A Novel Oncogene, v-ryk, Encoding a Truncated Receptor Tyrosine ...

JOURNAL OF VIROLOGY, OCt. 1992, P. 5975-5987

Vol. 66, No. 10

0022-538X/92/105975-13$02.00/0 Copyright © 1992, American Society for Microbiology

A Novel Oncogene, v-ryk, Encoding a Truncated Receptor Tyrosine Kinase Is Transduced into the RPL30 Virus without Loss of Viral Sequences RAN JIA, BRUCE J. MAYER, TERUKO HANAFUSA, AND HIDESABURO HANAFUSA* The Rockefeller University, 1230 York Avenue, New York, New York 10021-6399 Received 12 May 1992/Accepted 17 July 1992

MATERIALS AND METHODS Viruses, cells, and virus infection. The RPL25, RPL28, and RPL30 viruses (21) were kindly provided by L. B. Crittenden of the Avian Disease and Oncology Laboratory, East Lansing, Mich. The three viruses were injected into newly hatched chickens, and acutely transforming virus stocks were recovered from the sarcomas induced in the chickens. Preparation and maintenance of chicken embryo fibroblast (CEF) cultures and infection and propagation of viruses have already been described (23). Briefly, CEF were prepared from 11-day-old embryos and secondary cultures were infected with the virus stock. Oncogene blot screen of oncogenic viruses. Probes specific for 19 known viral oncogenes were prepared from molecularly cloned DNA, and they contained no retroviral sequences. The viral oncogene probes used were as follows: feline fms, 1.4-kb PstI fragment from pSM3 (14); chicken ros, 0.85-kb EcoRI-PvuII fragment from pROS (32); chicken erbB, 0.56-kb BamHI fragment from pERB5 (51); murine abl, 1.6-kb SacI-HindIII fragment from pAB3sub3 (35); feline fgr, 0.93-kb SmaI-KpnI fragment from pGR-FeSV (31); chicken src, 0.87-kb PvuII fragment from pTI107 (44); chicken fps, 0.41-kb BamHI fragment from pBR-FO4 (20); chicken yes, 0.43-kb BamHI fragment from pMR-YA (42); murine mos, 0.92-kb AvaI-HindIII fragment from pCMOS (46); chicken mil, 0.58-kb BamHI-SphI fragment from pMBS28 (27); rat H-ras, 0.46-kb EcoRI fragment from pBS9 (17); rat K-ras, 0.38-kb SacII-XbaI fragment from pHiHi3 (18); woolly monkey sis, 0.92-kb PstI-XbaI fragment from pSSV (13); chicken erbA-erbB, 1.2-kb SalI-BamHI fragment from pERBA (11); turkey rel, 0.97-kb EcoRI fragment from pEcoRI-rel (6); chicken ski, 1.2-kb SacI fragment from pSRski-1 (29); murine fos, 1.0-kb PstI fragment from pFOS1 (10); chicken myc, 0.8-kb ClaI-EcoRI fragment from

Acute oncogenic retroviruses have proven to be a rich source of various categories of oncogenes. More than 25 viral oncogenes have been molecularly cloned and studied (8). Since oncogenic retroviruses are the naturally selected genetic system in which important genes involved in regulation of proliferation and differentiation in normal cells were targeted, analysis of previously uncharacterized viral oncogenes will be valuable in identifying novel cellular genes in these pathways that otherwise would be difficult to find. Furthermore, identification of new oncogenes in retroviruses not only provides an immediate indication that the gene can be oncogenic, but it may also provide insights into the mechanism of activation of the oncogenic potential of its

cellular homolog. The RPL25, RPL28, and RPL30 viruses are three acute oncogenic avian retroviruses that were previously uncharacterized genetically. These viruses were first isolated from chicken tumors (21). Although they cause tumors in chickens (21), the transforming genes present in these viruses have not been identified. Here, we report the genetic analysis of these three RPL viruses and the molecular cloning and characterization of RPL30, which was found to contain a novel viral oncogene. This viral oncogene is a new member of the protein tyrosine kinase gene family and appears to encode a truncated receptor-type tyrosine kinase. It is interesting that unlike all oncogenic retroviruses studied so far, a cellular gene was transduced by the RPL30 virus without loss of any viral sequence.

* Corresponding author. 5975

Downloaded from http://jvi.asm.org/ on December 9, 2017 by guest

The RPL viruses are acute oncogenic avian retroviruses isolated from chicken tumors. We carried out a genetic analysis of three of the viruses, RPL25, RPL28, and RPL3O. While RPL25 and RPL28 were shown to contain the erbB oncogene, RPL3O appeared to contain a novel protein tyrosine kinase oncogene. This gene, v-ryk, was cloned and sequenced. The v-ryk oncogene contains a 1.39-kb nonretroviral sequence that includes a tyrosine kinase domain which was inserted into the viral envelope protein gp37-coding region and fused in frame with upstream gp37 to generate a p69m,37-y fusion oncoprotein. Unlike that of other acutely transforming retroviruses, transduction of the v-ryk gene into RPL30 did not result in deletion of viral sequences. Sequence analysis suggested that v-Ryk is more homologous to receptor-type tyrosine kinases than to nonreceptor-type kinases. By reconstitution of a virus from its cDNA, the v-ryk oncogene has been shown to be fully responsible for the transforming activity of the RPL30 virus. Antibodies specific to v-Ryk immunoprecipitated the v-Ryk oncoprotein from cells transformed by the RPL30 virus. The v-Ryk protein was shown to be first synthesized as a 150-kDa precursor and then cleaved into the mature 69-kDa gp37-Ryk fusion protein, both parts of which were found to be localized to the membrane fraction. As expected from the sequence of v-Ryk, immunoprecipitates of v-Ryk from RPL3O-transformed cells were found to display a protein tyrosine kinase activity in vitro, and the levels of tyrosine-phosphorylated proteins are elevated in v-yk-transformed cells.

5976

JIA ET AL.

sequenced. DNA sequencing. Complete DNA sequences from both strands of the clones identified above were determined by the dideoxy-chain termination method (38) by using the Sequenase sequencing system (United States Biochemical, Cleveland, Ohio). Overlapping fragments for sequencing were generated by restriction digestion and subcloning. DNA sequences and deduced amino acid sequences were used to search the GenBank data base by the FASTA program (33). Viral plasmid construction. Clones 8-1 and 3-1 (see Fig. 2B) were digested and ligated into a nonoverlapping RPL30 clone (gp85-5'gp37-ryk-3'gp37-3' long terminal repeat [LTR]). This construct was then digested with Sail (within gp85) and EagI (within the 3' polylinker) and ligated to SalIand EagI-digested pSR-REP (9), which is an RSV-derived retroviral vector containing 5' LTR-gag-pol-gp85 (to the Sall site). The resulting plasmid construct, pRV-ryk, represents the molecularly cloned RPL30 virus. Helper virus plasmid pUR2AV has been described previously (49). Transfection. Transfection of CEF was performed by the

calcium phosphate method. Briefly, 4 to 10 ,ug of plasmid DNA per 60-mm-diameter dish was digested with EagI (pRV-ryk) or SacI (pUR2AV), phenol-chloroform extracted, precipitated with ethanol, and dissolved in 0.5 ml of 1 mM Tris-HCl (pH 8.0)-0.1 mM EDTA. A mixture of 10 ,ug of calf thymus DNA and 55 p,l of 2.5 M CaCl2 was added to the plasmid DNA. A mixture of 0.5 ml of 50 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), 280 mM NaCl (pH 7.1), and 5 ,ul of 0.15 M Na2HPO4 was then added dropwise with mild vortexing. This solution was allowed to stand at room temperature for 30 min, and then the fine precipitate was added to 7 x 105 secondary CEF per 60-mm-diameter dish and left for 8 h at 37°C. After that time, the medium was replaced with fresh medium. Transformation, colony formation, and animal tumorigenicity assays. Transfected, infected, and mock-treated (same procedure except that no plasmid was added) cells were maintained in medium containing 0.375% agar at 37°C. Transformation was assessed by focus formation. To assay anchorage-independent growth, cells were trypsinized and replated in 10-cm-diameter dishes in 10 ml of medium containing 0.4% agar on a layer of 15 ml of the same medium containing 0.7% agar. The plates were incubated at 40°C. Tumorigenicity was assayed by injecting 0.1 ml of virus into the wing webs of newborn (7 days posthatching) White Leghorn chickens (SPAFAS, Inc.). Generation of specific antiserum. The C-terminal unique region of v-Ryk was expressed in E. coli BL21 (DE3)pLysS by using the T7 RNA polymerase expression system (36, 41). An 897-bp Sau3AI fragment containing the region from amino acid 463 to the C terminus was cloned into a compatible BamHI site in the linker region of the pET-3c vector. Plasmid-bearing bacteria were induced with isopropyl-P-Dthiogalactopyranoside for 2 h, spun out, and lysed on ice by sonication in RIPA buffer containing 1 mM phenylmethylsulfonyl fluoride. Proteins in the lysate were then separated by SDS-12% polyacrylamide gel electrophoresis (PAGE). The v-Ryk protein band was excised from the Coomassie blue-stained gel, washed with 10% methanol, dried, and homogenized in phosphate-buffered saline (PBS). Approximately 100 p,g of v-Ryk protein was emulsified with Freund's complete adjuvant and used to immunize rabbits, and 50 ,ug of protein was used for booster injections. Preimmune serum was collected for control experiments. The specificity of the antiserum was tested by an antigen competition assay. v-ryk-transformed CEF were labeled in vivo with 32Pi for 8 h (19) and then lysed in RIPA buffer (see below). Preimmune or immune serum (20 ,ul) was preincubated in 120 pl of RIPA buffer with or without 144 p,g of the immune antigen or control antigen (vector-bearing bacterial lysates) for 3 h at 4°C and then used to immunoprecipitate 14.4 p,g of 32P-labeled cell lysate (see below). Immunoprecipitated proteins were analyzed by SDS-7.5% PAGE. Immunoprecipitation and in vitro kinase assay. Cell cultures were washed with ice-cold Tris-glu buffer (25 mM Tris, 137 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4, 0.1% glucose, pH 7.4), and the cells were scraped off the plate, quickly centrifuged, and frozen in a dry ice-ethanol bath. Frozen pellets were suspended in RIPA buffer (10 mM Tris-HCl, (pH 7.4), 5 mM EDTA, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1% sodium deoxycholate, 1% Trasylol, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 0.1 mM sodium molybdate), incubated on ice for 20 min with periodic shaking, and spun in a microcentrifuge at 4°C, and the supernatants were recovered. Protein concentrations were assayed with a Protein Assay Kit (Bio-Rad Laborato-


