Cell transformation by a virus containing a molecularly constructed gag-erbB fused gene.

A provirus DNA that contains a gag-erbB fused gene as the sole and transforming gene was molecularly constructed from plasmid pSRA2 containing the entire genome of Rous sarcoma virus and pAE7.7 containing the entire genome of an avian erythroblastosis virus (AEV), AEV-H. A virus containing the gag-erbB fused gene (GEV) was recovered from chicken embryo fibroblasts transfected with the proviral DNA and a helper virus DNA. GEV could transform chicken embryo fibroblasts as efficiently as could AEV-H. Anti-erbB and anti-gag sera immunoprecipitated a protein with a molecular weight of about 110,000 from GEV-transformed cells. The erbB and gag-erbB fused-gene products in AEV-H- and GEV-transformed cells were analyzed.     

Transfection by DNAs of avian erythroblastosis virus and avian myelocytomatosis virus strain MC29.

Chicken embryo fibroblasts and NIH 3T3 mouse cells were transformable by DNAs of chicken cells infected with avian myelocytomatosis virus strain MC29 or with avian erythroblastosis virus. Transfection of chicken cells appeared to require replication of MC29 or avian erythroblastosis virus in the presence of a nontransforming helper virus. In contrast, NIH 3T3 cells transformed by MC29 or avian erythroblastosis virus DNA contained only replication-defective transforming virus genomes.     

A myristylated form of the sea oncoprotein can transform chicken embryo fibroblasts

The transforming gene product of the S13 avian erythroblastosis virus, v-sea, is a member of the growth factor receptor class of tyrosine kinases. In the virus genome, the sea sequences are fused in frame to the virus env gene, thereby generating an abnormally large envelope protein because of the presence of a cytoplasmic tyrosine kinase domain. To determine what role these envelope sequences play in v-sea transformation, we generated a myristylated form of v-sea which contains no envelope sequences. In this report, we show that this myristylated sea-encoded protein retained the ability to transform chicken embryo fibroblasts, indicating that envelope sequences are not essential for transformation by the v-sea tyrosine kinase.     

Immune response and resistance to Rous sarcoma virus challenge of chickens immunized with cell-associated glycoproteins provided with a recombinant avian leukosis virus.

The Rous-associated virus 1 env gene, which encodes the envelope gp85 and gp37 glycoproteins, was isolated and inserted in place of the v-erbB oncogene into an avian erythroblastosis virus-based vector, carrying the neo resistance gene substituted for the v-erbA oncogene, to generate the pNEA recombinant vector. A helper-free virus stock of the pNEA vector was produced on an avian transcomplementing cell line and used to infect primary chicken embryo fibroblasts (CEFs) or quail QT6 cells. These infected cells, selected with G418 (CEF/NEA and QT6/NEA, respectively) were found to be resistant to superinfections with subgroup A retroviruses. The CEF/NEA preparations were used as a cell-associated antigen to inoculate adult chickens by the intravenous route compared with direct inoculations of NEA recombinant helper-free virus used as a cell-free antigen. Chickens injected with the cell-associated antigen (CEF/NEA) exhibited an immune response demonstrated by induction of high titers of neutralizing antibodies and were found to be protected against tumor production after Rous sarcoma virus A challenge. Conversely, no immune response and no protection against Rous sarcoma virus A challenge were observed in chickens directly inoculated with cell-free NEA recombinant virus or in sham-inoculated chickens.     

Characterization of reticuloendotheliosis virus strain T DNA and isolation of a novel variant of reticuloendotheliosis virus strain T by molecular cloning.

Reticuloendotheliosis virus strain T (REV-T) is a highly oncogenic avian retrovirus which causes a rapid neoplastic disease of the lymphoreticular system. Upon infection, this virus gives rise to two species of unintegrated linear viral DNA, which are 8.3 and 5.5 kilobase pairs long and represent the helper virus (REV-A) and the oncogenic component (REV-T), respectively. Restriction endonuclease cleavage maps of these two DNA components indicate that REV-T DNA has a large portion of the genome deleted with respect to REV-A DNA and a substitution about 0.8 to 1.5 kilobase pairs long that is unrelated to REV-A DNA. These additional sequences comprise the putative transforming region of REV-T (rel). A chicken spleen cell line transformed by REV-T produced virus which upon infection gives rise to three species of unintegrated linear viral DNA (8.3, 5.5, and 3,3 kilobase pairs). We isolated the proviruses of the 8.3- and 3.3-kilobase pair species from this cell line by cloning in the phage vector Charon 4A. Restriction enzyme mapping showed that the two proviral clones are proviruses of REV-A and a variant of REV-T, respectively. A subclone of the variant REV-T provirus specific for the rel sequences of REV-T was used as a hybridization probe to demonstrate that the rel sequences are different from the putative transforming sequences of Schmidt-Ruppin Rous sarcoma virus strain A, avain myelocytomatosis virus, avian myeloblastosis virus, avian erythroblastosis virus, Abelson murine leukemia virus, and Friend erythroleukemia virus. In addition, the rel-specific hybridization probe was used to identify a specific set of sequences which are present in uninfected avian DNAs digested with several restriction enzymes. The corresponding cell sequences are not arranged like rel in REV-T.     

