Mixed infection with two types of Rous sarcoma virus

VIROLOGY

13, 158-163 (1961)

Mixed

Infection

with

Two Types

HOWARD Division

of Biology,

California

Virus’

M. TEMIN”

Institute

Accepted

of Rous Sarcoma

of l’echnology,

October

Pasadena,

California

14, 1960

A study was made of the consequences of the introduction, by superinfection or mutation, of a marked type of Rous sarcoma virus (RSV) into chicken embryonic cells slready infected with RSV. Characters controlling cell morphology (Temin, 1960) were used as markers. After superinfection, cells can release virus of the original type only, of the superinfecting type only, or of both the original and superinfecting types. Over ten generations after superinfection, single cells can release two types of virus. Cells which release both viruses can have the morphology of either type; the virus that controls cell type can be either the original or the superinfecting virus. It was found that the proportion of mutant virus and the proportion of mutant cells in clonal populations of Rous sarcoma cells was similar. INTRODUCTION

The morphology of a chicken embryonic cell infected in vitro by Rous sarcoma virus is partially controlled by the genetic character of the infecting virus (Temin, 1960). The virus does not kill the cell it infects, but changes certain of the characters of the cell. Therefore, it is possible to study after several generations the effects of the introduction of a second type of virus, by superinfection or mutation, into a cell already infected with RSV. The results of such experiments allow certain suggestions to be made about the number of inherited copies of virus and the nature of viral control of cell morphology. MATERIALS

AND

METHODS

This work was carried out with chicken embryo cells grown in vitro using techniques of cultivation, incubation and infection described previously (see Temin, 1960). Virus was assayed by its ability to produce foci of cells of characteristically altered morphology in secondary cultures of chicken embryo 1 Supported by grants from the American Cancer Society. ’ Present address : McArdle Memorial Laboratory, The University of Wisconsin, Ma.dison 6, Wisconsin. 158

cells (Temin and Rubin, 1958). Cells were cloned on X-irradiated feeder layers of duck cells. Since duck cells are relatively resistant to RSV before irradiation, and the capacity of cells to be infected with RSV is very sensitive to irradiation, the feeder layer does not contribute to the amount of virus released by the Rous cells. Cloning efficiency of the infected cells was about 10%. The two lines of virus used in these experiments were obtained from a single clone of Rous sarcoma cells and apparently differ only by a single mutational step in a character controlling cellular and, therefore, focal and clonal morphology: WWTP~~RSV causes appearance of foci and clones of round, refractile cells; morphf RSV, of foci and clones of long, fusiform cells (illustrated in Temin, 1960). These two types of RSV mutate and back mutate, are neutralized at the same rate by antiserum made against one of them, and make tumors similar in time of appearance and histology after injection intravenously or on the chorioallantoic membrane of chick embryos or into the wing web of young chickens (Temin, unpublished data). Virus release from single cells was studied by the microdrop technique of Lwoff m al. (1955). In the experiments reported here,

TWO

TYPES

OF ROUS

drops of medium containing about lo2 chicken embryo cells were placed under paraffin oil. Several hours later a single infected cell was placed in each drop. After a few days incubation, the cells and supernatant of each drop were added to the supernatant of an assay plate of secondary chick embryo cells. Agar was added after 16 hours. RESULTS

Superinfection Cells were infected with one strain of RSV and grown for one month. They were then infected with the second strain of RSV. The progeny cells appeared to release only the original type of RSV. In order to get superinfection, the following experimental plan was adopted. Secondary cultures of 2 to 4 x lo5 cells were infected with virus of one type at a multiplicity of l-4 focus-forming units (FFU) per cell. These cells and parallel uninfected ones were then cloned on feeder layers of X-irradiated duck cells. After 3 days, the agar overlay was removed and the cells were superinfected with approximately the same multiplicity of virus of the other type. Agar was overlaid. After 5 or 6 days of further incubation the plates with a small number of clones were examined and individual colonies showing a homogeneous morphology of the original type were marked. These were picked to test tubes and plated in parallel on feeder layers of X-irradiated TABLE

