey, 1965;
Lifchitz, 1974), and the displays of these Fourier
transforms were obtained with 20 intensity levels
442 A N A D E N O V I R U S - I N D U C E D P R O T E I N C R Y S T A L : A S T R U C T U R A L M O D E L symbolized by the printing of 20 combinations of
alphabetical and numerical characters (Epelboin &
Lifchitz, 1974).
Results
As observed on many electron micrographs, transverse sections through adenovirus-induced protein crystals reveal an approximately hexagonal lattice, limited by a polygonal convex contour, which shows that we are dealing with a real crystal [Fig. l(a)]. Fig. 2 presents the digitized image of a transverse section observed at higher magnification, as shown in Fig. l(b). The display of the Fourier transform (two-dimensional reciprocal space) performed by the Fast Fourier Transform algo- rithm on this digitized electron microscopic image presents a pseudohexagonal symmetry for the low- frequency harmonics. This symmetry disappears for the higher frequency harmonics. Along a principal direction, designated as b*, a systematic extinction occurs for odd harmonics, whereas such an extinction does not exist along the direction a*, perpendicular to b* (Fig. 3). This implies a glide-reflexion line with translation of one half the repeat distance along the line bo (Fig. 2). Hence the net is rectangular with a two- dimensional space group pg (No. 4), with two asym- metric units per unit cell and with the following param- eters obtained from measurements made directly on a series of original micrograph plates: a0 = 630 + 30, b0 =
J r o •
1200_ 60 A, 7 = 90 . The d~stances between the centres of the asymmetric units are respectively" Ii = 650 + 30;
l, = 610 + 30; la = 680 + 30 A. Although different, the/2 and 13 values are of the same order and the coordinate of the asymmetric unit centres is therefore approxi- m__ately ¼. The sectioned crystal habit is 01, 11, IT, 0T,
11 and ]1.
Longitudinal sections [Fig. 4(a)] show that the crystal habit is polygonal convex with an elongation axis parallel to the crystal fibres. As for the transverse sections, the existence of such a polygonal convex contour implies a two-dimensional periodicity, a characteristic of the periodical media, and therefore suggests a periodical modulation along the fibre axis.
Because the image seen on longitudinal sections cor- responds to the planar projection of a roughly cylin- drical fibre, the signal-to-noise ratio is low and the periodical modulation is scarcely visible. Nevertheless, if the longitudinal section micrographs are examined
with a tangential incidence, the signal-to-noise ratio
can be improved by physiological averaging of images on the retina, and this modulation appears clearly with an approximate wavelength of 10 3 /~. The Fourier transform of the digitized image of the longitudinal section confirms this period, giving the value Co=
930 + 60 A.
On the section presented in Fig. 4(a), one single fibre can be traced for its full length (up to 10 pm), proving that a net of fibres is totally contained in this section plane, for a section thickness of about 750 A. The i nterfibre distance is d = 6 3 0 A, which corresponds to
the/1 distance (Fig. 2). The interfacial angles (71 = 125, 72 = 145, 7a = 140, 74 = 120 °) allow the calculation of the axial ratio of the net, giving the respective values for the crystal parameters" /1 = 630 + 30, Co = 900 + 60/~,, ,6'=95+0.5 ° [Fig. 4(b)].
The three-dimensional characteristics of the crystal unit cell are therefore as follows" a 0 = 6 3 0 + 3 0 , b0=
1200+60, c 0 = 9 0 0 + 6 0 A; ~ = 9 0 , fl=95_+0.5, ),=90 °.
The crystal belongs to class 2 of the monoclinic system. The space group is P21, with two asymmetric units per unit cell.
From these crystallographic data, a value of 600 × 106 A 3 can be calculated for the volume of the unit cell, hence 300× 106 A 3 per asymmetric unit. Assuming a mean value of 2.37 A a for the volume of 1 Dalton of protein (Matthews, 1968), a molecular weight of 130 x
10 6 Daltons is obtained for the asymmetric unit. As- suming a density of 1.17 for crystal protein(s), as determined for adenovirus hexon crystals (Cornick, Sigler & Ginsberg, 1973), one calculates a water content of 64% (w/w) in the adenovirus-induced crystal.
