Cloning and characterization of an envelope-specific probe from xenotropic murine leukemia proviral DNA.

UR2 is a newly characterized avian sarcoma virus whose genome contains a unique sequence that is not related to the sequences of other avian sarcoma virus transforming genes thus far identified. This unique sequence, termed ros, is fused to part of the viral gag gene. The product of the fused gag-ros gene of UR2 is a protein of 68,000 daltons (P68) immunoprecipitable by antiserum against viral gag proteins. In vitro translation of viral RNA and in vivo pulse-chase experiments showed that P68 is not synthesized as a large precursor and that it is the only protein product encoded in the UR2 genome, suggesting that it is involved in cell transformation by UR2. In vivo, P68 was phosphorylated at both serine and tyrosine residues. Immunoprecipitates of P68 with anti-gag antisera had a cyclic nucleotide-independent protein kinase activity that phosphorylated P68, rabbit immunoglobulin G in the immune complex, and alpha-casein. The phosphorylation by P68 was specific to tyrosine of the substrate proteins. P68 was phosphorylated in vitro at only one tyrosine site, and the tryptic phosphopeptide of in vitro-labeled P68 was different from those of Fujinami sarcoma virus P140 and avian sarcoma virus Y73-P90. A comparison of the protein kinases encoded by UR2, Rous sarcoma virus, Fujinami sarcoma virus, and avian sarcoma virus Y73 revealed that UR2-P68 protein kinase is distinct from the protein kinases encoded by those viruses by several criteria. Our results suggest that several different protein kinases encoded by viral transforming genes have the same functional specificity and cause essentially the same cellular alterations.     

Membrane association of the transforming protein of avian sarcoma virus UR2 and mutants temperature sensitive for cellular transformation and protein kinase activity.

The localization of the transforming protein P68gag-ros of avian sarcoma virus UR2, which has a hydrophobic region at the N terminus of its ros-specific tyrosine kinase-encoding sequence, was examined by subcellular fractionation. P68 behaved as an integral membrane protein associated with the plasma membrane of transformed cells. P68 became membrane associated very rapidly in its biogenesis. Three temperature-sensitive mutants of UR2 were isolated and characterized. Cells infected with the mutants were temperature sensitive for morphological alteration and colony formation. The mutant P68 proteins were membrane associated in mutant-infected cells regardless of the temperature but were active as protein kinases only at the permissive temperature. The results suggest that P68 is a membrane-associated protein whose kinase activity plays a crucial role in UR2-mediated cell transformation.     

Nucleotide sequence of avian sarcoma virus UR2 and comparison of its transforming gene with other members of the tyrosine protein kinase oncogene family.

The genome of avian sarcoma virus UR2 was completely sequenced and found to have a size of 3,165 nucleotides. The UR2-specific transforming sequence, ros, with a length of 1,273 nucleotides, is inserted between the truncated gag gene coding for p19 and the env gene coding for gp37 of the UR2AV helper virus. The deduced amino acid sequence for the UR2 transforming protein P68 gives a molecular weight of 61,113 and shows that it is closely related to the oncogene family coding for tyrosine protein kinases. P68 contains two distinctive hydrophobic regions that are absent in other tyrosine kinases, and it has unique amino acid changes and insertions within the conserved domain of the kinases. These characteristics may modulate the activity and target specificity of P68.     

Specific inhibition of tyrosine kinase activity by an antibody to the v-ros oncogene product.

Antibodies present in two peritoneal exudates of rats bearing abdominal tumors induced by UR2-transformed rat cells were characterized. The ability to immunoprecipitate p68gag-ros and to inhibit the protein and phospholipid kinase activities of this protein was investigated. One of the exudates specifically inhibited tyrosyl phosphorylation by p68gag-ros but not the activity of other known tyrosyl kinases, such as p150gag-fps of UR1 avian sarcoma virus, p60src, and the insulin receptor. It precipitated p68gag-ros but not Pr76 or other gag-related proteins from UR2-infected cells. Phosphorylation of phosphatidylinositol was not affected by this exudate, suggesting that this activity is not intrinsic to p68gag-ros. Another exudate precipitated p68gag-ros but not gag-related proteins from UR2-infected cells or p140gag-fps from Fujinami sarcoma virus-infected cells. These results demonstrated that the antibodies in these exudates recognized epitopes present in the ros portion of the fused protein p68gag-ros, but only one of the two exudates inhibited the intrinsic tyrosyl kinase of p68gag-ros.     

