S13, a rapidly oncogenic replication-defective avian retrovirus

VIROLOGY 145, 141-153 (19%)

S13, a Rapidly Oncogenic Replication-Defective Avian Retrovirus

HARTMUT BEUG,* MICHAEL J. HAYMAN,? THOMAS GRAF,* STEPHEN H. BENEDICT,* ALFRED M. WALLBANK,$

AND PETER K. VOGT$’

*European Molecular Biolog2/ Laboratory, Postfach 10.2209, Meyerhofstrasse 1, 6909 Heidelberg, Federal Republic of Germany; +Impe&zl Ca- Research Fund Laborat&, St. Barthnknnews Hospital,

Dominion House, knwkm EClA ?‘BE, United Kingdom; $University of Southern California, School of Medicine, Department of Microbiology, 2011 Zonal Avenue, Los Angeles, Cal$iiia 90083; and Wnivertity of Manitoba, Department of Medical Microbiology, Winnepeg RSE OWS, Canada

Received March 20, 1985; accepted May 6, 1985

The avian leukemia sarcoma virus S13 transforms chicken and Japanese quail embryo fibroblasts and chicken erythroid cells in tissue culture. S13-induced erythroid transformation requires culture conditions suitable for the growth of normal erythroid precursors (H. Beug and M. J. Hayman (1984), Cell 36.963-972). Sit-transformed erythroid colonies contain a high percentage of cells that differentiate in absence of erythropoietin. S13 is defective in pal and onv functions but can code for a complete set of gag proteins. Nonproducer cell clones transformed by S13 release a noninfectious viral particle containing gag but no functional env or pal proteins. They also synthesize a transformation-specific protein of 155,660 molecular weight. This protein reacts with antibody to viral envelope glycoproteins and appears to represent one as well as env sequences. The 155,000-molecular weight env-linked protein does not cross react immunologically with an antiserum against the v-e& A and v-e& B gene products. o 1985 Academic press, IW

INTRODUCTION

Virus S13 was isolated by Stubbs and Furth in the course of an investigation of strain 1 avian erythroleukemia virus. In numerous animal passages strain 1 had caused exclusively acute leukemia with predominating erythroid elements, but in the study of Stubbs and Furth (1935) a single bird was found with a sarcoma at the site of injection. The virus recovered from this tumor was termed S13. It induced sarcoma, acute erythroleukemia, and, rarely, granulocytic leukemia within a short latent period. This multiple oncogenic potential was shown to reside probably in a single viral entity (Stubbs and Furth, 1935).

In this paper evidence will be presented that S13 represents a new erythroblasto-

’ Author to whom requests for reprints should be addressed.

sis-type virus unrelated in its oncogenic determinant to the erb oncogene containing avian erythroblastosis viruses ES4, R, and H (Engelbreth-Holm et ah, 1932; Ya- mamoto et aL, 1983; Lai et d, 1979; Bister and Duesberg, 1979; Roussel et al., 1979). The virus is rapidly oncogenic in chickens, and in vitro it transforms fibroblasts and-under specific conditions-also erythroblasts derived from chicken bone marrow. The transformed erythroblasts differ from cells transformed by the ES4 strain of avian erythroblastosis virus in that they require specific growth conditions and are only partially blocked in differentiation. Sl&transformed cells ex- press a transformation-specific, virus- coded glycoprotein, that can be detected by immunoprecipitation with antisera prepared against the envelope glycoproteins of avian retroviruses. This transformation-specific glycoprotein, termed gp155s’3, is not related to the v-e& A and

141 6042-6822185 $3.00 Copyright 0 1985 by Academic Press, Inc. AI1 rights of reproduction in any form reserved

142 BEUG ET AL.

v-erb B oncogene products of avian erythroblastosis virus ES4. S13 virus is defective for replication, since nonproducer erythroblasts expressing gp155’13 but not gp92”” have been isolated. This observation suggests that cell-derived 01~: sequences have replaced viral replicative information in the S13 genome. In an accompanying paper we will present data on the characterization of gp155’13, on the genome of S13, and on the nature of S13 cmc sequences.