Generation of transforming viruses in cultures of chicken fibroblasts infected with an avian leukosis virus.

During serial passages of an avian leukosis virus (the transformation-defective, src deletion mutant of Bratislava 77 avian sarcoma virus, designated tdB77) in chicken embryo fibroblasts, viruses which transformed chicken embryo fibroblasts in vitro emerged. Chicken embryo fibroblasts infected with these viruses (SK770 and Sk780) had a distinctive morphology, formed foci in monolayer cultures, and grew independent of anchorage in semisolid agar. Bone marrow cells were not transformed by these viruses. Another virus (SK790) with similar properties emerged during serial subcultures of chicken embryo fibroblasts after a single infection with tdB77. The 50S to RNAs isolated from these viruses contained a tdB77-sized genome (7.6 kilobases), 8.7- and 5.7-kilobase RNAs, and either a 4.1-kilobase RNA or a 4.6-kilobase RNA. These RNAs did not hybridize with cDNA's representing the src, erb, mac, and myb genes of avian acute transforming viruses. Cells transformed by any one of the Sk viruses (SK770, SK780, or SK790) synthesized two novel gag-related polyproteins having molecular weights of 110,000 (p110) and 125,000 (p125). We investigated the compositions of these proteins with monospecific antiviral protein sera. We found that p110 was a gag-pol fusion protein which contained antigenic determinants, leaving 49,000 daltons which was antigenically unrelated to the structural and replicative proteins of avian leukosis viruses. An analysis of the SK viral RNAs with specific DNA probes indicated that the 5.7-kilobase RNA contained gag sequences but lacked pol sequences and, therefore, probably encoded p125. The transforming ability, the deleted genome, and the induced polyproteins of the SK viruses were reminiscent of the properties of several replication-defective acute transforming viruses.     

Protein phosphorylation at tyrosine is induced by the v-erbB gene product in vivo and in vitro

The v-erbB gene product of avian erythroblastosis virus (AEV) has extensive homology with the receptor for epidermal growth factor (EGF). We report here that chicken embryo fibroblasts (CEF) transformed by AEV show enhanced tyrosine phosphorylation of a number of cellular polypeptides, including the 36 kd protein, which is phosphorylated in avian sarcoma virus-transformed fibroblasts, and the 42 kd protein, which is phosphorylated in mitogen-stimulated cells. CEF infected by AEV mutants with deletions in v-erbA showed enhanced tyrosine phosphorylation, whereas CEF infected by mutants with deletions in v-erbB did not. When membranes from AEV-transformed cells were incubated with gamma-32P-ATP, both the v-erbB gene product and the 36 kd cellular protein became phosphorylated at tyrosine. These results indicate that the v-erbB protein induces tyrosine phosphorylation in vivo and in vitro, and suggest that, like the EGF receptor, it possesses tyrosine-specific protein kinase activity.     

Development of avian sarcoma and leukosis virus-based vector-packaging cell lines.

We have constructed an avian leukosis virus derivative with a 5' deletion extending from within the tRNA primer binding site to a SacI site in the leader region. Our aim was to remove cis-acting replicative and/or encapsidation sequences and to use this derivative, RAV-1 psi-, to develop vector-packaging cell lines. We show that RAV-1 psi- can be stably expressed in the quail cell line QT6 and chicken embryo fibroblasts and that it is completely replication deficient in both cell types. Moreover, we have demonstrated that QT6-derived lines expressing RAV-1 psi- can efficiently package four structurally different replication-defective v-src expression vectors into infectious virus, with very low or undetectable helper virus release. These RAV-1 psi--expressing cell lines comprise the first prototype avian sarcoma and leukosis virus-based vector-packaging system. The construction of our vectors has also shown us that a sequence present within gag, thought to facilitate virus packaging, is not necessary for efficient vector expression and high virus production. We show that quantitation and characterization of replication-defective viruses can be achieved with a sensitive immunocytochemical procedure, presenting an alternative to internal selectable vector markers.     