Transforming properties and substrate specificities of the protein tyrosine kinase oncogenes ros and src and their recombinants.

To determine the sequences of the oncogenes src (encoded by Rous sarcoma virus [RSV]) and ros (encoded by UR2) that are responsible for causing different transformation phenotypes and to correlate those sequences with differences in substrate recognition, we constructed recombinants of the two transforming protein tyrosine kinases (PTKs) and studied their biological and biochemical properties. A recombinant with a 5' end from src and a 3' end from ros, called SRC x ROS, transformed chicken embryo fibroblasts (CEF) to a spindle shape morphology, mimicking that of UR2. Neither of the two reverse constructs, ROS x SRC I and ROS x SRC II, could transform CEF. However, a transforming variant of ROS x SRC II appeared during passages of the transfected cells and was called ROS x SRC (R). ROS x SRC (R) contains a 16-amino-acid deletion that includes the 3' half of the transmembrane domain of ros. Unlike RSV, ROS x SRC (R) also transformed CEF to an elongated shape similar to that of UR2. We conclude that distinct phenotypic changes of RSV- and UR2-infected cells do not depend solely on the kinase domains of their oncogenes. We next examined cellular proteins phosphorylated by the tyrosine kinases of UR2, RSV, and their recombinants as well as a number of other avian sarcoma viruses including Fujinami sarcoma virus Y73, and some ros-derived variants. Our results indicate that the UR2-encoded receptorlike PTK P68gag-ros and its derivatives have a very restricted substrate specificity in comparison with the nonreceptor PTKs encoded by the rest of the avian sarcoma viruses. Data from ros and src recombinants indicate that sequences both inside and outside the catalytic domains of ros and src exert a significant effect on the substrate specificity of the two recombinant proteins. Phosphorylation of most of the proteins in the 100- to 200-kDa range correlated with the presence of the 5' src domain, including the SH2 region, but not with the kinase domain in the recombinants. This corroborates the conclusion given above that the kinase domain of src or ros per se is not sufficient to dictate the transforming morphology of these two oncogenes. High-level tyrosyl phosphorylation of most of the prominent substrates of src is not sufficient to cause a round-shape transformation morphology.     

Enhancement of transforming potential of human insulinlike growth factor 1 receptor by N-terminal truncation and fusion to avian sarcoma virus UR2 gag sequence.

The human insulinlike growth factor 1 (hIGF-1) receptor (hIGFR) is a transmembrane protein tyrosine kinase (PTK) molecule which shares high sequence homology in the PTK domain with the insulin receptor and, to a lesser degree, the ros transforming protein of avian sarcoma virus UR2. To assess the transforming potential of hIGFR, we introduced the intact and altered hIGFR into chicken embryo fibroblasts (CEF). The full-length hIGFR cDNA (fIGFR) was cloned into a UR2 retroviral vector, replacing the original oncogene v-ros. fIGFR was able to promote the growth of CEF in soft agar and cause morphological alteration in the absence of added hIGF-1 to medium containing 11% calf and 1% chicken serum. The transforming ability of hIGFR was not further increased in the presence of 10 nM exogenous hIGF-1. The 180-kDa protein precursor of hIGFR was synthesized and processed into alpha and beta subunits. The overexpressed hIGFR in CEF bound hIGF-1 with high affinity (Kd = 5.4 x 10(-9) M) and responded to ligand stimulation with increased tyrosine autophosphorylation. The cDNA sequence coding for part of the beta subunit of hIGFR, including 36 amino acids of the extracellular domain and the entire transmembrane and cytoplasmic domains, was fused to the 5' portion of the gag gene in the UR2 vector to form an avian retrovirus. The resulting virus, named UIGFR, was able to induce morphological transformation and promote colony formation of CEF with a stronger potency than did fIGFR. The UIGFR genome encodes a membrane-associated, glycosylated gag-IGFR fusion protein. The specific tyrosine phosphorylation of the mature form of the fusion protein, P75, is sixfold higher in vitro and threefold higher in vivo than that of the native IGFR beta subunit, P95. In conclusion, overexpression of the native or an altered hIGFR can induce transformation of CEF with the gag-IGFR fusion protein possessing enhanced transforming potential, which is consistent with its increased in vitro and in vivo tyrosine phosphorylation.     

