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Fragmentation of the nucleus in Rous sarcoma virus-infected chick embryo cells
VIROLOGY
32, 73-83
(1967)
Fragmentation
of the
Nucleus Chick
in Rous Embryo
WARRES Department
of Zoology, dccepled
Sarcoma
Cells
LEVINSON’~ Universit~y January
Virus-Infected
College,
2 London,
England
27, 1367
Infection of chick embryo cells with Rous sarcoma virus causes fragmentation of the nucleus as early as 36 hours after infection. Neither Rous-associated virus nor Newcastle disease virus causes nuclear fragmentation. The presence of both tryptose phosphate broth and calf sertlm in the medium are required for fragmentation to occur. An assay procedllre for 110~1s sarcoma virus based upon nuclear fragmentation is described.. INTRODUCTION
embryos (avian leukosis virus free) by mincing and treatment with 0.25 % trypsin (Difco) in Tris-buffered saline. The cells (8 to 10 x 106) were seeded onto 90-mm plastic tissue culture dishes (Falcon Plastics, Los Angeles, California) in 12.5 ml of primary medium which contained 8.3 parts of single-skength Jledium 199 (Glaxo Laboratories, Greenford, England), 1.0 part of trypt’ose phosphate brot’h (Difco), 0.4 part of calf serum, 0.1 part of chick serum, and 0.2 part of 2.S% NaHC03. The cultures were incubated at 39’ in a humidified atmosphere containing air plus sufficient CO, to keep the medium at pH 7.3. Secondary cultures were made by treating the primary cultures with 0.05% trypsin followed by seeding 4 X lo5 cells in 2 ml of secondary medium ont’o 33-mm plastic tissue culture dishes. Secondary medium differed from primary medium only in that it contained 0.5 part of calf serum and no chick serum. The cells were infected either in suspension or as a monolayer on the surface of the dish and overlaid with 2 ml of an agar medium after a suitable adsorption period. The agar medium was composed of 4.2 parts of l.S % agar (Difco-Bacto) 4.2 parts of double strength Medium 199, 1.0 part tryptosc phosphat’e broth, 0.5 part wlf serum, and 0.2 part of 2.8 % SaHC03. Primary and seconda,ry cultures of mouse
In the course of recent experiments concerned with the behavior of Rous sarcoma virus-infected chick cells, extensive fragmentation of t#he nucleus was observed. Kuclear fragment,ation has been described in cells growing out of Rous sarcoma explant’s (Tenenbsum and Doljanski, 1942), and brief mention of fragment’ed nuclei was made in a study of the morphological changes in chick embryo cells following infection with Rous sarcoma virus (Gold& 1959). No information, however, has been published on the precise virus-cell interaction required for nuclear fragmentation. In view of the importance of proper nuclear function for the survival and multiplication of bot,h nonmalignant and malignant cells, experiments Were performed in order to determine the nature of the fragmentation process. The results of these experiments will be presented in this paper. MATERIALS
AND
METHODS
Tissue cultu~z. Primary cultures of chick embryo cells were prepared from lo-day-old 1 Holder of British-American Exchange Fellowship PF-288 awarded by the American Cancer Society. 2 Present address: Department of Microbiology, Medical School, 17niversitjy of California, San Francisco, California. 74
FRAGMENTJW
NUCLEUS
embryo cells were prepared from 14-day-old embryos in t,he same way as were the chick cell cuhures. I’irus. The Bryan high-titer strain of Rous sarcoma virus (RSV) and a Rous-associated virus (RAV-1) isolated from the RSV stock were used. In one series of experiments, the Schmidt-Ruppin and Bryan standard strains of RSV and the ARC virus isolated by Dougherty and Rasmussen(1964) were used. A noncytopathic strain (B-l Hitchner) and a cytopathic strain (Hertfordshire) of Newcastle disease virus (NDV) were employed to determine the effect of a virus which is not a member of the avian leukosis group. The focus assay for RSV and the plaque assay for KDV have been described previously (Levinson and Rubin, 1966). Histology. The cultures were fixed and stained 72 hours after infection unless otherwise stated. Prior to fixation, 2 ml of %[edium 199 was added to the cultures in order to loosen the agar. After incubation at’ 39” for 15 minutes, the agar was separated from the sides of the dish with a glass rod and discarded. The cells were fixed in 4?$ formaldehyde in phosphate-buffered saline (pH 7.2) for 10 minutes. The formal saline was removed and 33% ethanol was added for 5 minutes. After t’he removal of the 35% ethanol, t’he cultures were exposed to 70%. ethanol for 30 minutes followed by 35%~ ethanol for 1 minute The cultures were stained wit)h Harris’ hematoxylin for 10 minutes, “blued” with 6 rinses in tap water, and mounted in Farrant#‘s medium (G. Gurr, London, England). The fixation and staining procedures were carried out at room temperature. Microscopy. The cultures were examined under phase contrast at 430 X magnification using a 50 X oil immersion objective with a Wild AI-20 microscope. When quantitative result,s were desired, an ocular grid 49 mm” which circumscribed an arca of 2.64 X lo4 ,J‘Jof the tissue culture dish was used. The number of focal areas of cells containing fragmented nuclei were counted in at least 100 fields. The fields to be counted were determined by chance by moving the dish t,hrough 5 or more rows of 20 fields per row. Since the area of a dish 33 mm in diameter is S.55 X 108/$ and the area, of the dish
IN KS\:-INFECTED
CELLS
75
circumscribed by the grid is 2.64 X 104/$, there are 3.2 X lo4 fields per dish. If, for example, the number of areas of multilobed nuclei per 100 fields was counted, then the titer of the RSJ’ inoculum was calculated by multiplying the number of areas containing multilobed nuclei by 3.2 X 102. Antiserum. Antiserum against RAV and therefore against the RSV (RAV-1) in the Bryan stock was made by inoculating 4week-old White Leghorn chickens with lo6 infectious unit8s of RAV1 intravenously followed by monthly hleedings. Cltl-aviolet irradiation. A 2-ml sample of a 1: 10 dilution of RSV stock in phosphatebuffered saline was irradiated in a 50-mm plastic tissue culture dish. The out’put of the lamp was calibrated biologically using NDV as a standard and was shown to be approximately 15 ergs/mm2/sec at a distance of 30 cm from t,he lamp. RESULTS
Appearance of theFragmentedNuclei Secondary cultures of chick embryo fibroblasts were infect,cd wit,h the Bryan hightiter strain of Rous sarcoma virus (RSV) and grown under the conditions described in the Materials and Methods section. As early as 36 hours after infection, cells containing nuclei which had fragment,ed into two or more pieces were obscrvcd. Within 72 hours after infection, the nuclei fragmented into as many as 10 grapelike lobeswith a thin strand connecting each lobe. Many of the lobes contained a densely staining area resembling a nucleolus. lcigurc 1 illustrat,es the t,ypical appearance of a group of cells with fragmented nuclei. This figure also showsseveral nonfragmented nuclei which are represemative of those seen in normal, uninfected fibroblasts. If the infected cultures are overlaid with an agar medium in order to prevent recurrent cycles of virus infection, then the focal areas of cells containing fragmented nuclei can be counted and an accurate picture of the time of appearance of the fragmented nuclei can be obt,ained. As shown in Table 1, the first multilobed nuclei appear at 36 hours after infection, followed by a great incrcasc in the number of focal areas of
76
LEVINSON
FIG. 1. Microscopic appearance of fragmented nuclei can be SCCII on the left. Normal rells with ficntion: X 680.
nuclei. regular,
multilobed nuclei by GO hours, and n slower rate of increase during the succeeding 36 hours. Cells whose nuclei appear IO be in t.hc early stages of the fragmentat’ion prowss frequently surround the focal area of cells with obviously fragment,ed nuclei. Thrse cells in t,hr early stages contain nuclei n it,11a, scalloped or indented edge. In t#he later stages of the fragment’ation process, which a11 be seen at SC, hours after infe&on, t,hc strand c~onne6ng t’he many lobes of t,hc nucalei disappears and cells containing several small, da,rkly stained bodies cm1 bc observed. dt no stage in the fragmentat~ion process does any structure seem to bc intruding into the nwleus and the t80tal area of stained nuclear material does not appear to decrease, this indicates that t,hc fragmentation is not, due to dissolut,ion or loss of staining affinity of portions of the nucleus. The appearance of the fragmented nuclei is the same mhet,her the cult,ures are kept’ in fluid medium or overlaid with an agar medium. Ut,ilizing the data in Table 1, the st,andnrd
h focal area of cells containing fragmented oval llrlclei can be seen on the right. Magni-
conditions for a nuclear fragmenting unit (Xl%) were defined as a focal nrca of cells CYJIltaiIlhg fragmented nuclei formed on 4 X 1Oj cells at, 72 hours after infect,ion. In this case, since there were 3S focal areas in the 100 grid fields counted and 3.2 X lOA grid fields per dish (see Materials and I\Iethode section), there were 1.2 X lOA focal areas per dish. Since the culture was infected with 0.2 ml of a 1:40 dilution of RST’ stock, the titer of t,he stock was 2.3 X lo6 Sl~U/ml. The tit’er of the RSV stjock was approximately 2.5 to 3 X 10” focus-forming units (VIW)/ml giving an SE’U:FFU ratio of 5 to 10. This discrepancy between t)he results of the nuclear fragmentat~ion arid t)he focusformation assays will be invcstigat~cd nrld discussed in subsequent parts of this paper. Evidence That Nuclear Fragnmz fation Caused by Rous S’amma 17ims
Is
Some RST; st,ocks cont,ain other members of the avian leukosis group of viruses which function as helper viruses for the defect,ivc RSV (Hannfusn cf al., 1964). Mince the
FRAGMENTED TABLE: TIME
OF A\~~~.\~.\~~~
NUCLEUS
IN
I%\--1NFECTEI)
ii
CELLS
1 OF FRAGMENTEO
NT(‘LEI“
Hours
after 24 30 36 18 60 72 84 96
infection
Focal
areas/100
fields
0 0 1 6 30 38 53 64
tl Cultures were prepared by seeding 4 X lo5 cells in 2 ml of medium and were infected by adding 1.2 X lo3 FFU of Bryan “high titer” strain of RST- in 0.2 ml of medium. After a 12.hour incubation period at 39”, the cultures were overlaid with 2 ml of agar medium, then fixed and stained at intervals of 6 or 12 hours. The number of focal ‘areas contaitrirtg cells with fragmented nuclei was counted at 430 X magnification.