MATERIALS AND METHODS

CeuS and wiruses. Virus S13 was obtained from material passaged in chickens about 20 years ago (Wallbank and Stubbs, 1965). The sources of the Prague strain of Rous sarcoma virus, subgroup A (PR-A) of RSV(RAV-7) and of RAV-2 have been described (Duff and Vogt, 1969). Fertile chicken eggs were of the H & N Cross- Strain White Leghorn breed and were produced at H & N Farms in Redmond, Washington and Lohmann Farms, Cux- haven, West Germany, respectively. Fer- tile Japanese quail eggs came from Life Sciences Inc., St. Petersburg, Florida, under contract with the National Cancer Institute. Avian embryo fibroblast cultures were prepared according to standard techniques (Vogt, 1969). The methods for focus assays in chicken and quail cells as well as for plating infected cells in nu- trient agar have also been published (Vogt, 1969; Graf, 1973). Chicken bone marrow cultures were started from the femural marrow of l- to 14-day-old SPAFAS chicks as described previously (Graf, 1973). The continuous Japanese quail cell line 16Q infected with the defective Bryan high- titer strain of Rous sarcoma virus in the absence of helper virus was a kind gift of Dr. Jan Svoboda. The origin of the ts34 avian erythroblastosis virus (AEV)- transformed cell line LSCC HD3 has also been described elsewhere (Beug et aL, 198213).

Cell culture. Normal and virus-transformed fibroblasts as well as AEV-transformed erythroblasts were grown in stan-

dard growth medium (Dulbecco’s modified Eagle’s medium supplemented with 8% fetal calf serum, 2% chicken serum, and 10 mM HEPES, pH 7.3). S13-transformed erythroblasts were grown in CFU-E medium (Radke et aC, 1982; Beug and Hay- man, 1984).

In vitro transfcrrmation of he marrow cells. Chick bone marrow cells were infected with virus and seeded into Methocel under standard conditions or CFU-E conditions as described earlier (Graf et aL, 1981; Radke et aL, 1982). Colonies were isolated 6-10 days later with a drawn-out Pasteur pipet and expanded in standard growth medium (AEV-ES4-infected cells) or CFU-E medium (Sl&transformed cells).

Assays for ergthroid dQ72wntiation markers. Morphology and hemoglobin content of S13-transformed erythroblasts were analyzed by staining cytospin preparations with neutral benzidine plus histological dyes as described earlier (Beug et aL, 1982a). The more sensitive staining for hemoglobin content using acid benzidine was performed according to Orkin et aL (1975). Detection of erythroid-specific cell surface antigens by indirect immunofluorescence using antisera to mature erythroid cells and to immature erythroblasts (anti-Ery and anti-Mbl; Beug et aL, 1979) was done as described earlier (Beug et aL, 1982a). The possible presence of myeloid cells in the transformed cultures was assessed using the myeloid-specific monoclonal antibody MC512 (Kornfeld et aL, 1983).

Percoll fracticmation of S13 erythroblusts. S13-transformed erythroblasts (lo-20 X 106) (producer clone 6) were loaded on a discontinuous Percoll gradient (prepared as in Beug and Hayman, 1984; densities from top to bottom: 1.070; 1.072; 1.075; 1.085 g/cm3) and centrifuged for 10 min at 2OOOg. The least dense cells (1.070) and the cells banding at 1.072 and 1.075 g/cm were retrieved, seeded in CFU-E medium containing either normal or anemic chicken serum and analyzed by cytocen- trifugation- and staining as above. Alter- natively, cells from the least dense frac-

S13 AVIAN RETROVIRUS 143

tion were also seeded into plasma clot cultures, which were processed and stained 3-4 days later as described earlier (Beug et ak, 1982a).

Reverse transcriptuse assay. Aliquots (5 10 ml) of tissue culture supernatants from S13 erythroblasts were tested for reverse transcriptase activity as described (Hay- man et aL, 1979).

