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of the SR-D virus harvested in Brown Leghorns contains ap- proximately ..... Nicholas Stedman for useful discussion and analysis of the data, and. Dr. Oliviero ...
Proc. Nati Acad. Sci. USA Vol. 77, No. 5, pp. 3014-3018, May 1980 Microbiology

Recombinants between avian sarcoma virus genome and chicken helper factor gene of the host cell: Cloning by transfection (Rous sarcoma virus replication/DNA transfection/cloning of provirus/kinetics of recombination)

GUIDO CARLONI, MICHEL KACZOREK, AND MIROSLAV HILL* Department of Cellular and Molecular Biology and Equipe de Recherche, no. 148 du Centre National de la Recherche Scientifique, Institute of Cancerology and Immunogenetics, 94800 Villejuif, France

Communicated By Andre Lwoff, February 22, 1980

Chicken cells of chicken helper factor-positive ABSTRACT (chf+) phenotype were infected with a cloned (envE-free) Rous sarcoma virus, subgroup D, and examined for the presence of parent and recombinant proviruses by transfection in chicken and turkey cells, respectively. It was found that most parent virus DNA is integrated into the host cell genome during the first 18 hr after infection, and no significant integration occurs between 18 and 72 hr after infection. On the other hand, no recombinant virus DNA was detected at 18 hr, although both unintegrated and integrated (provirus) forms of this DNA occurred 72 hr after infection. Recombinant proviruses were also found in chronically virus-infected chf+ cells but not in chfcells lacking virus-related RNA. Our results show that recombinants between the exogenous virus and endogenous chf gene can be cloned from the DNA of the host cell by transfection and suggest that a second replicative cycle of the virus is required to generate such recombinants.

Avian sarcoma retroviruses are known to recombine with a high frequency (1-4) by a mechanism involving intramolecular crossing-over(s) (5, 6). The molecular features of this mechanism are, however, unknown. It has been reported that mixedly infected cells release heterozygote particles, suggesting that heterozygotes may be precursors of recombinants (2). The question remains, however, as to whether heterozygotes are an obligatory intermediate in the formation of recombinant viruses. Alevy and Vogt (7) gave evidence suggesting that wildtype recombinants between temperature sensitive (ts) early mutants could be isolated in the first 24 hr after infection. The low level of the wild-type viruses detected, however, could also be revertants of ts mutants rather than recombinants (8). In three-factor crosses Wyke and Beamand (8) have recently shown a number of recombinant viruses that appeared to segregate from virus particles heterozygous for all three genes under study. We have studied the genesis of recombinants by identifying the recombinant provirus by transfection (see ref. 9 for review). We have chosen a two-factor cross between the envelope gene envD of Rous sarcoma virus (RSV), subgroup D, and the chicken helper factor gene envE of chicken endogenous virus (10). In this cross the chf gene is endogenously carried in chicken cells. In transfection assays this gene is uninfectious (11) and, therefore, cannot interfere with subgroup E recombinants. We will show that the recombinant envE provirus occurring in chf+ chicken cells infected with the Schmidt-Ruppin strain of RSV, subgroup D (SR-D), may be transfected in turkey cells susceptible to envE viruses but resistant to the envD parent. According to our results the time required for recombinational events to occur between the exogenous and endogenous env genes is longer than one cycle of virus infection. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 3014