pCMYC1 (2); chicken myb, 0.35-kb EcoRI-SalI fragment from pVM2 (28). A 3.6-kb EcoRI fragment of Rous sarcoma virus (RSV) (non-src region) from pSRA2 was used as an avian retrovirus positive control (12). To prepare the oncogene blot, 50 ng of each oncogene fragment was denatured and blotted onto a nitrocellulose filter by using a slot blot manifold. This blot was then hybridized with 32P-labeled RPL virus cDNA generated by reverse transcription of viral genomic RNA harvested from infected cell culture media. Medium was collected at 4-h intervals, prespun to remove cells and debris, and then spun for 90 min at 40,000 rpm to pellet the virus. The viral pellets were suspended in 10 mM Tris-HCl (pH 7.4)-5 mM EDTA, 0.5% sodium dodecyl sulfate (SDS)-1 mg of proteinase K per ml (1 ml of extraction buffer per 25 ml of original medium collected) and incubated at 37°C for 1 h. From 5 to 10 ,ug of viral RNA was usually recovered from 100 ml of culture fluid. The viral RNAs were then subjected to reverse transcription in the presence of [a-32P]dCTP to generate 32Plabeled retrovirus cDNAs (37). cDNA library and cloning of the viral oncogene. Total RNA from RPL30 virus-transformed nonproducer cells (49) was isolated, mRNA was purified, and cDNA was synthesized by standard procedures (37). Internal EcoRI sites of the cDNA were methylated. The ends of the cDNA were blunted with T4 DNA polymerase and then ligated to synthetic EcoRI linkers. The cDNA was digested with EcoRI and run on a 0.8% agarose gel, and cDNA in the size range of 1 to 15 kb was recovered. This cDNA was ligated to AgtlO vector arms to construct an RPL30 cDNA library. The recombinant bacteriophage were packaged with Gigapack Gold Packaging Extract (Stratagene, La Jolla, Calif.) in accordance with the manufacturer's protocol and used to infect Escherichia coli C600. Three DNA probes derived from the gag, pol, and env regions of the genome of cloned helper virus UR2AV were used to screen 105 plaques from the RPL30 cDNA library (see Fig. 2A). Positive clones were subjected to Southern blot analysis with probes derived from other regions of the UR2AV genome. The sizes of the cDNA inserts from the clones and the distance between the hybridizing probes on the viral genome were compared. Those clones containing cDNA inserts that were larger than the distance between the hybridizing probes were subcloned into the pBluescript phagemid (Stratagene) by using standard techniques (37) and

J. VIROL.

VOL. 66, 1992

5977

RESULTS

Genetic analysis of three RPL oncogenic retroviruses. Three of the RPL series of acute oncogenic retroviruses, RPL25, RPL28, and RPL30, were first isolated from chicken tumors in farm flocks by Burmester's group at the Regional Poultry Research Laboratory (now named the Avian Disease and Oncology Laboratory) in East Lansing, Mich., in the 1950's and 1960's (21). These RPL viruses were shown to be able to induce chicken erythroblastosis, fibrosarcomas, endotheliomas, visceral lymphomatosis, nephroblastoma, osteopetrosis, and hemorrhages. The nature of the lesions was influenced by the virus dose inoculated, the age at inoculation, and the route of inoculation (21). Since these viruses had not previously been genetically characterized, we decided to analyze the transforming genes present in these three RPL viruses. Injection of the viruses into newly hatched chickens resulted in induction of sarcomas from which acutely transforming avian viruses were recovered. These RPL viruses were also shown to be able to transform CEF in culture and were used for further analysis. First, we determined whether they encode oncogenes that have already been identified in other oncogenic retroviruses. A screening method which allows rapid comparison of new retroviral genes to known viral oncogenes was developed. Oncogene-specific DNA fragments were isolated from a panel of 19 plasmids containing known viral oncogenes. Each DNA probe was carefully chosen so that it contained no virus-derived sequences that could hybridize nonspecifically with the retroviruses to be tested. These probes were bound to nitrocellulose filters, and this oncogene blot was then hybridized under conditions of moderate stringency with 32P-labeled cDNA made by reverse transcription of different RPL virus genomic RNAs. These viral RNAs were obtained from virion particles released from CEF transformed with the RPL viruses. If any of these RPL viruses contained a known oncogene, its cDNA would be expected to hybridize to the corresponding probe on the oncogene blot. The results of the screening are shown in Fig. 1. All three RPL virus cDNAs, as expected, hybridized to the positive control, a DNA fragment of the avian retrovirus structural gene. RPL25 and RPL28 also hybridized to the slots corresponding to the erbB and erbAerbB probes. Southern blotting of the restriction-digested original oncogene-containing plasmids with the RPL25 and RPL28 cDNAs demonstrated that the hybridization was specific to erbB sequences (data not shown). This kind of Southern blotting analysis also indicated that the low-level hybridization to the src and myb probes observed with RPL25 and RPL30 was due to contamination of the probe fragment with non-oncogene-derived viral sequences (data not shown). Furthermore, Northern (RNA) blots of RNAs from cells infected with RPL25 and RPL28 hybridized with an erbB probe, showing specific hybridization bands not present in RNA from uninfected CEF (data not shown). These results demonstrated that the RPL25 and RPL28 viruses contain the erbB oncogene, similar to avian erythroblastosis virus (AEV), whose cellular counterpart is the epidermal growth factor receptor (15, 51). More interestingly, no specific hybridization of RPL30 to any of the oncogene probes was detected, suggesting that the RPL30 virus contains a novel oncogene. Like almost all of the acute oncogenic retroviruses, RPL30 is also a replication-defective virus and needs a helper virus to produce infectious viral progeny (data not shown). The size of the RPL30 viral genome, 9 kb, is unusually large among avian sarcoma viruses and is exceeded only by


ries, Richmond, Calif.), and equal amounts of proteins were used in the subsequent assays. For immunoprecipitation, antibodies were added to the above-described cell lysates and incubated on a rotating wheel at 4°C for 1 h and then protein A-Sepharose (Pharmacia, Piscataway, N.J.) was added and the incubation was continued for another 30 min. The immunoprecipitates were washed three times with RIPA buffer containing 300 mM NaCl and twice with RIPA buffer containing 10 mM NaCl and then used directly either for SDS-PAGE analysis or for in vitro kinase assays. For in vitro kinase assays, the immunoprecipitates were washed twice with the kinase reaction buffer (see below) instead of RIPA buffer containing 10 mM NaCl, and then each was suspended in 30 ,ul of kinase reaction buffer (50 mM HEPES buffer [pH 7.5], 10 mM MnCl2). A total of 5 pCi of [-y-32P]ATP was added to each sample, and it was incubated at 30°C for 10 min. Kinase reactions were terminated by washing the samples twice with RIPA buffer containing 10 mM NaCl and then subjected to SDS-10% PAGE. Rainbow markers (14.3 to 200-kDa range; Amersham, Arlington Heights, Ill.) were used as molecular weight standards in all protein gels. The gels were dried and exposed to X-ray film at -70°C for 24 h with an intensifying screen. Western blotting (immunoblotting). Equal amounts of protein from cell lysates (see above) were subjected to SDS10% PAGE and then electrophoretically transferred to a polyvinylidene difluoride nylon filter (Millipore Corp., Bedford, Mass.) with transfer buffer (2.5 mM Tris-HCl [pH 8.3], 9.2 mM glycine, 10% methanol). The filters were then probed with anti-Ryk serum or antiphosphotyrosine (PTyr) polyclonal antibodies, followed by incubation with 1"Ilabeled protein A (Amersham) as described previously (22). Autoradiography was performed as described above. Partial protease mapping with Staphylococcus aureus V8 protease. 32P-labeled protein bands were excised from in vitro kinase assay gel and subjected to partial digestion with S. aureus V8 protease as described previously (7). Pulse-chase analysis. CEF transformed by the cloned v-ryk gene were labeled with [35S]methionine (110 ,uCi; Amersham) for 1 h (pulse) in MEM (without methionine) medium (GIBCO, Gaithersburg, Md.) after starvation in the same medium for 2 h. The cells were then washed, incubated with complete medium, and chased for 0, 15, or 30 min or 1, 2, 4, or 8 h. At those time points, cells were lysed as described above, immunoprecipitated with anti-Ryk antibodies, and analyzed by SDS-10% PAGE. Cell fractionation. Cells were swollen in hypotonic lysis buffer (20 mM borate, 0.2 mM EDTA [pH 10.1]) (0.45 ml/10-cm-diameter dish) for 10 min, collected, and homogenized in a Dounce homogenizer (15 strokes), and then 8 volumes of hypertonic solution (0.5 M borate [pH 10.21) was added. The lysate was spun at 500 x g and 4°C for 10 min, the supernatant was collected and spun at 20,000 x g and 4°C for 30 min, and the supernatant (soluble fraction) was collected. The pellets were dissolved in PBS and layered over sucrose gradients (40 and 20% [wt/vol] sucrose in PBS) and then spun at 100,000 x g and 4°C for 1 h. The plasma membrane-enriched fraction was collected, diluted in PBS, and spun at 100,000 x g and 4°C for 30 min, and the pellets were collected as the membrane fraction. The two fractions were then subjected to both Western blotting and the in vitro kinase assay. Nucleotide sequence accession number. The sequences reported here have been deposited in the GenBank data base under accession no. M92847.

THE v-ryk TYROSINE KINASE ONCOGENE

5978

J. VIROL.

JIA ET AL.

RPL25

RPL28

A LTR I

gag

pol

probes:

gag

B LTR

LTR

env

I

I

a

gag

pol

pol

env LTR

env

;..

