The surface of Gross virus-induced murine lymphoblasts and C-type virus particles budding from these cells were investigated under the scanning electron microscope (SEM). The cells appeared spindle-shaped or roughly-rounded with extensive surface features consisting of microvilli, blebs and ruffled membranes. C-type virus particles were detected on the cell membrane as small spherical particles, distinguishable from the microvilli. Clustered virions were observed in some cases. However, the distribution of virions appeared to be random. The surface of the virion was smooth and had no globular units at high magnification. These morphological observations were confirmed in ultrathin sections.</p

Fujio, Kouji

Ichikawa, Hiroyuki

Tawara, Jutaro

Tsutsui, Kiyoshi

Uno, Fumio

Okayama University Scientific Achievement Repository

Acta Medica OkayamaVolume 31, Issue 2 1977 Article 1APRIL 1977Morphological studies of Gross virus-inducedlymphoblasts by scanning electron microscopyHiroyuki Ichikawa∗ Fumio Uno† Kouji Fujio‡Kiyoshi Tsutsui∗∗ Jutaro Tawara††∗Okayama University,†Okayama University,‡Okayama University,∗∗Okayama University,††Okayama University,Copyright c©1999 OKAYAMA UNIVERSITY MEDICAL SCHOOL. All rights reserved.Morphological studies of Gross virus-inducedlymphoblasts by scanning electronmicroscopy∗Hiroyuki Ichikawa, Fumio Uno, Kouji Fujio, Kiyoshi Tsutsui, and Jutaro TawaraAbstractThe surface of Gross virus-induced murine lymphoblasts and C-type virus particles buddingfrom these cells were investigated under the scanning electron microscope (SEM). The cells ap-peared spindle-shaped or roughly-rounded with extensive surface features consisting of microvilli,blebs and ruffled membranes. C-type virus particles were detected on the cell membrane as smallspherical particles, distinguishable from the microvilli. Clustered virions were observed in somecases. However, the distribution of virions appeared to be random. The surface of the virion wassmooth and had no globular units at high magnification. These morphological observations wereconfirmed in ultrathin sections.∗PMID: 197797 [PubMed - indexed for MEDLINE] Copyright c©OKAYAMA UNIVERSITYMEDICAL SCHOOLActa Med. Okayama 31,95-101 (1977)MORPHOLOGICAL STUDIES OF GROSS VIRUS-INDUCEDLYMPHOBLASTS BY SCANNING ELECTRONMICROSCOPYHiroyuki ICHIKAWA, Fumio UNO, Kouji FUJIO,Kiyoshi TSUTSUI and Jutaro TAWARADepartmennt (If Virology, Okayama University Medical School,Okayama 700, JapanReceived December 4, 1976Abstract. The surface of Gross virus-induced murine lympho-blasts and C-type virus particles budding from these cells were inves.tigated under the scanning electron microscope (SEM). The cellsappeared spindle-shaped or roughly-rounded with extensive surfacefeatures consisting of microvilli, blebs and ruffled membranes. C-typevirus particles were detected on the cell membrane as small sphericalparticles, distinguishable from the microvilli. Clustered virions wereobserved in some cases. However, the distribution of virions appearedto be random. The surface of the virion was smooth and had noglobular units at high magnification. These morphological observa-tions were confirmed in ultrathin sections.Neoplastic cells transformed in vitro by x-irradiation appear pleomorphic anddisplay surface excrescences, such as microvilli, blebs and ruffles (I). Similarobservations were reported by chemically transformed mouse embryo cells (2).Porter, Todaro and Fonte (3) have employed SEM to study the topography ofvirally and spontaneously derived transformants of BALB/3T3 cells and reportedthat all transformants showed morphological differences from the parent celllines. These changes included increased numbers of microvilli, blebs and ruffles.In these reports budding virus were not observed on cell surfaces.Recently, high resolution SEM was used to study and identify the buddingparticles on the plasma membrane. Holmes (4) showed maturation of vesicularstomatitis virus (VSV) in cultured cells and pointed out that the major topologicalchanges in the cell surface, such as microvilli, filipodia and ruffled