stock of RSV used i11 this study is known to contain Rous-associated virus (RAY-l). (P. Simons, personal communication), cxperiments were performed to det,crmine which virus causes nuclear fragmentation. 1. Inhibition of f~agmenfation by RA T’ antiserum. Antiserum against8 RAV-1 will inactivate t#he infectivity of both RAY-1 and RSV with a RAV-1 out#er coat (Hanafusa, 1964). RAV :&serum cannot t-hereforc be used to distinguish between RSV (RAT:-1) and RAV-1 but can be used as an initial step in the identification of t’hese viruses. An experiment was performed to determine the rate of inactivation by RAT’ ant,iserum of t,he nuclear fragmenting ability and qf the focus-forming abilit,y of t,he Bryan high-titer stock of RSV. Appropriate dilutions of the RSV stock and of t,he RAV antiserum were mixed and incubated at 37”. Samples were taken at 7 and at 14 minutes and assayed for the formation of multilobed nuclei and for focus formation. The data presented in Table 2, demonstrate that RAY antiserum innct,ivatcd the ability to fragment nuclei and the ability t,o form foci at the same rate. Although this experiment) indicates that nuclear fragment,ation is wused by either RSTT or RAV, it cannot be ruled out that a third virus present in t,hc RAT’ stock used to
Minutes of incubation at Ili” 7 14
T‘raition
of RSV surviving antiserum
Fragmentation
Focus assay
aSa>
1 .5 X lo--“’ 5.0 x 10-z
1.2 7.5
x x
10-t 10-Z
‘6 A 0.1.ml sample of RS1. srtspetisioti containing lo5 FFU was mixed with 0.1 ml of a 1:30 diltttiott of ItA\. antiserum attd 0.8 ml of secotldary tnrdium. A control mixture containing 0.1 ml of the ItSI’ suspension and 0.9 ml of secondary medium was also made. These mislrtres were incubated at 37” for 7 attd 11 mittutes, at which time samples were removed a11d assayed for sttrvivittg virus by the fragmetrtatiott and by the foctts-assay methods. h k:arh number represeiits the sttrviving fractiott of RS\- calcrtlated by dividing the ttter of IiS\. exposed to attttscrrtm by the titer of IiS\. not, Psposd
to
&lltisel.llIll.
make t,hc ant,iserum and present in t,he Bryan st’ocak of RSS’ c-ould be t#he agent rcqonsiblc for nwlear fragmentation. 2. dbsenrc oj’ j’ra~mcntation in Rd Jr-inJ’~tctl cells. Two experiments were performed to d&ermine whether infwtion with RAY alone would product nuclear fragmentation. In t#he first experiment, cultures cont,aining 4 X 10” chick embryo cells wcrc infected with 4 X lo4 infect,ious units (NJ) of RAY-I, overlaid J\ ith agar medium, then fixed and stained at’ daily intwvnls for 1 days. Ko fragmented nwlei were seen at any t,imc in the RAY-infected wlls. l’arallel cult,urcs infwted with RSV developed t,he expec%cd number of foci of frugment,ed nuclei. In the swond experiment, cultures containing 4 X 10” cells Jvere infected wit,h an undiluted sample and with :L sample of 4 serial tenfold dilutions of a stock of RAY-1 n-hich titercd :tpproximat,cly 2 X IO6 IV/ml. The wlturcs wew incubated for 3 days, at, which time they were transferred and seeded at a (‘onwnt8rntion of 4 X 10” cells. The cultures were overlaid with ngar, incubated for 3 days, and fixed and stained. So nuclear fragmentation was observed in any of the Rr2V-infcctcd cultures. The nurlci of the
LEVINSON
7s TABLE
3
tain a contaminating virus capable of fragmenting nuclei, stocks of the Bryan standard strain, the Schmidt-Ruppin (SR) strain, and the Bryan high t,iter strain (prepared Relative independently from the high titer stock Focal areas sensitivity Log dilution per 100 fields to RSV of RAV used throughout this study) were tested for their ability to produce multilobed No RAV 3G 1 nuclei. It was found that suit’able dilutions 7 0.19 -5 of the stocks of all the strains tested pro1 -4.5 0.028 duced focal areas of typical fragmented 0 <0.028 -4 nuclei. The titer of the stocks of the Bryan (I Cultures were prepared by seeding 1 X 106 standard strain, the SR strain, and this ceils in 5 ml of medium onto 50-mm plastic tissue independently prepared Bryan high titer culture dishes. The cells were infected with 0%ml strain were 3.2 X lo’, 3.5 X 104, and 7.7 X samples of serial half-log dilutions of RAT- and 10’ NW/ml, respectively. The finding that incubated for 3 days. The cultures were then other strains of RSV produce fragmented transferred and 4 x lo5 cells seeded in 2 ml of nuclei supports the hypothesis that RSV is medium on 33-mm dishes. The cells were infected the cause of the nuclear fragmentation. with 1 X 103FFU of RSV in 0.2 ml of medium, then The SR strain of RSV can causesarcomas overlaid with 2 ml of agar medium. The cultures in mice (AhlstrBm, 1964). In view of this, were fixed and stained 72 hours after infection, and the ability of SR-RSV to fragment the the number of focal areas of cells with fragmented nuclei was counted. nuclei of mouse embryo cells was tested. So nuclear fragmentation was observed in RAV-infected cells appeared similar in every cultures of mouse embryo cells to which way to those of the cells in uninfected cul- SR-R.SV ha’d been added. High concentrations of the Bryan high titer strain of RSV tures treated in the same manner. Infection with an appropriate dilution of RSV stock also failed t,o cause fragmentation of the of these uninfected cells causedthe expected nuclei of t’hc mouse embryo cells. Dougherty and Rasmussen (1964) have number of focal areas of fragmented nuclei, demonstrating that the cells were capable of isolat,ed a virus which they have named hRC (another Rous contaminant) from t,he nuclear fragmentation. &cl; of the SR-RSV used in this study. 3. Interference with the formation of fraymcnted nuclei by preinfection with RAI’. One Since it is possible that the virus in the SRcriterion which can be used to determine stock which causes nuclear fragment’ation whether nuclear fragmentation is caused by was ARC rather than SR-RSV, a stock of RSV is the specific interference of RSV in pure ARC virus was tested for its ability to RAV-infected cells. Ot,hcr viruses, such as produce fragmentation. No nuclear fragKcwcastle disease viru;,, vaccinia and mentation was observed in arly culture inWestern equine encephahtls virus, are not, fertcd with ARC virus. 5. Sensitivity of the fragmentation ability to affected by the presence of RAV in the cells (St’eck and Rubin, 1966). In view of this uliraviolet Tadiation. Focus formation by specific interference, an experiment, was RSV is approximately 20 times more recarried out in which cultures were infectfed sistant t)o inactivation by ultraviolet (UV) wit,h serial half-log dilutions of RAV, in- radiation than is plaque formation by NDV cubated for 3 days, transferred, and chal- (Rubin and Temin, 1959; Levinson and lenged with RSV. The data in Table 3 Rubin, 1966). The reason for the extrademonstrate that preinfection by RAV ordinary resistance of RSV to inactivation inderferes with the formation of mult,ilobed by UV radiation is unknown. Since this nuclei ; this finding suggeststhat t,he causn- resistance to UV radiation is an important t.ive agent of nuclear fragmentation is RSV. characteristic of RSV (and of other nvian 4. Nuclear fragmentation by other strains oj lcukosis viruses), an experiment’ was done t,o RSV. Since it is unlikely t,hnt st,ocks of determine the UV sensit,ivity of the fragseveral different, strains of RST’ would con- mcnt’ation ability. Bs illustrat,ed in Fig. 2, INTERFERENCE WITH THE FORMATION FRAGMENTED NUCLEI BY RAVa
OF
FRAGMENTED
NUCLEUS
IN R.SV-INFECTED
CELLS
79
TABLE 4
Dilution of RSV ~~~~. ~~ 1:2 1:4
Focal areas per 100 fields
I:8
I:16 1:32
43 20
11 5 2.7
n Cult,ures were prepared by seeding 4 x 105 cells in 2 ml of medium and were infected with 0.2.ml samples of serial twofold dilutions of RSV. After incubation for 10 hours, the cultures were overlaid stained counted. I 2
I 1 1 4 6 8 UV Dose - minutes
with 2 ml of agar medium at i2 hours and the number
then fixed and of focal areas
1 10
Fra. 2. Ultraviolet sensitivity of the fragmentstion ability. A 2-ml sample of a 1:lO dilution of RSV stock in phosphate-buffered saline was irradiated and the surviving virus was assayed for its ability to form foci and to fragment nuclei according to the procedures in the met.hod section. A sample of NDV was irradiated in the same manner and assayed for plaque formation. The surviving fraction of virus was calculated by dividing the titer of the irradiated virus by the titer of the u11irradiated virus. Fragmentation ability of RSV (A); focus-forming ability of RSV (0); plaqueforming ability of NDV (m) the rate of inact,ivation by UV radiation of the inultilobe-forming ability and of t,he focus-forming ability was the same. In t,his experiment, the 37 % survival dose of the infect,ivity of ?;DV was approximately 30 times more sensitive to inactivation by UV radiation than was t’he 37% survival dose of the infectivity of RSV. Since it is highly unlikely t,hat another virus which is not a member of the avian leukosis group would have precisely the same, unusual, resistance to ultraviolet radiation as does RSV, these results strongly indicate that RSV is the agent which causes nuclear fragmentation. 6. Bjfect of dilution oj the virus stock on ihe Jbl,mation of fragmented nuclei. Since t’hc Bryan high titer stock of RSV contains both RSV and RAV, it is important to determine whether one infectious particle is sufficient to produce fragmentat;ion or
whether the cooperation of two or more particles is needed. Dulbecco and Vogt (19.54) have shown that if one part,icle is sufficient t,hen the response will be a linear function of the dose whereas if two or more particles are required, the response will be a fun&ion of a second or higher power of the dose. In this experiment, cultures were infected with samples of serial twofold dilutions of the RSV stock and overlaid with an agar medium. Three days later the cultures were fixed and stained and the number of focal areas of cells with multilobed nuclei were counted. As shown in Table 4, the number of areas is in linear proportion to the virus dose, indicating that’ one virus particle is sufficient t)o cause fragmentation. This suggests t,hat RSV is capable of fragmenting nuclei without the help of RllV and therefore that mature virus production is unnecessary for fragmentation to occur. 1’. Attempted isolation of a putative fi’aqmentation-inducing virus. As described previously, the NFU:lQ’U ratio is approximately 5 to 10. One possible explanat,ion of t,his ratio is the existence of another virus capable of fragmenting nuclei which is present, in a five- to tenfold higher titer t’han is RSJ’. An attempt to isolat,e t,his virus was mudc by irlf’ect8irlg cultures with samples of serial t#wofold dilutions of RSV stock at the end point of t,he focus-forming ability of RSV. The cultures were incubated for 3 days, transferred, and incubat,ed for 3 more days, at, which time the medium was assayed for it,s
Dilution
of RSV ~.~.~ I:2 1:4 1:8 1: 16 1:32
XFU/ml 2.7 9.0 0.0
x 10” x 10” x 102 0 0
FFU/ml CollAllellt 2.4 x 101 3.1 x lO> 5.5 x 10’ 0
u Cultures were prepared by seeding 1 X 10” cells in 5 ml of medirlm on 50.mm dishes and were infected with 0.2-ml samples of serial twofold dilutions around the end point of focus-forming ability of Its\‘. The ctdtllres were incltbated for 3 days, transferred, and incubated for 3 more dn>-s; at this time the virus in the medium was assayed for fragmenting ability atld for focus formation.