-+

;I-

clones:

1.7kb 2.2 kb 2.5 kb

*:

i

FIG. 1. Hybridization of three RPL virus cDNAs to oncogene probes. DNA probes were isolated from 19 known viral oncogenes and bound to nitrocellulose filters as diagrammed in the lower right panel (see the text for precise probe identities). 32P-labeled cDNAs prepared from RPL25, RPL28, and RPL30 virions were hybridized to filters as described in the text. ALV, avian leukosis virus.

that of RSV (9.31 kb), which contains the src oncogene in addition to all of the viral genes (48). To determine what, if any, viral genetic information had been deleted from the RPL30 virus during the process of oncogene transduction, as seen in almost all of the oncogenic retroviruses characterized so far, nine probes spanning the whole retroviral genome were prepared from cloned UR2AV DNA and then hybridized to the 9-kb RPL30 genome. The results showed, surprisingly, that all nine probes hybridized to the RPL30 genome (data not shown). This suggested that either no viral sequence deletion occurred during transduction of the oncogene or only a small deletion occurred. In other experiments, immunoprecipitation of lysates from RPL30-infected CEF with tumor-bearing rabbit serum showed elevated in vitro tyrosine kinase activity relative to lysates from uninfected CEF (data not shown). Tumor-bearing rabbit serum was raised in rabbits bearing RSV-induced tumors and recognizes avian retroviral structural proteins in addition to pp60-s'c (4). This preliminary characterization of the RPL30 virus strongly suggested that RPL30 contains a novel tyrosine kinase oncogene. Therefore, we decided to clone this oncogene molecularly. Cloning of the transforming gene in the RPL3O virus. Since we did not have a specific probe for the transforming gene in the RPL30 virus and did not know the precise location of the gene in the RPL30 genome, we decided to use three probes derived from UR2AV gag, pol, and env to screen a cDNA library constructed from RPL30-infected nonproducer CEF (Fig. 2A). The rationale of this strategy was to isolate RPL30

3-1 8-1 2-4

FIG. 2. Cloning of the transforming gene in the RPL30 virus. (A) Diagram of the UR2AV genome, a helper virus required for RPL30 replication. gag, pol, and env denote the viral structural genes. Three DNA probes used in the initial screening for the oncogene in RPL30 are represented by thick bars beneath the positions in the viral genome from which they were derived. (B) Cloning of the oncogene in the RPL30 virus. The three thick bars represent the three oncogene-containing clones that were isolated from the library and sequenced. Their sizes are indicated beneath the bars, and their names are indicated to the right of the bars. Above the bars is a diagram showing the location of the transforming gene in the RPL30 viral genome; this information was deduced from the cloning and sequencing analyses.

viral gene-oncogene fusion fragments. The fusion fragments could then be identified as clones containing inserts larger than those expected from prototypic retrovirus se uences. Six positive clones were isolated from 3 x 10 plaques from the RPL30 cDNA library with the env probe, eight were isolated with the pol probe, and none were isolated from 4 x 104 plaques with the gag probe. The inserts in positive clones were excised from the phage vectors and subjected to Southern blot analysis. The Southern blots were first hybridized with the probes used in the library screening to confirm the authenticity of the clones and then rehybridized with probes from regions in the UR2AV genome adjacent to those from which the screening probes were derived. The insert size was compared with the distance between the hybridizing probes on the UR2AV genome to determine whether the size was consistent with or larger than that distance. Three clones originally detected with the 3' env probe caught our attention. These three clones were later found to be identical to each other and are represented by clone 3-1 in Fig. 2B. The inserts from these clones were 1.7 kb long, and they also hybridized with an LTR probe but not with a 5' env probe (data not shown). However, the distance between the 5' end of the 3' env probe (0.8 kb) and the 3' end of the LTR probe (0.6 kb) is only 1.4 kb. The negative results obtained with the 5' env probe suggested that these 1.7-kb clones contain sequences in addition to the viral genes; the extra sequences were potentially those of the RPL30 oncogene. The remaining clones were not found to contain extra sequences. By subcloning and sequencing, clone 3-1 was found to contain a 1,665-bp insert. At the 3' end of this insert is the viral 3' env gp37 gene, followed by the LTR sequence. At the 5' end, however, the insert contains 818 bp of nonviral sequences which are homologous to the catalytic domain of protein tyrosine kinases (Fig. 2B). To isolate the full-length


RPL30

RPL30

onc

.1.

.f:`

UR2AV

env

iI i.

I

VOL. 66, 1992

5979

V. The full-length v-Ryk sequence was used to search for protein sequence homology in GenBank. The region of v-Ryk homologous to other proteins is limited to the kinase catalytic domain (Fig. 4). The highest homologies found in the kinase domain were to the axl-UFO oncogene product (67.3% amino acid identity), the ark gene product (67.3%), the v-sea oncogene product (47.4%), the human met oncogene product (43.2%), the human insulin receptor (41.4%), the bovine insulin-like growth factor 1 receptor (41.1%), and the human ret oncogene product (40.0%). axl (32a) and UFO (26a) are the same gene, isolated from human myeloid leukemia cells, which encodes a putative tyrosine kinase receptor. ark is a murine putative tyrosine kinase receptor gene (35a). The v-sea oncogene is the transforming gene of avian erythroblastosis retrovirus S13, and its cellular homolog is a receptor-type tyrosine kinase (40). Since these highly homologous kinases are receptor-type molecules and v-Ryk is only 36.1% homologous to c-Src in the kinase catalytic region, c-Ryk is more likely a receptor-type than a non-receptor-type tyrosine kinase. Another interesting feature of v-Ryk is that the 123 amino acids C terminal to the kinase domain show no significant homology to any other protein in GenBank. Since this domain contains three tyrosine residues, they may be potential autophosphorylation sites and thus may have some regulatory role(s), as proposed for tyrosine residues in the C-terminal region of the epidermal growth factor receptor (45) or the Neu oncoprotein (1). Biological activity of v-ryk. The RPL30 virus caused transformation of CEF 10 to 14 days after infection. The transformed CEF exhibited an elongated and fusiform morphology and grew to a higher cell density than normal CEF (Fig. 5D). These transformed CEF formed colonies, but control cells did not (Fig. 6D), reflecting anchorage-independent growth. The RPL30 virus induced chicken sarcomas 5 to 6 weeks after injection into the wing webs of 1-week-old chickens (data not shown). To determine whether the cloned v-ryk gene is fully responsible for the transforming ability of the RPL30 virus, we tested the biological activity of this putative oncogene. To do this, v-ryk was cloned into retrovirus vector pSR-REP to construct retrovirus pRV-ryk, which contains the 5' LTR, gag, pol, and gp85 from RSV and gp37-ryk and the 3' LTR from RPL30. When pRV-ryk was cotransfected into CEF with pUR2AV, the CEF became transformed and formed foci morphologically indistinguishable from those of RPL30-transformed cells after 10 to 14 days (Fig. SC), while neither pUR2AV itself nor mock transfection caused any transformation (Fig. 5A and B). When these transformed cells were plated on agar, colonies very similar to those induced by the original RPL30 virus formed (Fig. 6C). Virus collected from these pRV-ryktransformed cells induced sarcomas in chickens within 6 weeks, and histological examination of the sarcomas revealed that they were fibrosarcomas and rhabdomyosarcomas (data not shown). Therefore, the cloned v-ryk gene can fully reproduce the transforming capacity of the RPL30 virus. Biochemical analysis of the v-Ryk oncoprotein. To avoid cross-reaction with other tyrosine kinases, the unique region C terminal to the kinase catalytic domain of v-Ryk was chosen as an immunogen to generate specific anti-v-Ryk antibodies in rabbits (see Materials and Methods). The specificity of the serum was tested in an antigen competition assay. Figure 7 shows that in lanes 3, 5, and 7, cold immunogen competed successfully to remove a band with a molecular mass of 70 kDa immunoprecipitated from in vivo phosphate-labeled RPL30-transformed cells by sera from


cDNA for this putative transforming gene, the kinase fragment from clone 3-1 was used as a probe to rescreen the RPL30 cDNA library. Numerous positive clones were isolated, and two of them (clones 8-1 and 2-4), containing the longest inserts (2.2 and 2.5 kb, respectively), were chosen for subcloning and sequencing (Fig. 2B). Clone 8-1 contains 234 bp of the viral env gp85 gene at the 5' end, followed by 538 bp of the gp37 gene, 1,390 bp of nonviral sequences, and 10 bp of the gp37 gene. Clone 2-4 started with 378 bp of the env gp37 gene at its 5' end, followed by 1,390 bp of nonviral sequences and 77 bp of 3' gp37, and ended with 298 bp of the LTR sequence. Therefore, both clones 8-1 and 2-4 appeared to contain the full-length putative oncogene. Since this 1.39-kb nonviral sequence has a protein tyrosine kinase domain, we named this putative viral oncogene v-ryk, for the RPL30 virus tyrosine kinase. Sequence analysis of v-ryk. The sequence of the 3' region of the RPL30 virus, including the v-ryk oncogene, is shown in Fig. 3A. The v-ryk gene was inserted into the viral envelope protein gp37 coding region within the env gene. It contained an open reading frame of 442 amino acids, followed by a 62-bp 3' untranslated region containing an AATAAA polyadenylation signal. This open reading frame was fused in frame with the upstream viral gp37 gene, which contains a transmembrane domain. Thus, the putative oncogene in the RPL30 virus encodes a 621-amino-acid gp37-Ryk fusion protein with a predicted molecular mass of 69 kDa termed P69gp37-,yk. Interestingly, in contrast to almost all acute oncogenic retroviruses, v-ryk was inserted into the 3' region of the viral envelope protein gp37 gene without deletion of any viral sequence. The DNA sequences flanking the v-ryk oncogene in the RPL30 viral genome match perfectly the continuous sequence in the env gene of other similar avian retroviruses, like RSV (39) and UR2 (32). In the sequence C terminal to the transmembrane domain of gp37, p69WI37-ik has four amino acids from gp37, followed by the v-Ryk sequence, which consists of a 42-amino-acid N-terminal domain, a 277-amino-acid protein tyrosine kinase catalytic domain, and a 123-amino-acid C-terminal domain. The structure of v-Ryk is diagrammed in Fig. 3B. The boundaries of the kinase catalytic domain were defined as described by Hanks et al. (25). It contains the ATP-binding site consensus sequence G-X-G-X-X-G and a lysine residue at position 334 (Fig. 3A), which is invariant among all protein kinases (24). v-Ryk contains all 11 of the subdomains conserved in all of the known protein kinases (24). Further analysis revealed that the sequence of v-Ryk more closely resembles that of tyrosine kinases rather than that of serinethreonine kinases. Namely, 38 of 40 highly conserved amino acids over the whole catalytic domain of 42 tyrosine kinases (24) were present in v-Ryk. In the regions thought to be important in determining the substrate specificity of a kinase (20, 25), D-L-A-A-R-N in subdomain VIb of receptor-type tyrosine kinases was conserved in v-Ryk and P-I/V-KIR-WT/M-A-P-E in subdomain VIII of most tyrosine kinases was P-V-K-W-i-A-i-E in v-Ryk; both regions were quite different from those characteristic of serine-threonine kinases. This strongly suggested that v-Ryk is a tyrosine kinase. Tyr-469 corresponds to the conserved autophosphorylation site in other kinases. Furthermore, in the position corresponding to the kinase insert of the platelet-derived growth factor and colony-stimulating factor 1 receptors (25), v-Ryk contains a seven-amino-acid insert with the sequence LEMAPQF. The functional significance of this insert is not known. Beside this short kinase insert, v-Ryk contains an additional sixamino-acid insert, LSSQQI, between kinase domains IV and


JIA ET AL.

