Adenovirus type 12 is of human origin and it shows carcinogenic activity in experimental animals. With negatively stained particles of this virus electron
microscopic observations were carried out. As the result it was demonstrated that its capsid, like other adenoviruses, is an icosahedron and each capsomere
is of a hexagonal shape with a hollow in its center, each of which is surrounded by 6 adjacent capsomeres and is composed of numerous small subunit-like-particles.</p

Tawara, Jutaro

Okayama University Scientific Achievement Repository

Acta Medica OkayamaVolume 19, Issue 6 1965 Article 3DECEMBER 1965Fine structure of negatively stained adenovirustype 12Jutaro Tawara∗∗Okayama University,Copyright c©1999 OKAYAMA UNIVERSITY MEDICAL SCHOOL. All rights reserved.Fine structure of negatively stained adenovirustype 12∗Jutaro TawaraAbstractAdenovirus type 12 is of human origin and it shows carcinogenic activity in experimentalanimals. With negatively stained particles of this virus electron microscopic observations werecarried out. As the result it was demonstrated that its capsid, like other adenoviruses, is an icosa-hedron and each capsomere is of a hexagonal shape with a hollow in its center, each of which issurrounded by 6 adjacent capsomeres and is composed of numerous small subunit-like-particles.∗PMID: 4223616 [PubMed - indexed for MEDLINE] Copyright c©OKAYAMA UNIVERSITYMEDICAL SCHOOLActa Med. Okayama 19. 287-292 (1965)FINE STRUCTURE OF NEGATIVELY STAINEDADENOVIRUS TYPE 12Jutaro TAWARADepartment of Microbiology, Okayama University Medical School, Okayama(Director: Prof. S. Murakami)Received for publication, October 12, 1965Virus particles have a genetically fixed structure specific to the kind ofviruses, and this structure is dependent on capsid, and it is also determined bythe symmetrical arrangement of the capsomeres that are subunits of the capsid.The study on such fine structures of virus particles has made a rapid advancesince BRENNER and HORNE1 introduced the negative staining technique for electronmicroscopic observations of virus particles. According to the studies on thestructure of adenovirus conducted with this technique in various types it is saidthat the virus particle is an icosahedron consisted of 252-capsomeres.There are reports by ALMEIDA et al.2 and MAYOR et al.8 about the morpho-logy of adenovirus type 12 and they have demonstrated that the structure is thesame as that of other types of viruses. The present experiment was conductedwith the purpose to observe still finer structures of adenovirus type 12 particles.MATERIALS AND METHODSThe culture cells used were HeLa cells. At first cell suspension at theconcentrations of 10-15 X 104 cells/ml were prepared, and 1 ml of this suspen-sion in a culture tube was incubated at 37°C. Within 2-3 days culture the cellnumber increased to 20- 30 X 104 cells/ml. Then the virus was inoculated. Thegrowth medium for cells was composed of 2096 calf serum plus YLE solution,and the YLE solution without serum served as the maintenance medium. Thevirus sample used was adenovirus type 12 (Huie strain) passaged by HeLa cellsin our laboratory for a long time, and infected with the virus, 102- 8 TCIDllo/ml.When the culture is continued under these conditions, on about the thirdday after inoculation the virus infected cells show specific cytopathogenic effects(CPE), and this CPE is seen in as much as 70-8096 of the entire cells 5-7days after inoculation. At this stage the virus is purified from the infectedmaterial for electron microscopic observation. In other words, the virus mate-rial is repeatedly frozen and thawed 30 times, subjected to ultracentrifugation at2871Tawara: Fine structure of negatively stained adenovirus type 12Produced by The Berkeley Electronic Press, 1965288 J. TAwARA100,000 x g, and the pellets thus obtained are resuspended in 1% phosphotungsticacid (PTA) (pH 7.0) for 15-minutes. Then the stained material is mounted oncollodion membrane coated grids and observed in the electron micloscope, Hitachi,00-11 at the instrumental magnification of 50,000.RESULTS AND DISCUSSION.The negative staining method employed for the elucidation of fine structuresof the virus particles enables us to observe th fine structure by virtue of contrastproduced by the electron dense substance of PTA that has penetrated into inter-sp~ces of the material under observation.. , By this means it is not only possible to see the size and the shape of the-virus particle as a whole but also it enables us to observe readily and directlythe arrangement of capsomeres that are structural subunits making up the virusprotein shell as white capsomeres against the black background of PTA.Representative pictures of the virus particles observed from different anglesare as shown in Figures 1 to 3.Each one of the pictures on the left side shows a virion of adenovirus type12 surrounded by PTA, the middle picture is of particle with superimposedorientation, and 'the picture on the right is that of 252-capsomere model inorientations similar· to the virus particles. The capsid in each figure presents ahexagon from a plane view while it is an icosahedron at the three dimensionalview, It is to be noted that each plane surface is of a right triangle on eachedge of which are 6 capsomeres, and with the exception of the ones at apices allothers are each surrou,nded by 6 capsomeres (Fig. 1). Consequently, when acommon base of two