studies on the reproductive and cell-converting abilities of avian sarcoma visuses

VIROLOGY 43, 427-441 (1971)

Studies on the Reproductive and Cell-Converting Abilities of

Avian Sarcoma Visuses

THOMAS GRAF, HEINZ BAUER, HANS GELDERBLOM, AND

DAN1 P. BOLOGNESI’

Max-Plan&-Znstitut fiir Virusjorschung, Biologisch-Medizinische Abteilung, Ttibingen, Gerwbany

With an appendix by ROLF J. LORENZ

Bundesjorschungsanstalt jar Viruskrankheiten der Tiere, Tiibingen, Germany

Accepted October 13, 1970

In order to study whether or not only a part of the genome of avian sarcoma viruses (ASV) is sufficient for focus formation and/or for virus reproduction we attempted to isolate two classes of ASV mutants. A search was made for virus particles which have lost their ability to reproduce but are still able to convert chicken cells; and particles which have lost their ability to convert cells but are still able to reproduce.

Particles of the first class were not found. Single foci induced by three ASV strains treated with either hydroxylamine, G°Co-y-, X-, or UV-rays all produced converting virus. Another ASV strain studied (B77 virus) was exceptional in that hydroxylamine-treated virus induced foci which regressed after subsequent subcultivations. Although these foci revealed no virus detectable in the focus assay, electron microscopic studies showed the presence of C-type particles. In addition three low virus- producing mutants were isolated from the B77 strain, one of which induced the formation of regressing foci.

Mutants of the second class were found which, although infectious, do not convert chicken embryo fibroblasts. These nonconverting mutants were obtained from ASV strains of subgroups A and D and have envelope properties similar to their corresponding sarcoma virus strains. A tentative model for the genome of avian sarcoma viruses is proposed. Whereas the whole genome of ASV is needed for the induction of proliferating foci only a part of it is sticient for the production of virus particles.

INTRODUCTION Bauer, 1970). These results conflicted with In earlier work it was shown that several the work of Gold& and Latarjet (1966),

functions in the genome of avian myelo- who reported the isolation of mutants from blastosis virus could be selectively inac- 6oCo-irradiated Schmidt-Ruppin Rous sar-

tivated (Graf and Bauer, 1970). Similar coma virus (SRV), which were able to con-

studies on the relative target size for the vert cells in the chicken chorioallantois oncogenicity (defined as the focus forming membrane without production of converting ability in chicken embryo fibroblast cul- virus. These authors postulated that the tures) of an avian sarcoma virus, led us to oncogenicity of ASV is coded for by only a conclude that the entire genome is involved fraction of the genome. While our work wan in t,he expression of this function (Graf and in progress another report by Gold6 ap-

1 Recipient of Public Health Service Postdoc- peared, in which the isolation of non-virus-

toral Fellowship No. I-F2-CA-38 592-01 from the producing (NVP) cells from chicken em- National Cancer Institute. bryo fibroblast cultures was described

427

428 GRAF ET AL.

(Gold& 1970a). Since the SRV strain used by Gold4 is different from SRV-1, the strain which we had used in the earlier studies, the question arose whether the conflicting results were caused by differences in the virus strains. Therefore, this problem was reinvestigat,ed with three further ASV strains, including the strain of Gold& Extended data obtained with SRV-1 will be also presented. As shown in the first part of this work, NVP foci were not detected with any of the three SRV strains studied after mutagenization under various conditions, However, regressing NVP foci were obtained from the B77 strain.

In the second part of this communication the isolation and partial characterization of nonconverting mutants of ASV will be described.

MATERIALS AND METHODS

Cells. Chicken embryo fibroblast (CEF) cultures were prepared according to the method of Temin and Rubin (1958). They were grown at 38.5” in plastic tissue culture dishes (Greiner und Sohne, Ntirtingen, Germany) using a modified Eagle’s medium which contained 4-8 % calf serum. Cells were prepared from chickens from two sources: the line 15, maintained in our institute, was obtained from Dr. B. R. Burmester, East Lansing, Michigan, in 1964. The second flock, Spafas, which was originally obtained from Dr. R. Luginbuhl, Storrs, Connecticut, is maintained by Loh- mann & Co., Cuxhaven, Germany. We screened single embryos of both chicken lines over a period of several years by complement fixation, interference tests and electron microscopy and detected no leukosis virus.

In these studies C/O type cells from both chicken lines were used. They were susceptible to viruses from subgroups A, B, C, and D (Vogt and Ishizaki, 1965; Duff and Vogt, 1969). Cells resistant to infection with subgroup A viruses (C/A cells) and cells re- sist’ant to subgroup B viruses (C/B cells) were derived from the chicken line 15 and Spafas, respectively. About 50% of the embryos from line 15 chickens were susceptible to infection with Rous sarcoma virus 0 (RSV (O)), whereas all the 60 chicken

embryos tested from the Spafas line were resistant to this virus strain.

