Acta Medica OkayamaVolume 30, Issue 3 1976 Article 2

JUNE 1976

Rescue of Rous sarcoma virus (RSV) fromRSV-transformed human embryonic cells by

cell fusion with chick embryo fibroblasts usinglysolecithin

Hideo Ishii∗

∗Okayama University,

Copyright c©1999 OKAYAMA UNIVERSITY MEDICAL SCHOOL. All rights reserved.

Rescue of Rous sarcoma virus (RSV) fromRSV-transformed human embryonic cells by

cell fusion with chick embryo fibroblasts usinglysolecithin∗

Hideo Ishii

Abstract

Heterokaryon formation and Rous sarcoma virus (RSV)-induction were studied by fusionof RSV-transformed human embryonic cells with chick embryo fibroblasts in the presence oflysolecithin. Heterokaryon formation was observed by autoradiography. RSV-induction was iden-tified by focus formation, electron microscopy and density gradient centrifugation of 3H-uridine-labeled particles. The most effective concentration of lysolecithin for virus induction was 10mug/10(6) cells/0.1 ml. Efficiency of lysolecithin in virus induction was not less than that ofultraviolet-inactivated Sendai virus (UV-HVJ).

∗PMID: 187016 [PubMed - indexed for MEDLINE] Copyright c©OKAYAMA UNIVERSITYMEDICAL SCHOOL

Acta Med. Okayama 30, 153-162 (1976)

RESCUE OF ROUS SARCOMA VIRUS (RSV) FROMRSV-TRANSFORMED HUMAN EMBRYONIC CELLS

BY CELL FUSION WITH CHICK EMBRYOFIBROBLASTS USING LYSOLECITHIN

Hideo ISHIIDepartment of Biochemistry, Cancer Institute, Okayama University

Medical School, Okayama 700, Japan(Director: Prof. T. Oda)

Received for publication, March 22, 1976

Abstract. Heterokaryon formation and Rous sarcoma virus (RSV)­induction were studied by fusion of RSV-transformed human embryo­nic cells with chick embryo fibroblasts in the presence of lysolecithin.Heterokaryon formation was observed by autoradiography. RSV­induction was identified by focus formation, electron microscopy anddensity gradient centrifugation of 3H-uridine-Iabeled particles. Themost effective concentration of lysolecithin for virus induction was10 ,ug/106 cells/O.! ml. Efficiency of lysolecithin in virus induction wasnot less than that of ultraviolet-inactivated Sendai virus (UV-HVJ).

Infection of rats with avian tumor viruses was first reported by Svet­Moldavsky (1). It was soon confirmed in a variety of other mammalian speciesincluding primates (2,3,4,5,6). These mammalian cells transformed by Roussarcoma virus (RSV) are generally non-permissive. Many problems remain tobe solved: (a) how does the viral genome exist in virally transformed non­producing cells, (b) what factor(s) regulates the expression of early arid lategenes of viruses, (c) whether a repressor exists in mammalian cells and (d)what kind of viral information is required to maintain the transformed state.

In biological studies of these transformed non-permissive cells, successes inrescuing the viruses from these cells have been reported by various methods(7-16). The procedures are established for virus induction in heterokaryonsformed by virally transformed non-permissive cells and permissive chick embryofibroblasts using ultraviolet (UV) inactivated or p-propiolactone-treated Sendaivirus (HVJ) (6, 17-20). However, several disadvantages are present, as evenif the HVJ is inactivated, it remains a virus. Since the virus was passaged inchicken eggs for proliferation, contamination is possible of the Rous associatedvirus (RAV). There is no standard method for virus stocking, and it is in­evitable that the stocked virus lose some cell fusion capacity during" preserva­tion. Furthermore, the introduction of virus particles into a somatic cellduring the fusion process may cause host cell alterations which are unknown

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154 H. ISHII

at present. It is reported that the presence of inactivated HVJ may actuallyinhibit the rescue of related viruses (21).