ability to produce foci and multilobed nuclei. As shown in Table 5, focus formation and t#he production of multilobed nuclei end at approximately t,he same dilution. It’ is insidered doubtful, therefore that :I caontamnating virus capnblc of fragmenting nuclei is present in t,he RSV stock. This experiment also shows t’hnt RSV contained in :L tissue culture medium is capable of causing fr:lgmcntation; this indicates that’ a nonviral fact)or possibly present in RSV tumour homogenate is not, responsible for fragment:ltion. In view of t,he seven lines of evideme presented, it is concluded that RSV muses fragmentation of t,he nucleus of cshick embryo cells.
was observed. In the second experiment, serial t,enfold dilutions of the B-l Hitchner NDV stock (4 X log IU/ml) were used to infect cult’ures which were fixed and stained 3 d:iys aft,er infection. So nuclear fragmentation was observed at any virus concentration. In t,he third experiment,, 4 X lo4 I’FU of t,he cytopathic Hcrtfordshire strain of 9DV were used to infect, cultures. At 1.5, 5, and 20 hours after infection, cultures were fixed and stained. These t’imes were chosen t’o fall during the latent, period, the first log growth period, and after several cycles of reinfec%ion. No nuclear fragmentation was seen at any time. These results indicate t)hat NDV does not cause nuclear fragment,ntion. 1Zquirement of Both Calf Serum and l’ryptosc Phosphate Bf,ofh JOT* Nuclear Fra~pzcnlafion
Bader (1966) has report’ed that the presence of serum in t’he medium is required for the initiation of RSV synthesis. In order to determine whet#her serum is necessary for nuclear fragmentation to occur, an experiment was performed in which cells n-cre seeded and infected in Medium 199, then overlaid with a11 agar medium containing either 199 alone or 199 in combination with tither 10 %#tryptose phosphate broth (TPB) or w&h 5 (7 calf serum or with both TPB and calf serum. As illust’rated in Table 6, t’he omission of either TPB or calf strum redurcd the number of focal areas of fragmented nuclei to less t,han ;5%aof t,hc number in the cultures which contained both InaldifyofNe~ccastleDisease I’irus lo l~i~orlucc TI’B and calf serum. Parallel assays for Nuclear Fl.apnPntation focus formation performed in t’he samemanThe ability of a virus with no ltnon-n ner indicate t’hat focus formnt’ion is similarly oncogenic activity, Kewcastle disease virus inhibited. (I\‘DV), to produce fragm&ed nuclei in The number of cells per 100 fields was chick embryo cells was investigated. SDV count,ed to det,erminc whether cell division was chosen for comparison to RSV because could be correlated with nuclear fragmentabot#h viruses have a similar size, shape, and tion. It, was found that. (aellsin 199 alone and chemical composition and because xD1’ iii 199 plus 10c/: TPB did not grow at all, has both cytopnthic and noncyt,opat~hic that cells in 199 plus :5‘5 and 10 % calf serum strains. In the first experiment, cultures were grew moderately well, and that cells in 199 infcctcd with approximately 4 < 10’ infecplus .j ‘3 fet,al calf strum grew as well as tious u&s of the noncytopat,hic (B-l Hitc*h~11s in 199 plus 10 % TPB and Ti‘2 calf ner) strain of KDV. At, daily intjerv:& fol serum. Despite the moderate growth in 199 4 days after infection, cultures were fixed, plus .i % and 10 % calf serum and the good stained and examined for t,he presence of growth in 199 plus 5 7 fet’al calf serum, no fragmentation. So nuclear fragmentation nuclear fragment,at’ion n-as observed in cells
NUCLEIJS
FRAGMENTED TABLE REQUIREMENT TRYPTOSE
contained agar plus
(ILip’
L1
199 199 + lo<,‘, TPB 199 + Yj,s calf serum 199 + 105; calf serum l!l9 + 5’,‘; fetal calf serum 199 + lo<;; TPB + 5’; calf serum
CELLS
6
TABLE
OF BOTH CALF SERGM PHOSPHSTE BROTH FOR FRI(:MENTITIOW~
.AND
RSV
PRODUCTION
IN
kmber of foci Per dish
fragmented nuclei per 100 iields
940
0 0 2
45
81 7
DIFFEREN’P
MEDI.L~L
KSV produced Media
Areas of \klium
IN RSY-INFECTED
1020 1472 1380 2052
0 0 0 0 0
1956
70
L
‘I Cttltures were prepared by seedittg 4 X lo5 cells in 2 ml of Medium 199 and incubated for 3.5 hortrs. The cells were then infected with 1 X lOa FFU of RSV suspended in 0.2 ml of 199. After au incttbatiotr period of 1 ho~tr, the cultttres were overlaid with 2 ml of agar medirtm cout,ainittg diflercttt constituents. The cultttrcs were fixed atld slaitted after 72 hottrs aud couuted in the t~ertal tnatuter. The number of cells per 100 fields was also counted in order t,o determine the extent of t.he cell division. Parallel cult ttres 011 50 mm dishes were prepared it1 the same manner, infected wilh 1 X lo2 FFU in 0.2 ml of 199, and t,hc nttmber of foci formed in the different media was detertnitred.
grown in these media. These result,s demonstrate that cell division and r~~clear fragmentation are not correlated and that media which are able t,o support the growth of cells arc lacking certain factors required to produce fragmented nuclei. Production of RSV without Nuclear Fragmenfatima One explanation of the lack of nuclear fragmentation in the absence of TPB or calf serum is t,hat, RSV synthesis does not occur without these constituents. To test t#his hypothesis, cells were seeded and infcct,ed in Medium 199, mashed 3 times to remove unadsorbed virus, and overlaid with fluid medium containing the different COW stit’uents. Aft’er a 3-day incubation period, the medium was assayed for the presence of RSV by nuclear fragmentation and by focus formation. The results presented in Table 7 show that the amount of RSV synthesized
NW/ml
199 f 10% TPB + 5’x calf serum 199 f lo:;> TPB 199 + 5y0 calf serttm 199
FFU/ml
x
106
1.0
x
105
1.3 x 0.4 x 1.6 x
106 104 104
9.0 3.8 7.3
x X x
104 lo3 102
1.9
* Cultures were prepared by seeding 4 x 105 cells in 2 ml of Medium 199 and incubated for 4 hours. The cells were then infect,ed with 1 X lo1 FFU of RSV suspended in 0.2 ml of 199. rZfter an incubation period of 1 hour, the cultures were washed 3 times with 2 ml of 199 and overlaid wit,h 2 ml of fluid media of different composition. The cultttres were incrtbated for 72 hottrs, at which time t.he medium was harvested, centrifuged at 1500 rpm for 10 mittrttes to remove any cells, and assayed for the presence of RSV by the fragmentation and focus methods.