Discussion
In spite of many studies, the nature of the adenovirus- induced protein crystals in infected cells remained con- jectural. Recent immunological evidence (Henry &
Atchison, 1971; Marusyk, Norrby & Marusyk, 1972) indicates that the crystals contain antigenic deter-
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negative electron microscopic plate showing the unit cell
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text for 11, 12 and la correspond to an average of at least
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Fig. 1. (a) E l e c t r o n m i c r o g r a p h o f a t r a n s v e r s e section o f an a d e n o v i r u s t y p e 5 i n d u c e d p r o t e i n crystal in K B cell nucleus.
T h e crystalline lattice is limited by a c o n v e x c o n t o u r . T h e b a r r e p r e s e n t s 1 /zm. (b) A p o r t i o n o f t r a n s v e r s e l y s e c t i o n e d crystal at higher magnification. At the t o p left, a crystal o f a d e n o v i r u s particles n e i g h b o u r s the p r o t e i n crystal. T h e limits o f the a s y m m e t r i c unit are clearly d i s t i n g u i s h a b l e a l o n g the edge o f the crystal. This unit is f o r m e d o f a faintly-stained t u b u l e , p o l y g o n a l in s h a p e on section, c e n t r e d b y a d e n s e l y - s t a i n e d t u b u l a r fibre. T h e circle delineates an a s y m m e t r i c unit. T h e b a r r e p r e s e n t s 0.5 /~m.
[ T o f a c e p . 4 4 2
A. L I F C H I T Z , A. R I M S K Y , G. T O R P I E R A N D P. A. B O U L A N G E R 443 minants of adenovirus arginine-rich internal core. How-
ever, the failure to find the expected large quantities of arginine-rich proteins in the crystal, either by bio- chemical analysis of a cellular subfraction containing the crystal components (Boulanger, Torpier & Biserte, 1970; Torpier & Boulanger, 1971), or by a polyacryl- amide-gel electrophoresis study of the basic proteins isolated from adenovirus type-5 infected cells (Russel, 1971), led us to admit that the structure of this crystal is more complex than previously postulated. In addi- tion, it seems hazardous to assess the exact nature and structure of the crystal from immunofluorescent staining, inasmuch as some antigenic determinants of crystal components can be masked within the crystal- line matrix.
Nothing is known about the mechanism of forma- tion of the crystals within the cell nucleus, but a geo- metrical pathway has been recently proposed (Wach- man & Levitzki, 1972) for the assembly of spherical viruses, involving a cubic crystal of capsomers as an intermediate, which collapses to form virus particles.
If the collapse of the crystal structure does not occur
. . . 2---- 2-- __-_-'L_ __ -2_ _-_ .-_-__ "_-'2 - -
: . . . . . . = _ _ = ........... ~ + . . . . . . . = . - -
: .. ... . ~ - - ~ ---= -__=_ _ = .....
. . . . . . . . . . = - - , , = = . .... .... ... .... ...
- ~ . . . . . . = = ..... = . - _ = .... = ....... = . . . . .
. . . . . . . . . . . . ÷ = .... . = - = = - ==÷-÷ --==. .. .. . = - -
... .... .... .... ... . z = ÷ - - = . . . . . . . . . .
. - .... = _ -=.-- ~-_. = . - = . .. .. . =
. . . .
- =--_= .. ..... ......... = ..... =---
. . . . . . . . . . . . . . . . . . . . .
. . . . . . .
F i g . 3. D i s c r e t e F o u r i e r t r a n s f o r m c o r r e s p o n d i n g t o F i g s . l ( b ) a n d 2. A l t h o u g h a p s e u d o h e x a g o n a l l a t t i c e is r e v e a l e d b y t h e
intense diffraction spots, the true lattice appears rectangular, as materialized by the lines. A systematic extinction occurs for odd harmonics along the b* axis. The vertical affinity, due to the line printer display, has been intentionally correc- ted on Fig. 2, but not on Fig. 3 to prevent a loss of informa- tion. (As in Fig. 2, the code is given by Epelboin &
Lifchitz, 1974.)
around a nucleation centre, such as the DNA-contain- ing inner core of the adenovirus, but rather around a linear core, the collapse of the crystalline array is longitudinal and filamentary structures will result. Such filaments have been observed in KB cells infected with adenovirus type 5 (Torpier & Petitprez, 1968, Plate III), in the process of generating crystalline inclusions within the nucleus. Other spherical animal viruses such as polyoma (Mattern & De Leva, 1968) and herpes (Stack- pole & Mizell, 1968) also induce tubular inclusions in the host cell.
