The Structure of Tobacco Mosaic Virus as revealed in

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struktur mit einer Identitätsperiode von 25—30 Ä. Beim Eintrocknen der Virusteildien in Gegenwart ... scope and X-Ray Diffraction Studies of crystalline Insect.
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H. FERNÄ NDEZ-M ORÄ N UND G. SCHRAMM

weil bei gleichem chemischem U m satz der 0 2-Druck in dem kleinen Gefäß schneller steigt als in dem großen Gefäß. A nm erkung. Da Photosynthese = 0 2-Entwicklung im Licht + 0 2-Verbrauch der A tm ung ist, so w ar — bei einfacher Ü berkom pensation der A tm ung — der korrekte W ert der Photosynthese 2 • dp/dt • ko2 ■ D a sich aber der F aktor 2 überall heraushebt, w urde er fortgelassen.

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O. W a r b u r g , W . K l o t z s c h u . G. K r i p p a h l , Z. Naturforschg. 12b. 622 [1957]. 13b. 63 [1958].

Milchsäure W enn in Chlorella durch anaerobe M ilchsäure­ b ild u n g die R eaktion sau rer w ird, so zerfällt die G lutam insäure; und da die G lutam insäure 6 fü r die P hotosynthese notw endig ist, so kann bei anaerobiotischen Versuchen G lutam insäurezerfall und 0 2M angel bei der H em m ung der Photosynthese Z u ­ sam m enw irken. Bei vielen älteren Versuchen ist das tatsächlich der Fall gewesen. Bei unseren Versuchen w aren M ilchsäurebildung und G lutam insäurezerfall ohne Bedeutung, da wir Südfenster-Zellen benutzten, die w ährend unserer Versuchszeiten so wenig M ilchsäure bilden, daß fast keine G lutam insäure zerfällt.

The Structure of T obacco M osaic Virus as revealed in Ultrathin Sections by Electron M icroscopy By H. F e r n Án d e z -M o r Án and G. S chram m Ultrastructure Department, Instituto Venezolano deNeurologia e Investigaciones Cerebrales (IVNIC), Caracas-Venezuela and Max-Planck-Institut für Virusforschung, Tübingen, Germany (Z. N a tu rfo rsc h g . 13 b , 68— 71 [1958] ; e in g e g a n g e n am 18. N o v em b er 1957)

Orientierte Präparate des Tabakmosaikvirus wurden mit Uranylacetat, Lanthanacetat, Phosphor­ wolfram- und Phosphormolybdänsäure sowie Ammoniumvanadat behandelt und Dünnschnitte hier­ von elektronenmikroskopisch untersucht. In Längs- und Querschnitten konnte der im Innern des Virusstäbchens liegende Hohlkanal mit einem Durchmesser von 30 —40 Ä sichtbar gemacht werden. Die beiden ersten Reagentien werden vorwiegend in einem Abstand von 40 Ä von der zentralen Achse eingelagert, wo sich nach Röntgenuntersuchungen die Ribonucleinsäure befindet. Die anderen Reagentien scheinen vorwiegend den Eiweißanteil anzufärben. Dieser zeigt Andeutungen einer Fein­ struktur mit einer Identitätsperiode von 2 5 —30 Ä. Beim Eintrocknen der Virusteildien in Gegenwart von Uranylacetat füllt sich der Kanal mit Schwermetall, wie bereits H u x l e y beobachtete. Die elek­ tronenmikroskopischen Untersuchungen bestätigen das aus den Röntgendaten entwickelte Struktur­ modell.

C onsiderable inform ation is already available on the fine structure of the tobacco m osaic virus based on X -ray diffraction studies of oriented p re p a ra ­ tions 5’ 6’ n , which can be correlated w ith the re ­ sults of biochemical s tu d ie s 10. H ow ever, electron m icroscope observation perform ed hitherto on to ­ bacco m osaic preparations dried on the specim en films and treated w ith various reagents or shadow-

cast w ith heavy m etals "■ 8’ 10- 12, have revealed only certain details of the internal structure of the ro d ­ shaped particles. It ap peared therefore of interest to apply the tech­ niques of staining, em bedding and u ltrath in section­ ing to a study of the fine structure of tobacco m osaic virus particle by high resolution electron m icro ­ scopy. These techniques have been successfully em-