5980

A

TSC CCO An CSC phe

pro

11l

TOI

CGA

COG

loU pro

gly va1

GAC

OTC

ACA CM

LC

a.r thr

trp v-1 *ap

s O00 CTA CCI CCC OCA ATT TTC CTC ATr TOC &la leu pro pro Ile ph. ile eye

gin

aA ART TTC gly men

AMA CCA AAM

AIC

ph. thr lye

pro

lye

CCT

AOT

60

20

cia

COT CCA 0TA arg pro val

AT? CAC AAM ATA CTT OCT AT TCC TCO CAA asp ile his lys ile leu atla sn ner ser g1n

CAT CT? hi. loU

*-S TT?

BA TCT mer

ph.

&la

CTA

CC

loU

cla

cap

CTA leu leou

CT?

trp ser v-1

GAM CAC AOC cap

thr

CAG va1

gin

gly

MA

lys

gin

OCT

AAC

vai cys

gly pro

cla

can

ACT AT? CGA CAT 0CC lac

thr ser ile crg his

CAM hi. GM

glU

CM? gly

his

TOT

gly cye

A

CT?

loU thr thr

mer

leo

leo

OT?

CTC CAM AAC CCA C00

vcl

loU

gin

can

arg &ia

TT?

CAM

TTS

OTA TCT

leo

gin

ph.

vai

Tac ACA CCM AA

OTA

AAM

val

&an tyr GGA

his thr A

erg

60

240

C OT?

ser

v-1

60

thr aia arg ile

thr

CTC AGC

gly vcl

aer

cia

lys

100

tyr

eer

leo

A

COO crg

eye

CAM AAC CT? gin lys loU

AGC GAM CTC CAM mer glU

gly

CGM

asp loU

GM

TC CB

leo

ph.

TT?

480

leu

160

WA

aer

va1

vai

loU gin

AMA

Srg

0CC OTC

C

GM

cap

CTA 0TA

720

vcl

240

acT ACT

ACA

760

lye

GM

net thr thr thr 260 Iv-ryk -2, 640 CT? ACA TTO

v-1 giu

loU

S CTOT

AT?

v.1

660

220

TT

va1

ile

thr

leo

280

gly

AMA AAM aep arg

00

--> ATC GAM GA C0T 960

CT?

ala

loU

AGO aer

AAC GTC

CT?

loU gly lye

CTC ACC CMG COCAA GGC loU ner gin TT?

ph.

TCC

pro

glu

gly

vcl

CGM TTC

CT? CGA GAM lou gly glu

gly

Cac COC AAT CST his pro &an va1

glo

ile

ph.

TCA gly ser va1

met

glo

giy

arg

320

ACC CCA CaM AAC GT OCT C? AAC ACC AT? AAM TTG OAT AAC gin lys v.1 &la v.1 lys thr set lys lOU cap can thr

glu glu

leo ah

ph.

WcA BA TOC ATA AAC cilac& a eys 1il lya

GM

glo

Gac cap

CTT lou

lye

ocA cia

MG

ser

crg

CCC

CAC

pro his

CCC

AT?

lys

pro net

COG CTC CA0

aar

GAM cap

oCC

CCC CAM

arg loU glU met *ia pro AT?

1ie

0CC

CT?

Oa

&a loU gly

gin

TTC ph.

OT

vcl

CCC CT? CAM ATC CT? CT? mat leu l-u pro loU gin

ATC CGM TAC CTC AOC AM CO0 CA net glu tyr loU ser ser crg

gin

ila

GAC COA C00

1660

cap crg pro

560

I

C-tere ->

ATc TA GTC AAC ACC MCC CTO Cea GM GA aoc CCC GAC TCC Ace 1600 val ile tyr vcl can thr *ar leu pro glu glu ser pro cap eor thr 600 TCT

GM

cap sor

cap

G

OT?

v.1

TT? CGC CCC GMG GM 1660 lou *ap pro gly cap 620

COA OT? cal v.l

GM

asp

AT? CAC GAM 1920 ilo his cap 640

GT? CTT CAA AI GMA 0C aO CCC aA GM CAT CT TAC tyr vcl leu glu ser glu gly ser pro thr glu cap cal tyr

TAC

Too ACC GM GM 0O0 TCO glu gly ser cal trp thr glu

0CC cia

VT? vcl

COA

pro

1660 660

Aac TCG 2040 aMC ACC TTO CCT OT? aer thr leu pro vcl gly *er ser 680

CTG CTO TOA 2100 TOC CTC cM cM TCC GA CTC CCA TOT CT CAT cal gln leu pro eys cla csp gly cys leu glu cap *-r glu ala leu lou '" 699

C CAM aoC AOC

GT?

TOA ATC AMC ACA CM aA TTT TAT

TT

AA AAa CAM 2160

TCO A?C

GOT

TAM CAT ACM

CTA CAM TAM GCC

GCM TST AAM AGO TCO CT? AWT AWT OC CGA CA AM

AAC CT TGA CCO OCA

TT?. CT? COC OAT CTA C

TT?

TAM

341

CCA ACMG

0o

CT? CO

GOT

TTSS

T?S GOA TCA AQ AAM CT? AGo

CT?

MG

CC

AM 2340

2400

CCa TQA TAT 2460

TTT ACM COT AAC TOG 0o

TAC

TT?

T AMC C0

T?

MaG Ce C CTC A" AM TAM TOG 2520

AT

AWT CTT An CAM aM TA AM TOT ATA 2580 AA TOT AGO ^ U3 --> MAT AAM COA ATC 0CC TOA TOC ACC aAA AA COT ATT ATA TOA TCC CAT Too TOO 2640

AM COc TTT TOC ATA TT

ph.*

ATC AAM CTC CTA GOT GT? TOC ATC CM CT? AOC TCT CAM CAM ATC ile lye loU ioU gly vc& cys ile glu loU aer aer gin gin ile

AT?

OC

TAM TAM GCA 2220 TCT TAT TA TTC ACT CG CTA TCO COA GOA ATA CAM GM ATT Aea AGA ^3'env --> C CAT AC TO TT? CGM C0 TAM 2280 CCC TA AAM aMC acW ATA GOC CBO TTC CTC TAM TCO TOT

OTA

1060

GM

360

ap

TTa

1140

CCC

TOA

COA

AGO ACC CAC CT? AMG OCA TAT 00c

pro 360

CT? CT? COC CT CTT CTC CCA TTC AT? AAM TAT CT CM CTC CAC ACC T vcl vcl loU pro ph. met lys tyr gly cap 1lU hi. aer ph. loU loU crg

TCC

cla 500

OCT Ca

T?C CTC AT?C AAM

cap

T

AT? CM CCT

1020

pro

CAT AMA GAM ATA CM CM TTC CTC AMT

*er hi. arg

glu

¢cA 1500

G?

ATT OCT GM CCC TOC TOC ?CC CAC acM AAM GCA GCG CT? 1lo cal glu pro eys cye ser his thr lys c,la al leu

300

cen

kinasoI

GCC

mer

CAG CT? gln leu

GM

ile lou loU loU

WC CGA AAC ATC thr

arg *a1

gin

TTA

TCC AAC CAC

gly gly

glU

AAM 1440 vcl lye 460

OT?

ACC TT TCA CAG CT? aAA OT CAT CT? GMA Aac CT? TT? GA aCc CTT CCT 0CC C00 AG 1740 thr ph. ,er gin leu lye vcl his leu glu lys il- lou glu nor leu pro ala pro arg 560

.00

il*

gly

T?A CAT GAM CT? TAC OAT ATC AT? TCC TCC TOC TOO Ao OGCT GM OCT lou cap glu l-o tyr cap i1e met aer eer cye trp arg cal glu pro

200

ATS C0T

pro

cap

CMC GM TT? 0C0 TT? CAT TCOG GT ATC CCC CAA GMO gln cap l-u gly lou asp ser val 11. pro gln *1-

TT?

ph.

CCA

AT

lys met

AAC CAC GM AT? TAT GM TAT CTA TTC Cac GM CAM COO CTC AAA AAC CCT Am AAC TOC 1620 leu lye lye pro glu can eye 540 can hi. glu ile tyr glu tyr leu ph. hi. gly gln

gly

GM

A

ATA GM TCC CTC CT MAC COT GT TAC acc ACC AM ATOATT TOO ili glu ser lou cla csp arg v1l tyr thr thr lye ser csp val trp

140

ile*ap

gly gly

CO ATA CCA ATM TACT COT CM tyr tyr crg gin gly arg 1ie cal

loU

CAM OTC AAM AMG ATC llo can lys

TTA

cap

00c an ACC AT? TOO GMa ATA 0CO ACC AMA GO AT? ACT CCC TMAC cCA 0o OTC CAM 1560 thr erg gly met thr pro tyr pro gly val gin 620 ph. gly val thr met tap glu ilie a

420

TTA TT

glu his vl

CT? OTACT? ATC giy

ser ser

AAM TCC TAC TOC lye

Go

TOO ATT trp ile cal TT

AT? TOT TOOC TTC AAM CT? AMT 540 AC CACT? GC cap ile &la gSly ntCy. eye ph. can lou *er 180

CT ATA CAM AAC TTC?C CAM CTA AT? AMU c&a ile gin lys lys ph. gin loU net lye

CT? CCT TOC CTT ioU pro Cye ioU

vcl

lou

TT

CIU

160

ACA

CAT

pro &&n

40

lys

ATA CAC TTA CTA AAM CGA CT CTT TTS ile his loU ioU lye gly loU leo iou

OTe

ear

AAC

AT? cia

...........A .............................