For these reasons, the author tried to find an effective method for virusinduction by cell fusion without using HVJ. Howell and Lucy (22), Lucy (23),Keay et ai. (24), and Ahkong et ai. (25) observed that lysolecithin (LL), aphospholipid, was capable of fusing somatic cells and that viable heterokaryo­cytes and hybrid cells were produced through fusion in the presence of LL.However, no report is available on the use of this cell fusion method for tumorvirus induction. In the present study, this cell fusion method with LL wasapplied for rescuing the virus from RSV-transformed human cells.

MATERIALS AND METHODS

RSb cells. RSb cells were the clonal cell line (l3-RSb 8) established by Dr.T. Kuwata, Chiba University, Chiba, Japan, derived from human embryonic cells(HuE 13 RS cells) which were successively transformed by the Schmidt-Ruppinstrain of Rous sarcoma virus (SR-RSY) and simian virus 40 (SY40) (26). Thesecells were maintained in Eagle's minimum essential medium (MEM) supplement­ed with 10% calf serum. This cell line was not productive of SR-RSV nor SV4O.But the presence of RSV genome in this cell line was verified by detecting weakgs-antigen in the cell and by inducing RSV-producing Rous sarcoma by inocul­ating the cells into the chick wing web (27).

Chick embryo fibroblasts (CEF). Fertilized C/B eggs of specific pathogen freetype were kindly provided by the Kanonji Institute of the Research Foundationfor Microbial Diseases of Osaka University, Kanonji, Kagawa, Japan. Theculture and focus assay followed the techniques in principle described by Vogt(28). CuI turemedia were based on MEM with 10% tryptose phosphate broth,10% calf serum, 0.5% chick serum, and l,ug/ml of Fungisone (Amphotericin B).

Sendai virus (HV.!J. This strain of HVJ was kindly provided by Dr. YoshioOkada, Research Institute of Microbial Diseases, Osaka University, Osaka,Japan. The viruses were inoculated into chorioallantoic sacs of 10 day-oldchick embryos and incubated at 37°C for 72 hours. The eggs were maintainedat 0 to 4°C overnight and the allantoic fluid was collected and centrifuged at34,880 x g for 30 min. The sediment was resuspended in one twentieth theoriginal volume in modified Hanks solution, divided into 1mllots in test tubesand stored at 0 to 4°C.

inactivation of HVJ with UV-light. Two ml of virus suspension (33,000HAU/ml) in a plastic Petri dish (50mm in diameter) was placed at a distance of13 cm from one 15 W germicidal tube (50cm in length) and exposed to UV-lightfor 3min with shaking at 30 second intervals.

Preparation of lysolecithin solution. Preparation procedures were accordingto Croce et al. (29). Lysolecithin (5.0mg) dissolved in 5ml of absolute ethanolwas placed in a screw-capped tube and incubated at 70 to 80°C for lOmin. Thissol ution was stored at 0 to 4°C as a stock solution. Aliquots of 0.5ml of stock

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RSV Rescue using Lysolecithin 155

solution were transferred into a closed tube and the flow of nitrogen wasdirected into the tube until all the ethanol was evaporated leaving sterilized LLpowder at the bottom of the tube. Absolute ethanol (0.05ml) was added to dis­solve the LL and was followed with addition of PBS (-) solution containing5mg BSA (fraction V) per 1ml to give a final concentration of 500 pg/ml.

Fusion procedures. RSb cells (2.5 x 105) labeled with tritiated thymidine andunlabeled CEF (7.5X 105) were mixed with each other in Hanks solution andcentrifuged at l60x g for 3min. The pellets were treated with 0.1 ml of LL atvarious concentrations for 1min. The tubes were shaken to detach the pelletsfrom the bottom of the tubes, thus increasing the availability of the cells to LL.The action of LL was neutralized by addition of 0.1 ml of MEM containing 30%calf serum inactivated at 56°C for 30 min to each tube. The samples were cen­trifuged for 3 to 5min at l60x g at room temperature and incubated for an addi­tional 10 min at 38°C. The pellets were resuspended in 1ml of Hanks solutionand seeded in 60 mm Petri dish with 3ml of growth medium.