1 H. F e r n a n d e z - M o r a n , J. Biophysic. Biochem. Cytol. 2, No 4, suppl., 29. [1956]. 2 H. F e r n a n d e z - M o r a n , Ind. Diamond Rev. 16. 128 [1956]. 3 H. F e r n a n d e z - M o r a n and J. B. F i n e a n , J. Biophysic. Bio­ chem. Cytol. 3, No 5, 725 [1957]. 4 H. F e r n a n d e z - M o r a n and G. H. B e r g o l d , Electron Micro­ scope and X-Ray Diffraction Studies of crystalline Insect Virus Inclusions (in press).

5 R. E. F r a n k l i n and A. K l u g , Biochim. biophysica Acta [Amsterdam] 1 9 ,4 0 3 [1956]. 0 R. E. F r a n k l i n , A. K l u g and K . C. H o l m e s , CIBA Founda­ tion Symposium, 1956, Boston, Little. Brown and Co., 1957. 39.

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H.

F e r n a n d e z -M oran

and G.

S chramm ,

The structure of Tobacco Mosaic Virus as revealed in Ultrathin Sections by Electron Microscopy (p. 68).

Fig. 1. Ultrathin longitudinal section of tobacco mosaic virus particles stained with \% uranyl acetate and embedded in meth­ acrylate showing internal fine structure. 250 000 X.

Fig. 2 a, b. Longitudinal sections of TMV particles stained with \% uranyl acetate showing dense para-axial double lines with indications of fine structure, (a) 600 000 X, (b) 460 000 X. Z e its c h rift fü r N a tu rfo rsch u n g 13 b, S eite 68 a.

Fig. 3. TMV particle dried on specimen film and stained with \% uranyl acetate showing distinctly outlined central channel, 400 000 X.

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Fig. 4 a, b, c. Transverse ultrathin section of TMV particles from orientied preparation stained with 1% uranyl acetate show­ ing light central area surrounded by dense annular structure with indications of a rosette-type pattern (arrows) (a) 650 000 X , (b) 670 000 X , (c) 820 000 X .

Fig. 5. Longitudinal section of TMV particles stained with ammonium vanadate showing light axial area bounded by dense outer layers with indications of fine structure. 250 000 X .

Fig. 6. Longitudinal section of bundle of TMV particles stained with ammonium vanadate showing system of dense double bands alternating with lighter spaces. 330 000 X.

Z e its c h rift fü r N a tu rfo rsch u n g 13 b , S e ite 68 b.

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69

ST RUCT URE OF TOBACCO MOSAIC VIRUS

ployed in the investigation of the m acrom olecular stru ctu re of various types of virus particles, and are particu larly revealing when dealing w ith paracrystalline sy stem s4’ 9. By staining and u ltra th in section­ ing of oriented TM V p rep a ra tio n it has now been possible to observe certain reg u larly o ccurring feat­ ures of the in ternal structure of the virus particles which seem to be in general agreem ent w ith the m odel derived from X -ray diffraction studies 5’ 6.