membrane,result from virus infection. Another application of SEM was to examine theproduction and release of oncogenic RNA virus. Wong and MacLeod (5) usedtemperature sensitive mutants and demonstrated the budding process of the C-type virus. They calculated the budding particles on the surface with the aidof SEM. Panem and Kirsten (6) showed budding of murine oncornavirus at theplasma membrane of productively infected cells. Moreover, they pointed outthat the surface of the virion was composed of globular unit 20-28 nm in di-951Ichikawa et al.: Morphological studies of Gross virus-induced lymphoblasts byProduced by The Berkeley Electronic Press, 197796 H. ICHIKAWA, F. UNO, K. FUlIO, K. TsuTsUI and J. TawARAameter and that the distribution of the budding virus appeared to be random overthe cell surface. In this communication we report on the budding virion and sur-face morphology on Gross virus-induced murine lymphoblasts using high resolu-tion SEM. These observations were confirmed in ultrathin sections.MATERIALS AND METHODSCell cultures. Gross virus-induced lymphoblasts (7) were kindly providedby Dr. M. Nakai (Osaka Medical College, Osaka, japan). These lymphaticleukemia cells were maintained in our laboratory and were grown in suspen-sion culture in RPMI 1640 supplemented with 20% inactivated fetal calf serumor Eagle's medium as modified by Baluda and Goetz (8) supplemented with 5%inactivated fetal calf serum. The medium contained 200 units/ml penicillinand 200 ,ug/ml streptomycin. Cells were fed every 3 days, subcultured twice amonth and incubated at 37"C in an atmosphere of 5% CO2,Preparation of specimens for SEM. Cells were seeded on glass coverslips in6 cm plastic petri dishes and cultivated for 2-3 days. The coverslips were re-moved from the petri dishes, immediately fixed for 20 min with 2.5% glutaral-dehyde, rinsed twice with 0.1 M cacodylate buffer, postfixed for 15 min with 1%osmium tetraoxide, rinsed twice with the same buffer and then dehydraTed ina graded series of ethanol followed by critical point drying using liquid CO2 (9).The resulting specimens were lightly coated with platinum-palladium by asputter coating method and were observed by field emission type SEM (HFS-2,Hitachi Ltd., Japan).Preparation of specimens for ultrathin sections. Cells were harvested at ex-ponential growth. The collected cells were fixed with 2.5% glutaraldehyde for30 min and postfixed with 1'% osmium tetraoxide for 15 min. The specimenswere dehydrated through a graded series of ethanol and embedded for ultrathinsectioning. Thin sections were poststained with uranyl acetate and lead citrate.RESULTSMorphology of lymphatic leukemia cells by SEM. The cells were attached to theglass substrates and to each other by rigid filipodia. On a few days incubation,the cultured cells grew as a monolayer and detached from the glass into themedia. Fig. I shows the Iymphoblasts as spindle-shaped and rounded. Ruffledmembranes were observed at the margins of the fusiform cell. Small roundedprojections, which have been called blebs, were present on the surface of somecells. The cell surface was studded with curving, cylindrical, unbranched micro-villi with rounded tips. Fig. 2 demonstrates a rounded cell with extensive surfacefeatures: microvilli, ruffled membranes, blebs and some clustered virus parti-cles. These microstructures could be resolved at low magnification as illustratedin Figs. I and 2.2Acta Medica Okayama, Vol. 31 [1977], Iss. 2, Art. 1http://escholarship.lib.okayama-u.ac.jp/amo/vol31/iss2/1Gross Virus-induced Lymphoblasts 97Fig. 1. Gross virus-induced murine lymphoblast cells are fusiform or rounded in shapewith numerous microvilli (M), some blebs (B) and a few marginal ruffles (R). Bar: lpm.X 6,300.1:1Fig. 2. A SEM picture of a rounded lymphoblast cell. Note the microvilli, complicatedruffles and some clustered virions on the surface of the cell. Bar: I pm. X 12,600.3Ichikawa et al.: Morphological studies of Gross virus-induced lymphoblasts byProduced by The Berkeley Electronic Press, 197798 H. ICHIKAWA, F. UNO, K. FUlIO, K. TSUTSUI and J. TaWARAFig. 