Viruses. Schmidt-Ruppin strain 1 of RSV (SRV-1) a member of subgroup A of avian RNA tumor viruses; B77 virus (B77V) belonging to subgroup C; and RSV(0) were generously supplied by Dr. P. K. Vogt, Seattle, Washington. Two pseudotypes of the Bryan high titer strain of RSV, RSV(RAV-1) and RSV(RAV-2) of subgroup A and B, respectively, were obtained through the kindness of Dr. P. M. Biggs, Houghton, Huntingdon, U.K. Rous associated virus 1 (RAV-1) and RAV-2 were isolated from these virus stocks by the end- point dilution interference technique (Rubin and Vogt, 1962). A cloned SRV strain of subgroup D was kindly provided by Dr. A. Gold& Paris, France. SRV-H, another member of subgroup D, was isolated from SRV converted hamster cells as previously described (Bauer and Graf, 1969). Stocks of SRV-1 and SRV-H were prepared in CEF cultures infected by virus which had been cloned three times. No associated’ leukosis virus was detected in these stocks by the endpoint dilution interference technique.

Focus assay. The focus assay was performed according to the procedure described by Temin and Rubin (1958). CEF cultures containing 1 X lo6 cells per dish (5 cm in diameter) were infected within 5 hours after seeding by the addition of 0.1 to 1 ml of the sample to be tested. Foci were counted macroscopically 7-10 days after infection. In order to increase the sensitivity (Toyo- shima and Vogt, 1969) 2 pg polybrene (EGA-Chemie KG, Steinheim/Albuch, Ger- many) per milliliter were added to the assay dishes in most of the experiments with viruses of subgroups C and D.

Viral interference (Rubin, 1961) was determined by infecting CEF cultures with a nonconverting avian tumor virus and challenging with an avian sarcoma virus 7 days (two subcultivations) later. Non- converting viruses associated with ASV were isolated by the end-point dilution interference assay as described by Rubin and Vogt (1962).

Neutralizing sera were prepared by in- travenous injection of undiluted stock virus

AVIAN SARCOMA VIRUS INACTIVATION STUDIES 429

into 3-month-old chicken. The birds were bled 4 weeks later.

Neutralization test. Virus suspended in 0.4 ml of phosphate-buffered saline, pH 7.2, containing 10% calf serum was incubated with 0.1 ml of antiserum at 37”. CEF cultures were inoculated 40 min thereafter with the entire reaction mixture.

Cell-free solutions were prepared by filter- ing the sample through Millipore AP 2501300 prefilters. When 1 X lo6 chicken cells were filtered in repeated experiments, no cells were detected in the filtrate.

Cloning of avian sarcoma virus. Single foci of converted cells in CEF cultures containing less than 10 foci were aspirated through the agar with a narrow Pasteur pipette and transferred to a tube containing 1 ml of phosphate-buffered saline with 10 % calf serum. The material was then suspended, filtered cell-free and inoculated into CEF cultures. This procedure was repeated twice more with the foci appearing 7 days later. After the last cloning the infected cells were subcultivated until they were completely converted. Cell-free supernat’ants of these cultures were used as virus stocks.

Examination of the virus production of single foci. Single foci from dishes containing less than 15 foci were aspirated through the agar overlay with a narrow Pasteur pipette and seeded onto fresh CEF cultures in dishes (3 cm in diameter) containing 3 X lo5 cells. The cultures were subcultivated thereon at successive 3- to 4-day intervals until they became completely converted. (In cultures inoculated with foci induced by wild-type ASV, one to two subcultivations were necessary for complete conversion.) The supernatants of these cultures were filtered cell-free and assayed for focus- forming virus. In order to rule out a possible spread of converting virus under the agar overlay, cells of normal morphology were cloned from five different areas of a focus assay dish in every experiment. Converting virus was not detected in any of these controls.

Mutagenic treatments. (1) Hydroxylamine (HA): A 2 M solution of HA-hydrochloride (Merck AG, Darmstadt, Germany) was prepared in phosphate-buffered saline. The pH of the solution was adjusted to 7.5 wit,h

NaOH. Virus samples containing S-15% calf serum were distributed in several tubes and incubated in a water bath (Ultrather- mostat Colora) at a constant temperature of 20”. At various intervals thereafter equal volumes of 2 M HA solution were added to the tubes and the chemical reaction was terminated by a simultaneous transfer of the tubes to an ice bath. The samples were then dialyzed at 4” in phosphate-buffered saline until all HA was removed. This was tested with a micromethod which allows the detection of as little as lo+ M HA (Feigl, 1954). (2) The Vo-irradiation was kindly performed by the Willy Riisch Company, Waiblingen, Germany. The virus samples containing 10% calf serum were sealed in ampules and kept in dry ice (-76”) during irradiation. An average value of the dose rate was calculated from the result obtained after irradiation of several dosimeters to- gether with the virus samples. Before testing in the focus assay the virus samples were mixed with calf serum to give a 40% solution and incubated for 30 minutes at 37” in order to facilitate the dispersion of virus particles (Gold& and Latarjet, 1966). (3) X-irradiation: A Miiller RT 100 unit operating at 100 kV and 8 mA was used. Two milliliters of the virus, suspended in phosphate-buffered saline containing calf serum, were irradiated without filter from a distance of 2 cm in a closed plastic dish (3 cm in diameter). The delivery of the ma- chine under these conditions was about 10,000 roent,gen per minute. (4) For the ultraviolet (LTV)-irradiation, 2 ml of the virus suspension in Eagle’s medium containing 30% calf serum were added to an open plastic dish (8.5 cm in diameter) and irradiated under continuous agitation. The light source (Niederdruckbrenner NK 20/40, Quarzlampengesellschaft, Hanau, Germany) delivered about 13 erg/mm*/sec at a distance of 32 cm.

