Biological Activity of Cloned Rat Endogenous C-Type Virus DNA Transferred by Microinjecfion

J. gen. Virol. (1982), 63, 37-43. Printed in Great Britain 37

Key words: retrovirus DNA/intranuclear microinjection/cell transformation

Biological Activity of Cloned Rat Endogenous C-type Virus DNA Transferred by Microinjecfion

By STRINGNER S. YANG, 1. J. TAUB, 1 R. MODALI, 1 D. BROWN 2 AND E. MURPHY JR 2 1Laboratory of Cell Biology, National Cancer Institute, Bethesda, Maryland 20205, U.S.A. and 2Department of Tumour Virology, University of Texas System, Houston, Texas 77030, U.S.A. (Accepted 4 June 1982)

SUMMARY The biological activity of a molecularly cloned DNA of a rat endogenous C-type leukaemia helper virus, RHHV, was assessed by intranuclear microinjection into normal rat kidney cells (NRK 153). Release of rat C-type leukaemia helper viruses by the microinjected cells was examined by superinfection of Kirsten-transformed non- producer cells (K-NRK). Immediate release of helper leukaemia viruses at a very low

level was observed only in the ~DVlS3• - x~X~m3.5/ci r cells microinjected with the supercoiled form of RHHV DNA in toto, suggesting that the circular form of the virus DNA might have expedited the replication and expression of virus particles. Genome rescue experiments were also performed by co-cultivating the microinjected NRK~m53 cells carrying various linear RHHV DNAs, in toto or of subgenomic sizes, with K-NRK cells. The results indicated that both the total and the 5.8 to 6.2 kb DNA fragment proximal to the 5' terminus of the cloned RHHV 8.8 kb DNA were able to rescue successfully a transforming replication-competent pseudotype virus. Subgenomic DNA fragments derived from the centre or the 3' end of the RHHV DNA were ineffective in the genome rescue experiments. INTRODUCTION Recent transfection studies, using proviral DNA or high molecular weight DNA prepared from transformed cells, have shown that virus DNA alone, or cellular copies of it, is sufficient to induce cell transformation (Lowy et al., 1977; Copeland et al., 1979; Andersson et al., 1979), demonstrating that all necessary genetic information for morphological transformation of cells in culture is carried by the src genetic region of the sarcoma virus. Additional transfection experiments using subgenomic DNA fragments derived from both cellular and sarcoma virus sources (Tronick et al., 1979; Vande Woude et al., 1979; Haywood et al., 1981; Hager!et al., 1979) have been reported. In view of the diversity of the results obtained in the various systems employed, no unified thesis can yet be advanced. It has, however, been established that co-transfection of cellular src (c-src) or virus src (v-src) DNA with long terminal repeat sequences (LTRs), which flank retrovirus replication genes and which themselves bear RNA polymerase II promoter sequences capable of the activation of cellular oncogenes (Haywood et al., ~1981), would satisfy the molecular requirement for successful transformation of cells in culture. In spite of such concentrated research efforts towards understanding the biological potential of the c-src and v-src sequences, the role of the leukaemia helper virus DNA in cell transfOrma- tion and replication of virus particles has yet to be resolved fully. Recently, we successfully cloned the 8-8 kilobase (kb) proviral DNA of an endogenous rat C-type helper virus, RHHV, and have established a restriction map for the whole genomic DNA (Yang et al., 1982). Results of restriction enzyme mapping suggest that there are two LTRs in RHHV DNA demarcated by two TaqI sites, located at about 0-8 kb proximal to the 5' terminus and at about 0.8 kb proximal to the 3' terminus. Thus, the virus replicative genes, gag, pol and env, would be expected to extend from the 5' terminus, at about 0-8 kb, to 8 kb on the map. This has been confirmed by hybrid selection of virus-specific mRNA in cell-free translation experiments (Yang et al., 1982).