199 plus 10% TPB was approximately the same as t#hcamount of RSV synthesized in 199 plus 10 % TPB and 5 % calf serum, indicat,ing that the fragmentation process is not expressedin the absenceof the necessary factors in the medium even though large amounts of RSV are synthesized. Comparatively little RSV was made in 199 plus 5% calf serum and in 199 alone, so the failure to form fragmentred nuclei in these media could be due to a decreasein RSV synthesis. Focus assaysof the virus produced in the different media were made in order t,o ensure that it was indeed RSV that was synthesized. As shown in Table 7, the focus assays confirm the results obtained by the fragmentation assay. in
DISCUSSION
Rous sarcoma virus can cause nuclear fragmentation in chick embryo cells. The ability to fragment nuclei appears to be a specific characteristic of RSV since neither RAV nor NDV can fragment nuclei and this phenomenon has not been reported for any other virus (Enders, 1954; Pereira, 1961). It will be of interest to determine whether nuclear fragmentation can be caused by other sarcoma viruses. Fragmentation presents the problem of
a2
LEVINSON
whether morphological integrity is necessary for the proper functioning of the synthetic and control abilities of the nucleus. Is fragmentation a lethal change or is it a step in the malignant transformation? Studies are being carried out to investigate the biological properties and synthetic abilities of cells with fragmented nuclei. The first fragmented nuclei can be seen 36 hours after infection making this one of the earliest cytological changes resulting from tumour virus infect’ion to be noted. Since the latent period of RSV is approximately 12-14 hours (Temin and Rubin, 1959), fragmentation is not directly correlated to events in the replication of the virus. The fragmentation could be due to a factor carried by the virus or could be due t’o a product of viral growth. The extraordinary resistance of nuclear-fragmenting and of focus-forming ability to inactivation by UV light suggeststhat these abilit’ies are related to a small piece of RXA or protein which is carried by the virus. In this case virus production, which requires t’he whole, intact genome would be more sensitive to inactivation than fragmentation or focus formaCon. Experiments designed t,o investigate this hypothesis showed that’ virus growth and transforming abilit,y were equally resistant to UV light (Rubin and Temin, 1959; Levinson, unpublished results), thus demonstrating that the hypothesis is probably incorrect. The appearance of focal areas of cells containing easily recognizable fragmented nuclei provides a rapid, reliable, reproducible assay of the RSV. The titer of RSV in SFU is five- to tenfold higher than t,he titer in FFU. Since it is unlikely that a contaminating virus present in high titer is the cause of the fragmentation, an NFU:FFU ratio of 5 to 10 indicates a striking inefficiency in the focus assay, probably on the level of the growth of the focus to an easily recognizable size. The number of infectious units of RAV in the Bryan high titer strain of RSV is known to be approximately tenfold higher than is the titer of RSV in FFU’s (Hanafusa et al., 1963). The tit’er of RSV in NFU is therefore approximately the same as the titer of RAV in the stock. This 1: 1 relationship between infectious particles of RSV and
RAV in the stock is of interest in view of the helper relationship between RAV and RSV. The Schmidt-Ruppin strain of RSV (SR-RSV) is thought to be not defective since no helper virus has been isolated from SR-RSV stocks (Hanafusa, 1964). While it is not known definitely whether or not, the particular stock of SR-RSV used in these experiments contains a helper virus, the inability to isolate a helper virus (P. Simons, personal communication) suggeststhat there is no helper virus present. This indicates that nuclear fragmentation is caused by RSV alone and that the action of the helper is not necessary to produce fragmentation just as it is not necessary to produce transformation. This conclusion is supported by the observation that Dhe number of NE’U’s is a linear function of the virus dose, which indicates that one virus particle is sufficient to cause fragmentation. As mentioned in the introduction, two reports of nuclear fragmentation in Rous sarcoma cellshave appeared (Tenenbaum and Doljanski, 1942; Gold6 1959). In both cases the transformed cells had been carried, either as a sarcoma or in tissue culture, for long periods and the rela,tionship betvvecn RSV infection and fragmentation was unclear. In a recent paper Stenkvist (1966) described a clone of bovine lung cells transformed by the Engelbreth-Holm strain of RSV which contained cells with fragmented nuclei, indicating that fragmentation can occur in mammalian cells as well as in chick cells. Two extensive cytological studies of RSV infection in chick cells, however, failed to demonstrate nuclear fragmentation. Loomis and Prat’t (1956) inoculated RSV into the wing web of chickens and st,udied the morphological changes daily. Although nuclear changes were seen at 4s to 96 hours such as enlarged nucleoli, margination of chromatin and large, dense eosinophilic st’ructures, no fragmentation was described at any time. Lo et al. (19,55)reported that the first definite cytological change in RSV infect’ed cells occurred at S days after infection and did not observe nuclear fragmcn tation over a period of several mont’hs. It is difficult to say why fragment8ation was not seen in these two studies ; perhaps because
FRAGMENTED
NUCLEUS
the nutritional or physiological conditions, such as the presence of tryptose phosphate broth, were not correct. Nuclear fragmentation does not occur in cells infected with RSV if either TPB or calf serum is omitted from the medium. Bader (1966) has shown that in chick cells seeded in Eagle’s medium without serum, ItKA and protein synthesis continue normally but DNA synthesis is suppressed. If cells cultured in Medium 199 without serum function in a manner similar to cells in Eagle’s medium without serum, then we can conclude that’ it is not a failure of RNA or protein synthesis t,hat prevents nuclear fragmentation. It is possible that, as Bader has concluded, DNA synthesis is required for an early stage in t#he growth of RSV and that it is inhibition of RSV growth that prevents nuclear fragmentation in the absence of serum. However, this cannot be the case when cells are grown in 199 plus 10 ‘% TPB because large amounts of RSV were produced yet no fragmentation was seen. Further studies are in progress to define the role of RSV synthesis in nuclear fragmentation and to characterize the fact.ors in TPB and calf serum which are necessary for t,he expression of the fragmentation process. ACKNOWLEDGMENTS The hospit,ality of Professor M. Abercrombie and other members of his laboratory during the aut,hor’s stay in London is gratefully acknowledged. The technical assistance of Miss Nancy Fletcher and Miss Sue Bevan was greatly appreciat,ed. The avian leukosis-free embryos were supplied by Dr. J. Carr of the Poult.ry Research Centre, Edinburgh. The RSV and RAV stocks were kindly given by Dr. P. Simons of the Imperial Cancer Research Institute, London. The Hertfordshire and B-l Hitchner strains of NDV were kindly given by Dr. E. Martin of the National Institut,e for Medical Research, London, and by Dr. H. Spears of the Central \‘eterinnry Lsboiatory. Weybridge, Surrey, respect#ively. REFERENCES C. (1964). Neoplasms in mammals illduced by Rous chicken sarcoma material. lV:nll. Cancer Inst. Monograph 17, 299-319.
AHLSTROM,
IN
RSV-INFECTED
CELLS
53
BADER, J. P. (1966). Metabolic requirements for infection by Rous sarcoma virus. I. The transient requirement for DNA synthesis. ViroZog?l 29, 444-451. DOUGHERTY, R., and RASMUSSEN, It. (1964). Properties of a strain of Rous sarcoma virus that infects mammals. X&Z. Cancer Inst. Monograph 17 ) 337-350. DCI~BECCO, It., and [‘OGT, M. (1954). Plaque formation and isolation of pure lines with poliomyelitis virus. J. Ezptl. Med. 99, 167-182. ENDERS, J. (1954). Cytopathology of virus infections. Ann. ZZefl. Microbial. 8, 473-498. GOLDI?, A. (19-59). fitude quantitative des modifications cellulaires provoqukes par le virus du sarcome de ROUS dans les cultures de cellules embryonnaires de poulet. Ezpll. Cell Res. 18, 529-541. HANAFUSA, H. (1964). Nature of the defectiveness of Rous sarcoma virus. Nail. Cancer Znst. Monograph 17, 543-556. HANAFUSA, H., HANAFUSA, T., and RUBIN, H. (1963). The defectiveness of Rous sarcoma virus. Proc. L\rutZ. Acad. Sci. U. S. 49,572-580. HANAFUSA, H., HANAFUSA. T., and RUBIN, H. (1964). Analysis of the defectiveness of RSV II. Specification of IZS\antigenicity by helper virus. Proc. Xatl. dead. Sci. U. S. 51, 41-48. LEVINSON, W., and RUBIN, H. (196G). Radiation studies of avian tumor viruses and of Newcastle disease virus. l’irology 28, 533-542. Lo, W., GEY, G., and SHOPRAS, It. (1955). The cytopathogenic effect of the Rous sarcoma virus on chicken fibroblasts in tissue culture. Bull. Johns Hopkins Hosp. 9i, 248-264. LOOMIS, L., and PRAYIT, A. (1956). The histogenesis of Rous sarcoma I induced by partially purified virus. J. N&Z. Cancer Inst. 17, 101-120. YEREIRA, H. (1961). The cytopathic effect of animal viruses. Iitlvan. Virus IZes. 8, 245-285. RUBIN, H., and TEMIN, H. (1959). A radiological study of cell-virus interaction in the Rous sarcoma. virology 7, 75-91. STECK, F. T., and RUBIS, H., (1966). The mechanism of interference between an avian leukosis virus and Rous sarcoma virus. Virology 29, 628641. STENKVIST, B. (1966). Clonal analysis of bovine cells morphologically transformed in vitro by by Itous sarcoma virus. Acta. Pathol. Microbial. Stand. 67, 201-219. TEMIN, H., and RUBIN, H. (1959). A kinet,ic study of infect,ion of chick embryo cells in vitro by Rous sarcoma virus. Virology 8, 209-222. TENNENBAUM, E., and UOLJANSKI, L. (1942). Studies on Rous sarcoma cells cultivated in vitro. Cancer Res. 3, 585-603.