Radioactive labeling, immune precipitation, and gel ekctrophoresis of proteins. Cells (5-10 X 106) were labeled with lOO- 250 &i of [35S]methionine in methionine- free differentiation medium (Beug and Hayman, 1984) for 2 hr. They were then lysed and immunoprecipitated according to the procedures described by Hayman et al. (1979). Analysis was by SDS PAGE. Antisera used in the immune precipita- tions were as follows: rabbit anti-PR RSV-B, rabbit anti-p27, rabbit anti-gp85 + gp37 and rabbit anti-reverse transcriptase (Hayman et aZ., 1979) rat anti-v-e& A + v-e& B (Hayman et al., 1983). For immune precipitation of virus proteins from cell-free supernatants, S13 producer and nonproducer cells were labeled with r5S]methionine as above, washed twice in cold growth medium, and incubated in this medium for 6 hr. The supernatants were then clarified by centrifugation for

10 min at 2000 g and 30 min at 30,000 g, underlayed with 20% sucrose in Hank’s balanced salt solution, and centrifuged for 2 hr at 40,000 rpm (100,000 g) in a Beckman Ti50 rotor to pellet secreted virus. Virus pellets were lysed in 0.5 ml of lysis buffer and processed for immune precipitation as above.

RESULTS

Pathogenicity of Sl3 Virus in H & N Cross-Strain White Leghorn Chickens

S13 virus was injected intravenously and intramuscularly into l-day-old chicks of H & N cross-strain white leghorns (CSWL). Table 1 shows the results. S13 virus proved to be highly pathogenic, causing 100% mortality within 20 days. Reduction of the inoculum to 50 FFU/ bird did not moderate the course of the disease. Most birds died of acute anemia; small internal tumors in the liver, spleen, kidneys, and heart were common at the time of death. In one of the birds a tumor developed at the site of intramuscular inoculation. This tumor and eight of the small internal tumors were diagnosed histologically as fibromyxosarcomas. Blood smears of moribund birds showed ex-

TABLE 1

PATHOGENICITY OF S13 VIRUS IN ~-DAY-OLD H&N CSWL CHICKS

Virus Inoculum Route”

Number of animals injected Mortality Symptoms

Days postinfection

s13 10’ to 50 FFU iv 29 29/29 Anemia, internal tumors

11 to 20

S13 AV -104 inf. units iv

s13 5 x lop im

S13 AV -10’ inf. units im

a iv intravenous; im, intramuscular.

10 o/10

14 14/14

2 o/2

None

Anemia yellow liver fibromyxo sarcoma

None

30

6

21

144 BEUG ET AL.

FIG. 1. (A) Focus of S13-transformed cells in a chick embryo fibroblast monolayer. Fusiform- transformed cells in radial orientation extending from the focus. 100X bright field. (B) Focus of S13-transformed chick embryo fibroblasts with beginning detachment from the culture dish. 150X bright field. (C) A portion of the focus shown in Fig. 1A. 250X phase contrast. (D) Chick embryo fibroblast layer heavily infected with S13, demonstrating extensive detachment of cells and growth and contraction into clumps of transformed cells. 100X bright field.


tremely few erythrocytes. No overt erythroblastosis was seen in the SlQ-inoculated H & N chickens. This result was confirmed by inoculation of the same virus into SPAFAS chickens (Graf, 1973). The bone marrow of the infected moribund chicks, however, consisted almost entirely of erythroid cells at all stages of differentiation.

A replication-competent leukosis virus was isolated from the S13 stock by endpoint dilution (Rubin and Vogt, 1962). This virus, termed S13AV (for S13-associated virus), belongs to envelope subgroup A. It was assayed by interference with Rous sarcoma virus.

Injection of Sl3AV into young chickens did not have any pathogenic effects within the observation period of 20 to 30 days.

Transformatim of Chicken and Japanese Quail Ewdnyo Fibroblasts bg Asian Retrmirus Strain 13

Figure 1 shows foci of transformed cells induced in chicken embryo fibroblasts by S13 virus. The cells are of fusiform morphology and have a tendency to detach from the culture dish, to contract, and to

grow in tightly packed aggregates. Foci in Japanese quail cultures consisted of more adhesive cells and consequently sel- dom detached from their substrate.

