the xenotropic sequences in the region of the env gene. The to the env gene of mouse xenotropic type C virus.

Transcription

1 Proc. Natl. Acad. Sci. U$A Vol. 74, No. 10, pp , October 1977 Microbiology Friend strain of spleen focus-forming virus is a recombinant between ecotropic murine type C virus and the env gene region of xenotropic type C virus (murine leukemia virus/xenotropic virus/molecular hybridization) DAVID H. TROXLER*, DOUGLAS Lowyt, RICHARD HOWK*, HOWARD YOUNG*, AND EDWARD M. SCOLNICK** * Laboratory of Tumor Virus Genetics and t Dermatology Branch, National Cancer Institute, Bethesda, Maryland Communicated by Wallace P. Rowe, August 4, 1977 ABSTRACT The spleen focus-forming virus (SFFV), a replication-defective murine leukemia virus that causes the rapid transformation of certain hematopoietic target cells, has acquired specific xenotropic viral genetic information not contained in Friend helper virus. In the current studies, it is shown that a cdna that represents a xenotropic virus portion of SFFV detects genetic sequences derived from the env gene region of murine xenotropic virus. The significance of the acquisition of these xenotropic viral sequences by SFFV is discussed with regard to their possible role in the rapid leukemogenicity of SFFV, and an analogy is drawn between the formation of SFFV and the formation of the Kirsten and Harvey sarcoma viruses. Recently we examined the molecular composition of a murine type C virus that causes acute leukemia in order to define the viral gene(s) responsible for induction of leukemia. We chose to study the Friend strain of the spleen focus-forming virus (SFFV), which causes rapid splenomegaly and leukemia when injected as pseudotypes into susceptible mice (1, 3). Within 48 hr after injection of appropriate SFFV pseudotypes, transformed hematopoietic cells can be detected in the spleens of inoculated mice (2), and it is clear that, in this leukemia virus system, the genetic information responsible for rapid leukemogenicity is contained in the SFFV genome (3). To analyze the gene(s) of SFFV that might be responsible for its leukemogenicity, we first isolated SFFV free of replicating helper virus in nonproducer fibroblast cell lines (4, 5). Using these cell lines in molecular hybridization studies, we demonstrated that SFFV was a recombinant between a portion of ecotropic virus [NBtropic clone of Friend type C virus (F-MuLV)] and a portion of mouse xenotropic virus (5). Parallels were noted between the SFFV genome and the genomes of Kirsten and Harvey sarcoma viruses, which are recombinants between mouse ecotropic viruses and a class of endogenous rat type C virus (6, 7). As an extension of this work, we have attempted to define which portion(s) of the xenotropic virus genome recombined with F-MuLV to form SFFV. Using a cdna probe that detects xenotropic sequences contained in SFFV (cdnasffv), we have analyzed by molecular hybridization the genomes of various clonal isolates of replicating MuLV that have a broadened host range such that they grow on both mouse cells and cells permissive for xenotropic virus and thus potentially are recombinants between ecotropic and xenotropic viruses. We report here (i) positive hybridization of cdnasffv to viral RNA from certain recombinant viruses that demonstrate replicative properties associated with the envelope glycoprotein of xenotropic virus and (ii) mapping data on the genome of a recom- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C solely to indicate this fact. binant replicating MuLV that are consistent with a location of the xenotropic sequences in the region of the env gene. The results indicate that the formation of SFFV was accompanied by the acquisition of sequences eitherwithin or closely linked to the env gene of mouse xenotropic type C virus. MATERIALS AND METHODS Cells. The following cell lines were used in these studies