Proto-oncogene c-ros codes for a molecule with structural features common to those of growth factor receptors and displays tissue specific and developmentally regulated expression.

A recombinant DNA clone containing cellular sequences homologous to the transforming sequence, v-ros, of avian sarcoma virus UR2 was isolated from a chicken genomic DNA library. Heteroduplex mapping and nucleotide sequencing reveal that the v-ros sequences are distributed in nine exons ranging from 65 to 204 nucleotides on cellular ros (c-ros) DNA over a range of 11 kilobases. Comparison of the deduced amino acid sequences of c-ros and v-ros shows two differences: v-ros contains a three-amino-acid insertion within the hydrophobic domain presumed to be involved in membrane association, and (ii) the carboxyl 12 amino acids of v-ros are completely different from those of the deduced c-ros sequence. The deduced amino acid sequence of c-ros bears striking structural features similar to those of insulin and epidermal growth factor receptors, including the presumed hydrophobic membrane binding domain, amino acids flanking the domain, and the distance between the domain and the catalytic region of the kinase activity. The expression of c-ros appears to be under a very stringent control. When tissues at various stages of chicken development were analyzed, only kidney was found to contain a significant level of c-ros RNA. The level of c-ros RNA in kidney tissue is most abundant in 7- to 14-day-old chickens. Finally, nucleotide sequences of c-ros DNA and UR2-associated helper viral genome at regions corresponding to the gag ros recombination site suggest that the junction has been formed by RNA splicing.     

Molecular and biochemical bases for activation of the transforming potential of the proto-oncogene c-ros.

The transforming gene of avian sarcoma virus UR2, v-ros, encodes a receptor-like protein tyrosine kinase and differs from its proto-oncogene, c-ros, in its 5' truncation and fusion to viral gag, a three-amino-acid (aa) insertion in the transmembrane (TM) domain, and changes in the carboxyl region. To explore the basis for activation of the c-ros transforming potential, various c-ros retroviral vectors containing those changes were constructed and studied for their biological and biochemical properties. Ufcros codes for the full-length c-ros protein of 2,311 aa, Uppcros has 1,661-aa internal deletion in the extracellular domain, CCros contains the 3' c-ros cDNA fused 150 aa upstream of the TM domain to the UR2 gag, CVros is the same as CCros except that the 3' region is replaced by that of v-ros, and VCros is the same as CCros except that the 5' region is replaced by that of v-ros. The Ufcros, Uppcros, CCros, and CVros are inactive in transforming chicken embryo fibroblasts, whereas VCros is as potent as UR2 in cell-transforming and tumorigenic activities. Upon passages of CCros and CVros viruses, the additional extracellular sequence in comparison with that of v-ros was delected; concurrently, both viruses (named CC5d and CV5d, respectively) attained moderate transforming activity, albeit significantly lower than that of UR2 or VCros. The native c-ros protein has a very low protein tyrosine kinase activity, whereas the ppcros protein is constitutively activated in kinase activity. The inability of CCros and CVros to transform chicken embryo fibroblasts is consistent with the inefficient membrane association, instability, and low kinase activity of their encoded proteins. The CC5d and CV5d proteins are indistinguishable in kinase activity, membrane association, and stability from the v-ros protein. The reduced transforming potency of CC5d and CV5d proteins can be attributed only to their differential substrate interaction, notably the failure to phosphorylate a 88-kDa protein. We conclude that the 5' rather than the 3' modification of c-ros is essential for its oncogenic activation; the sequence upstream of the TM domain has a negative effect on the transforming activity of CCros and CVros and needs to be deleted to activate their biological activity.     

Molecular cloning and characterization of avian sarcoma virus UR2 and comparison of its transforming sequence with those of other avian sarcoma viruses.