AMC C?

CCC

120

*sr

pro

qP37 -> OCT CCT ACA BCA AA ATC 300

CTC

GMC AGC CAC CCA ATC CG ACT TOO CTCQGA GMA cap ser cap pro ile gly ser trp 1iU arg gly

GTC TOC

ATT Ile

hi. ph.

TCA

ACA

val

tap

erg

MA

TT?

oar

TOG GC

AMA CAM va1

CT? TG loU tap

hi.

0GC giy

TCT C? AAA CAM

cys

OCT

MCA OC

gly

BOA

ATC TTA CC CCA GOO OTA WCA OCC CC CAA OC CTA AX GM ATC CM AMA 360 ile leo al pro gly va1 &ia*- &la gin &la loU erg gln Ile glo erg 120 U TOO

vcl

gin

GM

AM

TTC

AT?

lye

ph.

met

1200

GT?

TCA TCT acM COG CAM AMT ATC

AT?

AAC TCT CTG TCC TSG C0T CAT TCC 2700

GA

OGA Ta CAM

400

COT 0GC COA TOC TCA TTG CT? TCA CTA ACG aWT TAT 0TA ACC CAT

1260

TOT AW TOC TAM CAA TAA AWI

vcl 420

CAT 0O

Tm CTT CAC AO OAT TT? GOG ph. loU hi. crg cap loU &la

BA

CCT TMA TMA

1320

440

COa

CG0

0CC

GM

BOC aMc ATT CAT 2760 TOT ACT TaA OCT 2620

ATT TTA CCT CCC ACC ACa TTC OT? TOC ACC TOO OTT 2660 A

R --> aCC OTC OAT TCC TCA C0G CTA CQA OA CCT GA

US

-->

TOA aoC aMA aGo CTT CAC 2940

0C0 CAT ?CT AT? ACW CCA TOA TCA AAA CT? AAA acc TAC ACT

AT

TT

CCC AAC

COO

3000

cell ->

OCT C00

ila arg

AAC TOC AT? TTA CC0 can cys met

leo

crg

GA? cap

CAC AT? ACG GTG TOT GT? WCA asp met thr vcl eye va1 &ca

GM

cap

TT?

c0

CT?

TCC 1360

ph. gly lou ser

CM OCA AT?

460

B

v-ryk

gag

l

I

LTR

M

pol

04r%27

gp85

I

.re#4

0 r xx TMso, catalytic C-term

gpif

17 LTR

FIG. 3. cDNA sequence and structure of the 3' portion of the RPL30 virus. (A) cDNA sequence and translated protein sequence of the 3' portion of RPL30, including the v-tyk oncogene. The starting site for each gene region is indicated by a single caret underneath the first nucleotide followed by the name and direction of that gene. A series of carets underlines the transmembrane domain of gp37, an asterisk marks the ATP-binding site at Lys-334 in the kinase catalytic domain of v-Ryk, and c-term denotes the C-terminal domain of v-Ryk (see panel B). A series of three asterisks indicates the stop codon for the protein encoded by this gene, and the AATAAA polyadenylation signal site is underlined. The numbers for nucleotides and amino acids are to the right of the corresponding lines. (B) Genomic structure of the RPL30 virus. The viral gene names are indicated above their locations, and the coding regions for the transmembrane domain, kinase catalytic domain, and C-terminal domain (C-term) of v-Ryk, as well as the stop codon, are labeled underneath. The triangles denote the kinase inserts. This diagram is drawn approximately to scale.


cap

GAT CA? ACA TOC TCA GAC GAA asp asp thr eye *ap glU

TOT

CAT CGMCC

&Ia trp

GMOC CCC TOC TATCTA WOA AAA CT? ACC ATO TTA OA 0C gly gly pro cya tyr loU giy lye leo thr met leu *la

CAT

TCA

TOG C>

OAT COC WA

*ap rg

giy

~-

AM AM ATC TA AOC GO lys lye il tyr aer gly

gp

J. VIROL.


VOL. 66, 1992 v-RYK NALSLGKVLGEGEFGSVMUGRLSQPEG--TPQKVAVETMKLDNFSHRIIEEFLSEAACIKDrDHPN AXL/UFO HKVALGKTLGEGEFGAVKEGQLNQDDS--- ILKVAVTMKIAICTRSELEDFLSEAVCMKEFDBPN ARK HKVALGKTLGEGEFGAVKEGQLNQDDS --- ILKVAVKTKIVIC;TRSELEDFLSEAVCMXEFDHPN v-SEA HIR MET IGF1R RET c-SRC

LITHRSRVIGRGHFGSVYHGTYMDPLL--GNLHCAVESLHRIT-DLEEVEEFLREGILMKGFHHPQ EKITLLRELGQGSFGMVYZGNARDIIKGEAETRVAVXTVNESA-SLRBRIEFLNEASVMKGFTCHH LIVHFNEVIGRGHFGCVYHGTLLDNDG--KRIHCAVKSLNRIT-DIGBVSQFLTEGIIIMKDFSHPN

v-RYK AXL/UFO ARE v-SEA HIR

VIELLGVCIELSSQQI-PKPMVVLPFXKYGDLHSFLLRSRLEMAPQF ----------------- VP VMRLIGVCFQGSERESFPAPVVILPFKHGDLEESFLLYSRLGDQPVYL ----------------- PT

MET IGF1R RET c-SRC

EKITMSRELGQGSFGMVYEGVAKGVVKDEPETRVAIKTVNEAA-SMRERIEFLNEASVMKEFNCHH KNLVLGKTLGZGZFGKVVEATAFHLKGRAGYTTVAVKMLKENA-SPSELRDLLSEFNVLKQVNHPH ESLRLEVKLGQGCFQEVWMGTWNG ----- TTRVAIETLEPGTM -- SPEAFLQEAQVMEKLRHEK

VMRLIGVCFQGSDREGFPEPVVILPFKKHGDLSLHLYSRLGDQPVFL-----------------PT VLSLLGVCLPRH ---- GLPLVVLPYNRHGDLRHFVRAQERS ----------------------- PT VVRLGVVSE G------ QPTLVVMELKAHGDLKSYLRSLRPEAENNPGRPP -------------- PT VLSLLGICLRSE----GSPLVVLPYKHODLRNFIRNETHN----------------------PT VVRLLGWSQG ----- QPTLVIMELKTRGDLKSYLRSLRPEMENNPVLAP -------------- PS

VIELYGACSQD ----- GPLLLIVEYAKYGSLRGFLRESRKVGPGYLGSGGSRNSSSLDHPDERALT LVQLYAVVSE ------- EPIYIVTEYKSKGSLLDFLKGETGKY ---------------------- LR

v-RYE LQMLLKFKVDIALQEYLSSRQFLHRDLAARNCHLRDDTVCVADFGLSKIKYSGDYYRQG--RIA AXL/UFO -QILVEFKADIASQIZYLSTKRFIHRDLEAARNCLNENKSVCVADFGLSEKIYNGDYYRQG--RIA ARE

v-SEA

-QMLVKFADIASQTLSTKRFITR tDLAA.RNC)LNENMSVCVADFGLSEEIYNGDYYAKG--RIA VKELIGFGLQVALGKEYLAQKKFVERDLAARNCNLDETLTVKVADFGLARDVFGKEYYSIRQHRHA

HIR

LQEMIQMAAEIADQIAYLNAEKFVHRDLAARNCXVAHDFTVKIGDFGMTRDIYETDYYRKG--GKG

MET

VKDLIGFGLQVAKANKYLASKKFVHRDLAARNCMLDEKFTVKVADFGLARDMYDKEYYSVHNKTGA

IGFlR

LSEMIQMAGEIAD(MAYLNANKFVSREAARNCNVAEDFTVKIGDFGMTRDIYETDYYRKG--GKG MGDLISFAWQISQQ4QYLAEMKLVHRDLAAREILVAEGRKMR ISDFGLSRDVYEEDSYVKR--SQG LPQLVDMAAQIASGKArVERMNYVHRDLRAANILVGENLVCKVADFGLARLIEDNEYTAR ---QGA

RET c-SRC

ARK

IKPVENlARIESLADRVYTTKSDWVAFGVTf NATRGMTPYPGVQNHEITEYLFHGQRLKKPENCL

v-SEA

HIR MET

IGF1R RET c-SRC

IKPVKWIAIESLADRVTTSKSDVNLFGVTD4NKIATRGQPYPGVENSEIYDYLRKGNRLKQPVDCL KLPVRNMALESLQTQEFTTKSDNSFGVIJNILLTRGASPYPEV DPYDMARYLLRGRRLPQPQPCP LLPVRHMAPESLEDGVFTTSSDWSFGVVLWITSLAEQPYQGLSNEQVLKFVMDGGYLDQPDNCP KLPVEWMALZSLQTQEFTTSDVWSFGVVLNZEMTRGAPPYPDVNTFDITVYLLQGRRLLQPEYCP LLPVRHMSPZSLKDGVFTTYSDVSFGVVLWEIATLAEQPYQGLSEEQVLRFVMEGGLLDKPDNCP RIPVEWMAIUSLFDHIY!TQSDVWSFGVLLWKIVTLGGNPYPGIPPERLFNLLKTGHRMERPDNCS KFPIEWTAPKAALYGRFTIKSDWSFGILLTULTTEGRVPYPGMVERBVLDQVERGYRMPCPPECP

v-RYE AXL/UFO ARE v-SEA

DELTDIMSSCNRAEPADRPTFSQELVHLUELLESL DGLYALSRCNELNPQDRPSFTELREDLZNTLKAL DGLYALKSRCNELNPRDRPSFAELREDLENTLKLC DTLYGVKLSCWAPTPEERPSFSGLVCELERVLASL

HIR

ERVTDLRRMCNQFNPNMRPTFLEIVNLLEDDLHPS

MET

DPL!EVNLKCWHPEAEMRPSFSELVSRISAIFSTF

IGF1R RET

DMLFELXIMCNQYNPEMRPSFLEIISSIKEEMEPG EEMYRLMLQCWKQEPDERPVFADISEDLEEMMVER

c-SRC

ESLHDLNCQCVREEPEERPTFEYLQAFI.DYFTST

FIG. 4. Homology of v-Ryk to other tyrosine kinases. The v-Ryk protein sequence is aligned with those of other tyrosine kinases (sequences are from GenBank). The name of each protein is on the left, dashes denote gaps in the sequences introduced for maximal alignment, and the residues in v-Ryk that are also present in at least two other kinases are in bold letters.