and have been described (4, 5): SC-1, BALB/c 3T3, and NIH 3T3 mouse embryo fibroblast cell lines, permissive for ecotropic MuLV; mink lung fibroblasts, permissive for the growth of xenotropic type C viruses; nonproducer cells containing the Friend strain of SFFV, BALB clone 4, and SFFV-NRK clone 1; normal rat kidney (NRK) cells; a subclone of NRK (V-NRK) that produces rat type C viruses; and NRK cells nonproductively infected with the p30+ strain of Moloney sarcoma virus (S+L- NRK) and Abelson virus (Ab-NRK) (8). Viruses Used. F-MuLV and BALB clone 4/F-MuLV, a virus complex containing SFFV and F-MuLV, have been described (5). Various clones of Moloney leukemia virus were used. Ecotropic clones: Mo-MuLV Cl H was obtained from Janet Hartley, National Institute of Allergy and Infectious Diseases, Bethesda, MD; Mo-MuLV Cl 2 was obtained from Nancy Hopkins, Massachusetts Institute of Technology, Boston, MA; Mo-MuLV Cl 35 was obtained from Alan Rein, National Cancer Institute, Bethesda, MD. Other clones: Mo-MuLV clones 4 and 83 were obtained in our laboratory by end-point dilution cloning in Microtest II culture plates on SC-i cells of an uncloned stock of Mo-MuLV obtained from Robert Bassin, National Cancer Institute (9). N-Tropic AKR MuLV, AKR- MCF no. 247, and amphotropic MuLV 1504A were obtained from Janet Hartley (10, 11). Finally, ATS-4, a xenotropic type C virus, has been described (). Virus Assays. Replicating type C virus (ecotropic, xenotropic, and amphotropic) was detected by assay of a 24-hr collection of supernatant medium from infected cells for reverse transcriptase activity by procedures that have been described (4). MuLV were also assayed by (i) the XC plaque assay (13) and (ii) assay for cytopathic effects on mink lung fibroblasts by procedures recently described (11). Preparation of cdna Probes. 3H-Labeled cdnasffv was Abbreviations: SFFV, Friend strain of spleen focus-forming virus; F-MuLV, NB-tropic clone of Friend type C virus; MuLV, murine leukemia virus; cdnasffv, cdna probe that detects xenotropic related sequences in SFFV; NRK, normal rat kidney; cdnagag, cdna probe homologous to RNA sequences that code for polypeptides of the gag gene of Mo-MuLV; cdna3', cdna prepared from the 3' end of ecotropic Mo-MuLV RNA. t To whom reprint requests should be addressed. 4671

2 4672 Microbiology: Troxler et al. prepared from sucrose density gradient-purified BALB clone 4/F-MuLV by utilizing viral RNA as a template for partially purified avian myeloblastosis virus reverse transcriptase with calf thymus DNA fragments as primer. The cdna was subsequently purified by hydroxylapatite column chromatography. The details of preparation of cdnasffv have been described (5). A [3H]cDNA probe for ecotropic Mo-MuLV was synthesized in the endogenous reaction with sucrose density gradient-purified Mo-MuLV clone 2 grown on SC-1 cells. All cdnas were labeled with [3H]dCTP to a specific activity of 2 X 107 cpm/,ug and protected 60% of viral [32P]RNA at 1:1 molar ratios and over 85% of the [32P]RNA at 3:1 ratios. From unfractionated Mo-MuLVeco cdna probe, a cdna fraction was prepared that was homologous to RNA sequences that code for polypeptides of the gag gene of Mo-MuLV, designated cdnagag, as described (8, 14). The cdnagag used in these studies was identical to fraction 5 (14). In addition, a cdna prepared from the 3' end of Mo-MuLV RNA was synthesized in an exogenous reaction by priming with oligo (dt) 18, a poly(a)-containing 4-6S piece of Mo-MuLV viral RNA; this cdna was designated cdna3'. The methods for preparation of both these cdna fractions have been fully detailed (14). Preparations of Poly(A)Containing Viral RNA. Different size classes of Mo-MuLV8s viral RNA containing poly(a) at the 3' end were obtained by procedures detailed by Wang et al. (15). Preparation of RNA. Total cellular RNA was extracted by a modified cesium chloride centrifugation