Electron microscopy. Single foci of converted cells were marked on the culture dish and fixed in a 5 % glutaraldehyde solution. Cell sections were prepared as described by Gelderblom et al. (1970). To screen the cell sections for the presence of virus particles, a Siemens Elmiskop, model Ia, was

430 GRAF ET AL.

used at an instrument magnification of 20,000.

RESULTS

Search for NVP Foci among Foci Induced by Mutagen- Treated SR V-l

A sample of about 106FFU/ml of SRV-1 was treated for 8 hours with HA, which resulted in an inactivation of focus formation of 2.6 logs. Single foci induced by this virus sample were tested for the production of converting virus. If only part of the ASV genome is necessary for focus formation, foci should be found which do not produce converting virus. No such NVP foci were found among t,he 104 foci examined in several experiments (Table 1). This result could be due to a neculiar mode of action

mammalian cells (Bauer et al., 1969; Vigier and Montagnier, 1966; Hlavayova et al., 1964). The results obtained with these viruses after HA-treatment and/or 6oCo- irradiation are presented in Table 2. While all the cultures inoculated with foci induced by SRV-H and SRV-Gold6 synthesized converting virus, 4 out of 13 of the cultures inoculated with foci induced by HA-treated B77V did not produce any virus detectable in the focus assay. However, a low virus production could not be excluded because of their relatively low content of converted

TABLE 1

SEARCH FOR NVP FOCI AMONG FOCI INDUCED BY MUTAGEN-TREATED SRV-1

of the HA. For exa*mple, it is possible that genes not needed for focus formation are protected against the reaction with the HA

Mutagen

solution. Therefore, the effect of other mutagens, namely 6oCo-y-, X-, and UV- rays, was tested. As was the case with HA, HAb irradiation of SRV-1 under these conditions 6oCo-y-rays

did not lead to the production of NVP foci xzt?’ “@ (Table 1). -

Dose

8 Hr 3.15 Mradd 240 Set

240 Set

Search for NVP Foci among Foci Induced $-fight by Three Other Mutagen-Treated ASV

8 Min

Strains a Focus-forming units.

Inactivation of FFUa

in log

2.6 f 0.2e 2.7 f 0.2 2.6 f 0.2

1.5 f 0.2

2.8 f 0.2

NW foci

total foci

o/104 O/26 o/19

o/20

o/35

The failure to detect NVP foci induced * Data summarized from seven experiments.

by mutagen-treated SRV-1 could reflect a c During irradiation the virus was suspended

peculiarity of this virus strain. Therefore, in low or high concentrations of calf serum (CS)

further ASV &rains were studied. SRV-H, in order to favor indirect or direct radiation

SRV-Gold& and B77V differ from SRV-1 effects (Luria and Exner, 1941), respectively.

not only in their envelope properties, but d Megarad. e Standard deviation estimated from repeated

also in that t’hey are capable of converting titrations of the same virus sample.

TABLE 2

SEARCH FOR NVP FOCI AMONG FOCI INDUCED BY MUTAGEN-TREATED SRV-H, SRV-GOLD&, AND B77V

ASV strain Mutagen Dose Inactivation of FFU (log)

NVP foci

total foci

SRV-H HA 8 Hr 2.0 f 0.2 O/52 6oCo-r-rays 3.15 Mrad 2.5 & 0.2 o/43

SRV-Gold4 G°Co-r-rays 2.87 Mrad 2.3 f 0.2 O/18 B77V HA 8 Hr 2.2 f 0.2 45/13

u The percentage of converted cells in the cultures inoculated with these foci was 70.1 at the time of test,ing for production of focus-forming virus.


cells. Moreover, the few converted cells dis- appeared completely after 4-5 subcultivations. Such a regression was not observed with foci of any of the SRV strains, although it was noticed that some of them had to be subcultivated more than twice in order to obtain conversion of all cells.

The next set of experiments was devised in order to clarify whether the inability to find regressing “NVP” foci with the SRV strains reflected a strain difference to B77V or if it was due to some unknown variable in the experimental conditions. Therefore, the ASV strains SRV-1, SRV-H, SRV-Gold& and B77V were compared in a single experiment. Cultures were inoculated with appropriate dilutions of HA-treated and control virus samples. Ten days later foci were isolated, suspended vigorously in 1 ml of phosphate-buffered saline in order to dis- perse the cells, and inoculated onto two dishes with chicken cells. One of these cultures was then observed for the spread of conversion after subsequent subcultivations as well as for its capacity to produce converting virus. The second culture was screened electron microscopically for the production of virus particles. Again, as shown in Table 3, neither regressing nor NVP foci were obtained from SRV, but two regressing “NVP” foci were found with mutagenized B77V.