0022-1317/82/0000-5178 $02.00©1982 SGM 38 S. S. YANG AND OTHERS The experiments to be reported here entailed the isolation and microinjection of defined RHHV DNA restriction fragments in order to assess their biological activity, reasoning that the results thereby obtained might prove helpful in the resolution of the molecular basis of cell transformation and virus replication in this particular rat C-type tumour virus system.

METHODS Preparation of cloned RHHV DNAfor microinjections. We chose to microinject (i) the 8.8 kb total RHHV DNA (DNA fragment 1, Fig. 1) and the following subgenomic DNA fragments: (ii) a 5-8 to 6-0 kb fragment (2, Fig. 1) containing an LTR and the RHHV gag and pol genes, (iii) a 4.0 kb fragment (3, Fig. 1) containing pBR322 sequences only, (iv) a 2-65 to 2.85 kb fragment (4, Fig. 1) containing mostly RHHV env gene and LTR sequences, (v) a 2.5 kb fragment (5, Fig. 1) containing mostly RHHV 2ool gene sequences and (vi) a circular (cir) DNA migrating at 3.5 to 3.7 kb fragment (6, Fig. 1), representing the supercoiled RHHV DNA. The cir DNA is a mixture of the circular forms of 8.8 kb (80~) and 5.8 to 6.0 kb (20~) RHHV DNA. These were isolated from the recombinant DNA as described below; they became self-ligated at the sticky BamHI ends upon storage at 0 to 2 °C. These DNAs were prepared from the 8/32 recombinant clone as described previously (Hager et al., 1979). Fragments of RHHV DNA were further purified by excision from pBR322 DNA, using either EcoRI or BamHI digestion, followed by electrophoresis of the DNA in 0-85 ~o low-melting agarose gels (Bio-Rad). The desired DNA was then localized by ethidium bromide staining and recovered by the borosilicate adsorption technique. The DNA was eluted from the borosilicate powder with sterile distilled water and adjusted to 0.01 N-Tris-HC1, 0.15 ta- NaC1 and 0.001 ra-EDTA, pH 8.1 (TEN), to be used in microinjection experiments. Microinjection. We chose NRK153 as target cells because these normal rat kidney cells showed an extremely low frequency of spontaneous transformation. They are adherent cultures and therefore facilitated microinjection. Ideally, transformed non-producer cells such as K-NRK carrying v-src or c-src sequences should be the appropri- ate target cells for microinjection studies of the leukaemia helper virus DNA biological activity. But K-NRK cells are a suspension culture, and proved to be impossible targets for microinjection. The target cells, NRK 153, were trypsinized, seeded on to individual coverslips and cultured in 35 mm plates under the conditions described below. Intranuclear microinjections were carried out on approximately 30 to 50~o-confluent NRK ls3 cells growing on coverslips, inverted for the purpose of microinjection, on a Leitz Laborlux II fixed-stage microscope. Micro- injections were controlled visually at x 400 magnification. Microneedles were forged on a modified DeFonbrune microforge (Stacey, 1981 ; Kopchik et al., 1981) to achieve an orifice of 0.1 to 0-5 gin. DNA samples in 0.1 ~ KC1 were prepared for microinjection by centrifugation in a sealed 20 btl glass capillary pipette at 40 000 rev/min for 40 min and 1 gl of each sample was loaded under pressure into the tip of a water-filled microneedle by displacement of water (Stacey, 1981). Movement of the microneedle was controlled by a micromanipulator. An estimated 10 femtolitres carrying 200 to 700 DNA molecules was microinjected, an efficiency compatible with documented microinjection reports (Stacey, 1981 ; Kopchik et al., 1981). One hundred ceils were intranuclearly microinjected at one edge of each coverslip. After this, uninjected ceils on the remainder of the coverslip were scraped away. The coverslip was then cultured in a Petri plate at 37 °C in a CO2 incubator until ready for trypsinization. Micro- injected cells were designated NRK~m53. Cells and tissue culture. NRK 153 and NRK a1°l, normal rat kidney cell lines derived from the original NRK line (Duc-Nguyen etal., 1966) were supplied by Biotech (Rockville, Md., U.S.A.). NRK cells were used from passages 11 to 14. NRKs, HTC-H1 (a rat hepatoma tissue culture line), HT-1 and K-NRK were cultured in Dulbecco's modified Eagle's medium supplemented with 5~o heat-inactivated (56 °C, 30 min) foetal calf serum (FCS), plus penicillin (50 units/ml) and streptomycin (25 gg/ml) in a 5~ CO2 atmosphere at 37 °C. Superinfection, focus-formation assay and genome rescue experiments. The superinfection experiment was carried out with 5 x 105 K-NRK cells cultured in a 75 cm 2 flask. Five ml of cell-free culture medium prepared from the various microinjected NRKlms3 clones was added to each K-NRK culture. After 2 h of infection at 37 °C, 20 ml of fresh medium was added to each culture. After 4 days of cultivation, release of virus in the various superinfection K-NRK culture media was determined by the focus-formation assay. The focus-formation assay was carried out on 1.5 x 105 NRK 153 cells cultured overnight in polybrene- supplemented (2 ~tg/ml) medium on a 60 x 15 mm Petri plate as described above. After the medium was with- drawn, the cells were infected with 0.5 ml of cell-free culture medium prepared from either the superinfected cultures or the genome rescue co-cultures. The Petri plates were gently rocked at 37 °C for 30 min. Fresh medium with 5~ FCS was then added to the cultures. Scoring of transformed foci was carried out on day 5 or 6. Genome rescue experiments were carried out by co-cultivating the various microinjected cells with various control and transformed non-producer cells, such as NRK B1°1, HT-1, HTC-H1 and K-NRK, at a ratio of 2 : 1 in cell number. The selected transformed, non-producer cell lines were known to carry different numbers of copies of the v-src sequence. Under the described co-cultivation conditions, either complementation or recombination with the rat helper leukaemia virus DNA sequence carried in the microinjected NRK~m53 cell was known to take place at a high frequency, resulting in the release of a pseudotype virus (Yang et al., 1976). Cell-free media were prepared Microinjection study of rat C-type helper virus DNA 39