MATERIALS AND METHODS Cells. Chicken embryo fibroblasts of the C/E phenotype, positive for chicken helper factor, were obtained from 11day-old embryos of Brown Leghorns supplied by Institute Gustave Roussy (Villejuif, France). chf- embryos of C/AE phenotype (Reaseheat line C), 11 days old, were obtained from L. N. Payne, Houghton Poultry Research Station, Huntingdon, England. Secondary cultures of chicken embryo cells of C/E phenotype [chf-, gs- (gs, group-specific antigen)] were kindly provided by B. Baker, Department of Microbiology, University of California, San Francisco, CA. T/B,D turkey embryo fibroblasts were obtained from H. Murphy, Imperial Cancer Research Fund Laboratories, London. Cells were grown in a modified Eagle's medium (12) supplemented with 10% (vol/ vol) tryptose phosphate broth (Difco) and 5% heated calf serum. In secondary and later passages of C/E cells, 5% calf serum was sometimes replaced by 5% fetal calf serum. Medium for virus-transformed cultures was supplemented with 1% dimethyl sulfoxide. The presence of the chf gene in chicken cell lines used in this study was assessed by the ability to complement noninfectious Bryan sarcoma virus [BH-RSV(-), in which the helper virus that provides envelope glycoprotein is given in parentheses] released by quail 16Q cells (13). In this assay 10616Q cells were cocultivated with 106 test cells, and 4 days later 5 ml of culture medium was filtered and assayed for infectious pseudotypes in T/B,D turkey cells. In positive assays 20-200 foci of transformed cells formed within 9-12 days, whereas no foci formed under the same conditions in negative assays. Viruses. SR-D no. 304 and its transformation-defective (td) derivative no. 300 were isolated after transfection of a focuscloned SR-D as described (14). These viruses were grown in Brown Leghorn chf + cells and crude stocks contained, therefore, envE recombinants belonging to subgroup E avian tumor viruses, as indicated by the superscript E. To remove these recombinants SR-D was cloned by triansfection in C/E chfchicken cells, using the DNA purified from SR-D-transformed chf + cells. In this proviral-DNA-cloning procedure the DNA was serially diluted and the virus was isolated from a culture transfected at the end-point dilution of the DNA, thus with all probability generated from the provirus(es) expressed in one transfected cell. Cloned SR-D was multiplied in C/E chf- cells grown in Corning 490-cm2 plastic roller bottles. Culture medium was harvested twice a day for several days, centrifuged at 10,000 X g and 4°C for 15 min, and stored at -700 C. Abbreviations: chf, chicken helper factor; env, gene for viral envelope; RSV, Rous sarcoma virus; SR, Schmidt-Ruppin strain of RSV; PR, Prague strain of RSV; BH-RSV(-), noninfectious Bryan sarcoma virus; ts, temperature-sensitive; td, transformation-defective; nd, nondefective; moi, multiplicity of infection; IU, infectious unit; FFU, focus-forming unit. * To whom reprint requests should be addressed.

Microbiology:

Carloni et al.

DNA Extraction. Chicken cells were infected with the virus at different multiplicities of infection (moi) as given in Results and cultured in roller bottles under previously described conditions (15). DNA from chronically virus-transformed cells was extracted about 15 days after infection by a slightly modified Marmur procedure (16, 17). In the case of DNA from 18- and 72-hr-infected cells, Hirt's fractionation was employed (18) and the DNA was purified from both Hirt pellet and supernatant fractions as described elsewhere (19). DNA Infectivity Assay. DNA in the presence of rat thymus carrier DNA at concentrations up to 10 ,g/ml was assayed for infectivity in Brown Leghorn chicken cells and turkey cells as described (19). Transfected cultures were examined for foci of transformed cells to detect nondefective (nd) virus and by reverse transcriptase assay (20) for td virus. The specific infectivity, in terms of infectious units (IU) per Atg of DNA or 106 cells, was determined at the end point dilution from the fraction of virus-positive cultures. Preparation of Whole-Cell RNA. RNA extraction was performed according to Deng et al. (21). Briefly, about 108 chicken cells grown in confluent monolayer were lysed in 0.15 M NaCl/0.05 M Tris-HCI (pH 7.4)/0.01 M EDTA solution containing Pronase at 500,gg/ml and 0.5% sodium dodecyl sulfate. The lysate was incubated for 1 hr at 370C, then extracted with phenol and precipitated with ethanol. The preparation was then treated with RNase-free DNase. RNA was extracted with phenol, precipitated with ethanol, and resuspended in 0.02 M Tris-HCI, pH 7.4. The 260/280-nm absorbance ratio of the RNA ranged from 1.8 to 2.0 in all preparations. RNA-DNA Hybridization. The RNA was diluted in 0.02 M Tris-HCI, pH 7.4, and several 1:2 dilutions were prepared, using the same buffer. Samples (40 ,l) of each RNA dilution were mixed with 10 pil of a probe mixture containing 3H-labeled cDNA of td Prague (PR) strain of RSV, subgroup C (1300 cpm, specific activity 25 X 106 cpm/flg) in an annealing buffer to obtain 0.6 M NaCl/0.02 M Tris-HCI (pH 7.4)/0.01 M EDTA and incubated in sealed 50-,ul Coming micropipettes at 68°C for several days. The cDNA probe was prepared as described (22) and kindly provided by D. Stehelin, Lille, France. After 60 or 120 hr the hybridization of cDNA was assayed by digestion with S1 nuclease. The contents of the micropipette were expelled into 0.03 M sodium acetate (pH 4.5)/0.2 M NaCl/ 0.001 M ZnS04 and sonicated and alkali-denatured calf thymus DNA at 10 ,ug/ml. The samples were split into two aliquots and incubated at 50°C for 2 hr in the presence and absence of S1 nuclease, then precipitated with trichloroacetic acid, and the precipitate was collected on Whatman GF/C filters and assayed for radioactivity. The percent hybridization was determined from the count ratio of Si-digested sample to undigested control. RESULTS Examination of Chicken Cell Lines for Expression of chf