TABLE 3

COMPARISON, IN ONE EXPERIMENT, OF THE PRO-

PORTION OF REGRESSING AND OF NVP FOCI

AMONG FOCI INDUCED BY FOUR DIFFERENT ASV STRAINS TREATED WITH HA

ASV strain

SRV-1

SRV-H

SRV-Gold6

B77V

HOUIS of

treatment

Inactivation of FFU (log)

egressin foe

otal foci

:r i.

t

\TVP foci

otal foci

0 0 Oh-5 O/6 8 2.6 f 0.2 o/13 o/13 0 0 O/8 O/8 8 2.0 f 0.2 o/14 o/14 0 0 O/23 O/23 8 2.8 f 0.2 O/28 O/28 0 0 o/10 o/10 8 2.2 f 0.2 2/17 la/17

- a The percentage of converted cells in the cul- a This number was calculated according to a

tures inoculated with these foci was 70.1 at the formula of Dougherty and di Stefano (1965) : virus time of testing for production of focus-forming particles per cell = virus particles per cell section virus. times 500.

In addition to these, three foci with abnormal properties were found. They will be described in more detail in the following section.

Studies on the Abnormal Foci Induced by HA-Treated B77V

Three types of foci with abnormal behavior were found. The foci of a first type (clones 31 and 40) regressed completely after 4 to 5 subcultivations on CEF culbures and did not produce any virus detectable in the focus assay. An electron microscopic examination of the foci, however, revealed the presence of C-type particles in low amounts (Table 4). The finding of virus particles in the regressing foci raised the question as to whether t,he latter were superinfected by nonconverting mutants of B77V which might inhibit the spread of conversion. In order to study this question, several experiments were performed with the two cultures in which the regression was observed and with a noninfected control culture. In a first experiment the cultures were subcultivated twice and divided in two parts. One part was infected with lo4 FFU of B77V, the other was kept as a control. After one further subcultivation the infected cultures were completely converted. This showed t,hat they mere susceptible to B77V. In a second experiment

TABLE 4

SEBRCH WITH THE ELECTRON MICROSCOPE FOR

VIRUS PARTICLES IN ABNORMAL FOCI INDUCED BY HA-TRE.ZTED B77V

Foci with abnormal properties

Focus induced by nontreated B77V

F ‘

-

‘ecus ~1OIle I

No CI

--

31

40 38 42 43 -

Num- ber of ell set tions

80 5 30 196 57 145 60 13 110 45 104 1150 56 167 1450 26 48 900

Total number of virus >articles

found

Virus particles Per

cella

432 GRAF ET AL.

the supernatanbs of the cultures subcultivated five times were tested for their content of leukosis virus in the interference assay. No agent interfering with sarcoma viruses of subgroups A, B, C, or D could be detected. That these cultures did not contain any avian RNA tumor virus in larger amounts was also indicated by the result of a complement fixation assay performed as described by Bauer et al. (1965). No group-specific (gs) -antigen could be detected in extracts of 2 X 10’ cells of cultures subcultivated seven times. Thus it seems unlikely that the observed particles represented infectious, nonconverting virus which might have superinfected t,he regressing foci.

Clone 38 represents a second type of focus with abnormal properties. In the cultures inoculated with this clone conversion spread very slowly until about 10% of the cells were converted after lo-12 subcultivations. A superinfection with RAV-1 or RAV-2 did not accelerate this process. The supernatants of the cultures were tested before

each subcultivation in the focus assay and remained negative until the 10th passage. From that time on, a focus-forming virus was detected which reached its highest titer after five further subcultivations. Foci induced by this virus were smaller than those from wild-type virus. They regressed when transferred to CEF cultures. Photographs of one of four foci studied, taken at different intervals after cloning on chicken feeder cells, are shown in Fig. 1.

The results obtained from host range and neutralization experiments indicate that clone 38 virus has envelope properties characteristic for subgroup C viruses. Chicken cells of types C/O, C/A, and C/B as well as cells either susceptible or resistant to RSV (0) were equally susceptible to both clone 38 virus and B77V. A chicken serum specific for subgroup C neutralized clone 38 virus to a similar extent as the wild type B77 virus (Table 5). The titers obtained from the supernatants of cultures converted wit,h clone 38 virus were lower by a factor of 20 to 50 than those obtained from the

FIG. 1. Growth on CEF cultures of cells converted by clone 38 virus. The numbers indicate the days after transfer of the focus. The black ring in each picture marks the position of the focus. X 30.


wild type of B77V. This was consistent with the virus production per cell as determined with the electron microscope (Table 4). While no associated leukosis virus could be detected in repeated experiments with stocks of this virus clone, the reproductive ability of clone 38 virus was strongly en- hanced when cells were infected in the presence of subgroup A helper virus (Table 6). A similar enhancement was observed when infected cells were maintained at relatively high temperatures (Fig. 2), indicating that the defective function of the clone 38 virus is not temperature sensitive in the range examined.

TABLE 5

NEUTRALIZATION ASSAY WITH VIRUS DERIVED FROM ABNORMAL Focus CLONES INDUCED

BY HA-TREATED B77V

Chicken sera”

AntiiB77V Normal serum

Clone 38 virus lb 107 Clone 42 virus 2 260 Clone 43 virus 2 75 RSV(RAV-1) (A)” 162 118 RSV(RAV-2) (B) 21 23 B77V (Cl 0 152 SRV-H 0) 22 44

a The sera were used in a dilution of 1:25 b Number of foci per dish. c Avian tumor virus subgroup.