32, 73-83
(1967)
Fragmentation
of the
Nucleus Chick
in Rous Embryo
WARRES Department
of Zoology, dccepled
Sarcoma
Cells
LEVINSON’~ Universit~y January
Virus-Infected
College,
2 London,
England
27, 1367
Infection of chick embryo cells with Rous sarcoma virus causes fragmentation of the nucleus as early as 36 hours after infection. Neither Rous-associated virus nor Newcastle disease virus causes nuclear fragmentation. The presence of both tryptose phosphate broth and calf sertlm in the medium are required for fragmentation to occur. An assay procedllre for 110~1s sarcoma virus based upon nuclear fragmentation is described.. INTRODUCTION
embryos (avian leukosis virus free) by mincing and treatment with 0.25 % trypsin (Difco) in Tris-buffered saline. The cells (8 to 10 x 106) were seeded onto 90-mm plastic tissue culture dishes (Falcon Plastics, Los Angeles, California) in 12.5 ml of primary medium which contained 8.3 parts of single-skength Jledium 199 (Glaxo Laboratories, Greenford, England), 1.0 part of trypt’ose phosphate brot’h (Difco), 0.4 part of calf serum, 0.1 part of chick serum, and 0.2 part of 2.S% NaHC03. The cultures were incubated at 39’ in a humidified atmosphere containing air plus sufficient CO, to keep the medium at pH 7.3. Secondary cultures were made by treating the primary cultures with 0.05% trypsin followed by seeding 4 X lo5 cells in 2 ml of secondary medium ont’o 33-mm plastic tissue culture dishes. Secondary medium differed from primary medium only in that it contained 0.5 part of calf serum and no chick serum. The cells were infected either in suspension or as a monolayer on the surface of the dish and overlaid with 2 ml of an agar medium after a suitable adsorption period. The agar medium was composed of 4.2 parts of l.S % agar (Difco-Bacto) 4.2 parts of double strength Medium 199, 1.0 part tryptosc phosphat’e broth, 0.5 part wlf serum, and 0.2 part of 2.8 % SaHC03. Primary and seconda,ry cultures of mouse
In the course of recent experiments concerned with the behavior of Rous sarcoma virus-infected chick cells, extensive fragmentation of t#he nucleus was observed. Kuclear fragment,ation has been described in cells growing out of Rous sarcoma explant’s (Tenenbsum and Doljanski, 1942), and brief mention of fragment’ed nuclei was made in a study of the morphological changes in chick embryo cells following infection with Rous sarcoma virus (Gold& 1959). No information, however, has been published on the precise virus-cell interaction required for nuclear fragmentation. In view of the importance of proper nuclear function for the survival and multiplication of bot,h nonmalignant and malignant cells, experiments Were performed in order to determine the nature of the fragmentation process. The results of these experiments will be presented in this paper. MATERIALS
AND
METHODS
Tissue cultu~z. Primary cultures of chick embryo cells were prepared from lo-day-old 1 Holder of British-American Exchange Fellowship PF-288 awarded by the American Cancer Society. 2 Present address: Department of Microbiology, Medical School, 17niversitjy of California, San Francisco, California. 74
FRAGMENTJW
NUCLEUS
embryo cells were prepared from 14-day-old embryos in t,he same way as were the chick cell cuhures. I’irus. The Bryan high-titer strain of Rous sarcoma virus (RSV) and a Rous-associated virus (RAV-1) isolated from the RSV stock were used. In one series of experiments, the Schmidt-Ruppin and Bryan standard strains of RSV and the ARC virus isolated by Dougherty and Rasmussen(1964) were used. A noncytopathic strain (B-l Hitchner) and a cytopathic strain (Hertfordshire) of Newcastle disease virus (NDV) were employed to determine the effect of a virus which is not a member of the avian leukosis group. The focus assay for RSV and the plaque assay for KDV have been described previously (Levinson and Rubin, 1966). Histology. The cultures were fixed and stained 72 hours after infection unless otherwise stated. Prior to fixation, 2 ml of %[edium 199 was added to the cultures in order to loosen the agar. After incubation at’ 39” for 15 minutes, the agar was separated from the sides of the dish with a glass rod and discarded. The cells were fixed in 4?$ formaldehyde in phosphate-buffered saline (pH 7.2) for 10 minutes. The formal saline was removed and 33% ethanol was added for 5 minutes. After t’he removal of the 35% ethanol, t’he cultures were exposed to 70%. ethanol for 30 minutes followed by 35%~ ethanol for 1 minute The cultures were stained wit)h Harris’ hematoxylin for 10 minutes, “blued” with 6 rinses in tap water, and mounted in Farrant#‘s medium (G. Gurr, London, England). The fixation and staining procedures were carried out at room temperature. Microscopy. The cultures were examined under phase contrast at 430 X magnification using a 50 X oil immersion objective with a Wild AI-20 microscope. When quantitative result,s were desired, an ocular grid 49 mm” which circumscribed an arca of 2.64 X lo4 ,J‘Jof the tissue culture dish was used. The number of focal areas of cells containing fragmented nuclei were counted in at least 100 fields. The fields to be counted were determined by chance by moving the dish t,hrough 5 or more rows of 20 fields per row. Since the area of a dish 33 mm in diameter is S.55 X 108/$ and the area, of the dish
IN KS\:-INFECTED
CELLS
75
circumscribed by the grid is 2.64 X 104/$, there are 3.2 X lo4 fields per dish. If, for example, the number of areas of multilobed nuclei per 100 fields was counted, then the titer of the RSJ’ inoculum was calculated by multiplying the number of areas containing multilobed nuclei by 3.2 X 102. Antiserum. Antiserum against RAV and therefore against the RSV (RAV-1) in the Bryan stock was made by inoculating 4week-old White Leghorn chickens with lo6 infectious unit8s of RAV1 intravenously followed by monthly hleedings. Cltl-aviolet irradiation. A 2-ml sample of a 1: 10 dilution of RSV stock in phosphatebuffered saline was irradiated in a 50-mm plastic tissue culture dish. The out’put of the lamp was calibrated biologically using NDV as a standard and was shown to be approximately 15 ergs/mm2/sec at a distance of 30 cm from t,he lamp. RESULTS
Appearance of theFragmentedNuclei Secondary cultures of chick embryo fibroblasts were infect,cd wit,h the Bryan hightiter strain of Rous sarcoma virus (RSV) and grown under the conditions described in the Materials and Methods section. As early as 36 hours after infection, cells containing nuclei which had fragment,ed into two or more pieces were obscrvcd. Within 72 hours after infection, the nuclei fragmented into as many as 10 grapelike lobeswith a thin strand connecting each lobe. Many of the lobes contained a densely staining area resembling a nucleolus. lcigurc 1 illustrat,es the t,ypical appearance of a group of cells with fragmented nuclei. This figure also showsseveral nonfragmented nuclei which are represemative of those seen in normal, uninfected fibroblasts. If the infected cultures are overlaid with an agar medium in order to prevent recurrent cycles of virus infection, then the focal areas of cells containing fragmented nuclei can be counted and an accurate picture of the time of appearance of the fragmented nuclei can be obt,ained. As shown in Table 1, the first multilobed nuclei appear at 36 hours after infection, followed by a great incrcasc in the number of focal areas of
76
LEVINSON
FIG. 1. Microscopic appearance of fragmented nuclei can be SCCII on the left. Normal rells with ficntion: X 680.
nuclei. regular,
multilobed nuclei by GO hours, and n slower rate of increase during the succeeding 36 hours. Cells whose nuclei appear IO be in t.hc early stages of the fragmentat’ion prowss frequently surround the focal area of cells with obviously fragment,ed nuclei. Thrse cells in t,hr early stages contain nuclei n it,11a, scalloped or indented edge. In t#he later stages of the fragment’ation process, which a11 be seen at SC, hours after infe&on, t,hc strand c~onne6ng t’he many lobes of t,hc nucalei disappears and cells containing several small, da,rkly stained bodies cm1 bc observed. dt no stage in the fragmentat~ion process does any structure seem to bc intruding into the nwleus and the t80tal area of stained nuclear material does not appear to decrease, this indicates that t,hc fragmentation is not, due to dissolut,ion or loss of staining affinity of portions of the nucleus. The appearance of the fragmented nuclei is the same mhet,her the cult,ures are kept’ in fluid medium or overlaid with an agar medium. Ut,ilizing the data in Table 1, the st,andnrd
h focal area of cells containing fragmented oval llrlclei can be seen on the right. Magni-
conditions for a nuclear fragmenting unit (Xl%) were defined as a focal nrca of cells CYJIltaiIlhg fragmented nuclei formed on 4 X 1Oj cells at, 72 hours after infect,ion. In this case, since there were 3S focal areas in the 100 grid fields counted and 3.2 X lOA grid fields per dish (see Materials and I\Iethode section), there were 1.2 X lOA focal areas per dish. Since the culture was infected with 0.2 ml of a 1:40 dilution of RST’ stock, the titer of t,he stock was 2.3 X lo6 Sl~U/ml. The tit’er of the RSV stjock was approximately 2.5 to 3 X 10” focus-forming units (VIW)/ml giving an SE’U:FFU ratio of 5 to 10. This discrepancy between t)he results of the nuclear fragmentat~ion arid t)he focusformation assays will be invcstigat~cd nrld discussed in subsequent parts of this paper. Evidence That Nuclear Fragnmz fation Caused by Rous S’amma 17ims
Is
Some RST; st,ocks cont,ain other members of the avian leukosis group of viruses which function as helper viruses for the defect,ivc RSV (Hannfusn cf al., 1964). Mince the
FRAGMENTED TABLE: TIME
OF A\~~~.\~.\~~~
NUCLEUS
IN
I%\--1NFECTEI)
ii
CELLS
1 OF FRAGMENTEO
NT(‘LEI“
Hours
after 24 30 36 18 60 72 84 96
infection
Focal
areas/100
fields
0 0 1 6 30 38 53 64
tl Cultures were prepared by seeding 4 X lo5 cells in 2 ml of medium and were infected by adding 1.2 X lo3 FFU of Bryan “high titer” strain of RST- in 0.2 ml of medium. After a 12.hour incubation period at 39”, the cultures were overlaid with 2 ml of agar medium, then fixed and stained at intervals of 6 or 12 hours. The number of focal ‘areas contaitrirtg cells with fragmented nuclei was counted at 430 X magnification.