(a) gag pol env A A A A LTR LTR

(b) 5' 3 r 0 8.8 13-16 kb ! ! t RHHV pBR322 DNA I , , I ...... r-i fragments B B R B RB kb 5 2-5 4 2.65-2.8 2 5.8-6.0 1 8.8 6 cir 3 4.0 Fig. 1. (a) Functional and (b) schematic representations of the subgenomic DNA fragments, which span the entire 8.8 kb of RHHV DNA restriction endonuclease map, used in microinjection studies. The total and subgenomic DNA fragments and the supercoiled 'cir' are shown separated on a gel in Fig. 3. Total or subgenomic 'Microinjection fragments of RHHV DNA ) 100 NRK~5~ cells Passage no.

Trypsinization ) Carrying P0 culture

Co-cultivation I ] at 2:1 ratio NRK B~°~ HT-1 HTC-H1 K-NRK P1

NSX°l/Nm HT-1/Nrn HTC-H1/N m K-NRK/N m CP0 J

) Carrying co-culture CP 1

Focus-formation assay on co-culture media or cell-flee extract for virus release

Single cell or penny-cylinder cloning of transformed cell

Focus-formation assay on transformed culture for perpetuating virus release Fig. 2. Flow scheme of genome rescue experiments with NRK 153 cells microinjected with total and subgenomic fragments of RHHV DNA. from each co-culture at intervals for focus-formation assays on NRK ls3 cells in order to determine any release of transforming viruses. A flow scheme of the genome rescue experiment is shown in Fig. 2. Restriction enzyme assays. All restriction enzymes were purchased from Boehringer-Mannheim, Bethesda Research Laboratories or New England Biolabs. Unless otherwise specified, all digestion conditions were according to the instructions provided by the suppliers.