TABLE 6

EFFECT OF HELPER VIRUS ON THE REPRODUCTION OF CLONE

38 VIRUSa

Virus Helper virus added

RAV-1 NC-SRV-lb

a Separate C/O type CEF cultures were infected with either 250 FFU of B77V or with both 250 FFU of B77V and about lo6 infectious units of helper virus. The supernatants were tested 3 days later in the focus assay.

0 This virus is described later in this paper (see Table 9).

c FFU per milliliter.

I I I 4 5 6

B?7VWl

I I I

3 L 5

Days attu infection -

FIQ. 2. Growth curves of clone 38 virus as compared to the wild type of B77V (B77V wt). Sepa- rate C/O type CEF cultures were infected with 250 FFU of virus and incubated at 36”, 38.5”, and 41”, respectively. The supernatants were har- vested 3, 4, and 5 days later and frozen at -70”; their virus content was determined in the focus assay.

The focus clones of a third type (42 and 43) resembled clone 38 in that the spreading of conversion in the inoculated CEF cultures was also delayed, but in this case the delay was only for 4 to 5 subcultivations. A focus-forming virus isolated thereafter was neutralized with a serum specific for subgroup C (Table 5). The titers of virus clone 43 were consistently lower than those of the wild type of B77V by a factor 5 to 10, whereas those of clone 42 were reduced only by a factor 2 to 5. In addition, an agent interfering with subgroup C virus was detected in these virus “clones” by end point dilution interference assay. This finding might explain the relatively high virus production as observed in the electron micro- scrape (Table 4). The experiments described in the following chapter suggest the possi-

434 GRAF ET AL.

TABLE 7

PROPERTIES OF ABNORMAL FOCI INDUCED BY HA-TREATED B77V

Focus tw

Clone No. Production Production of Production of

Behavior of foci on CEF cultures of virus focus-forming nonconverting

particle9 subgroup C virus

subgroup C virus

31, 40 Regression after 4-5 subcultiva- Yes No” No tions

38 Conversion of all cells after Yes Yes No 10-12 subcultivations

42, 43 Conversion of all cells after 4-5 Yes Yes Yes subcultivations

Q See Table 4. b See footnote in Table 3.

bility that the nonconverting viruses found evident that there was no major difference in clones 42 and 43 represent mutants of in titer between the nonconverting viruses B77V. and the corresnondina sarcoma viruses.

The properties of the five abnormal focus- clones are summarized in Table 7. Properties of NC-SRV-1 and NC-XRV-H

Isolation of Nonconverting Viruses from HA- Treated SRV-1 and SRV-H

If only a fraction of the genome of ASV is necessary for virus reproduction, it should be possible to obtain mutants which have lost their ability to convert chicken cells but, which are still able to reproduce. The following experiments were devised in order to detect such mutants. Samples of SRV-1 and SRV-H were incubated for eight hours with HA. This resulted in an inactivation of focus formation by a factor of about 102.5. One milliliter of t’hese samples was inoculated onto CEF cultures, and separate dishes received a dilution of the nontreat’ed virus containing about the same amount of FFU. After two subcultivations, when most of the cells in t’he cultures appeared to be converted, the supernatants were examined for the presence of nonconverting virus by the end-point dilution interference assay. In two separate experiments with SRV-1 and in three with SRV-H, nonconverting viruses interfering with SRV-1 and SRV-H, respectively, were isolated from progeny of HA- treated virus, but not from progeny of nontreated virus. These nonconverting interfering viruses were designated NC-SRV-1 and NC-SRV-H, respect’ively. The results obtained in one experiment with each of the SRV st,rains are shown in Table S. It is

Chicken embryo fibroblasts infected with either of the NC-SRV strains did not, form colonies in agar suspension in experiments performed with a modified technique of ,1 Iacpherson and Montagnier (1964)) although NC-SRV-H infected cells somewhat resembled converted cells morphologically, In contrast to CEF cultures infected with NC-SRV-1, cells infected with NC-SRV-H showed a cytopathic effect in that’ they divided at a much lower rate than noninfected controls. The supernatants of CEF cultures infected with either of the NC- SRV’s contained gs-antigen of avian RNA tumor viruses, as determined by the method of Bauer et al. (1965). Furthermore, an electron microscopic examinabion of these cells revealed the presence of particles typical of this virus group. The interference patterns of the two strains (Table 9) indicate that they have envelope properties similar to the corresponding sarcoma viruses. NC-SRV-1 interfered only with RSV (RAV-l), indicating that it belongs to subgroup A, as does SRV-1. Likewise, NC- SRV-H interfered best with SRV-H, although marked titer reductions were also observed on cells superinfected with A, B, and C viruses. That this was probably due to the cytopathic effect already mentioned is supported by serological data. A chicken serum prepa,red against NC-SRV-H neu-


TABLE 8

ISOL.ITION OF NONCONVERTING VIRUS FROM HA-TREATED SRV BY THE END POINT Drr INTERFERENCE TECHNIQUE

Sample derived from cultures ino- Dilutions of SRV-1 culated with treated&or not trea- ,(+5 ,65.0 ,65-5 ,$.o ,$.5 ,67.0 ,67.5 ,$.o

ted virus

nc )ntre ,ated

;reat ed

-

Dilutions of SRV-H

nontreated

treated

a SRV treated 8 hours with hydroxylamine.