3. A SEM micrograph of clustered virions (el. Note the virions piling up on thecell surface. The surface of the virion is smooth and has no globular units. Bar: 0.5,um.X 63,000.Fig. 4. A SEM micrograph of the cell surface at high magnification. Arrows indicatevirions budding from the cell membrane. Virions are distinguishable from microvilli (M)by their surface features. Bar: O.I.um. X86,000.4Acta Medica Okayama, Vol. 31 [1977], Iss. 2, Art. 1http://escholarship.lib.okayama-u.ac.jp/amo/vol31/iss2/1Gross Virus-induced Lymphoblasts 99Budding of C-type virus. Structures which were considered to represent vir-ions were identified at high magnification (Figs. 3 and 4). Virions were observedas small uniform spherical particles attached to the cell surface, distinguishablefrom microvilli (Fig. 4). The diameter of the virion was about 100 nm. Micro-villi were of varying lengths but most had a diameter of about 100-120 nm;therefore, some microvilli were confused with virions, especially when the micro-villi were viewed as short structures.Frequently, C-type virus were observed as clusters piling up on the surfaceof cells (Fig. 3). Clusters of three to five or more virions were observed by highresolution SEM. The surface of the virion was smooth and had no globular unit.We could not observe the budding virion attached on the ruffled membranes orblebs. Therefore, these excrescences appeared not related to the budding processof the C-type virus. Fig. 5 illustrates clusters of C-type virus particles with di-Fig. 5. A TEM micrograph of a lymphoblast cell. Note the cluster of virions withdense nucleoid. Bar: 0.5 pm. x 3,000. Inset shows a budding virus with a diameter of100 nm, from the plasma membrane. X 100,000.ameters of 100 nm and the electron dense nucleoid by transmission electron micro-scope (TEM). The inset of Fig. 5 shows an enveloped budding virion from theplasma membrane. No figures were observed budding from the microvilli orother surface projections. Ultrathin sections confirmed the budding virus ob-served by SEM.5Ichikawa et al.: Morphological studies of Gross virus-induced lymphoblasts byProduced by The Berkeley Electronic Press, 1977100 H. ICHIKAWA, F. UNO, K. FUlIO, K. TSUTSUI and J. TaWARADISCUSSIONSEM permits the study of a large number of whole cells in situ and is a valu-able tool for investigating the surface morphology of tissue culture cells. Westudied the morphology of the Gross virus-induced murine lymphoblasts with thebudding C-type virus. The surface of the cell was covered with microstructures,such as microvilli, blebs and rufHes. These microstructures on the cell surfacehave been observed on neoplastic cells transformed in vitro by x-irradiation (1)or by chemical carcinogens (2). These excrescences were not a characteristicfeatures of the transformants. The surface of cells infected with VSV but notoncogenic virus showed the same microstructures (4). Other observations pointedout that established cell lines synchronized in culture changed surface structuresat different stages of the cell cycle with large numbers of microvilli, blebs andrufHes being displayed in the Gl stage (10).The present SEM study demonstrated the budding virions on the plasmamembrane. We found virions revealed as small spherical particles associated withthe cell surface, distinguishable from microvilli. Virus budding from microvilliand from surface folds could not be observed in our experiments. Clusters of thevirions were observed on the murine lymphoblasts surface. The clustered virionswere the most striking features and were absent on cells infected with murineoncornavirus (6). The virion was smooth and we could not observed the globularunit. Therefore, we could not agree with Panem and Kirsten (6), who pointedout that the virion was composed of symmetrically arrayed globular unit 20-28nm in diameter. Wong and MacLeod (5) counted the virus particles, and anaverage of 1600 virions was observed on a single cell surface during the produc-tion of Moloney murine leukemia virus. They suggested that the distribution ofvirions appeared to be random. In the