Leukemogenicity of avian oncovirus S13

Avian oncovirus S13 induces erythroblastic and granulocytic leukemias in line 6 and Spafas chickens. It also causes anemia, sarcomas, and endothelial proliferation. The leukemic cells contain the transformation-specific protein of S13, gp155.     

Avian retrovirus S13: properties of the genome and of the transformation-specific protein

The avian retrovirus S13 codes for an env-linked transformation-specific glycoprotein with a molecular weight of 155,000 (gp155). Treatment of gp155 with endoglycosidase H or growth of S13-infected cells in the presence of tunicamycin reduces the molecular weight of gp155 to about 140K, but these gp155-related molecules may still contain sugar residues. The gp155 protein is not incorporated into virions; it is phosphorylated, but in immunoprecipitates does not show protein kinase activity. The genome of S13 is an 8.5-kilobase (kb) RNA; the helper virus genome is 7.5 kb in size. The putative onc sequences of S13 do not hybridize to DNA probes representing src, erb A, erb B, myc, myb, fps, fms, H-ras, B-lym, abl, rel, and ets.     

Requirement of the avian retrovirus pp32 DNA binding protein domain for replication

The NH2-terminal amino acid sequence of the pp32 DNA binding protein has been determined, thus establishing its precise coding region in the polymerase gene of Rous sarcoma virus. Specific mutations were constructed in molecularly cloned Prague A DNA near the NH2- and COOH-termini of pp32 and the effects were assayed by transfection on chick embryo fibroblasts. Out-of-frame deletions at both sites and an in-frame deletion near the NH2 terminus rendered the DNA noninfectious and transformation negative. Single point mutations near the NH2 terminus reduced the transfection efficiency and the rate of virus replication. Biochemical studies indicated that the RNA-directed DNA polymerase and RNase H activities of the mutant viruses were not affected but the processing of the viral beta polypeptide was altered.     

The involvement of a type-B retrovirus in the induction of thymic lymphomas

A highly leukemogenic virus (DMBA-LV) (in vivo leukemogenic titer 1-5 X 10(6) IU/ml, and 35-40 days to thymic lymphoma detection) is produced by a chemical carcinogen-induced transplanted thymic lymphoma. The virus preparation is a mixture of a type-B retrovirus highly related to exogenous type-B retroviral isolates and a biologically defective type-C retrovirus. The DNA of DMBA-LV-induced-tumors contains new type-B proviruses but no additional type-C proviruses could be detected. The leukemogenicity of DMBA-LV was completely neutralized by a monoclonal antibody against MMTV envelope glycoprotein, but was not affected by a broadly reacting Friend MuLV anti-gp70 serum which effectively neutralizes type-C ecotropic, xenotropic, and recombinant retroviruses and which completely abolishes the leukemogenic activity of Moloney leukemia virus. Three type-B mammary tumor-inducing retroviral isolates, while containing type-C retroviral sequences, were not leukemogenic. A further characterization of the type-C retroviral sequences present in DMBA-LV indicated that sequences characteristic of endogenous, nonxenotropic proviruses are present. In addition, using a variety of type-C-specific retroviral DNA probes, no evidence was obtained for the presence of a type-B-C-recombinant genome in DMBA-LV. Leukemogenesis was absolutely dependent upon the presence of a functional type-B retroviral envelope gp 52 and DMBA-LV does not appear to contain a leukemogenic retroviral type-C genome.     

Complete env gene deletions of three replication-defective strains of Rous sarcoma virus and a model for the origin of their genetic structures

Replication-defective deletion mutants of Rous sarcoma virus (RSV) have been described which transform cells in culture and elaborate envelope (-) defective particles. The env deletions of two clonal variants of the Bryan strain of RSV, RSV(-)3, and RSV(-)16, and of a replication-defective variant of Schmidt-Ruppin RSV (SRN8) were analyzed by fingerprinting oligonucleotides hybridized by a molecularly cloned env DNA probe that spans from near the 3' end of pol to the 3' end of env. It was observed that all three replication-defective RSV strains are essentially complete env deletions but retain the 3' end of pol. Based on a common pol-src junction oligonucleotide that may reflect a homologous sequence repeated at both ends of env in nondefective RSV, the env deletions of RSV(-)3 and 16 appear to be isogenic. The original deletion may have involved recombination between these sequences. The absence of this oligonucleotide in SRN8 indicates that the env deletion of SRN8 has different borders and represents an independent env deletion of nondefective RSV. All three defective RSVs have the genetic structure gag-pol-src. This genetic structure is consistent with the need for a complete gag to make a particle and with the assumption that an independent src gene rather than a gag- or gag-pol-src hybrid gene functions in transformation. It is suggested that a complete pol is not necessary for, but may assist, virus particle formation.     