Converted cultures.

JJTION

L-l Nonconverted cultures susceptible to viruses fran subgroup A,B,C, and D.

tralized group D virus and, to a slight extent, B virus, but failed to neutralize viruses of the other subgroups (Table 10). A cross reaction between subgroup B and D has been observed earlier (Bauer and Graf, 1969; Duff and Vogt, 1969).

In order to test whether the NC-SRV’s are capable of phenotypic mixing, separate C/O cells converted with RSV(0) were infected with about lo6 infectious units of

NC-SRV-1 or NC-SRV-H. Three days later the supernatants of these cultures were assayed on C/O type cells resistant to RSV(0). They contained 3 X lo4 and 2 X lo3 focus-forming virus per milliliter respectively, indicating that pseudotypes of RSV (Rubin, 1965) had been formed.

Marker rescue experiments were performed in order to determine whether NC- SRV-1 st,ill contains genetic information

456 GRAF ET AL

TABLE 9

INTERFERENCE PATTERNS OF NC-SRV-1 AND NC-SRV-H”

Cells preinfected with

NC-SRV-1 NC-SRV-H

Relative plating &i&n&&

0 C/O type CEF cultures were inoculated with about lo6 infectious units of NC-SRV and subcultivated twice before challenge with ASV.

b Plating efficiency in uninfected cells = 1.0. c Avian tumor virus subgroup.

TABLE 10

CROSS NEUTRALIZATION EXPERIMENT WITH A

CHICKEN SERUM PREPARED

AGAINST NC-SRV-H”

I / Chicken serab

RSV(RAV-1) A 60” 67 70 RSV (RAV-2) B 35 93 195 B77V C 124 166 130 SRV-H D 1 4 208 RSV (0) Unclassified 93 71 102

0 The samples were assayed on C/O cells from the line 15 chicken flock.

b The sera were used at a dilution of 1:25. c Number of foci per dish.

TABLE 11

INACTIVATION RATE OF IRRADIATED SRV ON

CELLS PREINFECTED WITH A NON-

CONVERTING MUTANT OF SRVa

Expt. No.

Inactivation of FFU (in log) as measured on

NC-SRV-1 cells Control cells

1 1.86 2.18 2 2.01 2.34

a A sample of SRV-Gold4 was exposed to 2.87 Mrad and titrated in comparison to a nonirradi- ated sample on cells preinfected with NC-SRV-1 and on uninfected control cells. The titers were calculated on the basis of focus counts obtained from 4 (Expt. 1) and 8 (Expt. 2) dishes for each dilution of the virus sample.

for cell conversion. The 6oCo-inactivation rate of SRV-Gold6 was determined on cells preinfected with NC-SRV-1 and compared to the rate determined on uninfected cells. The results of two such experiments show that the inactivation rate of SRV is lower when determined on preinfected cells (Table 11). This probably means that the nonconverting virus rescued sarcoma viruses defective in their ability for focus formation.

DISCUSSION

Work on DNA tumor viruses has shown that only part of their genome is sufficient to convert mammalian cells (Basilic0 and di Mayorca, 1965; Benjamin, 1965; Gershon et al., 1965; Latarjet et al., 1967; Defendi et al., 1968). Results are described in the present paper which indicate a different situation with avian RNA tumor viruses.

Single foci induced by four ASV strains treated with various mutagens (hydroxylamine ; ‘j°Co-y-, X-, and UV-rays) were tested for their ability to produce focus- forming virus. As in previous studies with SRV-1 (Graf and Bauer, 1970), non-virus- producing (NVP) foci could not be detested with the three SRV strains tested. How- ever, foci with abnormal properties were obtained from the B77 virus. While some of these regressed after subsequent subcultivations and did not produce focus-forming virus in detectable amounts, others showed a decreased proliferating ability and a reduced virus production. That such abnormal foci were obtained only with the B77 strain must not necessarily represent an absolute difference to the SRV strains. It is conceivable that under the culture conditions used, foci induced by low virus-producing mutants of SRV would proliferate even less than foci of similar mutants of B77V and would have been too small to be detected.

Concerning the origin of t)he regressing foci, two possible explanations may be con- sidered :

1. They are induced by mutants which reproduce poorly. This interpretation is supported by the finding of regressing foci containing low amounts of virus particles as determined by electron microscopy. In ad-


dition, mutants were isolated (B77V clones 38, 42, 43) which induce the formation of converted cells having a decreased proliferating ability. These mutants show a reduced ability to produce converting virus and might be partially defective in a gene function involved in virus reproduction. Whet)her mutants which are totally defective in this function would still be able to convert single cells cannot be decided on the basis of our studies.

2. Regressing foci are induced by mutants which produce noninfectious particles. This possibility is indirectly supported by results obtained with HA-treated AMV (Graf and Bauer, 1970). It would suggest that only a fraction of the genome is sufficient for the formation of regressing foci.

The mechanism of focus regression re- mains to be clarified. If this occurs as a result of segregation of converted cells into normal and converted daughter cells, this would not be consistent with the provirus- integration hypothesis of Temin (1964). However, preliminary experiments suggest that such a segregation does not occur and that converted regressing cells are actually outgrown by normal cells.