case of Gross virus-induced leukemia cells,however, the determination of the number of virus particles per cell was difficultto estimate because of the clustered virions. Therefore, some form of cell treat-ment, such as trypsinization seems to be necessary to harvest the extracellularvirions successfully from these leukemia cells. The release of Friend leukemiavirus was almost completely inhibited in low-ionic strength medium (11). Onthe other hand, the production of C-type virus is significantly enhanced by theaddition of dimethylformamide and bromodeoxyuridine (BUDR) on murine leu-kemia cells (12) or by adrenocorticoid in iododeoxyuridine (IUDR)-treated cells(13). IUDR and its bromo-analog have been Llseful as an inducer of murine leu-kemia virus from AKR mouse embryo cells (14, 15).From a practical standpoint, the SEM technology demonstrated here mayrepresent a screening method for virus-producing cell lines by examining cellsurfaces for budding particles, rather than the extracellular supernatants for freevirions.6Acta Medica Okayama, Vol. 31 [1977], Iss. 2, Art. 1http://escholarship.lib.okayama-u.ac.jp/amo/vol31/iss2/1Gross Virus-induced Lymphoblasts 101Acknowledgments. We wish to thank Dr. Masuyo Nakai for helpful discussion and interestin this study.REFERENCES1. Borek, C. and Fenoglio, C. M.: Scanning electron microscopy of surface features ofhamster embryo cells transformed in vitro by x-irradiation. Cancer Res. 36, 1325-1334,1976.2. Malick, L. E. and Langenbach, R.: Scanning eletron microscopy of in vitro chemicallytransformed mouse embryo cells. j. Cell Biol. 68, 654-664, 1976.3. Porter, K. R., Todaro, G. J. and Fonte, V.: A scanning electron microscope study ofsurface features of viral and spontaneous transformants of mouse BALB/3T3 cells. j.Cell Biol. 59, 633-642. 1973.4. Holmes, K. V.: Scanning electron microscopic studies of virus-infected cells. 1. Cyto-pathic effects and maturation of vesicular stomatitis virus in L2 cells. j. Virol. 15, 355-362, 1975.5. Wong, P. K. Y. and MacLeod, R.: Studies on the budding process of a temperature-sensitive mutant of murine leukemia virus with a scanning electron microscope. j. Virol.16, 434-442, 1975.6. Panem, S. and Kirsten, W. H.: Secondary scanning electron microscopy of cells in-fected with murine oncornaviruses. Virology 63, 447-458, 1975.7. Imagawa, D. T., Issa, H. and Nakai, M.: Cultivation of Gross virus-induced murinethymic lymphoma cells in vitro. Cancer Res. 28, 2017-2026, 1968.8. Baluda, M. and Goetz, 1. E.: Morphological conversion of cell cultures by avian myelo-blastosis virus. Vir~logy 15, 185-199, 1961.9. Anderson T. F.: Techniques for the preservation of three dimensional structure in pre-paring specimens for the electron microscope. Trans. N. Y. Acad. Sci. 13, 130-134.1951.10. Porter, K., Prescott, D. and Frye, J.: Changes in surface morphology of Chinese ham-ster ovary cells during the cell cycle. j. Cell Biol. 57, 815-836, 1973.11. Ussery, M. A., Ramirez-Mitchell, R. and Hardesty, B. A.: Inhibition of Friend murineleukemia virus production by low-ionie-strength medi urn. j. Virol. 17, 453-461, 1976.12. De Harven, E., Lampen, N. and Sato, T.: Scanning electron microscopy of cells infectedwith a murine leukemia virus. Virology 51, 240-243, 1973.13. Paran, M., Gallo, R. C., Richardson, L. S. and Wu, A. M.: Adrenal corticosteroids en-hance production of type C virus induced by 5-iodo-2'-deoxyuridine from cultured mousefibroblasts. Proc. Nat!. A cad. Sci. USA 70, 2391-2395, 1973.14. Lowy, D. R., Rowe, W. P., Teich, N. and Hartley, J. W.: Murine leukemia virus: High-frequency activation in vitro by 5-iododeoxyuridine and 5-bromodeoxyuridine. Science 174,1974.15. Teich, N., Lowy, D. R., Hartley, J. W. and Rowe, W. P.: Studies of the mechanism ofinduction of infectious murine leukemia virus from AKR mouse embryo cell lines by5-iododeoxyuridine and 5-bromodeoxyuridine. Virology 51, 163-173, 1973.7Ichikawa et al.: Morphological studies of Gross virus-induced lymphoblasts byProduced by The Berkeley Electronic Press, 1977

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