Preparation techniques Oriented samples were prepared by sucking the stiff gel of fresh tobacco mosaic virus into plastic or glass capillaries (0.2 mm internal diam eter). Specimens with random orientation of the particles were also obtained from droplets of the gel collected at the ends of plastic or wood fibers. Short segments of the samples were stained with cold (4° C) aqueous or alcoholic 1 per cent solutions of uranyl acetate, or lanthanum nitrate, phosphotungstic acid, phosphomolybdic acid, and am­ monium vanadate. Embedding in prepolymerized butylmethacrylate was carried out after dehydration by pas­ sage through a graded series of ethanol. Alternatively, gelatin em bedding3 was tried, thus avoiding the effects of ethanol treatment. The blocks containing the short segments of capillary tubing with the fixed TMV speci­ mens were readily oriented for sectioning in the desired plane. Ultrathin serial sections of 100 —200 Ä were prepared with a Morän ultramicrotome using a diamond knife. Confirming previous investigations1-3, the regular production of a large number of exceedingly thin sections devoid of compression and distortion, using the diamond knife, was found to be an essential tech­ nical requirement in interpreting structural relationships at the macromolecular level. The sections were mounted on fenestrated Formvar films with regularly distributed pores of 200 to 2000 Ä 3, which are particularly suitable for high resolution studies since the edges of the minute holes facilitate rapid focusing, and many areas of the sections can be examined free from the supporting sub­ strate. Control specimens were prepared by applying the same type of stains to suspensions of TMV dried on the specimen screens. An RCA EMU 3 B and a Siemens ELMISKOP 1 con­ nected to a specially regulated power supply were used in this study. The micrographs were taken at electron optical magnifications of 20 000 X to 42 000 X . The preliminary observations described here are based on evalution of 700 plates in which an average resolution of 10 to 20 A was consistently achieved. E. H u x ley , Proc. Stockholm Conf. Electron Microscopy, 1956, New York, Academic Press, Inc., 1957, 260. 8 R. E. F. M a t t h e w s , R. W. H o r n e and E. M . G r e e n , Proc. Stockholm Conf. Electron Microscopy, 1956, New York, Academic Press, Inc., 1957, 261. 9 C. M org a n , G . H . B erg o ld and D. H . M o o r e , J. Biophysic. Biochem. Cytol. 1, No 3, suppl., 187 [1955]. 7 H.

Results The appearance of the stain ed an d em bedded TM V particles in thin sections is very sim ilar to the well-known im ages of TMV specim ens d ried directly on the films, as reg ard s size and general co n fig u ra­ tion. The in dividual particles are ab o u t 3 0 0 0 Ä long and 150 —160 Ä wide, but asso ciatio n of the p a r t­ icles into ribbon-like structures appro x im ately 150 A w ide was m ore com m only encountered (F ig. 1 ). Closely packed bundles of particles in reg u la r a rra y are seen sectioned lo n g itu d in ally an d transversely in the oriented p rep aratio n s (Figs. 2, 4 ) . T he follow ing details of in tern al stru ctu re can be discerned in the particles according to the stain in g p ro ced u re em ­ ployed. Uranyl acetate and la nth anum nitrate staining In the m ajo rity of the lo n g itu d in al and oblique sections th ro u g h TM V particles stain ed w ith u ranyl acetate (Fig. 1 *, 2) two dense parallel lines can be seen ru n n in g along the center of each p article. T he dense lines have an average w idth of 20 Ä , w ith in ­ dications of a g ran u la r stru ctu re, an d are sep arated from each other by a light space of 25 to 30 Ä (Fig. 2 ) . The outer lay er of the particle has a low er density, but the m arg in s stan d o u t distinctly ag ain st the background of the em bedding m edium . T his characteristic p ara-ax ial double line system found in sectioned specim ens stained w ith u ran y l acetate or lan th an u m n itrate, is different fro m the central dense line first observed by H u x l e y 7 in stained TMV particles. A u n ifo rm central line ab o u t 20 — 30 A in d iam eter can be reg u larly observed in the virus p articles dried on the specim en film an d stain ed w ith uranlyl acetate (Fig. 3 ). H ow ever, in agreem ent w ith H u x l e y 7- it pro b ab ly represents the internal axial channel of the particle outlined by deposition of the stain. T herefore, by v irtu e of its dim ensions and axial location this single central line w ould c o r­ respond m ore closely to the lig h t in term ed iate space betw een the two in tern al dense lines of the virus p article observed in sectioned p rep aratio n s. 10 G. S chra m m , G. S chu m a ch er and W . Z il l ig , Z . Naturforschg. 10 b, 481 [1955]. 11 J. D. W a tso n . Biochim. biophysica Acta [Amsterdam] 13, 10 [1954], 12 R. C. W il l ia m s , CIBA Foundation Symposium, 1956, Boston, Little, Brown and Co., 1957, 19. * Figs. 1 —6 s. table p. 68 a u. b.