Avian sarcoma virus UR2 and its associated helper virus, UR2AV , were molecularly cloned into lambda gtWES X lambda B by using unintegrated viral DNAs. One UR2 and several UR2AV clones were obtained. The UR2 DNA was subsequently cloned into pBR322. Both UR2 and UR2AV DNAs were tested for their biological activity by transfection onto chicken embryo fibroblasts. When cotransfected with UR2AV DNA, UR2 DNA was able to induce transformation of chicken embryo fibroblasts with a morphology similar to that of parental UR2 . UR2 -specific protein with kinase activity and UR2 -specific RNA were detected in the transfected cells. Transforming virus, UR2 ( UR2AV ), was produced from the doubly transfected cells. Five of the six UR2AV clones tested were also shown to be biologically active. The insert of the UR2 DNA clone is 3.4 kilobases in length and contains two copies of the long terminal repeat. Detailed restriction mapping showed that UR2 DNA shared with UR2AV DNA 0.8 kilobases of 5' sequence, including a portion of 5' gag, and 1.4 kilobases of 3' sequence, including a portion of 3' env. The UR2 transforming sequence, ros, is ca. 1.2 kilobases. No significant homology was found between v-ros and the conserved regions of v-src, v-yes, or v- abl . By contrast, a significant homology was found between v-ros and v-fps. The v-fps-related sequence was mapped within a 300-base-pair sequence in the middle of ros.     

Monoclonal antibodies to the transforming protein of Fujinami avian sarcoma virus discriminate between different fps-encoded proteins

Two monoclonal antibodies have been obtained that recognize antigenic determinants within the C-terminal fps-encoded region of P140gag-fps, the transforming protein of Fujinami avian sarcoma virus (FSV). The hybridomas which secrete these antibodies (termed 88AG and p26C) were isolated after the fusion of NS-1 mouse myeloma cells with B lymphocytes from Fischer rats that had been immunized with FSV-transformed rat-1 cells. FSV P140gag-fps immunoprecipitated by either antibody is active as a tyrosine-specific kinase and is able to autophosphorylate and to phosphorylate enolase in vitro. The fps-encoded proteins of all FSV variants, including the gag- p91fps protein of F36 virus, are recognized by both monoclonal antibodies. However, the product of the avian cellular c-fps gene. NCP98, and the transforming proteins of the recently isolated fps-containing avian sarcoma viruses 16L and UR1 are recognized only by the p26C antibody. The 88AG antibody therefore defines an epitope specific for FSV fps, whereas the epitope for p26C is conserved between cellular and viral fps proteins. The P105gag-fps protein of the PRCII virus is not precipitated by p26C (nor by 88AG), presumably as a consequence of the deletion of N-terminal fps sequences. These data indicate that the fps-encoded peptide sequences of 16L P142gag-fps and UR1 P150gag-fps are more closely related to NCP98 than that of FSV P140gag-fps. This supports the view that 16L and UR1 viruses represent recent retroviral acquisitions of the c-fps oncogene. The P85gag-fes transforming protein of Snyder-Theilen feline sarcoma virus is not precipitated by either monoclonal antibody but is recognized by some antisera from FSV tumor-bearing rats, demonstrating that fps-specific antigenic determinants are conserved in fes-encoded proteins.     

Genetic structure, transforming sequence, and gene product of avian sarcoma virus UR1

We analyzed the genetic structure and gene products of the newly isolated avian sarcoma virus UR1, which recently has been shown to be replication defective and to contain no sequences homologous to the src gene of Rous sarcoma virus. The sizes of the genomic RNAs of UR1 and its associated helper virus, UR1AV, were determined to be 29S and 35S (5.9 and 8.5 kilobases), respectively, by gel electrophoresis and sucrose gradient sedimentation. RNase T1 oligonucleotide mapping of purified viral RNAs indicated that UR1 RNA contains eight unique oligonucleotides in the middle of the genome and shares four 5'-terminal and three 3'-terminal oligonucleotides with UR1AV RNA. The unique sequences of UR1 and Fujinami sarcoma virus were found to be closely related to each other by molecular hybridization of UR1 RNA with DNA complementary to the unique sequence of Fujinami sarcoma virus RNA, but minor differences were found by oligonucleotides fingerprinting. In the regions flanking the unique sequences, UR1 and Fujinami sarcoma viral RNAs contain distinct oligonucleotides, which are shared with oligonucleotides of the respective helper viral RNAs. Cell transformed with UR1 produce a single 29S RNA species which contains a UR1 unique sequence; this species is most likely the mRNA coding for the transforming protein. In UR1-transformed cells, a phosphoprotein fo 150,000 daltons (p150) was detected by immunoprecipitation with antiserum against gag proteins. p150 was associated with a protein kinase activity that was capable of phosphorylating p150 itself, immunoglobulin G of antiserum, and a soluble substrate, alpha-casein. This enzyme transferred phosphate exclusively to tyrosine residues of substrates in vitro, but p 150 labeled in vivo with 32P contained both phosphoserine and phosphotyrosine. The in vitro kinase reaction was not affected by the presence of cyclic AMP or cyclic GMP and strongly preferred Mn2+ over Mg2+. Thus, the properties of UR1 protein are almost identical to those of Fujinami sarcoma virus protein.     