Control

three immunized rabbits. This competition was specific, because the control antigen (vector with no insert) could not compete successfully for binding of the antibodies to the 70-kDa band (lane 1). In addition, preimmune serum could not precipitate the 70-kDa protein from 32P-labeled cells (lanes 8 and 9). This 70-kDa phosphoprotein was also immunoprecipitated from pRV-ryk-transformed CEF (Fig. 8, lane 4) but not from pUR2AV-transfected cells (lane 2). The size of this phosphoprotein, 70 kDa, is consistent with the predicted molecular mass of v-Ryk, as determined from its sequence. Therefore, this 70-kDa protein is believed to be the v-Ryk oncoprotein. Since the v-ryk gene encodes a putative tyrosine kinase, we performed in vitro kinase assays on immunoprecipitates of v-Ryk. Following incubation of anti-v-Ryk immunoprecipitates from v-ryk-transformed CEF and nontransformed CEF with [.y-32PjATP, two proteins with molecular masses of 70 and 150 kDa became phosphorylated in both pRV-ryk (Fig. 9, lanes 2 and 4)- and RPL30 (data not shown)transformed cells. These two phosphoproteins were not detected in mock-transfected (data not shown) or UR2AVtransfected (lane 6) CEF. Phosphoamino acid analysis of the two proteins showed that the phosphorylation was on tyrosine residues (data not shown). We concluded from these experiments that v-Ryk is indeed a tyrosine kinase. The 70-kDa protein is believed to be autophosphorylated v-Ryk, and the 150-kDa band is either a protein substrate which coimmunoprecipitated with v-Ryk or a v-Ryk-related protein which can also be recognized by the serum we used. v-Ryk antibodies were also used in Western blotting analysis. These antibodies detected a prominent 150-kDa

UR2AV

-c Ak

le BS ..:5

pRV-ryk + UR2AV RPL30 + UR2AV FIG. 5. Transformation of CEF by v-iyk. CEF were mock transfected (A), transfected with the helper virus plasmid pUR2AV (B) or the cloned v-ryk construct pRV-ryk plus pUR2AV (C), or infected with the RPL30 virus plus helper virus UR2AV (D). Cell morphology at 20 days after transfection or infection is shown. Magnification, x 100.


v-RYK

AXL/UFO IOIVEWIAIESLADRVTTSKSDVSFGVTRNNIATRGQTP!PGVENSEIYDYLRQGNRLKQPADCL

5981

5982

J. VIROL.

JIA ET AL.

UR2AV

Control

Contrut Construct

SeruLm111 Ao

303 ;--

305 Prc-lmm1ni

304 +

+ UR2AV pRV-ryk RPL30 UR2AV U2AV+UR2AV

±+

-

±

-

KD

±

Serum

KD

P I P I P I P P P1

aWWSk4WI'm -

200

t g

#_

97,4

6974

~

XW: TX4xEW -

69

-

46

-

30

3 4 5 6 7 8 9 FIG. 7. Antigen (Ag) competition assay. v-ryk-transformed CEF were labeled in vivo with 32Pi, lysed, immunoprecipitated with either preimmune (Pre-Imm) serum (lanes 8 and 9) or serum 303 (lanes 1, 2, and 3), 304 (lanes 4 and 5), or 305 (lanes 6 and 7) in the absence of antigen (lanes 2, 4, 6, and 8) or in the presence of immune antigen (lanes 3, 5, 7, and 9) or control antigen (lane 1), and analyzed on SDS-7.5% PAGE. The arrowhead shows the band that was specifically eliminated by competition with the immune antigen. 1

2

~~200

69

-

46

2 3 4 5 6 1 FIG. 8. Immunoprecipitation of the v-Ryk oncoprotein. CEF transfected with pRV-ryk plus pUR2AV (lanes 3 and 4) or pUR2AV alone (lanes 1 and 2) or infected with the original RPL30 virus mixed with UR2AV (lanes 5 and 6) were labeled in vivo with 32pi, lysed, immunoprecipitated with either preimmune serum (P) or immune serum (I), and analyzed by SDS-10% PAGE. The arrowhead indicates the protein that was present only in v-ryk-transformed cells.


RPL30 + UR2AV pRV-ryk + UR2AV FIG. 6. Colony formation of v-ryk-transformed cells. As for Fig. 5, cells were put on agar 20 days after transfection or infection, the plates were cultured at 40°C, colonies appeared 2 weeks later, and these pictures were taken 3 weeks later. Magnification, x 30.


VOL. 66, 1992 pRV-ryk +UR2AV P

I

ppRV-ryk 4

PUR2AV p

I

UR2AV p

I

KD

pRV-ryk RPL30 UR2AV

--

5983

+

KD 200

200 _t

_

97.4

_

69

_

46

-

30

97.4

69

_4r, .1

46

"'Wooole,qmpxmmwl

30

6 1 2 5 3 4 FIG. 9. In vitro kinase assay. Cell lysates from CEF transfected with either pRV-ryk plus pUR2AV (lanes 1 to 4) or pUR2AV alone (lanes 5 and 6) were immunoprecipitated with either preimmune serum (P) or immune serum (I), subjected to an in vitro kinase assay in the kinase reaction buffer, and analyzed by SDS-10% PAGE. The arrowheads indicate the two proteins that were present only in v-ryk-transformed cells.

protein in v-ryk-transformed CEF (Fig. 10). Upon careful examination of lanes 3 and 4 in Fig. 10, another specific band with a molecular mass of 70 kDa can be seen (indicated by the arrow at the lower position); this became obvious when the film was exposed longer (data not shown). These results were obtained with serum 303; the same results were obtained with two other antisera (data not shown). These data showed that pRV-ryk-transformed cells have the same biochemical properties as RPL30-transformed cells, further proving that the cloned v-ryk gene can fully recapitulate the transforming activity of the RPL30 virus. Biosynthesis of the v-Ryk oncoprotein. On Western blots (Fig. 10), the anti-Ryk antibodies predominately recognized a 150-kDa protein, suggesting that the 150-kDa protein is the precursor of the 70-kDa protein. To study the relationship between these two proteins, the 70- and 150-kDa bands were excised from the in vitro kinase assay gel and subjected to S. aureus V8 protease mapping. The V8 protease digestion patterns of the two proteins were similar (Fig. 11), suggesting that the 70-kDa protein is derived from the 150-kDa protein. To rule out the possibility that the 150-kDa protein arose from dimerization of the 70-kDa protein, v-ryk-transformed cells were pulse-labeled with [35S]methionine and then chased for various periods with cold methionine. The results (Fig. 12) demonstrate that the 70-kDa protein is derived from the 150-kDa protein. The 150-kDa protein peaked 15 min after the pulse-labeling step and then was

2 3 4 1 FIG. 10. Western blot of the v-Ryk oncoprotein. Cell lysates from CEF transfected with pRV-ryk plus pUR2AV (lane 3) or pUR2AV alone (lane 2), mock transfected (lane 1), or infected with the original RPI30 virus (lane 4) were run on SDS-10% PAGE, transferred to a polyvinylidene difluoride nylon filter, and probed with a 1:200 dilution of v-Ryk-specific antiserum. The arrowheads indicate the two proteins that were present only in v-ryk-transformed cells.

gradually chased into the 70-kDa protein, which peaked at 2 h. Because anti-Ryk antibodies were generated against the mature form of v-Ryk, other proteins derived from the precursor would not be recognized by this antiserum. Envelope proteins of retroviruses are known to be first synthesized as the env polyprotein precursor Pr95, which is then cleaved into two mature env proteins, gp85 and gp37, by a cellular protease (5). We believe that analogous to this, in the RPL30 virus, an Env-Ryk precursor fusion protein is first synthesized and then cellular protease cleaves this precursor into gp85 and P69W-37ryk (Fig. 13). The glycoprotein complex of RSV lacking the cytoplasmic domain of gp37 has been shown to be stably expressed on the cell surface in a manner similar to that of wild-type RSV (34). Therefore, the cytoplasmic tail of gp37 is not required for assembly of the envelope glycoproteins into virions. In Fig. 12, the labeling of the 150-kDa protein was stronger than that of the 70-kDa protein, consistent with results obtained from Western blotting experiments (Fig. 10), which show that the 150-kDa protein is more abundant than the 70-kDa protein. The in vitro kinase assay data (Fig. 9) show that the 70-kDa mature oncoprotein seems to exhibit higher kinase activity than the 150-kDa protein or that the 150-kDa protein autophosphorylates poorly. V8 mapping revealed that all of the phosphopeptides were derived from the 70-kDa protein (Fig. 11), as expected if the other protein (gp85) is extracellular. Cellular localization of the v-Ryk oncoprotein. The receptorlike structure of P69337ryk suggests that this oncoprotein is localized on the cell membrane. Cell fractionation experiments with sucrose gradients were performed to test this


.":... 4.11'

J. VIROL.

JIA ET AL.

5984

pRV-ryk Ip

RPL30

KD -

-

pol

200 97.4 69 46

I

gp85

.

gp37

v-ryk

gp37 I

RPL30

r..

stop

LTR

* 150 kD Env precursor-Ryk

-

-

30

*

21.5

Cellular protease

AGP37

v-Ryk

i

.,

-

GP37-Ryk GP85 FIG. 13. Biosynthesis of the v-Ryk oncoprotein. This diagram shows that the RPL30 virus (top line) first synthesizes a 150-kDa Env-Ryk precursor protein (middle line) and then has it cleaved into two mature proteins, gp85 and p69j37-yk (bottom line). TM, transmembrane domain.