method of Glisin et al. (16). Viral RNA was extracted from virus particles released into the supernatant medium of infected cells by a sodium dodecyl sulfate/phenol extraction procedure (5). The amount of RNA obtained with either technique was determined from the absorbance at 260 nm in a Beckman model 25 spectrophotometer. Analytical Hybridization Reaction. RNA [3H]cDNA hybridizations were performed at 660 for hr and were assayed with the use of SI nuclease (17, 18). Each hybridization 0.5-ml mixture contained 0.02 M Tris-HCI (ph 7.5), 1 mm EDTA, 0.1% sodium dodecyl sulfate, 0.75 M NaCl, 1.0 jig of calf thymus DNA, 10,ug of yeast ribosomal RNA, the indicated cellular or viral RNA, and trichloroacetic acidinsoluble cpm of [3H]cDNA. Hybridization results were ana- *lyzed as a function of RNA concentration and time and were expressed as Crt (mol sec liter-') (19). RESULTS Host Range Characteristics of Various Murine Leukemia Viruses. Two types of murine type C viruses have been reported that can grow both on mink cells and on various mouse cells. One class is comprised of viruses, such as the AKR-MCF virus and certain strains of Moloney leukemia virus, that appear to have acquired a portion of the envelope glycoprotein of xenotropic viruses based on interference and neutralization data (11, 20). The other class is illustrated by amphotropic virus 1504A, which was isolated from a wild mouse and which appears to have a distinct envelope based on neutralization and interference patterns (10). To gain insight into which portion(s) of the xenotropic virus genome was incorporated into SFFV, we attempted to determine whether there was a correlation between specific biologic parameters of these virus isolates and hybridization to cdnasffv. To do this, we characterized various murine leukemia virus (MuLV) isolates, some of which were potential recombinants between murine ecotropic and xenotropic vituses, according to host range on SC-1 and mink cells. The results of Table 1. Proc. Natl. Acad. Sci. USA 74 (1977) Biological properties of various murine leukemia viruses SC-1 cells XC Growth on: Mink cells Virus clones Polymerase* plaques Polymerase* CPE Mo-MuLV Cl <1 - Cl <1 - Cl <1 - Cl H <1 - Cl Cl AKR-MuLV <1 - AKR-MCF ATS MuLV 1504A SFFV/MuLV ND ND A SFFV/WoLVt ND ND The indicated viruses were inoculated onto SC-1 or mink cells at a multiplicity of infection of approximately 0.1. After 6 days of growth the medium was changed, and either an XC test was performed (13) or a 24-hr collection of supernatant fluid was assayed for viral reverse transcriptase by using poly(ra).oligo(dt) 18 (4). The mink cells that did not permit growth of the indicated viruses were subcultured from the original 60-mm plate to 75-cm2 flasks and retested after 7 more days for viral growth as judged by supernatant reverse transcriptase. CPE, cytopathic effects; ND, not done. * Shown as cpm (X1O-3) of [3H]TMP incorporated. t Woolly monkey leukemia virus. host range studies of certain Mo-MuLV clones, AKR-MCF virus, and amphotropic MuLV 1504A are shown in Table 1. Four clones of Mo-MuLV (clones 2, 10, 35, and H) grew well on SC-1 cells, caused XC plaque formation, and did not grow on mink cells. In other studies (not shown), each of these clones formed equal numbers of XC plaques when grown on both BALB/c 3T3 and NIH 3T3 cells and were thus NB-tropic ecotropic viruses (Mo-MuLVO). However, two other NB-tropic Mo-MuLV clones (clones 4 and 83) isolated from an uncloned preparation of Mo-MuLV (9) grew equally well on mink and SC-1 cells but failed to induce XC plaques; these clones are presumably similar to the HIX virus that was isolated in a similar fashion (20). Host range studies on the AKR-MCF (N-tropic, amphotropic), AKR-MuLV (N-tropic, ecotropic), and amphotropic MuLV 1504A (N-tropic, amphotropic) confirmed what has been previously reported by Hartley et al. (10, 11). Finally, a