A novel oncogene, v-ryk, encoding a truncated receptor tyrosine kinase is transduced into the RPL30 virus without loss of viral sequences.

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 RPL30. While RPL25 and RPL28 were shown to contain the erbB oncogene, RPL30 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 P69gp37-ryk 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 RPL30-transformed cells were found to display a protein tyrosine kinase activity in vitro, and the levels of tyrosine-phosphorylated proteins are elevated in v-ryk-transformed cells.     

A membrane-associated, carbohydrate-modified form of the v-abl protein that cannot be phosphorylated in vivo or in vitro.

Abelson murine leukemia virus encodes a transforming protein which contains tyrosine kinase activity and is phosphorylated in vivo and in vitro. We found that P160 and P160-derived virus strains expressed an additional, altered v-abl protein which could not be phosphorylated. The altered v-abl protein (L-v-abl) differed from the phosphorylated form (K-v-abl) in that it was glycosylated and localized exclusively to the membrane fraction. Tunicamycin inhibition of N-linked carbohydrate addition did not restore phosphorylation. It did, however, reveal that L-v-abl had additional sequences relative to K-v-abl. The coding sequences required for this region and for the expression of L-v-abl were identified by replacing sequences in the P120 virus genome, which did not express L-v-abl, with sequences from the P160 virus genome. The necessary sequences were localized to the Moloney murine leukemia virus-derived gag gene. Comparison between the in vitro altered P120 and wild-type P120 virus strains indicated that expression of L-v-abl did not increase the efficiency of lymphoid transformation. Although the biological role of L-v-abl is not clear, our analyses have revealed that a specific amino terminal gag sequence can prevent v-abl from acting as a kinase substrate and can alter the cellular localization and modification of v-abl. These properties distinguish L-v-abl from previously reported v-abl proteins.     

Two point mutations in the transmembrane domain of P68gag-ros inactive its transforming activity and cause a delay in membrane association.

The transforming protein of the avian sarcoma virus UR2 is a 68-kDa transmembrane tyrosine protein kinase. We examined the relationship between membrane localization and transforming activity of P68 by changing Val-168-Val-169 in its hydrophobic domain into Asp-168-Glu-169. The resulting transmembrane (TM) mutant (P68TM) lost transforming activity toward chicken embryo fibroblasts (CEF). We found that the mutant protein was expressed and rapidly degraded into a smaller form which was still membrane associated and kinase active. The instability of the TM mutant protein is a phenomenon only manifested in CEF, because the same mutant protein was expressed with efficiency and stability similar to those of the wild-type protein in a transient expression system in COS cells. However, there are several differences between the wild-type and the TM mutant proteins in COS cells. The wild-type protein is more heavily phosphorylated and associated with membrane fractions in a cotranslational manner. It is enzymatically active when recovered from membrane fractions. The TM mutant protein is less phosphorylated, more labile toward protease degradation, and delayed in membrane association, with a lag period of 30 min or longer, and has little kinase activity when recovered from membrane fractions. Most of the kinase-active TM mutant protein was found in the cytosol fractions. Despite the delay, most of the TM protein in COS cells was found to be membrane associated, and its orientation on the cell surface was similar to that of the wild-type protein. It is probable that loss of the CEF-transforming activity of the TM mutant protein is due to its susceptibility to protease degradation resulting from improper membrane association of the newly synthesized product. The differences in the kinetics of membrane association and the distribution of kinase activity in COS cells might not be directly applicable in explaining the inability of the TM mutant to transform CEF but are intriguing as regards protein biosynthesis and translocation. The difference between CEF and COS cells implies that different factors or pathways are involved in the biosynthesis and processing of the TM mutant protein in these two cellular environments. Changes of P68TM in the kinetics of membrane association indicate that the transmembrane domain of ros, besides functioning as a membrane anchor, also plays a role in directing initial membrane association.     