14.3

150 kD 70 kD 150 kD 70 kD FIG. 11. V8 mapping of two proteins from v-ryk-transformed cells. The 150- and 70-kDa proteins from either pRV-ryk- or RPL30-transformed cells were excised from the in vitro kinase assay gel, subjected to V8 mapping analysis as described in the text, and run on SDS-12.5% PAGE.

prediction. The results (Fig. 14) showed that on the basis of the data from both the in vitro kinase assay and Western blotting analysis, v-Ryk is indeed located primarily in the membrane-enriched fraction rather than in the cytosol fraction. This is consistent with the idea that P69gP37-ryk is a membrane-bound receptorlike molecule. Phosphotyrosine-containing proteins are increased in v-ryktransformed cells. Since the v-ryk gene is a tyrosine kinase oncogene on the basis of sequence analysis and the in vitro kinase assay, we examined whether there was an increase in phosphotyrosine levels in v-ryk-transformed cells. Antiphosphotyrosine antibodies were used in a Western blot analysis

0'

15' 30' 1h 2h

69 kD

of whole-cell lysates. The results in Fig. 15 show that in v-iyk-transformed CEF, there were multiple phosphotyrosine-containing proteins which were not detected in untransformed cells; among them were the 150- and 70-kDa proteins, which presumably correspond to v-Ryk oncoproteins. This suggested that v-Ryk tyrosine kinase activity is required for the transforming phenotype. The identities of those multiple tyrosine-phosphorylated proteins are not known, but they are presumably substrates for the v-Ryk kinase.

DISCUSSION In this study, we analyzed three previously uncharacterized RPL oncogenic retroviruses; two of them, RPL25 and RPL28, were shown to contain the erbB oncogene in their genomes. We then further identified, and subsequent molec-

S M

S M

MW -

200 kD

-

97.4

4h 8h .4

--

4-

....

150kD 70 kD 1

1

2

3

4

5

6

7

FIG. 12. Pulse-chase analysis of the v-Ryk oncoproteins. CEF transfected with pRV-ryk plus pUR2AV were metabolically labeled in vivo with [35S]methionine for 2 h, chased for the times indicated at the tops of the lanes, immunoprecipitated by v-Ryk-specific antiserum, and analyzed by SDS-10% PAGE.

-

69

-

46

..:

.-m

2

3

4

FIG. 14. The v-Ryk oncoprotein is localized on the cell membrane. CEF transfected with pRV-ryk plus pUR2AV were fractionated into soluble (S) and membrane (M) fractions as described in Materials and Methods. These two fractions were then subjected to either Western blotting analysis with v-Ryk-specific antiserum (lanes 1 and 2) or an in vitro kinase assay (lanes 3 and 4) and analyzed by SDS-10% PAGE. The arrows indicate the two forms of the v-Ryk protein. MW, molecular mass.


MKSN=~~~


VOL. 66, 1992

pRV-ryk RPL30 UR2AV

-

+

+

+

200

4r

.1

: -

97.4

.,F t 7

.

-*,:

:,-

-

o

-

69

46

ularly cloned, a novel transforming gene from the genome of the third virus, RPL30. This viral oncogene, v-ryk, is a member of the protein tyrosine kinase gene family. It inserted itself into the RPL30 virus envelope gene without deletion of any of the viral sequences and is fused in frame with the gp37 gene upstream to form a 69-kDa gp37-v-Ryk transmembrane protein, P69gp37-yk. v-ryk can transform CEF, form colonies, and induce tumors in chickens, fully recapitulating the transforming activity of the original RPL30 virus. The v-Ryk oncoprotein is first synthesized as a 150-kDa precursor protein and then cleaved into the mature p69gp37-ryk protein. v-Ryk has a tyrosine kinase activity in vitro and is phosphorylated in vivo in v-ryk-transformed cells. Furthermore, elevated phosphotyrosine-containing protein levels are found in these v-ryk-transformed cells. Viral oncogenes are transduced from cellular genes by nononcogenic retroviruses. The v-erbB oncogene was first identified in the AEV genome (strains AEV-ES4 and AEVR), together with another oncogene, erbA (47). The erbB gene, however, is not always cotransduced into retroviruses with the erbA gene, since erbB transducing viruses lacking erbA have also been isolated (3, 30, 50). The finding that RPL25 and RPL28 both contain only the erbB oncogene is consistent with the notion that the transforming activity of erbB is stronger. All acute oncogenic retroviruses lose part of their viral genes during the oncogene transduction process, thus becoming replication deficient and relying on helper viruses to replicate and assemble infectious viral particles (43). Although the Schmidt-Ruppin and Prague strains of RSV contain the v-src gene yet preserve all of the viral structural genes and are replication competent, there is evidence that

the original RSV was a replication-defective transforming virus (16). The current replication-competent strains of RSV were probably produced after a second recombination event. The RPL30 virus is the first example of a virus that did not lose any of the viral sequences in transducing a protooncogene, although the envelope protein gp37 coding region was disrupted by insertion of the v-ryk gene, rendering the virus deficient in replication. The reason that no viral genes were deleted in RPL30 is probably that these are homologous sequences between retroviral sequences and the c-ryk gene at the region of the insertion. This will become clear when the sequence for the c-ryk proto-oncogene is known. Some viral tyrosine kinase oncogenes were transduced from cellular receptors for peptide growth factors. For example, v-erbB in AEV was transduced from the epidermal growth factor receptor (15), and v-sea was presumably transduced from a cellular receptor (8, 26). Since v-Ryk more closely resembles receptor-type tyrosine kinases than nonreceptor tyrosine kinases, c-ryk may encode another cell surface receptor. We are currently cloning and characterizing the c-ryk gene, and results from preliminary experiments have confirmed that c-ryk is indeed a receptor-type tyrosine kinase (26b). Therefore, it is very likely that transduction of the c-ryk gene by the RPL30 virus rendered this kinase active in the absence of its ligand. The two oncogenes erbB and sea are particularly interesting because of their processes of viral transduction and the structures of their final oncogene products. These two oncogenic proteins are similar to v-Ryk in that their basic mechanism of activation is truncation of extracellular domains and the final products still maintain transmembrane domains, albeit the one of v-ErbB is derived from the epidermal growth factor receptor and those of v-Sea and v-Ryk came from viral env product gp37. Although relatively high amino acid identities with Axl-UFO and Ark (67.3%) in the kinase catalytic domains were found, the preliminary data on the cloning of c-ryk indicate that they are encoded by different genes (26b). In addition, the catalytic domain of v-Ryk contains sequences similar to polymerase chain reaction fragments JTKll (32b), tyro-12, tyro-7, and tyro-3 (28a). Particularly, tyro-12 has 53 of the total of 54 amino acids identical to those of v-Ryk. However, the relationships between v-Ryk and these polymerase chain reaction fragments are difficult to judge because these polymerase chain reaction fragments are only 50 to 60 amino acids long and are derived from the most conserved region within the tyrosine kinase catalytic domain. The results of Western blot analysis (Fig. 10) and the pulse-chase experiment (Fig. 12) indicate that the 150-kDa precursor protein is more abundant than p69w37-ryk in v-ryktransformed cells. This raises the interesting question of whether the Env-Ryk precursor protein has transforming ability or whether cleavage is required for transformation. Other unanswered questions about the v-ryk oncogene include the function of the C-terminal sequences of v-ryk and the small inserts in the kinase domain. We are currently addressing these questions by mutagenesis of the v-ryk oncogene.

ACKNOWLEDGMENTS We are particularly indebted to L. B. Crittenden of the Avian Disease and Oncology Laboratory, East Lansing, Mich., for generously providing us with the RPL30 virus. We sincerely thank K. A. Sokol of the Laboratory Animal Research Center for excellent assistance in pathology studies and many members of our laboratory for technical help. We are grateful to G. Scholz for critical reading of the manuscript.


4 3 2 1 FIG. 15. Phosphotyrosine-containing proteins are increased in v-ryk-transformed cells. A Western filter was prepared as described in the legend to Fig. 10 and blotted with polyclonal antiphosphotyrosine antibodies. The arrowheads indicate the positions of the two v-Ryk proteins.

5985

5986

JIA ET AL.

This work was supported by grant CA44356.

Nature (London) 292:506-511. 19. Feldman, R. A., T. Hanafusa, and H. Hanafusa. 1980. Characterization of protein kinase activity associated with the transforming gene product of Fujinami sarcoma virus. Cell 22:757765. 20. Foster, D. A., J. B. Levy, G. Q. Daley, M. C. Simon, and H. Hanafusa. 1986. Isolation of chicken cellular DNA sequences with homology to the region of viral oncogenes that encodes the tyrosine kinase domain. Mol. Cell. Biol. 6:325-331. 21. Fredrickson, T. N., H. G. Purchase, and B. R. Burmester. 1964. Transmission of virus from field cases of avian lymphomatosis. III. Variation in the oncogenic spectra of passaged virus isolates. Natl. Cancer Inst. Monogr. 17:1-29. 22. Hamaguchi, M., C. Grandori, and H. Hanafusa. 1988. Phosphorylation of cellular proteins in Rous sarcoma virus-infected cells: analysis by use of anti-phosphotyrosine antibodies. Mol. Cell. Biol. 8:3035-3042. 23. Hanafusa, H. 1969. Rapid transformation of cells by Rous sarcoma virus. Proc. Natl. Acad. Sci. USA 63:318-325. 24. Hanks, S. K., and A. M. Quinn. 1991. Protein kinase catalytic domain sequence database: identification of conserved features of primary structure and classification of family members. Methods Enzymol. 200:38-62. 25. Hanks, S. K., A. M. Quinn, and T. Hunter. 1988. The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science 241:42-52. 26. Hayman, M. J., G. Kitchener, P. K. Vogt, and H. Beug. 1985. The putative transforming protein of S13 avian erythroblastosis virus is a transmembrane glycoprotein with an associated protein kinase activity. Proc. Natl. Acad. Sci. USA 82:8237-8241. 26a.Janssen, J. W. G., A. S. Schulz, A. C. M. Steenvoorden, M. Schmidberger, S. Strehl, P. F. Ambros, and C. R. Bartram. 1991. A novel putative tyrosine kinase receptor with oncogenic potential. Oncogene 6:2113-2120. 26b.Jia, R., and H. Hanafusa. Unpublished data. 27. Kan, N. C., C. S. Flordellis, G. E. Mark, P. H. Duesberg, and T. S. Papas. 1984. Nucleotide sequence of avian sarcoma virus MH2: two potential onc genes, one related to avian sarcoma virus MC29 and the other related to murine sarcoma virus 3611. Proc. Natl. Acad. Sci. USA 81:3000-3004. 28. Klempnauer, K.-H., T. J. Gonda, and J. M. Bishop. 1982. Nucleotide sequence of the retroviral leukemia gene v-myb and its cellular progenitor c-myb: the architecture of a transduced oncogene. Cell 31:453-463. 28a.Lai, C., and G. Lemke. 1991. An extended family of proteintyrosine kinase genes differently expressed in the vertebrate nervous system. Neuron 6:691-704. 29. Li, Y., C. M. Turck, J. K. Teumer, and E. Stavnezer. 1986. Unique sequence, ski, in Sloan-Kettering avian retroviruses with properties of a new cell-derived oncogene. J. Virol. 57: 1065-1072. 30. Miles, B. D., and H. L. Robinson. 1985. High-frequency transduction of c-erbB in avian leukosis virus-induced erythroblastosis. J. Virol. 54:295-303. 31. Naharro, G., K. C. Robbins, and E. P. Reddy. 1984. Gene product of v-fgr onc: hybrid protein containing a portion of actin and a tyrosine-specific protein kinase. Science 223:63-66. 32. Neckameyer, W. S., and L.-H. Wang. 1985. Nucleotide sequence of avian sarcoma virus UR2 and comparison of its transforming gene with other members of the tyrosine protein kinase oncogene family. J. Virol. 53:879-884. 32a.O'Bryan, J. P., R. A. Frye, P. C. Cogswell, A. Neubauer, B. Kitch, C. Prokop, R. Espinosa III, M. M. Le Beau, H. S. Earp, and E. T. Liu. 1991. axl, a transforming gene isolated from primary human myeloid leukemia cells, encodes a novel receptor tyrosine kinase. Mol. Cell. Biol. 11:5016-5031. 32b.Partanen, J., T. P. Makela, R. Alitalo, H. Lehvaslaiho, and K. Alitalo. 1990. Putative tyrosine kinases expressed in K-562 human leukemia cells. Proc. Natl. Acad. Sci. USA 87:89138917. 33. Pearson, W. R., and D. J. Lipman. 1988. Improved tools for biological sequence comparison. Proc. Natl. Acad. Sci. USA 85:2444-2448.