Autoradiography. Two coverslips were placed in the 60mm Petri dish.Twelve hours after seeding, the samples were fixed and treated with 2% perch­loric acid at 4°C for 30min to remove non-polymerized nucleotides and washedwith running water for 1min. The coverslips were dried and dipped into 2-folddiluted Sakura NR-M2 emulsion at 45°C in a dark room. After exposure for10 days at room temperature, the samples were developed and fixed. Finallythey were stained with 1% Giemsa stain at pH 5.8, dried and mounted in Bioleit(Oken Shoji Co., Tokyo, Japan).

Fusion procedures using UV-HV]. The cell fusion method followed in prin­ciple the procedures described by Okada (30). RSb cells (2.5X 105) and CEF(7.5x 105) were mixed with each other in a conical flask containing Earle's solu­tion, then 0.2ml of UV-HVJ suspension (1 HAU/103 cells) was added. Thesample was kept in an ice water bath for 20min and shaken mildly for an addi­tional 20 min at 37°C. The reaction mixture was transferred into 60mm Petridishes containing 4ml of growth medium and incubated at 38°C in a 5% CO2

atmosphere.Electron microscopy. Monolayer culture of cells 9 days after the cell fusion

reaction was washed with PBS solution, and the cells were fixed in 2.5% gluta­raldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, for 30 to 60min for elec­tron microscopy.

Focus assay of released viruses. Assay procedures followed in principlethose of Vogt (28). Nine days after the cell fusion reaction, the culture mediawere replaced with 3ml of growth medium per 60mm plastic Petri dish. After2 hrs incubation, the culture medium was collected and centrifuged at l60xgfor 3 min at room temperature, and 2ml of the supernatant were inoculatedinto a monolayer culture of CEF allowed to settle 2 hours earlier. Two pg/mlof polybrene were added to the assay system.

Virus purification. Labeling of viruses released from transformed foci with3H-uridine was performed in growth medium. The labeled medium collectedat 4 hour intervals was frozen at -80°C until purification was performed.

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156 H. ISHII

These pooled media were centrifuged at 1,300 x g for 10 min to remove thecellular debris. The viruses were purified from this supernatant according tothe method of Green et ai. (31). The concentrated virus solution was layeredon top of a linear sodium tartrate gradient (4 to 40% sodium tartrate, 0.15 MNaGl, 0.015 M sodium citrate, pH 7.0) and centrifuged in a Hitachi RPS-65TArotor at 33,000 rpm for 2 hours. The 10 drop fractions were collected from thebottom of the tube. Samples of 0.15ml each were added to 7.5ml of Bray'sscintillating solution for a radioactivity count with an Aroca spectrophoto­meter.

RESULTS

Effect of LL on virus induction. Effects of LL concentration on virus induc­tion are shown in Table 1. The effective concentration of LL on virus rescue

TABLE 1. EFFECTS OF UV-HVJ AND LYSOLECITHIN ON INDUCTIONOF RSV FROM MIXED CULTURES OF RSb CELLS AND CEF

ExperimentUV-HVJa Lysolecithin ffu/2mlno. (/Lg/l()6cells)

1 None (- ) 0

2 103 (-) 3

3 103 (- ) 1

4- 103 (- ) 0

5 103 (- ) 5

6 None 5 3

7 None 10 78 None 15 79 None 25 0

10 None 50 0

a, Hemagglutination unit/106cells

ranged from 5 to 15 ,ugj106 cells/D.1ml on observation of focus formation in­duced by these rescued viruses. Concentrations of more than 25,ug LL/106

cells/D.1m1 caused a cytolytic effect which resulted in stoppage of cell growth.The transforming titer of cultured media from these mixed cells in the presenceof LL was low as shown in Table 1. But the effect of UV-BVJ on virus rescuewas not more than that of 10 to l5,ug LL. In cultures untreated with UV-BVJor LL, no focus formation was induced in the monolayer of CEF by additionof polybrene. No addition of this compound resulted in no foci. Recently theauthor succeeded in increasing the transformation titer of these viruses to 180ffu/ml.