Modulating effects of the extracellular sequence of the human insulinlike growth factor I receptor on its transforming and tumorigenic potential.

We reported previously that an N-terminally truncated insulinlike growth factor I receptor (IGFR) fused to avian sarcoma virus UR2 gag p19 had a greater transforming potential than did the native IGFR, but it failed to cause tumors in vivo. To investigate whether the 36 amino acids (aa) of the IGFR extracellular (EC) sequence in the gag-IGFR fusion protein encoded by the retrovirus UIGFR have a modulatory effect on the biological and biochemical properties of the protein, four mutants, NM1, NM2, NM3, and NM4 of the EC sequence were constructed. NM1 lacks the entire 36 aa residues; NM2 lacks the N-terminal 16 aa residues (aa 870 to 885), including two potential N-linked glycosylation sites of the EC sequence; NM3 contains a deletion of the C-terminal 20 aa residues (aa 886 to 905) of the EC sequence; and NM4 contains N-to-Q substitutions at both N-linked glycosylation sites. NM1 was the strongest of the four mutants in promoting anchorage-independent growth of transfected chicken embryo fibroblasts, while NM2 and NM4 had weaker transforming potential than did the original UIGFR virus. Only NM1 and NM3 were able to induce sarcomas in chickens. The four NM mutant-transformed cells expressed the expected proteins with comparable steady-state levels. The in vitro tyrosine kinase activity of P53NM1 was about fourfold higher than that of the parental P57-75UIGFR, whereas NM2 and NM4 proteins exhibited four- to fivefold-lower kinase activities. Despite lacking the IGFR EC sequence, P53NM1 formed covalent dimers similar to those formed by the parental P57-75UIGFR. Increased phosphatidylinositol (PI) 3-kinase activity was found to be associated with the mutant IGFR proteins. Among NM4 proteins. Elevated tyrosine phosphorylation of cellular proteins of 35, 120, 140, 160, and 170 kDa was detected in all mutant IGFR-transformed cells. We conclude that the EC 36-aa sequence of IGFR in the gag-IGFR fusion protein exerts intricate modulatory effects on the protein's transforming and tumorigenic potential. The 20 aa residues immediately upstream of the transmembrane domain have an inhibitory effect on the tumorigenic potential of gag-IGFR, whereas N-linked glycosylation within the EC sequence appears to have a positive effect on the transforming potential of UIGFR. Increased in vitro kinase activity and, to a lesser extent, in vivo tyrosine phosphorylation as well as the elevated association of PI 3-kinase activity with IGFR proteins seem to be correlated with the transforming potential of IGFR mutant proteins.     

Analysis of gag proteins from mouse mammary tumor virus.

Structural proteins designated p10gag, p21gag, p8gag, p3gag, p27gag, and p14gag from the C3H strain of mouse mammary tumor virus (MMTV) were purified by reversed-phase high-pressure liquid chromatography. The N- and C-terminal amino acid sequences and amino acid composition of each protein were determined and compared with the amino acids encoded by the proviral DNA sequences for the MMTV gag gene. The results show that each of the purified proteins is a proteolytic cleavage product derived from the predicted primary translational product of the gag gene (Pr77gag) and that their order in Pr77gag is p10-pp21-p8-p3-n-p27-p14 (where n represents 17 predicted residues that were not identified among the purified proteins). Purified p10gag lacks the initiator methionine and has a myristoyl group attached in amide linkage to the N-terminal glycine residue predicted by the second codon of the gag gene. The cleavage products are contiguous in the sequence of Pr77gag, and the C-terminal residue of p14gag is encoded by the last codon of the gag gene. By analogy with other retrovirus, p14gag is the viral nucleocapsid protein, p10gag is the matrix protein, and p27gag is the capsid protein of mature MMTV. Proteolytic cleavage sites in MMTV Pr77gag bear a striking resemblance to cleavage sites in the gag precursors of D-type retroviruses, suggesting that these viral proteases have similar specificities.     