prototype xenotropic virus, ATS-4, grew only on mink cells. Each MuLV isolate that grew on mink cells was also assayed for cytopathic effects on mink cells, as described (11). The AKR-MCF virus was able to induce typical cytopathic effects of vacuolization and "piling-up" of cells. Interestingly, the two clones of Mo-MuLV that grew on mink cells (clones 4 and 83) also produced similar effects on mink cells. However, ATS-4, amphotropic MuLV 1504A, and pseudotypes of SFFV failed to induce any morphological change when grown on mink cells. Hybridization of RNA from Different Viruses to cdnasffv. To determine whether or not any of the viruses listed in Table 1 contained xenotropic sequences homologous to cdnasffv, viral RNA was extracted from virions released into the supernatant medium from SC-1 or mink cells infected

3 .Microbiology: TroxIer et al. Table 2. Analysis of replication-competent MuLVs for xenotropic virus-specific genetic sequences contained in SFFV cmp hybridized, with Source of RNA Mo-MuLV.co cdn4 cdnasffv Mo-MuLV ecotropic clones Clone Clone Clone Clone H Other Mo-MuLV clones Clone Clone Other MuLV AKR-MuLV AKR-MCF 247 ND 888 ATS MuLV 1504A SFFV pseudotypes SFFV/ 1504A (mink) ND 990 SFFV/WoLV (mink) ND 975 Fibroblast transforming viruses MoSV (S+L-NRK) AbLV (Ab-NRK) 532 Each hybridization 0.05-ml reaction mixture was incubated for 30 hr at 660 and contained 0.02 M Tris-HCl (ph 7.2),0.75 M NaCl, 0.1% sodium dodecyl sulfate, 1 mm EDTA, approximately 2200 trichloroacetic acid-insoluble cpm of the cdnasffv or 2500 cpm of Mo- MuLVeco cdna, and the indicated RNA. Reactions with viral RNAs were incubated to Crt values of 100 mol-sec-liter-1 and cellular RNAs to Crt values of 5000 mol-sec-liter-1. ND, Not done. with each MuLV isolate. Each of these viral RNAs was then hybridized to two cdna probes: (i) Mo-MuLVe<, cdna prepared from Mo-MuLV clone 2 and (ii) cdnasffv. The results of these studies are shown in Table 2. Mo-MuLVeco cdna hybridized equally well (>90%) to RNA from each of the Mo-MuLV clones at saturating levels of RNA. In addition, positive hybridization was obtained with this cdna to RNA from partially homologous viruses AKR-MuLV, ATS-4, and amphotropic MuLV 1504A, indicating that adequate levels of RNA from each of these viruses were tested with each cdna. cdnasffv failed to hybridize to RNA from any of the ecotropic Proc. Natl. Acad. Sci. USA 74 (1977) 4673 Mo-MuLV clones; however, it did hybridize well to the two Mo-MuLV clones that grew on mink cells (clones 4 and 83). Similarly, cdnasffv did not hybridize to AKR-MuLV (5) but did hybridize to RNA from AKR-MCF virus. As previously reported (5), cdnasffv hybridized well to ATS-4. Importantly, RNA from amphotropic MuLV 1504A grown on either SC-1 cells or mink cells failed to hybridize to cdnasffv. Finally, RNA from pseudotypes of SFFV grown on mink cells hybridized completely to cdnasffv, indicating that SFFV had infected mink cells without producing morphological alterations in these cells. Other controls for this experiment included RNA from cells nonproductively transformed by either Moloney sarcoma virus (S+L- strain) or Abelson virus, each of which was derived from Mo-MuLV; neither of these hybridized to cdnasffv. These results indicate that Mo-MuLV clones 4 and 83 and AKR-MCF virus contain xenotropic viral sequences that are highly related to those contained in SFFV, whereas amphotropic MuLV 1504A does not contain such sequences. Thus, Mo-MuLV clones 4 and 83 and AKR-MCF are likely to be recombinants between their respective ecotropic parents and xentropic virus. Furthermore, in these recombinant viruses with a broadened host range, the detection of xenotropic viral sequences with cdnasffv correlates with the ability of the virus to replicate on mink cells, suggesting that the xenotropic sequences present in SFFV were derived from within the env gene which determines the