stock of RSV used i11 this study is known to contain Rous-associated virus (RAY-l). (P. Simons, personal communication), cxperiments were performed to det,crmine which virus causes nuclear fragmentation. 1. Inhibition of f~agmenfation by RA T’ antiserum. Antiserum against8 RAV-1 will inactivate t#he infectivity of both RAY-1 and RSV with a RAV-1 out#er coat (Hanafusa, 1964). RAV :&serum cannot t-hereforc be used to distinguish between RSV (RAT:-1) and RAV-1 but can be used as an initial step in the identification of t’hese viruses. An experiment was performed to determine the rate of inactivation by RAT’ ant,iserum of t,he nuclear fragmenting ability and qf the focus-forming abilit,y of t,he Bryan high-titer stock of RSV. Appropriate dilutions of the RSV stock and of t,he RAV antiserum were mixed and incubated at 37”. Samples were taken at 7 and at 14 minutes and assayed for the formation of multilobed nuclei and for focus formation. The data presented in Table 2, demonstrate that RAY antiserum innct,ivatcd the ability to fragment nuclei and the ability t,o form foci at the same rate. Although this experiment) indicates that nuclear fragment,ation is wused by either RSTT or RAV, it cannot be ruled out that a third virus present in t,hc RAT’ stock used to
Minutes of incubation at Ili” 7 14
T‘raition
of RSV surviving antiserum
Fragmentation
Focus assay
aSa>
1 .5 X lo--“’ 5.0 x 10-z
1.2 7.5
x x
10-t 10-Z
‘6 A 0.1.ml sample of RS1. srtspetisioti containing lo5 FFU was mixed with 0.1 ml of a 1:30 diltttiott of ItA\. antiserum attd 0.8 ml of secotldary tnrdium. A control mixture containing 0.1 ml of the ItSI’ suspension and 0.9 ml of secondary medium was also made. These mislrtres were incubated at 37” for 7 attd 11 mittutes, at which time samples were removed a11d assayed for sttrvivittg virus by the fragmetrtatiott and by the foctts-assay methods. h k:arh number represeiits the sttrviving fractiott of RS\- calcrtlated by dividing the ttter of IiS\. exposed to attttscrrtm by the titer of IiS\. not, Psposd
to
&lltisel.llIll.
make t,hc ant,iserum and present in t,he Bryan st’ocak of RSS’ c-ould be t#he agent rcqonsiblc for nwlear fragmentation. 2. dbsenrc oj’ j’ra~mcntation in Rd Jr-inJ’~tctl cells. Two experiments were performed to d&ermine whether infwtion with RAY alone would product nuclear fragmentation. In t#he first experiment, cultures cont,aining 4 X 10” chick embryo cells wcrc infected with 4 X lo4 infect,ious units (NJ) of RAY-I, overlaid J\ ith agar medium, then fixed and stained at’ daily intwvnls for 1 days. Ko fragmented nwlei were seen at any t,imc in the RAY-infected wlls. l’arallel cult,urcs infwted with RSV developed t,he expec%cd number of foci of frugment,ed nuclei. In the swond experiment, cultures containing 4 X 10” cells Jvere infected wit,h an undiluted sample and with :L sample of 4 serial tenfold dilutions of a stock of RAY-1 n-hich titercd :tpproximat,cly 2 X IO6 IV/ml. The wlturcs wew incubated for 3 days, at, which time they were transferred and seeded at a (‘onwnt8rntion of 4 X 10” cells. The cultures were overlaid with ngar, incubated for 3 days, and fixed and stained. So nuclear fragmentation was observed in any of the Rr2V-infcctcd cultures. The nurlci of the
LEVINSON
7s TABLE
3
tain a contaminating virus capable of fragmenting nuclei, stocks of the Bryan standard strain, the Schmidt-Ruppin (SR) strain, and the Bryan high t,iter strain (prepared Relative independently from the high titer stock Focal areas sensitivity Log dilution per 100 fields to RSV of RAV used throughout this study) were tested for their ability to produce multilobed No RAV 3G 1 nuclei. It was found that suit’able dilutions 7 0.19 -5 of the stocks of all the strains tested pro1 -4.5 0.028 duced focal areas of typical fragmented 0 <0.028 -4 nuclei. The titer of the stocks of the Bryan (I Cultures were prepared by seeding 1 X 106 standard strain, the SR strain, and this ceils in 5 ml of medium onto 50-mm plastic tissue independently prepared Bryan high titer culture dishes. The cells were infected with 0%ml strain were 3.2 X lo’, 3.5 X 104, and 7.7 X samples of serial half-log dilutions of RAT- and 10’ NW/ml, respectively. The finding that incubated for 3 days. The cultures were then other strains of RSV produce fragmented transferred and 4 x lo5 cells seeded in 2 ml of nuclei supports the hypothesis that RSV is medium on 33-mm dishes. The cells were infected the cause of the nuclear fragmentation. with 1 X 103FFU of RSV in 0.2 ml of medium, then The SR strain of RSV can causesarcomas overlaid with 2 ml of agar medium. The cultures in mice (AhlstrBm, 1964). In view of this, were fixed and stained 72 hours after infection, and the ability of SR-RSV to fragment the the number of focal areas of cells with fragmented nuclei was counted. nuclei of mouse embryo cells was tested. So nuclear fragmentation was observed in RAV-infected cells appeared similar in every cultures of mouse embryo cells to which way to those of the cells in uninfected cul- SR-R.SV ha’d been added. High concentrations of the Bryan high titer strain of RSV tures treated in the same manner. Infection with an appropriate dilution of RSV stock also failed t,o cause fragmentation of the of these uninfected cells causedthe expected nuclei of t’hc mouse embryo cells. Dougherty and Rasmussen (1964) have number of focal areas of fragmented nuclei, demonstrating that the cells were capable of isolat,ed a virus which they have named hRC (another Rous contaminant) from t,he nuclear fragmentation. &cl; of the SR-RSV used in this study. 3. Interference with the formation of fraymcnted nuclei by preinfection with RAI’. One Since it is possible that the virus in the SRcriterion which can be used to determine stock which causes nuclear fragment’ation whether nuclear fragmentation is caused by was ARC rather than SR-RSV, a stock of RSV is the specific interference of RSV in pure ARC virus was tested for its ability to RAV-infected cells. Ot,hcr viruses, such as produce fragmentation. No nuclear fragKcwcastle disease viru;,, vaccinia and mentation was observed in arly culture inWestern equine encephahtls virus, are not, fertcd with ARC virus. 5. Sensitivity of the fragmentation ability to affected by the presence of RAV in the cells (St’eck and Rubin, 1966). In view of this uliraviolet Tadiation. Focus formation by specific interference, an experiment, was RSV is approximately 20 times more recarried out in which cultures were infectfed sistant t)o inactivation by ultraviolet (UV) wit,h serial half-log dilutions of RAV, in- radiation than is plaque formation by NDV cubated for 3 days, transferred, and chal- (Rubin and Temin, 1959; Levinson and lenged with RSV. The data in Table 3 Rubin, 1966). The reason for the extrademonstrate that preinfection by RAV ordinary resistance of RSV to inactivation inderferes with the formation of mult,ilobed by UV radiation is unknown. Since this nuclei ; this finding suggeststhat t,he causn- resistance to UV radiation is an important t.ive agent of nuclear fragmentation is RSV. characteristic of RSV (and of other nvian 4. Nuclear fragmentation by other strains oj lcukosis viruses), an experiment’ was done t,o RSV. Since it is unlikely t,hnt st,ocks of determine the UV sensit,ivity of the fragseveral different, strains of RST’ would con- mcnt’ation ability. Bs illustrat,ed in Fig. 2, INTERFERENCE WITH THE FORMATION FRAGMENTED NUCLEI BY RAVa
OF
FRAGMENTED
NUCLEUS
IN R.SV-INFECTED
CELLS
79
TABLE 4
Dilution of RSV ~~~~. ~~ 1:2 1:4
Focal areas per 100 fields
I:8
I:16 1:32
43 20
11 5 2.7
n Cult,ures were prepared by seeding 4 x 105 cells in 2 ml of medium and were infected with 0.2.ml samples of serial twofold dilutions of RSV. After incubation for 10 hours, the cultures were overlaid stained counted. I 2
I 1 1 4 6 8 UV Dose - minutes
with 2 ml of agar medium at i2 hours and the number
then fixed and of focal areas
1 10
Fra. 2. Ultraviolet sensitivity of the fragmentstion ability. A 2-ml sample of a 1:lO dilution of RSV stock in phosphate-buffered saline was irradiated and the surviving virus was assayed for its ability to form foci and to fragment nuclei according to the procedures in the met.hod section. A sample of NDV was irradiated in the same manner and assayed for plaque formation. The surviving fraction of virus was calculated by dividing the titer of the irradiated virus by the titer of the u11irradiated virus. Fragmentation ability of RSV (A); focus-forming ability of RSV (0); plaqueforming ability of NDV (m) the rate of inact,ivation by UV radiation of the inultilobe-forming ability and of t,he focus-forming ability was the same. In t,his experiment, the 37 % survival dose of the infect,ivity of ?;DV was approximately 30 times more sensitive to inactivation by UV radiation than was t’he 37% survival dose of the infectivity of RSV. Since it is highly unlikely t,hat another virus which is not a member of the avian leukosis group would have precisely the same, unusual, resistance to ultraviolet radiation as does RSV, these results strongly indicate that RSV is the agent which causes nuclear fragmentation. 6. Bjfect of dilution oj the virus stock on ihe Jbl,mation of fragmented nuclei. Since t’hc Bryan high titer stock of RSV contains both RSV and RAV, it is important to determine whether one infectious particle is sufficient to produce fragmentat;ion or
whether the cooperation of two or more particles is needed. Dulbecco and Vogt (19.54) have shown that if one part,icle is sufficient t,hen the response will be a linear function of the dose whereas if two or more particles are required, the response will be a fun&ion of a second or higher power of the dose. In this experiment, cultures were infected with samples of serial twofold dilutions of the RSV stock and overlaid with an agar medium. Three days later the cultures were fixed and stained and the number of focal areas of cells with multilobed nuclei were counted. As shown in Table 4, the number of areas is in linear proportion to the virus dose, indicating that’ one virus particle is sufficient t)o cause fragmentation. This suggests t,hat RSV is capable of fragmenting nuclei without the help of RllV and therefore that mature virus production is unnecessary for fragmentation to occur. 1’. Attempted isolation of a putative fi’aqmentation-inducing virus. As described previously, the NFU:lQ’U ratio is approximately 5 to 10. One possible explanat,ion of t,his ratio is the existence of another virus capable of fragmenting nuclei which is present, in a five- to tenfold higher titer t’han is RSJ’. An attempt to isolat,e t,his virus was mudc by irlf’ect8irlg cultures with samples of serial t#wofold dilutions of RSV stock at the end point of t,he focus-forming ability of RSV. The cultures were incubated for 3 days, transferred, and incubat,ed for 3 more days, at, which time the medium was assayed for it,s
Dilution
of RSV ~.~.~ I:2 1:4 1:8 1: 16 1:32
XFU/ml 2.7 9.0 0.0
x 10” x 10” x 102 0 0
FFU/ml CollAllellt 2.4 x 101 3.1 x lO> 5.5 x 10’ 0
u Cultures were prepared by seeding 1 X 10” cells in 5 ml of medirlm on 50.mm dishes and were infected with 0.2-ml samples of serial twofold dilutions around the end point of focus-forming ability of Its\‘. The ctdtllres were incltbated for 3 days, transferred, and incubated for 3 more dn>-s; at this time the virus in the medium was assayed for fragmenting ability atld for focus formation.