Two autonomous myc oncogenes in avian carcinoma virus OK10.

The oncogenic avian retrovirus OK10 has the genetic structure gag-delta pol-myc-delta-env. The myc sequence is transduced from a cellular gene, proto-myc, while gag, pol, and env are essential retrovirus genes. By analogy with other directly oncogenic retroviruses, the specific myc sequence of OK10 is thought to be essential for transforming function. However, unlike the specific sequences of all other transforming retroviruses that encode unique transforming proteins, the myc sequence of OK10 encodes two potential transforming proteins, p58 and p200. p200 is translated from the gag-delta pol-myc region of genomic RNA, while p58 is thought to be translated from the gag leader and the open reading frame of myc via a subgenomic mRNA. In this paper, we ask whether both myc genes of OK10 are autonomous transforming genes. By differentially inactivating the p200 myc gene of OK10 provirus in vitro and analyzing transforming function in quail embryo cells, it was found that mutants expressing only p58 transformed like wild-type OK10. Further, it was shown that p58 with and without the gag leader had transforming function and that p58 of wild-type OK10 is initiated from the gag leader. Mutants expressing only p200 were also transforming but less efficiently than mutants that express only p58. A mutant OK10 virus in which the native frameshift of retroviruses between gag and pol was deleted expressed a shortened p200 (delta p200). Although this virus expressed more delta p200 than wild-type OK10 did, it transformed cells less efficiently. It follows that OK10 expresses two autonomous transforming genes, which is unique among retroviruses with onc genes.     

Molecular and biological properties of c-mil transducing retroviruses generated during passage of Rous-associated virus type 1 in chicken neuroretina cells.

IC1, IC2, and IC3 are novel c-mil transducing retroviruses generated during serial passaging of Rous-associated virus type 1 (RAV-1) in chicken embryo neuroretina cells. They were isolated by their ability to induce proliferation of these nondividing cells. IC2 and IC3 were generated during early passages of RAV-1 in neuroretina cells, whereas IC1 was isolated after six consecutive passages of virus supernatants. We sequenced the transduced genes and the mil-RAV-1 junctions of the three viruses. The 5' RAV-1-mil junction of IC2 and IC3 was formed by a splicing process between the RAV-1 leader sequence and exon 8 of the c-mil gene. The 5' end of IC1 resulted from homologous recombination between gag and mil sequences. Reconstitution experiments showed that serial passaging of IC2 in neuroretina cells also led to the formation of a gag-mil-containing retrovirus. Therefore, constitution of a U5-leader-delta c-mil-delta RAV-1-U3 virus represents early steps in c-mil transduction by RAV-1. This virus further recombined with RAV-1 to generate a gag-mil-containing virus. The three IC viruses transduced the serine/threonine kinase domain of the cellular gene. Hence, amino-terminal truncation is sufficient to activate the mitogenic property of c-mil. Comparison of the transforming properties of IC2 and IC1 showed that the transduced mil gene, expressed as a unique protein independent of gag sequences, was weakly transforming in avian cells. Acquisition of gag sequences by IC1 not only increased the rate of virus replication but also enhanced the transforming capacity of the virus.     

Protein stabilization explains the gag requirement for transformation of lymphoid cells by Abelson murine leukemia virus.

The single protein encoded by Abelson murine leukemia virus is a fusion of sequence from the retroviral gag genes with the v-abl sequence. Deletion of most of the gag region from the transforming protein results in a virus capable of transforming fibroblasts but no longer capable of transforming lymphoid cells. Smaller deletions in gag reveal that p15 gag sequences are responsible for this effect, whereas deletion of p12 sequences had no effect on lymphoid transformation. In transformed fibroblasts, p15-deleted and normal proteins had similar activities and subcellular localization. When the p15-deleted genome was introduced into previously transformed lymphoid lines, its protein product exhibited a marked instability. The tyrosine-specific autophosphorylation activity per cell was less than 1/20th that of the nondeleted protein. Although pulse-Ia-beling showed that the p15-deleted protein was synthesized efficiently, immunoblotting demonstrated that its steady-state level was less than 1/10th that of the nondeleted Abelson protein. The specific instability of the p15-deleted protein in lymphoid cells explains the requirement of these sequences for lymphoid but not fibroblast transformation.