S13 also conferred upon chicken and quail embryo fibroblasts the ability to grow in soft agar. The cells of agar colonies cultivated on a solid substrate showed the same fusiform morphology as those from transformed foci in monolayers. No transformation was seen in macrophage cultures prepared from l&day-old chicken embryo yolk sacs.

Transformation of Bone Marrow Cells with s13

Initial attempts to transform bone marrow cells with S13 under standard conditions (Graf et oL, 1981) were negative. Inspection of the bone marrow of infected chicks, however, suggested a virus-induced proliferation of erythroid cells. Therefore, bone marrow was infected with S13 virus harvested from transformed fibroblasts and the cells seeded into Methocel under CFU-E conditions (Radke et d, 1982; Beug and Hayman, 1934). These conditions allow

TABLE 2

S13 ERYTHROID COLONIES

Cell type

Hemoglobin-positive Cells classified cells asLR+E

(percentage)n (percentage)b

Percentage cells stained with

cxEryc*d aEb1 aMb1

s13 Clone 1 48 18.5 29 (5) 90 0.1 Clone 3 37 1.0 5 (1) 95 ND Clone 4 80 11.2 29 (4) 90 ND Clone 6 ‘76 2.1 4 (1) 95 0.1

AEV-ES4 Clone 4 1 0.1 0.1 99 0.1 Clone 6 2 0.1 0.1 99 ND

0 Determined by staining with acid benzidine (Orkin et al, 1975). b Cell types were defined by neutral benzidine plus histological staining (Beug et aL, 1982a, b). LR, late

reticulocytes; E; erythrocytes. c Characterization of these antisera has been described elsewhere (Beug et aL, 19’79; Kornfeld et uL, 1983);

aEb1, anti-erythroblast serum; aEry, antierythrocyte serum; aMb1, anti-myeloblast monoclonal antibody MC51/2.

d Numerals in parentheses; cells strongly stained in a ring-like fashion.

146 BEUG ET AL.

the in vitro growth and differentiation of normal erythroid progenitors and were essential for successful in vitro transformation of erythroid cells by the WC (Kahn et aL, 1984), e&s (Radke et al, 1982), and erb B oncogenes (Beug et aL, 1985). After 6-10 days, compact colonies were obtained that could be isolated and grown into mass cultures using CFU-E medium (Radke et uL, 1982). Colony-forming titers were variable both with fibroblast- and erythroblast-grown virus, ranging from 5 X IO1 to 5 X lo3 colony-forming units/ml. As expected, Sl&induced erythroid colonies could not be propagated in standard growth medium.

Charactemation of SlbTransfimned Bone Marrow Cells

Mass cultures of Sl3-transformed bone marrow cells, when kept in CFU-E me-

dium grew with doubling times of 24 hr or less and exhibited an in vitro life span of 20-30 population doublings, depending on the clone used. Table 2 shows the characterization of several clones of Sl&transformed cells for various hematopoietic differentiation markers. The transformed cells were exclusively erythroid, consisting of a mixture of immature erythroblasts and more mature reticulocytes and erythrocytes. In this they clearly differed from erythroblasts transformed by AEV-ES4 which consisted entirely of immature cells (Fig. 2, Table 2).

In order to determine how the apparent heterogeneity of S13 erythroblasts with respect to erythroid differentiation was maintained, S13 cells from a clone containing a high proportion of mature cells were fractionated according to density using Percoll-step gradients. After characterization (see Materials and Methods)

FIG. 2. Characterization of S13-transformed bone marrow cells. (A) Cytospin preparation of a Sl3-transformed erythroblast clone (No. 6) stained with neutral benzidine and photographed under blue light to reveal hemoglobin staining (Beug et al, 1982a). Ebl, erythroblast, LR, late reticulocyte, E, erythrocyte as defined in Beug et al (1982a). (B, C, D) Living cells of S13 erythroblast clone No. 6 stained with anti-erythroblast serum (B), anti-reticulocyte monoclonal antibody (C) and anti-erythrocyte serum (D) by indirect immunofluorescence (Beug et al, 19’79, 1982a).