Identification and biochemical analysis of DNA replication-defective large T antigens from SV40-transformed cells

Nine commonly studied Simian virus 40 (SV40)-transformed rodent cell lines were screened for tumor (T) antigens defective in SV40 DNA replication using a simple polyethylene glycol-mediated cell fusion assay. Each line contained a functional origin of SV40 DNA replication, as shown by fusion with Cos 1 cells. Fusion with uninfected monkey cells revealed that T antigens from two lines lacked detectable replicative activity, while T antigens from five other lines exhibited only very weak replicative activity. One line, and a tumor cell line derived from it, expressed T antigen with wild-type replication activity. Biochemical analysis of these proteins revealed defects in DNA binding activity and ATPase activity. One line expressed large T antigen defective in both activities. All of the lines contained complexes of T antigen with the cellular protein p53 and all of the T antigens exhibited nucleotide-binding activity. The results indicate that some of these lines may constitute a useful source of new replication-defective T antigens.     

Subcellular localization of the v-erb-B protein,the product of a transforming gene of avian erythroblastosis virus

Avian erythroblastosis virus (AEV) is an oncogenic retrovirus capable of transforming both fibroblasts and immature erythroid cells. The v-erb-B locus within the AEV genome encodes a glycosylated protein, expression of which is required for oncogenic transformation of either cell type. Subcellular localization of the v-erb-B glycoprotein in AEV-transformed cells is reported here. Results indicate that the v-erb-B protein is synthesized on dense membrane fractions and appears to possess the properties of an integral membrane protein. The bulk of the v-erb-B protein remains with dense membranes after synthesis, although a small quantity may slowly become associated with the plasma membrane. The biogenesis and subcellular location of the v-erb-B protein are thus quite different from those of the transforming proteins that display protein kinase activity. These differences are especially provocative because the amino acid sequences of the v-erb-B protein and the protein kinases are closely related to one another.     

Defective cleavage of a precursor polypeptide in a temperature-sensitive mutant of avian sarcoma virus.

Noninfectious virus particles released at the nonpermissive temperature from cells infected with the temperature-sensitive mutant LA334 of avian sarcoma virus B77 have been investigated. These particles bud atypically, have improperly formed core structures, are heterogeneous in size and on a density gradient, and are also slightly more dense than wild-type virus. One major new viral-coded protein of 23,000 molecular weight, p23, and several minor abnormally cleaved and/or partially degraded viral-specific peptides are incorporated into the noninfectious particles. As a result of tryptic peptide analyses of some of these new proteins, it could be proposed that the defect of this mutant is in the cleavage of pr76, the 76,000 molecular weight precursor for the internal viral proteins of avian tumor viruses (ATV). In addition, we have detected one methionine-labeled viral tryptic peptide with an altered amino acid composition, which may represent the site of the temperature sensitive lesion.     

Molecular and biological properties of MH2D12, a spontaneous mil deletion mutant of avian oncovirus MH2

Avian oncogenic retrovirus MH2 carries two cell-derived oncogenes, v-mil and v-myc. From an infectious stock of MH2 a spontaneous deletion mutant, MH2D12, that has lost most of the v-mil gene but has retained a complete and functional v-myc gene, has been isolated. Nonproducer quail embryo cells transformed by MH2D12 in the absence of helper virus contain two virus-specific proteins: a gag-related protein of 53,000 Da (p53gag), and a v-myc gene product of 59,000/61,000 Da (p59/61v-myc) indistinguishable from the v-myc protein encoded by MH2. MH2D12 viral RNA contains all T1-oligonucleotides specific for the MH2 v-myc gene but none of those characteristic for the v-mil gene. The genetic structure of molecularly cloned proviral DNA of MH2D12 was revealed by restriction mapping, blot hybridization, heteroduplex analysis, and nucleotide sequencing. The MH2D12 provirus is homologous to the MH2 genome but has suffered a deletion of 1271 nucleotides from the central region encompassing the 3' end of delta gag and all of v-mil except the very 3' 31 nucleotides directly adjacent to the v-myc gene. A nine-nucleotide overlap of homology to gag or mil at the delta gag/delta mil junction suggests that recombination between homologous sequence elements of the delta gag and v-mil domains of MH2 was involved in the genesis of MH2D12. The nucleotide sequence analysis predicts that the carboxyterminal 17 amino acids of p53gag are encoded by the residual v-mil sequences and by intron-derived v-myc sequences. Transformation of quail embryo cells by MH2D12 can be assayed by focus and colony formation of transformed cells. This indicates that the v-mil gene is not essential for these activities. However, size and morphology of foci and colonies, and cellular morphology of cultured MH2D12-transformed cell lines can easily be distinguished from those observed in cell transformation by MH2 and resemble more those seen in cell transformation by viruses containing the myc oncogene only.     