host range specificity of murine xenotropic type C viruses or possibly from regions of the xenotropic viral genome closely linked to the env gene. Interference Pattern of MuLVs. Because cdnasfv is a xenotropic virus-specific cdna probe, the lack of hybridization to amphotropic MuLV 1504A noted above suggests that this virus has not acquired its host range properties as a result of recombination with the env gene of xenotropic virus, but rather that it represents a different class of virus with an amphotropic host range. This was suggested by previously reported data on interference patterns between amphotropic MuLV 1504A with both the HIX virus and AKR-MCF which indicated that xenotropic virus, but not amphotropic virus 1504A, interferes with AKR-MCF virus and the HIX virus (11, 20). Although we did not retest AKR-MCF because it was the same virus (no. 247) recently tested by Hartley et al. (11), we did determine the interference pattern between Mo-MuLVe, Mo-MuLV8s, ATS-4, and amphotropic MuLV 1504A (Table Table 3. Interference patterns between Ki-SV pseudotypes and different MuLV Source of SC-1 cells infected with: challenge virus, Ki-SV pseudotype Uninfected Mo-MuLV Cl 2 Mo-MuLV Cl 83 MuLV 1504A Mo-MuLV Cl Mo-MuLV Cl MuLV 1504A Mink cells infected with: Uninfected ATS-4 MuLV 1504A ATS Mo-MuLV Cl MuLV 1504A SC-1 mouse cells or mink cells were infected with the indicated type C viruses and then challenged in focus assays with approximately 200 focus-forming units of the Ki-SV pseudotypes. 0 indicates less than 2 foci per plate. Assays were carried out for 8 days, at which time foci were counted. * Refers to percentage of foci seen on infected cells relative to uninfected cells (100%). Foci*

4 4674 Microbiology: TroxIer et al. 3). Consistent with earlier reports, ATS-4 blocked infection with Mo-MuLV83, whereas amphotropic MuLV 1504A did not. Conversely, amphotropic virus 1504A did not interfere with either Mo-MuLV83 or ATS-4. Thus, viruses that have an amphotropic host range and contain sequences homologous to cdnasffv are interfered with by xenotropic virus. In contrast, amphotropic MuLV 1504A neither hybridized to cdnasffv nor crossinterfered with ATS-4. These results provide additional evidence that xenotropic sequences detected with cdnasffv were derived from a region within or closely linked to the env gene of xenotropic virus. LocatiQn of Xenotropic Sequences in Mo-MuLV83. To identify the map location of the xenotropic virus-specific sequences contained in Mo-MuLV8s, we prepared poly(a)-containing viral RNA from Mo-MuLV8s and fractionated it into different size classes by sucrose gradient sedimentation. Then, using three separate cdna probes-(i) cdnaa, (ii) cdnasffv, and (iii) cdnas'-prepared as describec in Materials and Methods, we tested different size classes of poly(a)-containing viral RNA to determine the kinetics of reassociation with each cdna (Fig. 1) cdnag9 hybridized more rapidly to 30-35S RNA than to 26S, 18S, S, or 4S RNA species. These results are consistent with the location of the gag gene of Mo-MuLV at the 5' end of the genome (23). In contrast, cdna3' hybridized much faster to 4S and S RNA than to larger species, consistent with the way in which this probe was made. Finally, cdnasffv hybridized most rapidly to 18-26S RNA in size. Based on a 26S RNA fragment having a molecular weight of 1.5 X 106(15), these mapping data indicate that xenotropic sequences in Mo-MuLV83 are located in the 3' half of the genome but are not immediately adjacent to the 3' end. Thus, if recombination between ecotropic and xenotropic MuLV occurred between similar genetic regions of each virus, these data suggest that the xenotropic sequences in Mo-MuLV83 were derived from the env gene region of xenotropic MuLV. This conclusion would be consistent with host range and interference data presented above which suggested that cdnasffv detects xenotropic