RESULTS

On the basis of the detailed restriction map for the RHHV genomic DNA (Yang et al., 1982), it became feasible to isolate defined subgenomic DNA fragments by precise restriction enzyme cleavages for microinjection studies. Fig. 1 provides a schematic and functional representation 40 s. s. YANG AND OTHERS

kb kb kb 23.7 9.6 8-8 • 6.0 6.0 • 6.6 4.3 4.0 • 3'5 3.7 • 2.65 • 2.5 2.80 2.3 Fig. 3. Agarose gel electrophoresis of RHHV DNA after exhaustive digestion by EcoRI and/or BamHI endonuclease for 20 h at 37 °C. Concentrated specific DNA fragments were recycled by low-melting agarose gel (0'9~o) electrophoresis at 50 V for 7 h. DNA bands marked by arrows are 8.8 kb, 6.0 kb, 4.0 kb, 2.65 to 2.80 kb, 2.5 kb and cir DNA, which migrated to the 3.5-3.7 kb region. They were visualized by u.v. illumination, excised, and extracted by the borosilicate technique (Yang et al., 1980) for microinjection. HindllI-digested lambda DNA mol. wt. markers are indicated on the right-hand side.

Table 1. Release of type C helper viruses by various microinjected NRK~ 53 clones DNA fragment number 1 2 3 4 5 6 DNA fragment microinjected into N RK 1s 3 nucleus (kb)* 8.8 5.8-6.0 4.0 2.65-2.85 2-5 3.5-3.7 Approximate number of DNA moleculest 205 260 500 700 400 100 Virus titre (f.f.u./ml) of conditioned mediums 0 0 0 0 0 5(280) * All microinjections were done in duplicate. t The number of DNA molecules was estimated on the basis of size and concentration of the particular DNA molecule or fragment microinjected (Stacey, 1981). :~ K-NRK cells were superinfected with cell-free culture media from the various microinjected NRK~m53 clones. The conditioned K-NRK culture media were then used as virus sources and assayed for focus-forming units (Yang et al., 1976). All assays were done in triplicate. The no. of f.f.u./ml represents averaged values. The number in parentheses represents averaged f.f.u, per culture. of the 8.8 kb total genomic DNA, the four subgenomic fragments 5.8 to 6.0 kb, 4.0 kb, 2-65 to 2.85 kb and 2.5 kb and the supercoiled recombinant DNA all recovered, recycled, concentrated and purified from agarose gels (Fig. 3) for microinjection studies. Since the subgenomic DNA fragments used in this study spanned the entire 8.8 kb, biological activities transferred by any of the fragments should provide some insight into both the gene function and its order. No significant change in cell morphology occurred during the initial growth of some 24 microinjected cell clones (Fig. 4a). During the second passage, one particular clone microinjected with the supercoiled RHHV DNA, ~,~vls3l'q tXXXm 3.5/cir, demonstrated morphological changes, such as a disoriented and overlapping growth pattern and a loss of contact inhibition (Fig. 4b). Morph- ology of N ~v153xXXXm 3.5/cir culture resembled that of chemically or spontaneously transformed cultures and persisted with the NRK15335/~ir clone. Release of type C helper leukaemia virus in microinjected NRK 153 clones Immediate release of helper leukaemia viruses by the various microinjected cells into the culture medium, as determined by superinfection of K-NRK cells, was observed only in the N RK ~mS335/circlone microinjected with the supercoiled form of the RHHV DNA (Table 1). Super- infection of the K-NRK cells with cell-free medium from the NRK~m5335/circulture resulted in the rescue of a pseudotype transforming virus even though the number observed in the assay was low (5 f.f.u./ml, 280 foci in all). The morphology of one transforming focus, cloned out by penny- cylinder selection, is presented in Fig. 4 (c). It had the character of a KSV(RHHV)-transformed focus. This observation suggested that the circular form of the virus DNA might have expedited expression of virus particle synthesis. All other cell cultures microinjected with either full size linear (8-8 kb) RHHV DNA or subgenomic restriction fragments of RHHV DNA, as well as the Microinjection study of rat C-type helper virus DNA 4 1

Fig. 4. Morphology of normal rat kidney NRK ls3 cells (a), which underwent changes after intranuclear microinjection of the supercoiled RHHV DNA (b). Transformed foci were induced by the infectious, transforming and replication competent virus released by the co-culture of K-NRK ceils with NRK~Ss3s (c) or with NRK mS.S-6-O~s3 (d) cells in the genome rescue experiment.