The results presented indicat,e that no particles capable of inducing proliferating NVP foci exist in populations of mutagenized ASV. Since it is known that a focus grows predominantly by the multiplication of primarily infected and converted cells, not by superinfection of adjacent cells (Temin and Rubin, 1958), it seems likely that proliferating NVP foci would have the same size as virus-producing foci and therefore would not have been overlooked in our studies.

In order to determine whether the amount of foci tested under the various conditions of mutagenization is sufficient to just.ify the conclusion that t’he target size for the ability t,o induce proliferating foci cf> is the same as the target size for the ability to produce converting virus (T), a statistical calculation was performed.

Under the assumption that the whole ASV genome is needed for r, the relative target size for the formation of proliferating foci: F ( = f/r) would be equal to 1. The

observed value of F is dependent on the degree of inactivation of the focus-forming virus (K) and the number of foci (n) tested. If no NVP foci are found, the increase in K or n leads asymptotically to the value of 1. A lower value of F can be calcuIated from the upper limit of the proportion of NVP foci (p,J still compatible with the number of foci studied which were all found to produce: virus. The derivation of a formula

Ft = K

K - log (1 - pu)

in which this relation is expressed is described in an appendix to this paper. The calculation of F on the basis of K is dependent on the assumption that the inactivation kinetics of the virus follows a first-order reaction. This has been shown to be the case with the infectivity of avian myeloblastosis virus and with the focus- forming ability of SRV-1 after HA-treatment (Graf and Bauer, 1970).

The results of the calculations in which the data from HA- and 6oCo-treated SRV-1, SRV-H, and B77V are included are very close to 1 (Table 12). The maximal difference between both target sizes is in all cases be- low 5%. This supports the conclusion that for ASV the target size for the ability to induce proliferating foci (which may be defined as oncogenicity) is identical to the target size for its ability to induce the production of converting virus (that is, the whole genome) .

There are at least three possibilities to explain this result in functional terms, of which we favor the first:

1. All the gene products of ASV are involved directly or indirectly (e.g., as regu- latory functions at the level of transcription or translation) in the process of formation of proliferating foci. This explanat,ion seems to conflict with the fact that virus production is not a prerequisite for the conversion of mammalian cells (Gelderblom et al., 1970), However, it is not excluded that in converted mammalian cells all the ASV gene products are synthesized and that, for example, a cellular factor essential for the assembly or maturation of virus particles is missing.

2. The mutagenic treatment employed leads mainly to breaks in the viral RNA.

438 GRAF ET AL.

TABLE 12 LOWER LIMIT OF THE RELATIVE TlRGET SIZE FOR THE ABILITY TO INDUCE THE FORMATION OF

PROLIFERATING FOCI OF VARIOUS ASV STRAINS”

Mutagen Virus strain Inactivation of FFU in logb

Proliferating NVP foci

(total prolifer- sting foci)

Pu FL

HA SRV-1 2.6 f 0.2 o/117 0.031 0.994 SRV-H 2.0 * 0.2 O/66 0.054 0.987 SRV-Gold& 2.8 f 0.2 O/52 0.069 0.988 B77 2.2 f 0.2 O/24 0.143 0.967

6OCo-r-rays SRV-1 2.8 f 0.2 O/26 0.132 0.972 SRV-H 2.5 f 0.2 o/43 0.082 0.984 SRV-Gold4 2.3 z!z 0.2 O/18 0.183 0.959

a The calculations were performed on the basis of the data contained in Tables 1, 2, and 3. b The lower values for the inactivation given by the standard deviation was used for the calculation

of F1 (e.g., 2.4 log for HA-treated SRV-1).

If a sequential transcription or translation is assumed and the genes involved with focus formation are at the end of the genome, any deletion would lead to an inactivation of this function. At least in the case of HA this possibility seems not very likely because under the conditions used this mutagen does not induce a significant decrease in the amount of high molecular weight RNA as contained in avian RNA tumor virus particles (Graf, 1969; unpublished).

3. A specific structure of the viral RNA must be present as a prerequisite for the cell-converting capacity of the virus.

Consistent with our work is the recent description of regressing foci obtained from W-irradiated B77V by Toyoshima et al. (1970). Also in agreement with our work is that these authors did not find any NVP foci which were able to proliferate. The dis- crepancy of the findings thus far discussed to those reported by Gold6 and Latarjet (1966) and Gold6 (1970b) is difficult to explain. It is not due either to differences in the virus strains used or to differences in the conditions of mutagenization, since we were not able to detect any NVP foci among foci induced by 6oCo-irradiated SRV-Gold6 in chicken embryo fibroblasts. However, it is conceivable that the NVP cells found by Gold6 were induced by mutants with a low ability for reproduction. Foci induced by such mutants possibly would have been very small under our culture conditions and might

therefore have been overlooked. It would be interesting to know whether the described proliferating NVP cells contain gs-antigen, whether they produce any virus particles, and whether they induce tumor formation in birds. Possibly the ehorioallantois membrane of the chicken embryo as used by Gold6 and Latarjet (1966) represents a better cell system for the isolation of NW cells from CEF cultures. In experiments performed under conditions in which these authors detected 37 % NVP pocks induced by 6oCo-irradiated SRV, all of the six pocks which we isolated were found to produce virus. However, our result was not conclu- sive because even after incubation of mem- branes containing less than 10 pocks with chicken serum able to neutralize 100 FFU of SRV-Gold&, the overlying fluid still contained focus-forming virus. Therefore, no further attempts to isolate NVP cells from the chorioallantois membrane were made. Very recently another author (Hanafusa, 1970) reported the detection of mutants of RSV(0) which were able to convert chicken cells without producing a focus-forming virus in amounts comparable to the wild type. It was not clear whether or not cells converted by these mutants proliferated.