Characterization of two strains of avian sarcoma virus isolated from avian lymphatic leukosis virus-induced sarcomas

Two replication-defective avian sarcoma viruses, S1 and S2, which were independently isolated from tumors of chickens inoculated with avian lymphatic leukosis virus (LLV) were characterized. The genomes of S1 and S2 contain src-related sequences and are, respectively, about 3.9 and 4.5 kilobases long. pp60src-related proteins with molecular weights of 62,000 (p62) were detected in cells infected with these viruses, and protein kinase activity was found to be associated with these proteins. No other viral proteins, such as gag, pol, and env gene products, were detected. These results suggested that the c-src sequence in normal chicken cells was incorporated into LLV genomes by recombination at the expense of most of the viral genes to generate highly defective new sarcoma viruses.     

Isolation of three new avian sarcoma viruses: ASV 9, ASV 17, and ASV 25

The newly isolated avian sarcoma viruses, ASV 9, 17, and 25, cause fibrosarcomas in young chickens and induce foci of transformed cells in chick embryo fibroblast cultures. They are defective in replication and belong to envelope subgroup A. The sizes of their genomes are 6 kb (ASV 9), 5 kb (ASV 17), and 6 kb (ASV 25), respectively. All three contain long terminal repeat (LTR) and gag sequences but lack pol. env is absent from ASV 9 and ASV 25, but some env sequences are detectable in ASV 17. None of the defective viral genomes hybridized to selected onc probes representing src, fps, yes, myc, myb, and erb A. erb B appears absent from ASV 9 and ASV 17, but some hybridization between the erb B probe and the RNA of ASV 25 was detected. ASV 9 codes for a transformation-specific gag-linked protein of 130kDa. Multiple gag-linked transformation-specific proteins are seen in ASV 17 and 25; they require further study.     

The defective maturation of viral progeny with a temperature-sensitive mutant of avian sarcoma virus.

LA334 (previously ts 75) has been identified as a mutant exhibiting two genetic lesions affecting late functions of the avian sarcoma virus genome. One causes the transformed phenotype to be temperature sensitive, and the other induces a rapidly reversible inhibition of progeny virus production at the nonpermissive temperature. The nature of the latter defect has been investigated in this study with the following findings: (1) no new protein synthesis was needed to initiate synthesis of infectious virus after a shift from the nonpermissive to the permissive temperature; (2) significant amounts of physical particles (20 to 70% of that produced at the permissive temperature) were produced at the nonpermissive temperature; and (3) intracellular accumulation of structures resembling budding virus, approximately 80% of which were aberrant, were observed in mutant-infected cells at the nonpermissive temperature.The possibility that a defect in the synthesis of the viral envelope glycoprotein, gp85, would account for these findings prompted an investigation of the expression of gp85 in LA334-infected cells. Intracellular synthesis of gp85 in normal amounts was shown by immunoprecipitation; however, only inefficient interference against superinfecting virus could be observed with mutant-infected cells at the nonpermissive temperature. Immunoferritin electron microscopic observations also indicated the presence of envelope glycoprotein in association with budding virions of normal morphology, but in contrast, little or no glycoprotein was associated with the budding structures of aberrant morphology.It is concluded that the primary virus defect is not related to expression of gp85; rather, on the basis of the abnormal core assembly, it is proposed that a defect exists in one of the core structural proteins. This hypothesis is discussed in the light of known properties of the mutant and of certain recent observations on the composition of the noninfectious virus produced by infected cells at the nonpermissive temperature.     

Temperature-sensitive mutants of avian sarcoma viruses: genetic recombination between multiple or coordinate mutants and avian leukosis viruses.