Autoradiography. Fig. 1 shows that LL induces multinucleated cell forma­tion between RSb cells and CEF. The evidence for the multinucleated cells to

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157RSV Rescue using Lysolecithin

Fig. 1. Autoradiograph of a heterokaryocyte of a tritiated thymidine-labeledRSb cell and an unlabeled chick embryo fibroblast.

be heterokaryocytes is based on observations that nuclei with grains from3H-thymidine incorporation were derived from RSb cells and other un­labeled nuclei were derived from CEF. The percentage of polykaryocytes ob­tained by LL treatment was approximately 17.5%. About 5.7 to 7.4% of RSbcells were involved in heterokaryocyte formation with CEF in the presence ofLL. The frequency of spontaneously formed heterokaryocytes was negligible.On the other hand, the frequency of heterokaryocyte formation induced byUV-HVJ was at a level similar to that induced by LL, but the recovery ofviruses was low.

Electron microscopy. RSb cells and CEF were fixed in glutaraldehyde forelectron microscopic observations. Virus particles were not found in either typeof cell. Co-cultivation of RSb cells and CEF in the absence of LL or UV-HVJwas also examined by electron microscopy. C-type virus particles were notfound. On the contrary, mixed cultures of both types of cells in the presenceof LL were fixed on the ninth day after cell fusion reaction. Electron micro­scopic examination revealed the presence of C-type virus particles which werein various phases, such as budding, immature and mature, as shown in Figs. 2and 3. These C-type virus particles had spikes on their surfaces and their

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158 H. IsHII

Figs. 2-3. Mature and budding C-type virus particles released from mixedcultures of RSb cells and chick embryo fibroblasts in the presence of lysolecithin(10 JLg/106cells/O.l mil. x 60,000.

diameters were about 100 mf.1.Ultracentrifugal and enzymatic analyses. Ultracentrifuga1 analysis of cultured

media containing 3H-uridine was performed according to the procedures de­scribed in Materials and Methods. It was proved that RNA viruses werereleased from the foci formed on CEF by demonstrating the peak of radio­activity at a density of l.17g/m1 (Fig. 4). The culture media fractions between

8.4u

NIo'X-3"0

Q.J....,ttl~

o0..

; 2uI::

5 10 15 20Fraction No.

Fig. 4. Sedimentation analysis of viruses released by cell fusion with lysoleci­thin. Viruses were layered on a 4-40% w/v sodium tartrate gradient in sse andcentrifuged at 33,000 rpm for 2 hours at 4°e in a Hitachi 65P RPS 65 T A rotor.

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RSV Rescue using Lysolecithin 159

5,000 X g and 100,000 X g had reverse transcriptase activity. These two pro­perties are characteristics of RNA tumor viruses.

DISCUSSION

There are many procedures for rescuing viruses from virally transformednon-productive mammalian cells, such as treatment with halogenated pyrimi­dine (7, 10, 12, 13), UV-irradiation (14), decreased temperature (9), DNAsynthesis inhibitors (8, 11), co-cultivation with permissive cells (15, 16), cellfusion with permissive cells in the presence of UV- or ,8-propiolactone treatedHVJ (17-20,32,33), inoculation of transformed cells directly into permissivehost animals to obtain the virus producing tumor (34) and transfection of iso­lated DNA from transformed non-producing cells to monolayer culture of per­missive cells (35-37).

Although these procedures have advantages they also have disadvantages.It is thus necessary to select the method of virus rescue depending upon thepurpose of the experiment. Among these methods the one generally used iscell fusion between transformed cells and permissive cells using UV-irradiatedHVJ. However, it has several disadvantages, especially in rescuing Rous

sarcoma virus. Since HVJ is a virus, other unexpected factors are presentwhich may cause some alterations in the cells. A loss of cell fusion capacitymay occur during storage. Meulen et al. (21) reported that the presence of UV­HYJ inhibited the rescue of virus. For these reasons, the LL mediated cellfusion technique introduced by Poole, Howell and Lucy (38) and Lucy (23)was applied to rescue RSV from RSV-transformed human embryonic cells(RSb).