SARCOMA

159

VIRUS

duck cells and assay plates of secondary chick embryo cells. At times the subcolonies on the feeder layer were replated. No release of the superinfecting virus was found at 3 days in about 50% of the clones tested. The other clones, which released both types of virus upon plating, fell into two classes: those giving rise only to subcolonies of the original type, and those giving subcolonies of both original and superinfecting type. The frequency of these two classes appeared to differ in different embryos. Superinfection with No Change in Morphological Type A secondary culture made with cells from a single embryo was infected with morphi virus, cloned, and superinfected with morph virus. Although the control clones of originally uninfected cells became converted by the morph’ virus there was no change in the f clones. Four such clones were picked. These remained f in appearance and released morphf and morph’ virus (Table 1). More than 2 weeks after superinfection, single cells were put into microdrops. After incubation for a few days, the entire contents of the drop was plated on an assay plate. Occasionally the assay plate was transferred if there was doubt about the results. The results of such experiments with cells from two of the clones from Table 1 and two other clones that behaved similarly are seen in Table 2. About a third of the 1

SUPERINFECTION WITH No CHANGE OF MORPHOLOGICAL TYPES Clone

Morphology of subcolonies Virus released After

third transfer: Morphology of cells Virus released

1

2

Confluent j 500 morph’ 50 morphr

50.f 44 morphf 9 morph’

lWf 500 morphf 100 morph’

40.f 40 morph’ 8 morphr

f 800 morphf 1400 morph’

f 500 morphf 300 morph’

f 200 morphf 110 morph’

f NDb ND

3

4

(1A secondary culture of cells from a single embryo was infected with morphf virus, cloned, and superinfected with morph’ virus. Four clones were picked and the cells plated for virus release and morphology. This procedure was repeated. b Not done.

160

TEMIN TABLE VIRUS

RELEASED

FROM

they can do this fifteen generations after infect,ion and superinfection, and that the cells have the morphology typical of cells carrying the original virus.

2 SINGLE CELLP

Number of cells Releasing only original virus

Clone

Releasing only sup- Releasing both erinfecting virus

Releasing no virus

Total cells

i -- ---- --6 18 36 9

t C

d

0 0 0 2h

1;: I17 '5 I-

12 (1.2%‘,),56

~ “8 23 I- 9 (34%),46

ii 7G 25 (23%) 173

I

a Single cells from clones superinfected 2 weeks previously were distributed in microdrops. The entire contents of the drop were then plated on an assay plate. b Only two and three foci on assay plate.

cells released both types of virus. The amounts of virus of both types was approximately equal. The results are notable for the almost complete absence of cells releasing only the superinfecting virus. (The two cells found of this type released so little virus that they may have been able to release both types.) Controls were done to evaluate the possibility that adsorbed virus contributed to the yield. Since a third of the cells were found to release both types of virus, there would have to be more than this proportion of cell-associated virus. Freezing-thawing experiments revealed 1 FFU per 3 cells to 1 FFU per 50 cells, most cultures having the smaller amount. As an additional control, cells on glass from one of these clones were treated for 30 minutes with 0.2 ml of undiluted serum from a chicken injected with Rous sarcoma cells. This serum had a 12 value of 5 (McBride, 1959). The cells were then washed and trypsinized and single cells were tested for virus release by placing in microdrops with uninfected chick cells. The proportion of cells releasing both viruses, 9/38, was about the same as that found previously, and no cells releasing only the superinfecting virus were found. We, therefore, can conclude that cells can release more t)han one type of RSV, that

Szlperinfection with Change of Morphological Type In other experiments, with cells from different embryos, clones releasing virus of the superinfecting type gave rise to subcolonies of t,his type. This result was found after first infection with either morphf or morphr virus. Chick embryo cultures were infected, cloned, and subcloned as previously described. When the original virus was morphf and the superinfecting virus morph’, some subcolonies of f and some subcolonies of r type were found. The colonies of T type were picked and plated on an assay plate of chick embryo cells. The foci that appeared are TABLE