Table 2. Rescue of pseudotype transforming virus in genome rescue experiments with NRKXm53 cells co-cultivated with various src-carrying cell lines Cells co-cultured DNA fragment microinjected into NRK~m53 cell* with NRK 153 clones 1 2 3 4 5 6 Expt. 1 NRK-B101 0t 0 HTC-H1 0 0 K-NRK 42 (3150) 15 (1025) Expt. 2 NRK ls3 0 0 0 0 0 0 HTC-H1 0 0 0 0 0 0 K-NRK 6 (450) 3 (225) 0 0 0 8 (600) * All microinjected samples were described in Table 1 and assayed in duplicate. -~ All samples were assayed in triplicate. Results are expressed as the number of f.f.u./ml in cell-free medium which represent averaged values; numbers in parentheses represent f.f.u./culture.

0.1 ~ KC1 control, failed to demonstrate release of helper leukaemia viruses into the culture media. These cultures were then assayed by genome rescue experiments as described below.

Rescue of a transforming pseudotype virus by microinjected RHHV DNA sequences To test the biological activity of the various microinjected DNA fragments in the NRK153 cell cultures, the cells were co-cultured at the first passage with cell lines carrying various copies of v- src and c-src sequences in their cellular DNA in a genome rescue experiment, as shown in the flow scheme (Fig. 2). Media from these co-cultures were then assayed for the release of transforming viruses by a focus-formation assay (Yang et al., 1976) on NRK 153 cells. The results from two independent microinjection series and subsequent genome rescue experiments are summarized in Table 2. Release of infectious transforming virus occurred 3 or 4 days after cocultivation of K-NRK cells with the NRK~ 53 cells microinjected with either 8.8 kb RHHV DNA (NRKm8.8)153 or the 5.8 to 6.0 kb of RHHV DNA ~tNRK 153mS.8.6.0j.~ No foci were observed using media prepared from any other co-cultures. As shown in Fig. 4 (d), the morphology of the transformed NRK foci resembled that of a Kirsten murine sarcoma virus-transformed focus. These results showed that the 5.8 to 6.0 kb of RHHV DNA proximal to the-5' terminus (containing in all probability the RHHV gag, pol, part of the env genes and an LTR; see Fig. 1) is sufficient to rescue a transforming virus from K-NRK cells. It also suggested that, if recombina- 42 S. S. YANG AND OTHERS

Table 3. Progeny virus replication in culture media of cloned transformed NRK Is3 cells Virus titre* Virus source Primary transformation in NRK t53 cells NRK~3s/K-NRK co-culturet 43 (36-52):~ NRK~m~630/K-NRK co-culture 18 (10-26)~. NRKlmS335/eirsuperinfected K-NRK culture 9 (1-17):~ Secondary transformation in NRK ls3 cells Transformed NRK clone a 236 (95-377) Transformed NRK clone b 24 (7-41) Transformed NRK clone c 13 (2-24) * All assays were performed in triplicate. The number of f.f.u./ml represents averaged values. Numbers in parentheses represent the range of values obtained from the triplicate samples. t Co-culture is described according to the cell strains used; e.g. 'NRK~Ss3.s/K-NRK' represents NRK 153 cells microinjected with the 8-8 kb DNA and then co-cultured with K-NRK cells. :~ Transformed NRK ~53 cells were designated clone a, b or c (top to bottom) and were isolated by either single- cell cloning or penny-cylinder cloning. tion had occurred between the helper DNA sequence in the microinjected cells with the v-src sequences carried in the K-NRK cells then it occurred during the first or second cell division. Alternatively, the independent expression of the helper leukaemia virus could have provided the virus polypeptide(s) essential for the encapsidation of the sarcoma virus genome and resulted in the production of a pseudotype virus. Since transforming viruses were found within 3 to 4 days, the expression of the infectious transforming virus probably occurred at the second or third generation of the co-cultured cells. When compared with the titre of transforming viruses released by the co-culture of K-NRK cells with the NRK153.s. cells, ~rRV1531,"m5.S-6.0 cells showed a much lower efficiency in the rescuing of the v-src sequence in K-NRK cells (Table 2). Transformed cells infected by viruses released by this co-culture seemed to undergo spontaneous autolysis and were difficult to keep in culture. Nevertheless, the success of the genome rescue experiment using the NRKms.s-6.0153 cells suggested that all leukaemia helper virus information essential for the rescue of an infectious transforming virus in the K-NRK cells resided within the RHHV 5.8 to 6.0 kb DNA fragment proximal to the 5' terminus. The stability of the transforming virus released in the various co-cultures was examined~ Cells from transformed loci (Fig. 4c, d) were isolated by penny-cylinder or single-cell cloning. As shown in Table 3, transformed daughter clones, designated 'a' and 'b', continued to produce progeny infectious viruses at high titre when their culture media were assayed for cell transformation. This increase in virus titre was probably due to virus gene amplification, resulting from multiple integrations during cell division, an observation based on preliminary results obtained from restriction enzyme analyses.