A chimeric avian retrovirus containing the influenza virus hemagglutinin gene has an expanded host range.

We have investigated what protein sequences are necessary for glycoprotein incorporation into Rous sarcoma virus (RSV) virions by utilizing the hemagglutinin (HA) protein of influenza virus. Two chimeric HA genes were constructed. In the first the coding sequence for the signal peptide of the RSV env gene product was fused in frame to the entire HA structural gene, and in the second the hydrophobic anchor and cytoplasmic domain sequences of the HA gene were also replaced with those from the RSV env gene. Both chimeric genes, expressed from a simian virus 40 expression vector in CV-1 cells, yielded functional HA proteins that were transported to the cell surface and were able to bind to erythrocytes. When the genes were expressed in combination with the RSV gag-pol gene region in QT6 cells by using a vaccinia virus-T7 expression/complementation system, virions that efficiently incorporated either chimeric protein were assembled. This result indicated that the presence of the RSV env membrane anchor and cytoplasmic sequences did not facilitate HA glycoprotein incorporation into virions. The presence of the RSV env signal sequence allowed the chimeric HA genes to be substituted into the RSV-derived BH-RCAN.HiSV viral genome in place of the RSV env gene. Both chimeric genomes yielded infectious virus that could infect human and avian cells with equal efficiency. These experiments demonstrate that a foreign glycoprotein, efficiently incorporated into virions lacking a native glycoprotein, can confer a broadened host range on the virus. Moreover, because the HA of influenza virus requires the acidic pH of the endosome in order to be activated, these results imply that foreign proteins can modify the normal route of entry of this avian retrovirus.     

Cooperation of v-jun and v-erbB oncogenes in embryo fibroblast transformation in vitro and in vivo.

Retroviral vectors carrying either the v-jun and v-erbB sequences or the v-jun gene linked to the neomycin resistance gene were constructed on the basis of the structural genome organization of avian erythroblastosis virus (AEV). These viruses, called JB and JN, respectively, were rescued as Rous-associated virus-1 pseudotypes, and they were shown to successfully transform chicken embryo fibroblasts in vitro. However, in agar, colonies developed from JB-infected fibroblasts were three to five times larger than those obtained after infection with JN or with AEV Pst124 carrying only a functional v-erbB gene. In vivo, on chorioallantoic membrane (CAM) assays, JB produced fibrosarcomas that were more rapidly growing and much larger than those induced by JN or AEV Pst124. Moreover, in chickens infected in ovo with JB, multiple fibrosarcomas arose in different organs a few days after birth, whereas no tumor could be detected in parallel experiments in either JN- or AEV Pst124-infected animals. These results demonstrate that in embryo fibroblast cells, v-jun and v-erbB can act synergistically to enhance the transformation potential of either oncogene alone both in vitro and in vivo.     

The family of env genes of avian retroviruses: molecular analysis of Rous sarcoma virus adapted to duck cells

For the elucidation of the molecular basis of RSV adaptation to conditionally permissive host from the genome library of duck embryo fibroblasts, transformed by Rous sarcoma virus in 30 passages on these cells, recombinant bacteriophages that include provirus sequences, were obtained. Complete and transformation-defective proviruses were characterized, nucleotide sequences of their env-genes were compared with their counterparts the original RSV (Pr-RSV-C) and with viruses of other subgroups (A, B, D and E). The possible relation of the revealed changes in domains coding gp85 and gp37, with the changes of chicken RSV characteristics during adaptation to duck cells is discussed.     

Marker rescue of endogenous cellular genetic information related to the avian leukosis virus gene encoding RNA-directed DNA polymerase.

Endogenous cellular genetic information related to the avian leukosis virus gene encoding RNA-directed DNA polymerase was studied, using a marker rescue assay to detect biological activity of subgenomic fragments of virus-related DNAs of uninfected avian cells. Recipient cultures of chicken embryo fibroblasts were treated with sonicated DNA fragments and were infected with a temperature-sensitive mutant of Rous sarcoma virus that encoded a thermolabile DNA polymerase. Wild-type progeny viruses were isolated by marker rescue with fragments of DNA of uninfected chicken, pheasant, quail, and turkey cells. The DNAs of these uninfected avian cells, therefore, appeared to contain endogenous genetic information related to the avian leukosis virus DNA polymerase gene.     