env gene sequences. DISCUSSION The current studies have extended previous work on the molecular composition of SFFV, a murine type C acute leukemia virus. In these recent studies, SFFV was shown to be a recombinant between a portion of the ecotropic type C virus F-MuLV and a portion of murine xenotropic virus (4, 5). The current studies were designed to determine what portion of the xenotropic viral genome is contained in SFFV. Two lines of evidence have been obtained to suggest that cdnasffv detects RNA sequences that were derived from the env gene region of xenotropic virus. First, cdnasffv was found to hybridize to certain isolates of Moloney MuLV as well as of the AKR-MCF virus, each of which has acquired host range properties of xenotropic virus by virtue of having undergone recombination with xenotropic virus. Because host range properties of MuLV are determined by the gp7o product of the env gene, these results indicated that cdnasffv detected sequences contained within or closely linked to the env gene of xenotropic virus. Second, by hybridization to different size classes of poly(a)- containing viral RNA, we have shown that sequences detected with cdnasffv are located on the 3' half of the RNA genome of one of these recombinant viruses, Mo-MuLV83, near the middle of the molecule. Oligonucleotide fingerprint studies of the RNA of Mo-MuLV8s show the set of sequences derived from xenotropic virus to be approximately 350,000 daltons in size and located approximately kilobases from the Proc. Natl. Acad. Sci. USA 74 (1977) cdna SFFV o80 35 co N t ffg cdna g Crt, mol sec liter' FIG. 1. Mapping of xenotropic viral sequences in recombinant Mo-MuLV. Each hybridization reaction mixture contained approximately 2000 trichloroacetic acid-insoluble cpm of the indicated cdna and hybridization was assayed as indicated in Materials and Methods. Numbers at the ends of the curves are sizes in S. poly(a) end of the viral genome (T. Y. Shih, D. H. Troxler, and E. M. Scolnick, unpublished data). The results of these molec-.ular studies place the xenotropic sequences in Mo-MuLV83 in the approximate region of the env gene of type-c viruses (15) and are therefore consistent with the above host range and interference data that suggest that the cdnasffv detects sequences derived from within or closely linked to the env gene region of xenotropic virus. The significance of the xenotropic viral sequences contained in SFFV is unclear at present. The acquisition of the xenotropic RNA sequences by SFFV might be unrelated to the rapid leukemogenicity of SFFV compared to F-MuLV, because recombination between xenotropic and ecotropic viruses might occur very frequently in the mouse. On the other hand, the xenotropic env gene sequences incorporated into SFFV by recombination with ecotropic F-MuLV might be directly or indirectly related to the enhanced leukemogenicity of SFFV and could function in one of several ways. (i) The xenotropic sequences could cause SFFV to integrate into cellular DNA at a specific site(s) that differs from the usual integration site(s)

5 Microbiology: Troxler et al. of ecotropic virus; SFFV could then indirectly cause leukemia by altered transcription of nearby cellular genes. (ii) The xenotropic env gene sequences in SFFV might be analogous to RNA sequences (designated "src") contained in avian and mammalian sarcoma viruses which are not present in the replicating viruses used to derive these viruses but which seem to endow these recombinants with new pathogenic potential. In this model, the xenotropic genetic sequences in SFFV, either alone or in combination with adjacent F-MuLV sequences, would encode for a gene product (possibly a glycoprotein) that would be necessary for leukemic transformation of hematopoietic target cells, and such sequences would thus represent the "leuk" gene of SFFV. (iii) Finally, the "Ieuk" gene of SFFV, acquired