DISCUSSION We have successfully transferred by intranuclear microinjection the rat leukaemia helper virus total or subgenomic DNA sequences into NRK 153 cells. Expression of the helper virus function was positively identified in some of these cells microinjected with RHHV in toto or subgenomic DNAs. The DNA sequence coding for complete helper virus biological activity, which proved essential in the recombination with the v-src DNA sequence carried in K-NRK cells for the evolution of an infectious transforming virus, resided within the 5.8 to 6.0 kb proximal to the 5' end of the rat helper virus DNA. Alternatively, the independent expression of the 5-8 to 6-0 kb DNA sequence could have provided the virus polypeptides (gag, pol and env) essential for the encapsidation of the virus genome, i.e. via a genetic complementation process. DNA fragments consisting primarily of LTR sequences such as the 2.65 kb proximal to the 3' terminus, or of incomplete virus information such as the 2.5 kb in the middle of the RHHV DNA, failed to elicit complete helper biological activity when tested in genome rescue experiments. Moreover, the observation that the transforming virus continued to replicate, and preliminary results from restriction enzyme analysis and Southern blot filter hybridizations, suggests that the microinjected DNA sequence became integrated into the host cell genomic Microinjection study of rat C-type helper virus DNA 43

DNA. A recent paper (Kopchik et al., 1981) on microinjection studies of avian viras DNA into RSV-transformed cells, a far more sensitive system, has documented successful release of infectious transforming virus 3 h after microinjection. In view of the rapid response in virus particle synthesis, it was proposed that integration of the virus DNA sequence was probably not necessary for the expression of viruses. In this rat cell system, the earliest expression of the rat helper leukaemia virus was within the 24 h after intranuclear microinjection. This implies that the microinjected DNA sequence had gone through at least one complete cell cycle prior to its expression, although it should be stressed that the superinfection assay for rat leukaemia helper virus is comparatively inefficient. In conclusion, rat helper virus total or subgenomic DNA sequences were successfully microinjected into NRK 153 nuclei and expressions of helper virus function were positively identified in the ceils. As shown by the long-term stability of the RHHV genome in the microinjected cells and by Southern blot hybridization experiments, the microinjected DNA sequence was found to be integrated into the host cell genomic DNA. The RHHV DNA sequence that proved essential in the rescue of an infectious transforming virus resided within the 5-8 to 6-0 kb proximal to the 5' terminus of the RHHV DNA. Our results are perhaps best explained as being those of a classical pseudotype rescue experiment in which the helper virus functions in complementing the replication-defective transforming virus by providing polypeptides essential for replication or encapsidation. However, our observations could also be explained as the result of a successful recombination with either the v-src or c-src sequences in the K-NRK cells, thus resulting in the evolution of an infectious, transforming and replication-component virus. Experiments to distinguish between these two possibilities are now in progress. This research was supported, in part, by grants from the Robert A. Welch Foundation (G-854) and Biomedical Research Support Grants (BR-50 and BR-51). The authors gratefully thank Gall Kington for manuscript preparation.