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STRUCTURE OF TOBACCO MOSAIC VIRUS

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In transverse sections the m ost notable feature is the d istinct appearance of a clear circu lar area, w ith a diam eter of 25 —30 A , in the center of each p article (Figs. 4 a, b, c ) . T his central opening or hole is su rro u n d e d by a concentric ring of dense granules, 15 to 20 A in diam eter, w ith indications of a regular arran g e m en t in a rosette-type of pattern. In suitably oriented oblique sections th ro u g h the virus particles the double para-axial lines which stain densely with u ran iu m salts are seen to correspond to segm ents of th is dense an n u lar stru ctu re su rro u n d in g the central hole. In general, the cross-sectioned TM V particles ex hibit a circ u lar or polygonal outline w ith an average diam eter of 150 Ä . H ow ever, in closely packed bundles there seems to be an intim ate adherence of the outer layers of the particles, ob­ literatin g the indiv id u al outlines.

niques fu rn ish su p p lem en tary in fo rm atio n , since the co n trast in tro d u ced is n ot due to the su rface de­ position effects o perative in th e usual d ried and stained virus suspensions. M oreover, the thin sectioning m ethods m ake it possible to observe the fine texture of the virus particles in any desired plane, while d isp lay in g the repetitive featu res of in tern al stru ctu re which are p resen t in reg u la r p a ra ­ crystalline array s. The general p ictu re of a cy lin d rical particle with a hollow axial core su rro u n d e d by a characteristic

A m m o n iu m van ad at e and p h o sp h o t u n g s t ic acid In lo ngitudinal sections of oriented sam ples of TM V virus stained w ith aqueous solutions of am ­ m onium v anadate or phosphotungstic acid the bundles of TM V particles packed in paracry stallin e array a p p ear as a reg u la r system of parallel dense double b ands uniform ly separated by lighter spaces (Figs. 5, 6 ) . Each of the dense double bands is 30 —35 Ä w ide an d shows indicatio n s of a g ran u la r fine stru c­ tu re of the o rd er of 25 — 30 Ä . In m any areas the p articu late com ponents of ad jac en t double lines are found to be in lateral register, which m ay be due to o rien tatio n effects intro d u ced by the p reparatio n procedures. The light spaces betw een the bands are approxim ately 70 —80 A wide. W hen TM V speci­ m ens are stained first w ith uranyl acetate, and then w ith am m onium vanadate, a fine dense line of 20 —25 A is com m only encountered coursing through the m iddle of the light bands. It w ould ap p ear th ere­ fore th a t there is a com plem entary relationship bet­ ween the effects of the two types of reagents, the u ranyl acetate staining the central region of the virus particles m ore intensely,, while the am m onium vanadate em phasizes the outer layer.

Maximum radius Mean radius

_L

“x Pitch of y helix

//

D iscussion

Radius o f hole

Nucleic acid

In terp reta tio n of these prelim inary findings is su bject to the num erous sources of artefacts inherent in the p re p a ra tio n procedures. The described tech-

Fig. 7. Schematic diagram of the structure of a tobacco mosaic virus particle as revealed in thin sections by electron microscopy, compared with the model (above) postulated by F r a n k l in , K lug and H o lm es based on X-ray diffraction studies.

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V E RHA LTEN DER ALKALISCHEN UND SAUREN PH OSPHATASE

p ara-ax ial com ponent which em erges from these p re ­ lim in ary observations (Fig. 7 ) , is in good agreem ent w ith the m odel postulated by F r a n k l in , K lug and H olm es 6 on the basis of X -ray diffractio n studies. T he dim ensions of 30 —40 Ä fo r the diam eter of the central core, and 50 —60 Ä fo r the thickness of the particle wall determ ined by electron m icroscopy, are likew ise in satisfactory agreem ent w ith the co r­ resp o n d ing p aram eters deduced from the X -ray diffraction patterns. T he a n n u la r stru ctu re su rro u n d ­ ing the central channel which stains intensely with u ranyl acetate or lanthanum n itra te m ight correspond to the RN A com ponent em bedded in the protein at a ra d ia l distance of about 40 Ä from the p article axis, although it is realized of course th a t these reagents are not specific fo r nucleic acids. The p referen tial stain in g of the outer lay er of the TM V particles

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with am m onium vanadate or phosp h o tu n g stic acid, as well as the indications of fine stru ctu re observed in the reg u la r double line system are of in terest in relatio n to the postulated grooved external surface of the pro tein m antle 6. It is evident from these p relim in ary investigations th a t the described techniques fo r u ltra th in sectioning com bined w ith selective stain in g of the virus com ­ ponents will prove highly useful in elu cid atin g the m acrom olecular stru ctu re of the v iru s p articles by electron m icroscopy. W ork is co n tin u in g and full details will be published elsew here. We wish to express our sincere thanks to Engs. J. S uter , W. R awyler , R . H auser , K . M unz , G . G eislek and H . K abe for the essential technical collaboration in the various aspects of this work.