the fractions were cultivated for 2-3 days and again tested for erythroid markers. The lightest fraction containing exclusively immature cells regenerated the initial amount of mature cells within 2 days. In contrast, fractions of intermediate density which contained partially mature erythroid cells differentiated into erythrocytes within 2 days (data not shown). In a second set of experiments, Percoll- fractionated, immature S13 erythroblasts were seeded into plasma clot cultures to determine what kind of colonies they would form. Figure 3 demonstrates that CFU-E-like erythrocyte colonies as well as heterogeneous colonies and colonies consisting entirely of immature cells developed from the initially immature S13 erythroblasts can either undergo self-renewal or enter the pathway of terminal erythroid differentiation, the frequency of these two events being characteristic for each clone.

In order to test whether or not either self-renewal or differentiation of S13 erythroblasts required exogenously added erythropoietin, the above experiments were repeated in presence or absence of anemic chicken serum or partially purified chicken erythropoietin (Beug et aL, unpublished). No effect of the hormone on either self-renewal or differentiation frequency was observed (Fig. 3 and unpublished results). This observation indicates that S13 erythroblasts have the capacity to differentiate terminally in a hormone- independent fashion.

Transfinmation of Erythroblasts and Fi- brob1oA.s Is Caused by the Replicatim- Defective, Transforming Compawnt of S13 Virus

In order to determine whether or not S13 virus was capable of transforming erythroblasts in the absence of its helper virus (S13AV), bone marrow cells were infected with different dilutions of S13 virus harvested from transformed erythroblasts. After 8 days, colonies obtained with the lowest positive dilution (lo-‘) were isolated, grown into mass cultures,

A B

I + Epo I I

- Epo I

mature

FIG. 3. Ability of purified, immature S13 erythroblasts to form colonies. Representative types of colonies obtained from immature, Percoll-purified S13 erythroblasts in plasma clot cultures are shown. Cells were cultivated in presence (A) or absence (B) of erythropoietin (EPO) for 3 days, the clots processed as described in Beug et aL (1982a), stained with neutral benzidine, and photographed under blue light to reveal staining for hemoglobin (Beug et al, 1982a).

and tested for reverse transcriptase (Hay- man et a& 1979). From more than 25 clones analyzed only two clones were reverse transcriptase negative. Table 3 shows that these clones were nonproducers, since infectious virus could be rescued from the erythroblasts by superinfection with a standard helper virus (RAV-2). This presumptive S13(RAV-2) pseudotype was capable of transforming both erythroblasts and fibroblasts in culture. Fur- thermore, virus harvested from erythroblasts transformed with the above pseudotype induced foci in C/O fibroblasts, but not in WBDE fibroblasts, showing that the natural subgroup A helper virus SI3AV had been replaced by the subgroup B helper virus RAV-2 (Table 3).

Replication-Defectiveness of S13 Virus in Fibroblasts

Table 4 shows the results of an infectious center test with several stocks of

148 BEUG ET AL.

TABLE 3

DEFECTIVENESS OF S13 IN REPLICATION

Transforming titer on

Virus harvested from Bone marrow

(CFU/ml)a

C/E fibroblasts (FFU/ml) *

C/BDE fibroblasts (FFU/ml) *

S13/S13AV-producer erythroblasts (cl 6)

Sl3-nonproducer erythroblasts NP cl 4’

S13-nonproducer erythroblasts NP cl 9”

S13 NP cl 9 superinfected with RAV-2

Erythroblasts transformed by S13(RAV-2) virus

MHa(RAV-2)

PR-A

2.4 X l@ ND ND

0 0 0

0 0 0

2.5 X 102 ND ND

ND 5 x lo2 0

1 x lo4 0

1 x lo7 2.5 X lo6

o Colony-forming units in CFU-E Methocel. *Focus-forming units determined as described by Graf (1973). ‘These colonies were negative for reverse transcriptase activity.