3

VIRUS RELEASED FROM CLONES SHOWING SUPERINFECTION WITH CHANGE OF M~RPH~LoGY~ Virus type Clone

Origina, Sup&n- of SuperinfectIng fecting

a

f

1

b C

Virus released from subcolopy

r ;

1

morpholog)

5oor 410r 750r 520r

2001 200r 2001 zoor

d

1

J

e

1

f

f

r

f

500j 5ooj 500s

Of V 41oj 421j OJ W

2ooj 2oojr or

0, Or

28.f

93r

3J

1Wf

9r 4r

1OQf

100,

1ooj

50r

a Cells were infected with RSV of one t,ype, cloned, and superinfected with virus of the second type. Clones showing the morphology characterist.ic of the original virus were subcloned. Subclones showing the morphology characteristic of the superinfecting virus were plated for virus release.

161

TWO TYPES OF ROUS SARCOMA VIRUS

given in Table 3, a, b, c. Parallel experiments were carried out at the same time with morph’ as the original virus, and morphi as the superinfecting virus. Some of the clones of f type were plated. The foci appearing are given in Table 3, d, e, f. Some of the clones having the morphology of the superinfecting virus still released the original virus. In a subsequent experiment cells were infected with morphf virus, cloned, superinfected with morph’ virus, and subcloned. Single cells from this plate, mainly from areas of r type were tested as described above for virus release (Table 4, a, b) . In a parallel experiment morph’ virus was used for the original infection and morphf for superinfection. Clones of r type were subcloned and single cells, mainly from areas of f type, were tested for virus release (Table 4, c). As before, single cells appeared to release both types of virus. This result shows that cells can release more than one type of RSV several generations after infection and that some of the cells have the morphology typical of cells carrying the superinfecting virus. Since in the different experiments the virus release from single cells was studied at different times after infection, it appears that the ratio of morphr to morphf virus released from a single cell was constant, insofar as this technique gave quantit’ative data about release rates. Mutation As a control on the effects of superinfection in producing these results, a study was made of the frequency of LLspontaneous” change of cell morphology in clonal populations of cells infected with one type of RSV. Clones of f cells were grown and subcloned. The subcolonies were then scored for r cells. It will be recalled that cloning was done on irradiated duck cell layers, on which the RSV virus is unable to grow. Areas containing cells classified as r were then picked to test whether or not they released morph’ virus. The results of two such experiments (Table 5) indicate that 6/36 clones of about 500 cells each gave rise to

subcolonies of mutant morphology. The actual virus released from these areas is given (Table 6). The virus released was morpti, usually with morphf. This morphi virus may have been released by f cells which were picked together with the neighboring r cells or it may have been released by the r cells themselves. The frequency of mutants in the virus population going from morphf to morph’ was similar (Table 7). This similarity suggests that a single event leading to a mutant in the virus population may also cause a corresponding change in cell morphology. TABLE 4 VIRUS RELEASED FROM SINGLE CELLS FROM CLONES SHOWING SUPERINFECTION WITH CHANGE OF MORPHOLOGY~ R&FClone

“;;;

-

superin Jriginal f&ing vnlls virus 1 3 1

Releasing both viruses

3 3 0

Releasing no virus

8 3 17

-

Total

7 10 1

19 19 19

a Cells from a single embryo were infected with virus of one type, cloned and superinfected with virus of the other type. Clones showing morphology of the original type (f for a and b; T for c) were subcloned. Single cells from areas showing morphology of the superinfecting type (r for a and b; f for c) were plated for virus release. TABLE

5

FREQUENCY OF MUTANTS THAT CAUSE CHANGE OF CELL MORPHOLOGY~ Clones with Experiment