Das Verhalten der alkalischen un d sauren Phosphatase in Zellkulturen aus Affennieren nach Infektion mit ECHO-Virus Serotyp 9 un d Poliovirus hominis Typ 1* V on W . A l b r e c h t , H. K. M it t e l s t r a s s und R. S a u t h o f f Aus der Univ.-Kinderklinik Freiburg i. Br. (Direktor: Prof. Dr. W.

K

eller)

(Z . N a tu rfo rs c h g . 13 b , 71— 74 [1958] ; e in g e g a n g e n am 13. S e p tem b e r 1957)

Nach Infektion in vitro gezüchteter Rh-Affennierenzellen mit ECHO-Virus Serotyp 9 und P olio­ myelitis-Virus Typ 1 sinkt die Aktivität der sauren Phosphatase in der Zelle stark ab, um nach 9 und 12 Stdn. wieder einen langsamen Anstieg zu zeigen. Die Aktivität der alkalisdien Phosphatase bleibt nach Infektion mit ECHO-Virus zunächst unverändert, um nach 9 und 12 Stdn. signifikant an­ zusteigen. Gleichzeitig ist nach anfänglichem Abfall eine Zunahme der RNS in Beziehung zur DNS zu beobachten. Nach Infektion mit Poliomyelitis-Virus zeigt die Aktivität der alkalischen Phosphatase ein ähnliches, jedoch nicht so ausgeprägtes Verhalten wie nach ECHO-Virus Infektion. Diese Verschiebungen der Phosphatase-Aktivität in Abhängigkeit von der Zeit sind mit der Virus­ multiplikation in Zusammenhang zu bringen.

Die A ktivität d er alkalischen P h o sp h atase (A P ) und der sau ren P hosp h atase (S P ) verschiedener O r­ gane v erä n d ert sich nach Infek tio n m it bestim m ten V iru sarten. So fü h rt die Infek tio n m it CoxsackieV iren der G ruppe A — T yp 1, 2 und 3 — zu einer A k tivitätssteigerung von A P u n d /o d e r SP im M uskel von Säuglings- oder ausgew achsenen M äusen Die Z eitspanne zwischen Infektion u n d U ntersuchung ist dabei von ausschlaggebender B edeutung. Im R ahm en solcher Beobachtungen konnte unerw arteterw eise

nachgew iesen w erden, daß der m aus-ad ap tierte Stam m L ansing des P o lio v iru s hom inis T yp 2 bei in tra cere b ra ler V erim pfung auf S äuglingsm äuse am N ervengew ebe bei nichtgelähm ten T ieren einen d eu t­ lichen positiven A usfall d er A P h e rv o rrie f2. Im G egensatz hierzu verursachte die In fek tio n m it dem P R 8-Influenzavirus eine signifikante H erabsetzung der A P-A ktivität im D ü n n d arm d er M a u s 3. L eider ist das S tudium des Effektes von V iru sin fek tio n en auf die E nzym aktivität und an d ere Stoffwechsel-

* Diese Arbeit wurde aus Mitteln der D e u t s c h e n F o r ­ s c h u n g s g e m e i n s c h a f t und der D e u t s c h e n G e s e l l s c h a f t zur B e k ä m p f u n g der K i n ­ d e r l ä h m u n g gefördert.

1 W. A l b r e c h t , Z. Naturforschg. 11b, 633 [1956]. 2 G. A . K a u s c h e , C h . L an dsch ütz u . R. S a u t h o f f . Z. Natur­ forschg. 9 b, 340 [1954]. 3 W. A l b r e c h t , Z. Kinderheilkunde 79, 270 [1957].

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