S13. At high virus dilutions, when most foci arose from infection with a single virus particle, there were substantially

TABLE 4

DEFECTIVENESS OF S13 IN REPLICATION AS INDICATED BY INFECFIOUS CENTER TESTS

Focus assaya Infectious

Virus center assay”

dilution Virus dilution

Virus 10-l lo-* 10-l 10-a

S13 2047-4-2 155 13 13 0 S13 2047-5-2 195 20 14 1 s13 2012-l-2 35 2 0 0 RSV(RAV-7) 20 1 0 0 PR-A 720 700

“Virus plated onto chick embryo fibroblast (Vogt, 1969). Average focus count of two plates.

*Virus plated onto chick embryo fibroblasts, cultures were treated with 5 pg mitomycin C for 2 hrs at 24 hrs after infection to prevent focus development by cell division. After the mitomycin C was washed from the cultures, a later of fresh cells was seeded on top of the infected layer. Infection of this indicator layer by progeny virus results in focus development with each focus representing an infectious center. Average focus count of two plates.

more focus-forming units than infectious centers, suggesting the occurrence of nonproducing transformed cells. Therefore, single foci were picked at very high virus dilutions (between 2 and 5 FFU/GO-mm plate). Upon picking, each focus was di- vided into two 8-mm wells of a multiple culture dish. One well was superinfected with avian leukosis helper virus RAV-2, the other did not receive helper virus. Wells were inspected microscopically for the presence of growing Sl&transformed cells. At 10, 12, and 14 days after picking, supernatant samples were assayed for infectious virus. Three types of foci were identified by these tests: (i) foci producing infectious S13 in the absence of helper virus, (ii) foci that did not produce infectious S13 and could not be activated by superinfection with helper virus, and (iii) foci that were nonproducers but released S13 when infected with helper virus. In a typical experiment we found 90 producers, 32 nonactivable nonproducers, and only 4 activable nonproducers. Further analysis of these nonproducers was hindered by the fact that very few of the single foci, especially the nonproducers, grew well enough to allow transfer from the El-mm wells. Similar data were obtained when


single agar colonies of Sl&transformed SlS-transforming activity, corresponding chick embryo fibroblasts were tested for to 1.3 X lo6 FFU/ml. A second possible S13 production. Use of Japanese quail explanation for the difficulty to obtain cells also gave the same results. The ob- nonproducers could be virus clumping, served preponderance of producer foci leading to the majority of foci being in- could result from an excess of helper virus fected with S13 and helper virus. Immu- in the virus stocks, leading to double nofluorescent staining of S13-infected cells infection of most foci by helper virus and showed indeed that the virus was present S13. To test this possibility, we determined at the cell surface in comparatively large helper virus concentrations in three S13 aggregates up to lo3 nm in diameter (data preparations by a dilution endpoint pro- not shown). The apparent contradiction cedure, testing for the ability of the virus between infectious center tests showing a to complement the env-defective Bryan preponderance of nonproducers and single high-titer strain of Rous sarcoma virus focus or colony analysis in which only and determining independently S13-in- very few nonproducers were found is at duced transformation. All three stocks present unresolved and will require ad- had the same endpoint for helper and for ditional work.

A B C D w- -

:

SW P ‘= &A gp 66 eda

,s13

1 2 3 4 5 12345 1 2 1 2

FIG. 4. Characterization of a putative transforming protein of S13 virus, g~155~‘~. (A) S13 producer erythroblasts (clone 6) were labeled with pS]methionine and extracts immunoprecipitated with antisera to various virus structural proteins. Lane 1, normal rabbit serum; lane 2, rabbit anti-PR RSV-B, lane 3, rabbit anti-p27; lane 4, rabbit anti-gp85 + gp37, lane 5, rabbit anti-reverse transcriptase (Hayman et al, 1979). Positions of major virus structural proteins and of g~155~‘~ are indicated by arrows. (B) SlS-producer erythroblasts (clone 6) were labeled and extracted as in A, but immunoprecipitated with two different rat antisera to p’75@@&A and gp’74”bB (Hayman et al, 1933) in absence (lanes 1, 2) or presence (lanes 3,4) of competing cold virus (Hayman et a& 1933). Lane 5 shows an extract of AEV-transformed erythroblasts immunoprecipitated for p7F &A and the 66 to 68 kDa precursors of gp74 erb B (Hayman and Beug, 1934) by one of the anti- erb sera in presence of competing virus. (C) S13 nonproducer erythroblasts (NP clone 9) were labeled and extracted as in A and immunoprecipitated with anti gp35 + gp37 (lane 2) or normal rabbit serum (lane 1). (D) [SSS]Methionine-labeled virus from S13 producer (lane 1) or nonproducer (lane 2) erythroblasts was produced, lysed, and immunoprecipitated with rabbit anti-PR-RSV-B serum as described under Materials and Methods. Arrowheads with small numerals indicate the position of molecular weight markers. Position of the same markers is indicated in panels A-C by arrowheads only.