ability to produce foci and multilobed nuclei. As shown in Table 5, focus formation and t#he production of multilobed nuclei end at approximately t,he same dilution. It’ is insidered doubtful, therefore that :I caontamnating virus capnblc of fragmenting nuclei is present in t,he RSV stock. This experiment also shows t’hnt RSV contained in :L tissue culture medium is capable of causing fr:lgmcntation; this indicates that’ a nonviral fact)or possibly present in RSV tumour homogenate is not, responsible for fragment:ltion. In view of t,he seven lines of evideme presented, it is concluded that RSV muses fragmentation of t,he nucleus of cshick embryo cells.
was observed. In the second experiment, serial t,enfold dilutions of the B-l Hitchner NDV stock (4 X log IU/ml) were used to infect cult’ures which were fixed and stained 3 d:iys aft,er infection. So nuclear fragmentation was observed at any virus concentration. In t,he third experiment,, 4 X lo4 I’FU of t,he cytopathic Hcrtfordshire strain of 9DV were used to infect, cultures. At 1.5, 5, and 20 hours after infection, cultures were fixed and stained. These t’imes were chosen t’o fall during the latent, period, the first log growth period, and after several cycles of reinfec%ion. No nuclear fragmentation was seen at any time. These results indicate t)hat NDV does not cause nuclear fragment,ntion. 1Zquirement of Both Calf Serum and l’ryptosc Phosphate Bf,ofh JOT* Nuclear Fra~pzcnlafion
Bader (1966) has report’ed that the presence of serum in t’he medium is required for the initiation of RSV synthesis. In order to determine whet#her serum is necessary for nuclear fragmentation to occur, an experiment was performed in which cells n-cre seeded and infected in Medium 199, then overlaid with a11 agar medium containing either 199 alone or 199 in combination with tither 10 %#tryptose phosphate broth (TPB) or w&h 5 (7 calf serum or with both TPB and calf serum. As illust’rated in Table 6, t’he omission of either TPB or calf strum redurcd the number of focal areas of fragmented nuclei to less t,han ;5%aof t,hc number in the cultures which contained both InaldifyofNe~ccastleDisease I’irus lo l~i~orlucc TI’B and calf serum. Parallel assays for Nuclear Fl.apnPntation focus formation performed in t’he samemanThe ability of a virus with no ltnon-n ner indicate t’hat focus formnt’ion is similarly oncogenic activity, Kewcastle disease virus inhibited. (I\‘DV), to produce fragm&ed nuclei in The number of cells per 100 fields was chick embryo cells was investigated. SDV count,ed to det,erminc whether cell division was chosen for comparison to RSV because could be correlated with nuclear fragmentabot#h viruses have a similar size, shape, and tion. It, was found that. (aellsin 199 alone and chemical composition and because xD1’ iii 199 plus 10c/: TPB did not grow at all, has both cytopnthic and noncyt,opat~hic that cells in 199 plus :5‘5 and 10 % calf serum strains. In the first experiment, cultures were grew moderately well, and that cells in 199 infcctcd with approximately 4 < 10’ infecplus .j ‘3 fet,al calf strum grew as well as tious u&s of the noncytopat,hic (B-l Hitc*h~11s in 199 plus 10 % TPB and Ti‘2 calf ner) strain of KDV. At, daily intjerv:& fol serum. Despite the moderate growth in 199 4 days after infection, cultures were fixed, plus .i % and 10 % calf serum and the good stained and examined for t,he presence of growth in 199 plus 5 7 fet’al calf serum, no fragmentation. So nuclear fragmentation nuclear fragment,at’ion n-as observed in cells
NUCLEIJS
FRAGMENTED TABLE REQUIREMENT TRYPTOSE
contained agar plus
(ILip’
L1
199 199 + lo<,‘, TPB 199 + Yj,s calf serum 199 + 105; calf serum l!l9 + 5’,‘; fetal calf serum 199 + lo<;; TPB + 5’; calf serum
CELLS
6
TABLE
OF BOTH CALF SERGM PHOSPHSTE BROTH FOR FRI(:MENTITIOW~
.AND
RSV
PRODUCTION
IN
kmber of foci Per dish
fragmented nuclei per 100 iields
940
0 0 2
45
81 7
DIFFEREN’P
MEDI.L~L
KSV produced Media
Areas of \klium
IN RSY-INFECTED
1020 1472 1380 2052
0 0 0 0 0
1956
70
L
‘I Cttltures were prepared by seedittg 4 X lo5 cells in 2 ml of Medium 199 and incubated for 3.5 hortrs. The cells were then infected with 1 X lOa FFU of RSV suspended in 0.2 ml of 199. After au incttbatiotr period of 1 ho~tr, the cultttres were overlaid with 2 ml of agar medirtm cout,ainittg diflercttt constituents. The cultttrcs were fixed atld slaitted after 72 hottrs aud couuted in the t~ertal tnatuter. The number of cells per 100 fields was also counted in order t,o determine the extent of t.he cell division. Parallel cult ttres 011 50 mm dishes were prepared it1 the same manner, infected wilh 1 X lo2 FFU in 0.2 ml of 199, and t,hc nttmber of foci formed in the different media was detertnitred.
grown in these media. These result,s demonstrate that cell division and r~~clear fragmentation are not correlated and that media which are able t,o support the growth of cells arc lacking certain factors required to produce fragmented nuclei. Production of RSV without Nuclear Fragmenfatima One explanation of the lack of nuclear fragmentation in the absence of TPB or calf serum is t,hat, RSV synthesis does not occur without these constituents. To test t#his hypothesis, cells were seeded and infcct,ed in Medium 199, mashed 3 times to remove unadsorbed virus, and overlaid with fluid medium containing the different COW stit’uents. Aft’er a 3-day incubation period, the medium was assayed for the presence of RSV by nuclear fragmentation and by focus formation. The results presented in Table 7 show that the amount of RSV synthesized
NW/ml
199 f 10% TPB + 5’x calf serum 199 f lo:;> TPB 199 + 5y0 calf serttm 199
FFU/ml
x
106
1.0
x
105
1.3 x 0.4 x 1.6 x
106 104 104
9.0 3.8 7.3
x X x
104 lo3 102
1.9
* Cultures were prepared by seeding 4 x 105 cells in 2 ml of Medium 199 and incubated for 4 hours. The cells were then infect,ed with 1 X lo1 FFU of RSV suspended in 0.2 ml of 199. rZfter an incubation period of 1 hour, the cultures were washed 3 times with 2 ml of 199 and overlaid wit,h 2 ml of fluid media of different composition. The cultttres were incrtbated for 72 hottrs, at which time t.he medium was harvested, centrifuged at 1500 rpm for 10 mittrttes to remove any cells, and assayed for the presence of RSV by the fragmentation and focus methods.