Phenotype. The chf phenotype of three different lines of chicken cells was determined from the ability to complement BH RSV(-) and to synthesize virus-related RNA as shown in Table 1 and Fig. 1, respectively. In the BH-RSV(-) complementation assay, chicken cells were cocultured with 16Q cells releasing noninfectious Bryan sarcoma virus. Table 1 shows that in this assay only cells derived from Brown Leghorns provided envelope glycoprotein and thus rendered the virus infectious. These cells were scored as chf + in distinction to the cells from two other sources. Molecular hybridization experiments were performed according to Hayward and Hanafusa (23) to gain additional information about the expression of endogenous viral genes. In these experiments cell RNA in excess was hybridized

Proc. Natl. Acad. Sci. USA 77 (1980)

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50 ., 50

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20 10 10-1

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Crt, mol . sec/liter

FIG. 1. Hybridization of td PR-C [3H]cDNA to the RNA isolated from uninfected chicken embryo cells. The sources and initial concentrations of RNA were as follows: Brown Leghorn C/E chf+, 5 mg/ml (0); line C C/AE chf-, 5.9 mg/ml (0); C/E chf-, gs , 4.25 mg/ml (-).

to virus-specific cDNA. Under these conditions the concentration of virus-specific nucleotide sequences is related to the rate of hybrid formation as expressed by the Crt value (product of RNA concentration in moles of nucleotide per liter and incubation time in seconds) at which the reaction is half complete (24). Fig. 1 shows that about 75% of the cDNA hybridized to the RNA of Brown Leghorn chf + cells and a lesser amount (about 50%) to that of line C chf- cells. The Crtl/2 value of the hybridization reaction indicated a virus RNA content in line C cells of approximately 0.2% of that determined in Brown Leghorns. In contrast, little (2-5%) of the cDNA hybridized to the RNA of C/E chf - cells, indicating that in these cells chf gene was absent or strongly inhibited in expression. Cloning by Transfection to Isolate SR-D Virus Devoid of chf Information. The cells with different chf phenotypes were infected with SR-D and the virus progeny were examined for heterozygotes and recombinants. Table 1 shows that the stock of the SR-D virus harvested in Brown Leghorns contains approximately 0.15% phenotypically mixed particles. This was determined from the titer of the virus in C/E chicken cells and T/B,D turkey cells. The SR-D virus was then cloned by transfection in C/E chf- chicken cells. After the cloning the virus contained virtually no (less than 0.001%) envE components when grown in the same C/E chf- cells and only small amounts (about 0.01%) when grown in line C chf- cells. We have concluded that the cloned SR-D is virtually devoid of the host range marker encoded by the chicken helper factor gene and can be used, therefore, in envD X chf recombination experiments.

Transfection of envD X chf Recombinants. Preliminary experiments were performed to test that the amount of recombinant proviruses synthesized in SR-D-infected chf + cells may be detected by transfection. In these experiments the transfection of the SR-D parent was looked for in chicken cells and that of the recombinant provirus in turkey cells. These turkey cells have a T/B,D phenotype and select for envE recombinants. The turkey cells were also chosen because the transfection efficiency of SR-E provirus in these cells (unlike, for instance, in quail cells) is about the same as that of SR-D in chicken cells (unpublished data). In the first experiment (Table 2) chf + chicken cells were chronically infected with crude SR-D and td SR-D viruses previously grown in the same chf + cells. The DNA purified

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Proc. Natl. Acad. Sci. USA 77 (1980)

Microbiology: Carloni et al.

Table 1. Host range determination of virus harvested from chronically infected chicken cells of different chf phenotypes Titer (FFU/ml) of the Cells used for chf virus harvest* in Phenotypic growing virus phenotype Chicken C/E cells Turkey T/B,D cells mixing,t % + Brown Leghorn C/E cells 8000 0.15 5,100,000 Line C C/AE cells 250 0.011 2,100,000 0 Secondary C/E cells