REFERENCES ANDERSSON,P., GOLDrAItB,M. P. &WEI~ERG, R. A. (1979). A defined subgenomic fragment of in vitro synthesized Moloney sarcoma virus DNA can induce cell transformation upon transfection. Cell 16, 63-75. COPELAND, N. G., ZELENETE,A. D. & COOPER, G. ~l. (1979). Transformation of NIH/3T3 mouse cells by DNA of Rous sarcoma virus. Cell 18, 993-1002. DUC-NGUYEN, n., ROSENBLtna, E. N. & ZEIGEL, R. F. (1966). Persistent infection of a rat kidney cell line with Rauscher murine leukemia virus. Journal of Bacteriology 92, 1133-1140. HAGER, G. L., CHANG, E. H, CHAN, H. W., CAR.ON, C. F., ISRAEL, M. A., MARTIN, M. A., SCOLNICK, E. M. & LOWY, D. R. (1979). Molecular cloning of the Harvey sarcoma virus-closed circular DNA intermediate: initial structural and biological characterization. Journal of Virology 31, 795-809. HAYWOOD, W. S., NEEL, B. G., ASTRIN, S. & FANY, S. (1981). Activation of a cellular onc gene by promoter insertion in ALV-induced lymphoid leukosis. Nature, London 290, 475-480. KOPCIJIK,J. J., m, G., St(ALKA,A. M. &SrACEY, O. W. (1981). Biologicalactivity of cloned retrovirus DNA in microinjected cells. Proceedings of the National Academy of Sciences of the United States of America 78, 4383-4387. LOWY, D. R., ~NDS, E. & SCOLNICK, E. M. (1977). Helper-independent transformation by unintegrated Harvey sarcoma virus DNA. Journal of Virology 26, 291 298. STACEY,W. S. (1981). Microinjection of mRNA and other macromoleculesinto living cells. Methods in Enzymology (in press). TRONICK, S. R., ROBBINS, K. C., CANAANI, E., DEVARE, S. G., ANDERSON, P. R. & AARONSON, S. A. (1979). Molecular cloning of Moloney sarcoma virus: arrangement of virus-related sequences within the normal mouse genome. Proceedings of the National Academy of Sciences of the United States of America 76, 6314-6318. VANDE WOUDE, G. F., OS~N, M., MeCLEMENTS, W. L, ENQUIST, L. W., BLAIR, D. G., FISCHINGI~R, P. J., MAIZEL, J. V. & SULLIVAN, i. (1979). Characterization of integrated Moloney sarcoma proviruses and flanking host sequences cloned in bacteriophage 2. CoM Spring Harbor Symposia on Quantitative Biology 44, 735-745. YANG, S. S., CHAN,C. &THOMPSON, E. B. (1976). Rescue of a rat tropic hepatoma virus pseudotype Kirsten sarcoma virus by cocultivation of hepatoma tissue culture cells with K-NRK cells. Journal of General Virology 32, 239- 243. YANG, S. S., YEH, L-S., TAUB, I., MILLER, N. & GARDNER, M. B. (1980). Integrated and proviral DNA sequences for wiid rat tumor virus and for rat hepatoma helper virus in various laboratory and wild rat tumor cells: a restriction enzyme analysis. In Viruses in Naturally Occurring Cancers, pp. 1083-1092. Edited by M. Essex, G. Todaro & H. Zur Hausen. New York: Cold Spring Harbor Laboratory. YANG, S. S., MODALI,R. &MURPHY, E., JR (1982). Cloning of rat endogenous helper leukemia virus DNA sequence and expression of the helper activity encoded by the cloned sequence in normal rat kidney cells. In Cell Culture in Support of Bioscience. Edited by R. Acton. New York: Plenum Press (in press).

(Received 28 March 1982)