Five coordinate temperature-sensitive mutants of avian sarcoma viruses which fail to transform or produce infectious progeny at 41° have been analyzed by genetic recombination. Four, namely LA334, 336, 338, and 343, carry multiple mutations. One of these mutations is always in the src gene affecting initiation and maintenance of transformation. The other mutations have not been mapped, but our data suggest that in LA338 there is no linkage of the second mutation with env, whereas in LA343 there is some linkage to env. LA336 has a second mutation affecting an early transient function in accordance with physiological data which have shown a thermolabile polymerase in this virus. The data on LA334 are in accord with previous studies which have indicated lesions in src and gag. For LA337 our data confirm the existence of single coordinate lesion segregating from env.     

Endogenous leukosis viruses in the avian family Phasianidae.

Helper virus. activity for the defective Bryan high titer strain of Rous sarcoma virus (RSV) has been found in normal embryo fibroblast cultures of Ghighi, green, Mongolian, silver, and Swinhoe pheasants, and of Chinese quail and chukar. The helper viruses from Chinese quail and from Swinhoe pheasant were isolated from their respective RSV pseudotypes. Chinese quail, Ghighi, and golden pheasant cultures were also found to synthesize endogenous virus spontaneously. No such spontaneous production was seen with the other avian species investigated. The helper activity demonstrated in Chinese quail, chukar, Mongolian pheasant, and Swinhoe pheasant appears to specify envelope determinants which are different from those previously described. Viruses from Ghighi and silver pheasant have the G envelope, and those from green pheasant the F envelope specificity. The RSV pseudotype formed with Chinese quail virus shows a high plating efficiency on fibroblasts from mouse and European field vole. Chinese quail virus and Swinhoe pheasant virus also form plaques in chicken cells. Whether these pheasant viruses belong to established avian oncovirus groups, or whether they represent new ones, will have to be decided by immunological and biochemical studies now in progress.     

A mutation at the major phosphotyrosine in pp60v-src alters oncogenic potential

Previously (M.A. Snyder, J.M. Bishop, W.W. Colby, and A.D. Levinson, 1983, Cell 32, 891-901) a mutant was constructed in v-src in which the major phosphotyrosine site, tyr-416, was converted to phenylalanine. This mutant has now been examined both for tumorigenicity and a number of in vitro parameters relating to the transformed state and to the known properties of pp60v-src, the product of v-src. Mouse cells transformed by this mutant gene, which are called RSV-SF1, are tumorigenic only if tested in immunodeficient mice, whereas cells transformed by the wild-type parent are tumorigenic in either syngeneic or immunodeficient animals. When examined in vitro, RSV-SF1-transformed cells are virtually indistinguishable from cells transformed by wild-type pp60v-src. These findings raise the possibility that the protein kinase activity of pp60v-src may not be fully responsible for tumorigenesis by v-src, and moreover suggest that evasion of the host immune response is a necessary step in tumorigenesis by v-src.     

Cell lines derived from avian lymphomas exhibit two distinct phenotypes

Lymphoid cell lines were derived from three avian leukosis virus (ALV)-induced lymphomas. These cell lines contained proviral DNA sequences integrated upstream from the c-myc proto-oncogene, expressed increased levels of c-myc RNA, and were tumorigenic in syngeneic animals. While cell surface immunoglobulin (IgM) was expressed by all three cell lines, only one of the lines secreted IgM into the culture medium. Further, analysis by light microscopy and flow cytometry demonstrated that these cell lines exhibited two distinct morphological and light-scattering profiles. The two nonsecreting lines exhibited a lymphoblastoid phenotype, whereas, the secreting line possessed a more differentiated plasmacytoid phenotype. These findings implicate the activation of c-myc in the pathogenesis of tumors representing two distinct stages of B-cell differentiation within a single animal species.     

Sequence homology of the simian retrovirus genome with human T-cell leukemia virus type I

A retrovirus highly related to human T-cell leukemia virus type I (HTLV-I) was isolated from a T-cell line established from a seropositive pig-tailed monkey and the provirus genome was molecularly cloned using HTLV-I as a probe. The monkey virus (STLV) had the genomic structure of the LTR-gag-pol-env-pX-LTR. Analysis of the env-pX-LTR region revealed the 90% homology of the nucleotide sequence with that of HTLV-I in each region. This high homology of the sequence indicates that STLV is a member of the HTLV family, but apparently different from HTLV-I. This suggests that the possibility of recent interspecies transmission from monkeys to humans in the endemic area is very small. From its similarity to HTLV, STLV should be useful as an animal model in studies on natural HTLV infection and leukemogenesis of HTLV in humans.