150 BEUG ET AL.

Transformation-Spe&tic Protein Expressed bg SlbTransfmd Erythroblasts Is Unrelated to v-erbGene Products

Immunoprecipitation analysis of S13- transformed fibroblasts with antisera to the avian retroviral envelope protein Pr92”” revealed the existence of a glycoprotein of 155 kDa, termed gp155’13 (see accompanying paper of Benedict et aL, 1985). This protein was not found in S13AV-infected cells nor in cells infected with other retroviruses. To study whether or not the same protein was expressed by S13-transformed erythroblasts, S13 nonproducer erythroblasts and S13(RAV-2)- transformed erythroblasts were labeled with [35S]methionine and immunoprecipitated with various antisera to virus structural proteins. Figure 4A shows that gp155’13 is immunoprecipitated from S13(RAV-2) erythroblasts by anti-whole virus and anti-env sera, but not by anti- gag or anti-pal sera. gp155’13 also proved to be unrelated to both p75DaB-erbA and gp74&B (Hayman et ak, 1979, 1983; Hay- man and Beug, 1984) since antisera to both these proteins did not immunopre- cipitate gp155’13 or any other transformation-specific protein (Fig. 4B). Our con- clusion, that S13 contains oncogenic sequences unrelated to those of avian erythroblastosis virus and does not transform erythroblasts by activation of erb- related cellular oncogenes is strengthened by the finding that S13 erythroblasts expressed only very low amounts of c-erb A and c-erb B-related RNA (B. VennstrSm and H. Beug, unpublished results).

When Sl&nonproducer erythroblasts were analyzed by immune precipitation with the same antisera, the only protein detected by anti-gp92 and gp85 sera was gp155=3, indicating that the defective, transforming component of S13 is negative for gp92”” synthesis (Fig. 4C). The same cells, however, expressed apparently normal amounts of pr768aB, suggesting the existence of noninfectious particles (data not shown). By immuneprecipitation of cell-free supernatants of SlS-nonproducer erythroblasts with anti-gag sera, the

presence of mature virus core proteins could indeed be demonstrated (Fig. 4D). The same supernatants contained only the RNA of the defective, transforming S13 virus, but no helper virus RNA (B. Vennstrijm and H. Beug, unpublished).

DISCUSSION

S13 was among the first avian acute leukemia viruses discovered about 50 years ago. Initially, at least, it was able to induce erythroleukemias and rarely granulocytic leukemias besides sarcomas in the chicken. The virus was lyophylized in 1938 and further investigations on S13 did not begin until 20 years later (Stubbs and Sperling, 1960). This work established basic physical and biological properties of S13 (Stubbs and Sperling, 1961; Stubbs et al, 1961; Wallbank et al, 1962a,b, 1964; Wallbank and Stubbs, 1965, 1966). Inter- estingly, during these studies S13 proved to be poorly, if at all, leukemogenic (Hub- ben et al, 1967), causing mainly sarcomas, endotheliomas, and anemia. The possibility that the original isolate of S13 contained two distinct viruses, a leukemogenic and a sarcomagenic one, and that the former had been lost during serial passage of sarcomas was considered. Another explanation for the loss of leukemogenic potential was a change in chicken breed which had been Plymouth Rock in the early experiments and White Leghorn in the work performed during the 1960s. The S13 seed inoculum used for the present studies was derived from the stocks that had been primarily sarcomagenic in the experiments of Hubben and co-workers (1967). In our animal inoculations using a white leghorn strain, S13 was also non- leukemogenic, inducing sarcomas and anemia. However, further passages in other chicken lines have recovered a leukemogenic potential that will be described in another publication (Moscovici et aL, in preparation).