In the second part of this work it was shown that not all the genes of ASV are needed for the production of nonconverting infectious viral particles. In five separate experiments nonconvert’ing viruses were iso-


lated from CEF cultures infected at a low multiplicity with two different HA-treated SRV strains. For several reasons it seems unlikely that they originated from leukosis virus previously associated with the SRV stocks. End-point dilut.ion interference tests performed with the untreated SRV indicated that the SRV stocks did not contain a leukosis virus in excess. On the other hand, it is unlikely that they contained a leukosis virus in equal or lower concentrations t’han the focus-forming virus because they were derived from SRV cloned serially three times.

Experiments described in another study (Bolognesi and Graf, 1971) indicate that the high molecular weight RNA of one of the mutant.s, NC-SRV-1, is smaller than t,he corresponding RNA of SRV-1. A similar difference was not found between bhe RNAs from another mutant and its corresponding sarcoma virus (NC-SRV-H and SRV-H) .

Marker rescue experiments indicated that the possible deletion mutant NC-SRV-1 still contains genetic information for cell conversion. It is not yet clear whether the described rescue of focus formation is based on a complementation or on a recom- binat.ion between the nonconverting and the defective sarcoma viruses. Since significant enhancement of focus formation of mutagenized ASV on cells infected with different RAV strains was not observed by several authors (Levinson and Rubin, 1967; Toyoshima et al., 1970; our unpublished result,s), it, is possible that the described capacity for marker rescue distinguishes the nonconverting mutants from common leukosis and Rous-associated viruses. A preliminary experiment suggests that cells can be converted by a coinfection with both nonconverting mutants and that t,hese cells produce a converting “recombinant” virus. However, more studies must be done in order to confirm and extend this observation.

In all the other properties tested the NC-SRV’s are similar to avian leukosis and Rous-associated viruses. In this con- nection it appears interesting that from stocks of ASV strains nonconverting viruses were isolated which possessed the same en-

velope properties as the corresponding helper independent sarcoma virus (Hanafusa and Hanafusa, 1966; Duff and Vogt, 1969). Therefore, it might be that avian leukosis viruses originate(d) from avian sarcoma viruses by spontaneous mutation.

From the data presented, a tentative model for the genome of ASV is proposed. The entire genome (supposedly needed for the production of converting virus) is necessary for the formation of proliferating foci. However, only a fraction of it is sufficient for the production of nonconverting virus particles.

APPENDIX

Let. f be the target size for the ability to induce the formation of proliferating foci and r the t.arget size for the ability to produce converting progeny of ASV. Assuming that, r is represented by the whole viral genome, the relative target size for f is defined as

F=f r

According to the target theory (Lea, 1946; TimoffeBff-Ressovsky and Zimmer, 1947) the probability P of survival of a focus at a given dose D of mutagen is

p (foci) = e-fXD = e--FX?‘XD

This probability is equal to the sum of the probabilities of survival of NVP and virus- producing (VP) foci separately

P (foci) = P(VP) + P(NVP)

The probability of survival for VP foci is

P(VP) = eprXD

and the probability of survival for NVP foci

p(NVp) = (1 - e-(‘f) X D) X e-fXD

= e-FXZ’XD - e-‘XD

If p is defined as the fraction of NVP foci among the totality of foci, then

P( NVP) ’ = P(VP> + P(NVP)

= 1 _ e-“-F’rXD

And if x is taken as an estimate of p

5 = (vP;N+v;;vP)

GRAF ET AL. 440

then

F=l+ log (1 - 5)

r X D X log e

The probability of survival of a focus at a given dose D may be expressed in terms of the logarithm of inactivation (K) observed:

e-fxD = 10-1~

Therefore K/F Xlog X e can be substi- tuted for r X D, and, because x represents the value which was observed for p, a point estimate F of F is obtained:

If no NVP focus is observed among n foci, then p = 1. However, it does not necessarily follow that F = 1. Because of sampling variation, the case of F = 1 - C, where E is small, cannot be excluded. Therefore, if an appropriate upper confidence limit p, for p is used in case of z = 0, a certain lower limit F1 of F is obtained:

Fl = K

K - log (1 - pa>

97.5 % confidence limits (one-sided) for p

were taken from Documenta Geigy, Base1 (1967).

ACKNOWLEDGMENTS

The authors wish to thank Professor W. Schafer for his support and interest throughout this work and for reading the manuscript. We are grateful to Dr. H. B. Strack for many helpful discussions and Dr. P. K. Vogt for the gift of a preprint. Thanks are also due to Miss P. Troxell, A. Seydel, E. Kreutzer, and D. Willutzki for excellent technical assistance and Dr. K. H. Peter and the Willy Rtisch Company, Waiblingen, for help with the @Co-irradiation.

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studies on the reproductive and cell-converting abilities of avian sarcoma visuses

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