Expression of the v-erbA oncogene in chicken embryo fibroblasts stimulates their proliferation in vitro and enhances tumor growth in vivo

In contrast to uninfected chicken embryo fibroblasts (CEFs), CEFs infected with a retroviral vector that carries the v-erbA gene of avian erythroblastosis virus displayed new properties. These included limited anchorage-independent growth in soft agar, growth without latency in serum-supplemented medium, ability to overcome quiescence induced by serum deprivation, growth at low cell density, and an extended life span in vitro. Furthermore, when explanted in vivo onto the chorioallantoic membrane of chicken embryo, the transformed CEFs expressing v-erbA in addition to v-erbB exhibited a high proliferative rate, giving rise to fibrosarcoma tumors that were ten times larger than those developed from transformed CEFs expressing v-erbB alone. All these data show that CEFs expressing the v-erbA oncogene display activated growth and suggest that the v-erbA product interferes with the mechanisms regulating the growth and/or differentiation of primary CEFs.     

Characterization of c-lil, a chicken cellular sequence associated with a stock of B77 avian sarcoma virus.

Using biochemical methods, we have shown that a new specific sequence, v-lil, is associated with a given stock of B77 avian sarcoma virus (clone 9). We prepared a DNA complementary to v-lil sequences, using substractive hybridizations, and investigated the properties of this sequence. v-lil has a genetic complexity of ca. 2,000 nucleotides and is not present in various stocks of avian sarcoma virus, avian leukosis virus, or defective leukemia virus. v-lil is not associated with B77 avian sarcoma virus isolated from the original tumor and thus has been acquired by in vitro passage of the virus on chicken embryo fibroblasts. A search for the origin of the v-lil sequence among the DNAs of different avian species has shown that a similar sequence, c-lil, is present in normal chicken DNA (1 to 2 copies per haploid genome). c-lil is not highly conserved but is present in the DNA of all chickens from the genus Gallus. The c-lil sequence is transcribed at a low level (1 to 3 copies per cell) in normal chicken embryo fibroblasts. The biological function, if any, of v-lil or its cellular equivalent has yet to be determined.     

A single amino acid substitution in v-erbB confers a thermolabile phenotype to ts167 avian erythroblastosis virus-transformed erythroid cells.

A library of recombinant bacteriophage was prepared from ts167 avian erythroblastosis virus-transformed erythroid precursor cells (HD6), and integrated proviruses from three distinct genomic loci were isolated. A subclone of one of these proviruses (pAEV1) was shown to confer temperature-sensitive release from transformation of erythroid precursor cells in vitro. The predicted amino acid sequence of the v-erbB polypeptide from the mutant had a single amino acid change when compared with the wild-type parental virus. When the wild-type amino acid was introduced into the temperature-sensitive avian erythroblastosis virus provirus in pAEV1, all erythroid clones produced in vitro were phenotypically wild type. The mutation is a change from a histidine to an aspartic acid in the temperature-sensitive v-erbB polypeptide. It is located in the center of the tyrosine-specific protein kinase domain and corresponds to amino acid position 826 of the human epidermal growth factor receptor sequence.     

Nuclear location of the putative transforming protein of avian myelocytomatosis virus

The putative transforming protein of avian myelocytomatosis virus MC29 is a 110,000 dalton (P110gag-myc) polyprotein comprised of sequences derived from both the gag region and the MC29-specific myc region. Two approaches have been taken to determine the location of the MC29 gag-related proteins in transformed cells: subcellular fractionation and immunofluorescence. Analysis of subcellular fractions of MC29-transformed cells by immunoprecipitation indicates that the majority of the gag-myc polyprotein is found in the nuclear fractions of Q8 cells (a nonproducer line of MC29-transformed quail embryo fibroblasts) and nonproducer cells derived from a liver tumor of MC20-infected quail. This is in contrast to the distribution of gag-related helper virus proteins lacking myc, which are found only in nonnuclear fractions of superinfected Q8 cells. The purity of unlabeled nuclei was assessed by electron microscopy and enzyme assays, revealing little contaminating material from other subcellular fractions. Immunofluorescence experiments using monospecific anti-gag serum showed specific, intense immunofluorescence in the nuclei of fixed Q8 cells. In contrast, the majority of P75gag-erb, a candidate transforming protein produced by avian erythroblastosis virus (AEV), is absent from the nuclei of nonproducer AEV-transformed chick embryo fibroblasts. The nuclear association of the MC29 transforming protein may be related to some of the unique properties of MC29-transformed cells.