199 plus 10% TPB was approximately the same as t#hcamount of RSV synthesized in 199 plus 10 % TPB and 5 % calf serum, indicat,ing that the fragmentation process is not expressedin the absenceof the necessary factors in the medium even though large amounts of RSV are synthesized. Comparatively little RSV was made in 199 plus 5% calf serum and in 199 alone, so the failure to form fragmentred nuclei in these media could be due to a decreasein RSV synthesis. Focus assaysof the virus produced in the different media were made in order t,o ensure that it was indeed RSV that was synthesized. As shown in Table 7, the focus assays confirm the results obtained by the fragmentation assay. in
DISCUSSION
Rous sarcoma virus can cause nuclear fragmentation in chick embryo cells. The ability to fragment nuclei appears to be a specific characteristic of RSV since neither RAV nor NDV can fragment nuclei and this phenomenon has not been reported for any other virus (Enders, 1954; Pereira, 1961). It will be of interest to determine whether nuclear fragmentation can be caused by other sarcoma viruses. Fragmentation presents the problem of
a2
LEVINSON
whether morphological integrity is necessary for the proper functioning of the synthetic and control abilities of the nucleus. Is fragmentation a lethal change or is it a step in the malignant transformation? Studies are being carried out to investigate the biological properties and synthetic abilities of cells with fragmented nuclei. The first fragmented nuclei can be seen 36 hours after infection making this one of the earliest cytological changes resulting from tumour virus infect’ion to be noted. Since the latent period of RSV is approximately 12-14 hours (Temin and Rubin, 1959), fragmentation is not directly correlated to events in the replication of the virus. The fragmentation could be due to a factor carried by the virus or could be due t’o a product of viral growth. The extraordinary resistance of nuclear-fragmenting and of focus-forming ability to inactivation by UV light suggeststhat these abilit’ies are related to a small piece of RXA or protein which is carried by the virus. In this case virus production, which requires t’he whole, intact genome would be more sensitive to inactivation than fragmentation or focus formaCon. Experiments designed t,o investigate this hypothesis showed that’ virus growth and transforming abilit,y were equally resistant to UV light (Rubin and Temin, 1959; Levinson, unpublished results), thus demonstrating that the hypothesis is probably incorrect. The appearance of focal areas of cells containing easily recognizable fragmented nuclei provides a rapid, reliable, reproducible assay of the RSV. The titer of RSV in SFU is five- to tenfold higher than t,he titer in FFU. Since it is unlikely that a contaminating virus present in high titer is the cause of the fragmentation, an NFU:FFU ratio of 5 to 10 indicates a striking inefficiency in the focus assay, probably on the level of the growth of the focus to an easily recognizable size. The number of infectious units of RAV in the Bryan high titer strain of RSV is known to be approximately tenfold higher than is the titer of RSV in FFU’s (Hanafusa et al., 1963). The tit’er of RSV in NFU is therefore approximately the same as the titer of RAV in the stock. This 1: 1 relationship between infectious particles of RSV and
RAV in the stock is of interest in view of the helper relationship between RAV and RSV. The Schmidt-Ruppin strain of RSV (SR-RSV) is thought to be not defective since no helper virus has been isolated from SR-RSV stocks (Hanafusa, 1964). While it is not known definitely whether or not, the particular stock of SR-RSV used in these experiments contains a helper virus, the inability to isolate a helper virus (P. Simons, personal communication) suggeststhat there is no helper virus present. This indicates that nuclear fragmentation is caused by RSV alone and that the action of the helper is not necessary to produce fragmentation just as it is not necessary to produce transformation. This conclusion is supported by the observation that Dhe number of NE’U’s is a linear function of the virus dose, which indicates that one virus particle is sufficient to cause fragmentation. As mentioned in the introduction, two reports of nuclear fragmentation in Rous sarcoma cellshave appeared (Tenenbaum and Doljanski, 1942; Gold6 1959). In both cases the transformed cells had been carried, either as a sarcoma or in tissue culture, for long periods and the rela,tionship betvvecn RSV infection and fragmentation was unclear. In a recent paper Stenkvist (1966) described a clone of bovine lung cells transformed by the Engelbreth-Holm strain of RSV which contained cells with fragmented nuclei, indicating that fragmentation can occur in mammalian cells as well as in chick cells. Two extensive cytological studies of RSV infection in chick cells, however, failed to demonstrate nuclear fragmentation. Loomis and Prat’t (1956) inoculated RSV into the wing web of chickens and st,udied the morphological changes daily. Although nuclear changes were seen at 4s to 96 hours such as enlarged nucleoli, margination of chromatin and large, dense eosinophilic st’ructures, no fragmentation was described at any time. Lo et al. (19,55)reported that the first definite cytological change in RSV infect’ed cells occurred at S days after infection and did not observe nuclear fragmcn tation over a period of several mont’hs. It is difficult to say why fragment8ation was not seen in these two studies ; perhaps because
FRAGMENTED
NUCLEUS
the nutritional or physiological conditions, such as the presence of tryptose phosphate broth, were not correct. Nuclear fragmentation does not occur in cells infected with RSV if either TPB or calf serum is omitted from the medium. Bader (1966) has shown that in chick cells seeded in Eagle’s medium without serum, ItKA and protein synthesis continue normally but DNA synthesis is suppressed. If cells cultured in Medium 199 without serum function in a manner similar to cells in Eagle’s medium without serum, then we can conclude that’ it is not a failure of RNA or protein synthesis t,hat prevents nuclear fragmentation. It is possible that, as Bader has concluded, DNA synthesis is required for an early stage in t#he growth of RSV and that it is inhibition of RSV growth that prevents nuclear fragmentation in the absence of serum. However, this cannot be the case when cells are grown in 199 plus 10 ‘% TPB because large amounts of RSV were produced yet no fragmentation was seen. Further studies are in progress to define the role of RSV synthesis in nuclear fragmentation and to characterize the fact.ors in TPB and calf serum which are necessary for t,he expression of the fragmentation process. ACKNOWLEDGMENTS The hospit,ality of Professor M. Abercrombie and other members of his laboratory during the aut,hor’s stay in London is gratefully acknowledged. The technical assistance of Miss Nancy Fletcher and Miss Sue Bevan was greatly appreciat,ed. The avian leukosis-free embryos were supplied by Dr. J. Carr of the Poult.ry Research Centre, Edinburgh. The RSV and RAV stocks were kindly given by Dr. P. Simons of the Imperial Cancer Research Institute, London. The Hertfordshire and B-l Hitchner strains of NDV were kindly given by Dr. E. Martin of the National Institut,e for Medical Research, London, and by Dr. H. Spears of the Central \‘eterinnry Lsboiatory. Weybridge, Surrey, respect#ively. REFERENCES C. (1964). Neoplasms in mammals illduced by Rous chicken sarcoma material. lV:nll. Cancer Inst. Monograph 17, 299-319.
AHLSTROM,
IN
RSV-INFECTED
CELLS
53
BADER, J. P. (1966). Metabolic requirements for infection by Rous sarcoma virus. I. The transient requirement for DNA synthesis. ViroZog?l 29, 444-451. DOUGHERTY, R., and RASMUSSEN, It. (1964). Properties of a strain of Rous sarcoma virus that infects mammals. X&Z. Cancer Inst. Monograph 17 ) 337-350. DCI~BECCO, It., and [‘OGT, M. (1954). Plaque formation and isolation of pure lines with poliomyelitis virus. J. Ezptl. Med. 99, 167-182. ENDERS, J. (1954). Cytopathology of virus infections. Ann. ZZefl. Microbial. 8, 473-498. GOLDI?, A. (19-59). fitude quantitative des modifications cellulaires provoqukes par le virus du sarcome de ROUS dans les cultures de cellules embryonnaires de poulet. Ezpll. Cell Res. 18, 529-541. HANAFUSA, H. (1964). Nature of the defectiveness of Rous sarcoma virus. Nail. Cancer Znst. Monograph 17, 543-556. HANAFUSA, H., HANAFUSA, T., and RUBIN, H. (1963). The defectiveness of Rous sarcoma virus. Proc. L\rutZ. Acad. Sci. U. S. 49,572-580. HANAFUSA, H., HANAFUSA. T., and RUBIN, H. (1964). Analysis of the defectiveness of RSV II. Specification of IZS\antigenicity by helper virus. Proc. Xatl. dead. Sci. U. S. 51, 41-48. LEVINSON, W., and RUBIN, H. (196G). Radiation studies of avian tumor viruses and of Newcastle disease virus. l’irology 28, 533-542. Lo, W., GEY, G., and SHOPRAS, It. (1955). The cytopathogenic effect of the Rous sarcoma virus on chicken fibroblasts in tissue culture. Bull. Johns Hopkins Hosp. 9i, 248-264. LOOMIS, L., and PRAYIT, A. (1956). The histogenesis of Rous sarcoma I induced by partially purified virus. J. N&Z. Cancer Inst. 17, 101-120. YEREIRA, H. (1961). The cytopathic effect of animal viruses. Iitlvan. Virus IZes. 8, 245-285. RUBIN, H., and TEMIN, H. (1959). A radiological study of cell-virus interaction in the Rous sarcoma. virology 7, 75-91. STECK, F. T., and RUBIS, H., (1966). The mechanism of interference between an avian leukosis virus and Rous sarcoma virus. Virology 29, 628641. STENKVIST, B. (1966). Clonal analysis of bovine cells morphologically transformed in vitro by by Itous sarcoma virus. Acta. Pathol. Microbial. Stand. 67, 201-219. TEMIN, H., and RUBIN, H. (1959). A kinet,ic study of infect,ion of chick embryo cells in vitro by Rous sarcoma virus. Virology 8, 209-222. TENNENBAUM, E., and UOLJANSKI, L. (1942). Studies on Rous sarcoma cells cultivated in vitro. Cancer Res. 3, 585-603.