As a result of this experiment, more viruses were recovered by LL thanby UV-HVJ, as described above. These findings show that lysolecithin, simplephospholipid, is useful for virus rescue by cell fusion because of its relativelysimple action mechanism. The incidence of heterokaryocyte formation beingrelatively low in contrast to homokaryocyte formation of both parent strain cellsmay depend on the origin of the cell line. In the results of Poole, Howell andLucy, higher fusion efficiency was found with LL in an acid conditions (pH5.5). Croce et ai. (29) found that hybrid formation after fusion was higher atpH 7.2 than pH 5.5. The author has not performed the present experimentunder acid conditions. The LL concentrations used in the experiment of Croceet ai. ranged from 200 to 600 ,ug/l 07 cells/O.l ml. This LL level was too highin my experimental system. The difference in the two studies may be due tovariations in the cell fusion capacity of LL itself.

It is important to determine the cytotoxicity of LL for every type of cellused in cell fusion experiments. Another paper (39) reports that UV- or

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160 H.~IsHU

ft-propiolactone treated HVJ induced greater fusion efficiency and rescued moreviruses by using anti-HVJ serum.

In this experiment, RSb cells which contain low gs-antigen were used asvirogenic cells for the purpose of virus recovery from human cells. Thepresence or absence of gs-antigen in CEF does not influence the outcome ofvirus rescue (40). Gs-antigen negative non-productive RSV-transformed mam­malian cells, however, cannot induce viruses by cell fusion even if the hetero­karyocytes are superinfected with helper virus or treated with BUDR (40-41).The limiting factor in virus rescue seems to be the degree of gs-antigen synthesisin the mammalian cell (42-43). The mechanism of RSV rescue from hetero­karyocytes is not yet clear. Chicken cell components of a heterokaryocyte mightcomplement certain functions of the mammalian cell which are necessary forSR-RSV formation and/or they might cause de-repression of the same functionsof the mammalian cell.

Although subgroup determination of these rescued viruses has not beencompleted, preliminary data indicates that the viruses may be classified in sub­group D.

Acknowledgment. The author wishes to express profound thanks to Prof. Takuzo Oda forenthusiastic guidance and encouragement throughout this work. Thanks are also due toDr. Osamu Hatase, Dr. Yoshito Kanzaki, Mr. Takashi Nakamura, Miss Sekiko Watanabeand Miss Tamie Yasui for their valuable advice and assistance. This investigation was sup­ported partly by a Grant-in-Aid for group study of tumor virus from the Japan Ministry ofEducation, Science and Culture.

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RSV Rescue using Lysolecithin 161

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29. Croce, C. M., Sawicki, W., Kritchevsky, D. and Koprowsky, H.: Induction of homo­karyocyte, heterokaryocyte and hybrid formation by lysolecithin. Exp. Cell Res. 67, 427­435, 1971.

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35. Hill, M. and Hillova, J.: Production virale dans les fibroblastes de poule traites parl'acide desoxyribonucleique de cellules XC de rat transformees par Ie virus de Rous.C. R. Acad. Sci. 272, 3094-3097, 1971.

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41. Donner, L., Sainerova, H., Svoboda, ]. and Sherneck, S.: Potentiation of RSV rescuefrom RSV rescue from RSV-transformed "poorly" virogenic cell lines by 5-bromode­oxyuridine treatment before fusion with chick embryo fibroblasts. Int. J. Cancer 13, 37­42, 1974.

42. Boettiger, D.: Reversion and induction of Rous sarcoma virus expression in virus­transformed baby hamster kidney cell. Virology 62, 522-529, 1974.

43. Vigier, P.: Persistence du genome du virus de Rous dans les cellules du hamster con­verties in vitro, et action du virus Sendai inactive sur sa transmission aux cellules depoule. C. R. Acad. Sci. 264, 422-425, 1967.

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