right triangles is viewed side-wise at the equator, thesetwo surfaces form a diamond shape (Fig. 2), and it is also possible to observefive triangles adjacent to each other when viewed from their common apex (Fig.3). These findings verify that the virion in the picture is an icosahedron con-sisted of twenty equal surfaces each of which is a right triangle.These are negative stain pictures of complete virus particles and most ofthe purified virus particles show such a structure but sometimes there are par-ticles with incomplete structure. Possibly due to insufficient purification of virusparticles. occasionally there can be recognized tissue debris in the vicinity ofparticles in (Fig. 4), a picture showing aside from the array of white capsomeres,there can be seen a white core with stronger contrast as if it is about to emigrateFigs. -1-3 Show the virus particles observed in three different views. The pictures on themiddle are of particles with superimposed orientation, and the pictures on the right are those of252-capsomere models in orientations similar to the pictures of virus ~articles on the left side.x 450, 0002Acta Medica Okayama, Vol. 19 [1965], Iss. 6, Art. 3http://escholarship.lib.okayama-u.ac.jp/amo/vol19/iss6/3Fine Structure of Adenovirus Type 12 289Fig. 1Fig. 2Fig. 33Tawara: Fine structure of negatively stained adenovirus type 12Produced by The Berkeley Electronic Press, 1965290 J. TAWYRA4Acta Medica Okayama, Vol. 19 [1965], Iss. 6, Art. 3http://escholarship.lib.okayama-u.ac.jp/amo/vol19/iss6/3Fine Structure of Adenovirus Type 12 291from the vmon (Fig. 5), and sometimes empty shells without core m whichPTA penetrates deep into the virion (Fig. 6).As regards capsomeres themselves, each capsomere has a round hollow inits center, and probably because being surrounded by six other capsomeres, itgives somewhat a hexagonal appearance (Fig. 7). Fig. 8 shows a virion ob-served at a higher magnification, in which each capsomere seems to be anaggregation of numerous finer granules of a stronger contrast.It is not possible to describe precisely about the structural arragement ofthese finer granules. However. they seem to have a close connection to chemi-cal structural units in some way.Generally, it is thought that capsomeres which are the structural units ofa virus as observed in electron microscope would be composed of still finer sub-units. According to HORNE et al." as in the case of poliovirus, there areobserved subunits smaller than capsomeres, but the structural arrangement ofcapsomeres themselves is sometimes not distinct. According to ALMEIDA et al.and MAYOR et al. who observed fine structures of negatively stained adenovirustype 12 in the electron microscope, they seem to have not yet recognized thepresence of subunits with still finer structure.In comparing the results obtained in the present observations on the nega-tively stained pictures of adenovirus type 12 with those of others, it seems thatdifferences in the electron micrographs arise even with identical materials becauseof the difference in the degree of washings of virion during the purificationprocess, the difference in the intensity of negative staining, and the differencein the developmental stage of virion itself.SUMMARYAdenovirus type 12 is of human origin and it shows carcinogenic activityin experimental animals. With negatively stained particles of this virus electronmicroscopic observations were carried out. As the result it was demonstratedthat its capsid, like other adenoviruses, is an icosahedron and each capsomereis of a hexagonal shape with a hollow in its center, each of which is surroundedby 6 adjacent capsomeres and is composed of numerous small subunit-likt:-particles.Figs. 4-6 Show the virus particles with incomplete structure in different stages of develop-ment. The virus particle with tissue debris in the vicinity of it in Fig. 4 and the one with around white core in Fig. 5 are seen. The particle in Fig. 6 shows an empty shell without core.Fig. 7 shows that each capsomere of virus particle has a round hollow in its center, and shapeof each capsomere shows a hexagonal appearance. x 450,000Fig. 8 Shows an aggregation of numerous finer granules on capsomeres at a higher magni-fication of virus particles. x 1057, 0005Tawara: Fine structure of negatively stained adenovirus type 12Produced by The Berkeley Electronic Press, 1965292 J. TAwARAACKNOWLEDGEMENTThe author expresses his thanks to Dr. Yoshiro YABE for supplying him with adenovirus type12 (Huie strain), to Mr. Nobuo !lAYASHI for his assistance in taking electron micrographs, and toProf. Sakae MURAKAMI for his advices and guidance throughout this work.REFERENCES1. S. BRENNER and R. W. HORNE: A negative staining method for high resolusion electronmicroscopy of virus. Biochim. Biophys. Acta 34, 103, 19592. J. D. ALMEIDA and A. F. HOWATSON: A negative staining method for cell associated virus.]. Cell Bioi. 16, 616, 19633. H. D. MAYOR, B. POWELL and ]. J. TRENTIN: Structure of the viral capsids of adenovirustype 12 and 18. Virology 23, 614, 19644. R. W. HORNE and J. NAGINGTON: Electron microscope studies of the development and struc-ture of poliomyelitis virus. ]. Mol. Bioi. 1, 333, 19596Acta Medica Okayama, Vol. 19 [1965], Iss. 6, Art. 3http://escholarship.lib.okayama-u.ac.jp/amo/vol19/iss6/3

Fine structure of negatively stained adenovirus type 12

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