S13 transforms chicken and Japanese quail embryo fibroblasts in tissue culture. The morphology of the transformed cell is fusiform, and full focus development


requires about 2 weeks of incubation. S13 also transforms bone marrow cells. This effect of S13 infection requires culture conditions (CFU-E medium) that support the growth and differentiation of normal erythroid progenitor cells. In contrast to chick fibroblasts, chicken erythroid precursors are dependent on a slightly alka- line pH (‘7.6), carefully controlled osmo- larity, and chick and fetal calf serum batches pretested for their ability to pro- mote growth of these precursors (Samarut and Nigon, 1976). In case of erythroblasts transformed with erb B alone, careful control of pH and Na+ ion concentration have proven to be most important for growth of these cells, which also require CFU-E medium (Beug et aL, 1985 and unpublished). S13-transformed bone marrow colonies consist of erythroid cells. These colonies contain a high percentage of more mature erythroid elements. They share this property with erythroid cells transformed by RSV and FSV (Kahn et al, 1984) as well as with AEV-H containing only erb B (Beug et cd, 1985). In contrast, bone marrow colonies transformed by avian erythroblastosis virus ES4 (containing erb A in addition to erb B) consist exclusively of immature erythroblasts. Avian erythroblastosis virus ES4-induced bone marrow colonies also differ from those of S13 by not requiring CFU-E medium for growth. We have not seen transformation of myelomonocytic cells by S13, either in bone marrow cultures or in cultures of chick embryo yolk sac macrophages.

S13 codes for a transformation-specific protein that is linked to env sequences and can be precipitated with an antiserum against gp92”““. It is likely that this protein contains cmc sequences as well, because its molecular weight exceeds that of the env precursor protein by more than 60,000. Because both S13 and avian erythroblastosis virus strain ES4 affect erythroid cells in viva and in vitro, it was important to test for the possible presence of the erb A and erb B cmc sequences in gp155 . ‘13 No immunological cross reaction between gp155’13 and sera directed against

the v-erb A and v-erb B gene products could be detected, suggesting that the putative cmc sequences in gp155’13 are unrelated to erb B or erb A.

S13 is defective in replication. Nonpro- ducing S13 transformed erythroid colonies do not contain gp92 but synthesize the complete gag precursor pr’76@‘@. They also release noninfectious particles that contain the gag proteins but are devoid of reverse transcriptase activity. pr180 was also not seen in Sl&transformed nonproducer cells. S13 thus appears to be defective in both the pol and env genes. Whether only one or both of these defects are caused by the insertion of one sequences in the S13 genome is not known. The defects of S13 are reminiscent of those seen with RSVa! (Hanafusa et al., 1972) which also effect pol and env. Like S13, RSVa produces envelope-defective noninfectious viral particles that lack reverse transcriptase activity. However, in RSV~Y all of the env gene is deleted; the genome of S13 retains env sequences expressed in gp155’13. It would be interesting to determine whether the noninfectious S13 particles are, like RSV~P, defective in tRNA primer selection (Sawyer and Han- afusa, 1979).

At high virus dilutions infectious center tests of S13 predict a frequent occurrence of nonproducing transformed cell clones. Yet the incidence of such nonproducers isolated from infectious fibroblasts or bone marrow cultures has been remarkably low. This discrepancy could result from a rapid spread of helper virus that could infect most of the single foci or single colonies by the time they are isolated. It could also have less trivial reasons, e.g., the presence of some nondefective virus that replicates slowly in solitary infection or virus that can be complemented by endog- enous viral sequences. These possibilities require further study.

ACKNOWLEDGMENTS

This work was supported by U. S. Public Health Service Research Grants CA 13213 and CA 29777, awarded by the National Cancer Institute. The au-

152 BEUG ET AL.

thors thank Lester Luck, Gahi Doederlein, Sigrid Grieser, and Gay Kitchener for excellent technical assistance and Glennis Harding for most competent help with the manuscript.

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S13, a rapidly oncogenic replication-defective avian retrovirus