by recombination with xenotropic virus, might be composed of genetic sequences closely linked to, but distinct from, the env gene of xenotropic virus, analogous to the "src" gene of avian sarcoma virus, which is closely linked to the env gene of avian sarcoma virus (15). Whether the xenotropic viral sequences in SFFV are responsible for the rapid leukemogenicity of SFFV by any of the above models is difficult to determine at the present time, for several reasons. First, little is known about the specificity of integration of type C RNA viruses into cellular DNA. Second, a protein(s) coded for by xenotropic viral sequences in SFFV has not yet been found. Finally, there is no genetic evidence for the existence of a "leuk" gene of SFFV; thus, one can only speculate on the nature of such a gene or its potential gene product. However, the molecular composition of the SFFV genome and the rapid transformation of hematopoietic cells by SFFV (2) are analogous to the molecular compositions and rapid transformation of fibroblasts by the Kirsten and Harvey sarcoma viruses, which are recombinants between ecotropic MuLV and endogenous type C viral sequences derived from rats. Prior work on these FT+ viruses (6, 7) might be interpreted as support for the existence of a "leuk" gene in SFFV, as defined above, which contains at least in part xenotropic viral sequences. Furthermore, because the xenotropic sequences in SFFV include env gene sequences, it might be predicted that recombination between murine helper viruses and endogenous rat viral sequences to form the Kirsten and Harvey sarcoma viruses might have occurred between the 3' portion of these MuLVs and the genetic region of the endogenous rat viral genome which is topographically and functionally equivalent to the env gene region of xenotropic virus. If this can be demonstrated, then strong evidence would exist for a common mechanism by Proc. Natl. Acad. Sci. USA 74 (1977) 4675 which acute transforming RNA tumor viruses are formed. This might in turn shed further light on the nature and mode of action of the potential transforming gene products that result from recombination within specific genetic regions. 1. Friend, C. (1957) J. Exp. Med. 105, Tambourin, P. & Wendling, F. (1971) Nature New Biol. 234, Steeves, R. A. (1975) J. Nati. Cancer Inst. 54, Troxler, D. H., Parks, W: P., Vass, W. C. & Scolnick, E. M. (1977) Virology 76, Troxler, D. H., Boyars, J. K., Parks, W. P. & Scolnick, E. M. (1977) J. Virol. 22, Scolnick, E. M., Rands, E., Williams, D. & Parks, W. P. (1973) J. Virol., Scolnick, E. M., Goldberg, R. & Williams, D. (1976) J. Virol. 18, Scolnick, E. M., Howk, R., Anisowicz, A., Peebles, P., Scher, C. & Parks, W. P. (1975) Proc. Nati. Acad. Sci. USA 72, Bassin, R. H., Tuttle, N. & Fischinger, P. J. (1970) Int. J. Cancer 6, Hartley, J. W. & Rowe, W. P. (1976) J. Virol. 19, Hartley, J. W., Wolford, N., Old, L. J. & Rowe, W. P. (1977) Proc. Natl. Acad. Sci. USA 74, Todaro, G. J., Arnstein, P., Parks, W. P., Lennette, E. H. & Huebner, R. J. (1973) Proc. Natl. Acad. Sci. USA 70, Rowe, W. P., Pugh, W. E. & Hartley, J. W. (1970) Virology 42, Parks, W. P., Anisowicz, A., Howk, R. & Scolnick, E. M. (1976) J. Virol. 18, Wang, L. H., Duesberg, P., Beemon, K. & Vogt, P. (1975) J. Virol. 16, Glisin, V., Crkvenjakov, R. & Byers, C. (1974) Biochemistry 13, Leong, J. M., Garapin, A., Jackson, N., Fanshier, L., Levinson, W. & Bishop, J. M. (1972) J. Virol. 9, Benveniste, R. & Scolnick, E. M. (1973) Virology 51, Birnsteil, M. L., Seels, B. H. & Purdom, I. F. (1972) J. Mol. Biol. 63, Fishinger, P. J., Nomura, S. & Bolognesi, D. P. (1975) Proc. Natil. Acad. Sci. USA, Peebles, P. T., Gerwin, B. I., Papageorge, A. G. & Smith, S. G. (1975) Virology 67, Parks, W. P., Scolnick, E. M., Ross, J., Todaro, G. J. & Aaronson, S. A. (1972) J. Virol. 9, Kerr, I. M., Oshevsky, V., Lodish, H. & Baltimore, D. (1976) J. Virol. 18,