E

No cell mutants

Some cell mutants

13 17

4 2

Q Cells were infected at a multiplicity of l-4 FFU per cell of morphf virus and cloned. After S-10 days growth each clone was plated on a separate plate of X-irradiated duck cells. These plates were then examined for r cells

162

TEMIN

TABLE

6

VIRUS RELEASE FROM MUTANT SUBCOLONIES" Virus released Subcolony

; : e f g

~morplzr

morph’

65 210 700 270 220 380 22

0 0 28 90 110 190 46

a Clones of cells infected with morph’ virus were plated on separate plates of X-irradiated duck cells and examined for I’ cells. Areas containing cells classified as r were then plated on regular assay plates to test whether or not they released morphr virus. TABLE

7

FREQUENCY OF VIRUS MUTANTP Clones with Experiment

it

C d e

No virus mutants

Some virus mutants

37 10 17 8 2

4 3 1 0 2

a Cells were infected at a multiplicity of l-4 FFU per cell of morph’ virus and cloned (experiments a and d); or an assay plate was infected with about 50 FFU of morph’ virus (experiments b and c). After 8-10 days growth (about lo3 cells per clone) the cells were X-irradiated with about 4000 r to stop cell division and whole individual clones or foci were plated on regular assay plates where the effect of the virus released by them could be studied. These foci were picked and replated to make sure that this identification was correct. In an additional experiment, an assay plate was infected with 6 FFU of morph’ virus and the foci plated without irradiation (experiment, e).

For instance, if two such events were required to give rise to a change of cell type, the frequency of the latter would have been at least a thousand times smaller than the observed frequency.

DISCCSSION

This paper presents data concerning the events occurring in a chicken embryonic cell infected in vitro with Rous sarcoma virus following the introduction by superinfection or mutation of a second type of RSV. Superinfection occurs if the second infection is within a few days of the first. The superinfected cells can release only the original virus, only the superinfecting virus, or they can release both the original and the superinfecting virus in approximately equal amounts. The ability to release two types of virus appears to be inherited in a stable fashion. Single cells can release two types of virus many generations after superinfection. These cells, which release both morph’ and morphf virus, can be either f or r in appearance. Which virus was the original and which was the superinfecting does not appear to be important. Experiments on the frequency of viral and cell mutants show that they occur with similar frequency. From these results it can be concluded that there is no physiological dominance between morph’ and morphf RSV; that RSV is inherited in a stable fashion; that in a cell carrying two types of viral genomes, one can reproduce without affecting cellular morphology; and that after superinfection and after mutation a cell can lose the original virus. From these general conclusions two characteristics of the virus-cell complex in chicken embryo cells infected with Rous sarcoma virus can be specified: (1) When there are two types of viral genomes in a cell they are approximately equal in number and only one type controls the morphology of the cell. This situation is stable. (2) After superinfection or mutation, the new virus multiplies either to establish condition (1) or to replace the original virus. The high frequency of superinfection leading to replacement of the original virus suggests that there are only a very small number of inherited copies of the virus at the time of superinfection. The stability of inheritance of two types of virus suggests that there is some special mechanism in the

TWO

TYPES

OF ROUS

cell for transmission of the virus equally to daughter cells. ACKNOWLEDGMENTS The author is pleased to acknowledge many helpful discussions with Renato Dulbecco and the technical assistance of LaVerne Wenzel. REFERENCES A., DULBECCO, R., VOGT, M., and LWOFF, M. (1955). Kinetics of the release of poliomyelitis virus from single cells. Virology 1, 128-139.

LWOFF,

SARCOMA

VIRUS

163

W. D. (1959). Antigenic analysis of polioviruses by kinetic studies of serum neutralization.

MCBRIDE,

Virology

7,45-58.

H. M. (1960). The control of cellular morphology in embryonic cells infected with Rous sarcoma virus in vitro. Virology 10, 18%197. TEWN, H. M., and RUBIN, H. (1958). Characteristics of an assay for Rous sarcoma virus and Rous sarcoma cells in tissue culture. Virology 6, 669688. TEMIN,