REFERENCES 1. Akiyama, T., S. Matsuda, Y. Namba, T. Saito, K. Toyoshima, and T. Yamamoto. 1991. The transforming potential of the c-erbB-2 protein is regulated by its autophosphorylation at the carboxyl-terminal domain. Mol. Cell. Biol. 11:833-842. 2. Alitalo, K., J. M. Bishop, D. H. Smith, E. Y. Chen, W. W. Colby, and A. D. Levinson. 1983. Nucleotide sequence of the v-myc oncogene of avian retrovirus MC29. Proc. Natl. Acad. Sci. USA 80:100-104. 3. Beug, H., M. J. Hayman, M. B. Raines, H. J. Kung, and B. Vennstrom. 1986. Rous-associated virus 1-induced erythroleukemic cells exhibit a weakly transformed phenotype in vitro and release c-erbB-containing retroviruses unable to transform fibroblasts. J. Virol. 57:1127-1138. 4. Brugge, J. S., and R. L. Erikson. 1977. Identification of a transformation-specific antigen induced by an avian sarcoma virus. Nature (London) 269:346-348. 5. Buchhagen, D. L., and H. Hanafusa. 1978. Intracellular precursors to the major glycoprotein of avian oncoviruses in chicken embryo fibroblasts. J. Virol. 25:845-851. 6. Chen, I. S. Y., T. W. Mak, J. J. O'Rear, and H. Temin. 1981. Characterization of a reticuloendotheliosis virus strain T DNA and isolation of a novel variant of reticuloendotheliosis virus strain T by molecular cloning. J. Virol. 40:800-811. 7. Cleveland, D. W., S. G. Fischer, M. W. Kirschner, and U. K. Laemmli. 1977. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J. Biol. Chem. 252:1102-1106. 8. Cooper, G. M. 1990. Oncogenes. Jones & Bartlett Publishers, Boston. 9. Cross, F. R., and H. Hanafusa. 1983. Local mutagenesis of Rous sarcoma virus: the major sites of tyrosine and serine phosphorylation of p6Orc are dispensable for transformation. Cell 34:597607. 10. Curran, T., G. Peters, C. Van Beveren, N. M. Teich, and I. M. Verma. 1982. FBJ murine osteosarcoma virus: identification and molecular cloning of biologically active proviral DNA. J. Virol. 44:674-682. 11. Debuire, B., C. Henry, M. Benaissa, G. Biserte, S. Saule, P. Martin, and D. Stehelin. 1984. Sequencing the erbA gene of avian erythroblastosis virus reveals a new type of oncogene. Science 224:1456-1459. 12. DeLorbe, W. J., P. A. Luciw, H. M. Goodman, H. E. Varmus, and J. M. Bishop. 1980. Molecular cloning and characterization of avian sarcoma virus circular DNA molecules. J. Virol. 36:50-61. 13. Devare, S. G., E. P. Reddy, K. C. Robbins, P. R. Anderson, S. R. Tronick, and S. A. Aaronson. 1982. Nucleotide sequence of the transforming gene of simian sarcoma virus. Proc. Natl. Acad. Sci. USA 79:3179-3182. 14. Donner, L., L. A. Fedele, C. F. Garon, S. J. Anderson, and C. J. Sherr. 1982. McDonough feline sarcoma virus: characterization of the molecularly cloned provirus and its feline counterpart. J. Virol. 41:489-500. 15. Downward, J., Y. Yarden, E. Mayes, G. Scrace, N. Totty, P. Stockwell, A. Ullrich, J. Schlessinger, and M. D. Waterfield. 1984. Close similarity of epidermal growth factor receptor and v-erb-B oncogene protein sequences. Nature (London) 307:521527. 16. Dutta, A., L.-H. Wang, T. Hanafusa, and H. Hanafusa. 1985. Partial nucleotide sequence of Rous sarcoma virus-29 provides evidence that the original Rous sarcoma virus was replication defective. J. Virol. 55:728-735. 17. Ellis, R. W., D. DeFeo, J. M. Maryak, H. A. Young, T. Y. Shih, E. H. Chang, D. R. Lowy, and E. M. Scolnick. 1980. Dual evolutionary origin for the rat genetic sequences of Harvey murine sarcoma virus. J. Virol. 38:408-420. 18. Ellis, R. W., D. DeFeo, T. Y. Shih, M. A. Gonda, H. A. Young, N. Tsuchida, D. R. Lowy, and E. M. Scolnick 1981. The p21 ras genes of Harvey and Kirsten sarcoma viruses originate from divergent members of a family of normal vertebrate genes.

J. VIROL.

VOL. 66, 1992

5987

cloned genes. J. Mol. Biol. 189:113-130. 42. Sudol, M., and H. Hanafusa. 1986. Cellular proteins homologous to the viral yes gene product. Mol. Cell. Biol. 6:2839-2846. 43. Swain, A., and J. M. Coffin. 1992. Mechanism of transduction by retroviruses. Science 255:841-845. 44. Takeya, T., and H. Hanafusa. 1982. DNA sequence of the viral and cellular src gene of chickens. II. Comparison of the src genes of two strains of avian sarcoma virus and of the cellular homolog. J. Virol. 44:12-18. 45. Ullnch, A., and J. Schlessinger. 1990. Signal transduction by receptors with tyrosine kinase activity. Cell 61:203-212. 46. Van Beveren, C., J. A. Galleshaw, V. Jonas, A. J. M. Berns, R. F. Doolittle, D. J. Donaghue, and I. M. Verma. 1981. Nucleotide sequence and formation of the transforming gene of a mouse sarcoma virus. Nature (London) 289:258-262. 47. Vennstrom, B., and J. M. Bishop. 1982. Isolation and characterization of chicken DNA homologous to the two putative oncogenes of avian erythroblastosis virus. Cell 28:135-143. 48. Wang, L.-H., and H. Hanafusa. 1988. Avian sarcoma viruses. Virus Res. 9:159-203. 49. Wang, L.-H., H. Hanafusa, M. F. D. Notter, and P. C. Balduzzi. 1982. Genetic structure and transforming sequence of avian sarcoma virus UR2. J. Virol. 41:833-841. 50. Yamamoto, T., H. Hihara, T. Nishida, S. Kawai, and K. Toyoshima. 1983. A new avian erythroblastosis virus, AEV-H, carries erbB gene responsible for the induction of both erythroblastosis and sarcomas. Cell 34:225-232. 51. Yamamoto, T., T. Nishida, N. Miyajima, S. Kawai, T. Ooi, and K. Toyoshima. 1983. The erbB gene of avian erythroblastosis virus is a member of the src gene family. Cell 35:71-78.


34. Perez, L. G., G. L. Davis, and E. Hunter. 1987. Mutants of the Rous sarcoma virus envelope glycoprotein that lack the transmembrane anchor and cytoplasmic domains: analysis of intracellular transport and assembly into virions. J. Virol. 61:29812988. 35. Reddy, E. P., M. J. Smith, and A. Srinivasan. 1983. Nucleotide sequence of Abelson murine leukemia virus genome: structural similarity of its transforming gene product to other onc gene products with tyrosine-specific kinase activity. Proc. Natl. Acad. Sci. USA 80:3623-3627. 35a.Rescigno, J., A. Mansukhani, and C. Basilico. 1991. A putative receptor tyrosine kinase with unique structural topology. Oncogene 6:1909-1913. 36. Rosenberg, A. H., B. N. Lade, D. Chui, S.-W. Lin, J. J. Dunn, and F. W. Studier. 1987. Vectors for selective expression of cloned DNAs by T7 RNA polymerase. Gene 56:125-135. 37. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 38. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463-5467. 39. Schwartz, D. E., R. Tizard, and W. Gilbert. 1983. Nucleotide sequence of Rous sarcoma virus. Cell 32:853-869. 40. Smith, D. R., P. K. Vogt, and M. J. Hayman. 1989. The v-sea oncogene of avian erythroblastosis retrovirus S13: another member of the protein-tyrosine kinase gene family. Proc. Natl. Acad. Sci. USA 86:5291-5295. 41. Studier, F. W., and B. A. Moffatt. 1986. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of