The adenovirus-induced crystal presents a tubular architecture: it consists of a crystalline array of faintly contrasted parallel tubules, polygonal in shape on transverse sections [Fig. l(b)], centred by a densely stained tubular filament which seems connected to the peripheral tubule by a thin reticulum. The limits of the external tubular unit, and hence the limits of the asym- metric unit, are clearly distinguishable along the edges of the crystal [Fig. l(a,b)] as well as on isolated tubules in the course of assembling to form a crystalline array (Torpier & Petitprez, 1968, Plate III).
If it is supposed that the adenovirus-induced protein crystal in the KB cell consists of viral capsid compo- nents, and particularly of hexon capsomers, the major adenovirus morphological subunits, collapsing around a filamentary axis of inner densely stained protein(s) (the formerly called 'tubules' of the crystal) to form longitudinal tubular fibres associating in a crystalline array, then it would be feasible to reconstruct from capsid components a three-dimensional structure com- patible with the crystallographic data previously ob- tained. This is shown in Fig. 5(a): a plane hexagonal surface lattice of adenovirus icosahedron faces (equi- lateral triangles of 350 A edge), constituted of 123 hexon capsomers, can be folded round upon itself to form a helical polyhedral tubule with an external dia- meter of 550 A and an axial periodicity of 930 A. A unique structure possibility is thus found [Fig. 5(a,b)], giving a tubular unit containing 28 triangles. In this hexagonal arrangement there is no room for pentons.
From the most accurately determined molecular weight for hexon capsomer (298000, Cornick et al., 1973) a value of 104 x 106 is obtained for the molecular weight of this tubular unit. As the volume of the internal densely-stained tubular fibre is 56 x 106 A 3, a molec- ular weight of 2 4 x 106 is calculated as mentioned above. The molecular weight of 128 x 106 for the whole tubular unit is in good agreement with the value of 130 x 106 inferred from crystallographic data.
The adenovirus-induced crystal would thus be
formed of a crystalline array of helical polyhedral tu-
bules 550A in diameter, whose tubular unit consists of 28
triangular facets of 123 subunits with hexagonal sym-
metry and molecular weight about 300 000, helicoidally
disposed with a 930 A period. These hollow tubules
contain a dense tubular fibre of basic protein(s). On the
hypothesis that these hexagonal subunits are true
adenovirus hexon capsomers, the occurrence of such
ACTA C R Y S T A L L O G R A P H I C A , VOL. B 3 1 , 1975--L[FCH[TZ, RIMSKY, TORPIER AND BOULANGER PLATE 3
?,',.3,1t~ ~ " ," "*' " :P.
:':':'~"t" ".
: . . - "." • ,.,,i. i. :"~lill~ :'L' ': ~ "
: ". . " , ~ , - -. . " . .-"(a)
r .. . . '~'~ \
(b)
Fig. 4. (a) Electron m i c r o g r a p h of a longitudinal section of an a d e n o v i r u s - i n d u c e d protein crystal in the KB cell nucleus.
The limits of the external t u b u l a r unit are visible at the periphery of the sectioned crystal. The bar represents 1 ,urn.
(b) D e t e r m i n a t i o n o f the crystal p a r a m e t e r s by the crystal m o r p h o l o g y . T h e reciprocal lattice is c o n s t r u c t e d f r o m the directions p e r p e n d i c u l a r to the crystal faces. This reciprocal lattice permits the calculation o f the direct p a r a m e t e r s by the following e q u a t i o n s : tg 0 = 2 sin ~,~. sin ~,3/sin (72 - h ) ; a0 = d/sin 0, d being the interfibre distance; Co = d . sin (~,~ +
~,~)/sin 7, • sin }'3.
A I o I
5 0 0 A
(a)
(b)
Fig. 5. (a) Plane hexagonal net of triangular facets (350 ,~ in edge; 123 hexagonal subunits per facet) foldable r o u n d u p o n itself to form a helical polyhedral tubule, 550 ,~, in diameter, 930 ,~ in axial periodicity, ,4C c o m i n g on BD.
(b) A p r o p o s e d three-dimensional model for tubular units of a d e n o v i r u s - i n d u c e d crystal, reconstructed from crystallo- graphic data. Each tubule is f o r m e d by helical e n w i n d i n g of triangular plane facets o f 12~ hexagonal subunits (28 tri- angles per a s y m m e t r i c unit). The inner hollow fibre repre- sents the densely-stained central filament o f basic proteins.
[To face p.
