Avian Oncoviruses Research Articles

Expression analysis of programmed death ligand 2 in tumors caused by the avian oncovirus Marek’s disease virus

PD-L2 is a ligand of the immunoinhibitory receptor PD-1. Here, we report functional and expression analyses of PD-L2 in tumor lesions and spleens from chickens infected with gallid herpesvirus 2 (GaHV-2, Marek's disease virus), which induces malignant lymphomas in chickens. We show that the expression of IFN-γ protein was decreased in PBMCs and splenocytes co-cultured with PD-L2-expressing cells and that the expression of PD-L2 mRNA was significantly higher in the spleens of infected chickens in the latent phase and in tumor lesions caused by GaHV-2. These results suggest that chicken PD-L2 has an immunoinhibitory function and is involved in the establishment of latency and tumor formation by GaHV-2.

Reflections on the pathogenesis of diseases caused by the acute avian leukosis/sarcoma viruses with special reference to avian erythroblastosis.

The various diseases that follow experimental infection with the acute and non-acute avian oncoviruses are discussed with special reference to the pathogenesis of avian erythroblastosis. One view, based on in vitro studies, sees erythroblastosis as the product of a failure in the differentiation of virus-infected stem cells to mature erythrocytes, as a result of cell 'transformation'. The results of some in vivo studies, however, point to a resemblance of the disease to a haemolytic anaemia, where cellular death is an important component. It seems probable that the disease is the result of transformation of cells of the erythroblastic series followed by the death of many of these cells due to influences that have not yet been determined. Determination of the causes of this cellular death may prove to be as important for our understanding of the problem of leukaemia as the work that has already been accomplished in explaining the causes of cell transformation. It is also suggested that the tendency of gs amino acid sequences of the avian leukosis viruses and mouse leukaemia viruses to form fusion proteins with a variety of proto-oncogenes may be part of a wider phenomenon, and that these sequences may fuse with other proteins, altering their properties. More work is required on the possibility that there is an undiscovered immunological component in the progression of the L/S diseases.

Structure and transforming function of transduced mutant alleles of the chicken c-myc gene.

A small retroviral vector carrying an oncogenic myc allele was isolated as a spontaneous variant (MH2E21) of avian oncovirus MH2. The MH2E21 genome, measuring only 2.3 kilobases, can be replicated like larger retroviral genomes and hence contains all cis-acting sequence elements essential for encapsidation and reverse transcription of retroviral RNA or for integration and transcription of proviral DNA. The MH2E21 genome contains 5' and 3' noncoding retroviral vector elements and a coding region comprising the first six codons of the viral gag gene and 417 v-myc codons. The gag-myc junction corresponds precisely to the presumed splice junction on subgenomic MH2 v-myc mRNA, the possible origin of MH2E21. Among the v-myc codons, the first 5 are derived from the noncoding 5' terminus of the second c-myc exon, and 412 codons correspond to the c-myc coding region. The predicted sequence of the MH2E21 protein product differs from that of the chicken c-myc protein by 11 additional amino-terminal residues and by 25 amino acid substitutions and a deletion of 4 residues within the shared domains. To investigate the functional significance of these structural changes, the MH2E21 genome was modified in vitro. The gag translational initiation codon was inactivated by oligonucleotide-directed mutagenesis. Furthermore, all but two of the missense mutations were reverted, and the deleted sequences were restored by replacing most of the MH2E21 v-myc allele by the corresponding segment of the CMII v-myc allele which is isogenic to c-myc in that region. The remaining two mutations have not been found in the v-myc alleles of avian oncoviruses MC29, CMII, and OK10. Like MH2 and MH2E21, modified MH2E21 (MH2E21m1c1) transforms avian embryo cells. Like c-myc, it encodes a 416-amino-acid protein initiated at the myc translational initiation codon. We conclude that neither major structural changes, such as in-frame fusion with virion genes or internal deletions, nor specific, if any, missense mutations of the c-myc coding region are necessary for activation of the basic oncogenic function of transduced myc alleles.

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.

Molecular cloning of proviral DNA and structural analysis of the transduced myc oncogene of avian oncovirus CMII.

Molecularly cloned proviral DNA of avian oncogenic retrovirus CMII was isolated by screening a genomic library of a CMII-transformed quail cell line with a myc-specific probe. On a 10.4-kilobase EcoRI fragment, the cloned DNA contained 4.4 kilobases of CMII proviral sequences extending from the 5' long terminal repeat to the EcoRI site within the partial (delta) complement of the env gene. The gene order of CMII proviral DNA is 5'-delta gag-v-myc-delta pol-delta env-3'. All three structural genes are partially deleted: the gag gene at the 3' end, the env gene at the 5' end, and the pol gene at both ends. The delta gag (0.83 kilobases)-v-myc (1.50 kilobases) sequences encode the p90gag-myc transforming protein of CMII. In comparison with the p110gag-myc protein of acute leukemia virus MC29, p90gag-myc lacks amino acids corresponding to additional 516 bases of gag sequences and 12 bases of 5' v-myc sequences present in the MC29 genome. Nucleotide sequence analysis of CMII proviral DNA at the delta gag-v-myc and the v-myc-delta pol junctions revealed significant homologies between avian retroviral structural genes and the cellular oncogene c-myc precisely at the positions corresponding to the gene junctions in CMII. Furthermore, the delta gag-v-myc junction in CMII corresponds to sequence elements in gag and C-myc that are possible splicing signals. The data suggest that transduction of cellular oncogenes may involve RNA splicing and recombination with homologous sequences on retroviral vectors. Different sequence elements of both the retroviral vectors and the c-myc gene recombined during genesis of highly oncogenic retroviruses CMII, MC29, or MH2.

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 (p53 gag ), and a v- myc gene product of 59,000/61,000 Da (p59/61 v- myc ) indistinguishable from the v- myc protein encoded by MH2. MH2D12 viral RNA contains all T 1-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 Δ 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 Δ gag/Δ mil junction suggests that recombination between homologous sequence elements of the Δ 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 p539 gag 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.

Immunological analysis of v-myc gene products using antibodies against a myc-specific synthetic peptide

Rabbit antisera were prepared against a synthetic peptide corresponding to the carboxyterminal amino acid sequences of the transforming protein p110 gag-myc of avian oncovirus MC29. Analysis of immunoprecipitates formed with these sera (anti- mycC) demonstrated that the gag-myc hybrid proteins encoded by the avian leukemia viruses MC29, CMII, and OK10 were recognized by the anti-peptide sera, but not the gag or pot precursor proteins of helper viruses or the MH2-encoded p100 gag-mil protein. In cells transformed by OK10 or MH2, putative v- myc proteins of 60K (OK10) and 59/61K (MH2) were also precipitated by anti- mycC. In addition, the anti-peptide sera reacted specifically with the gag-myc proteins encoded by three partially transformation-defective mutants of MC29, td10A, td10C, and td10H, and by MC29 variant HBI.

Avian oncovirus MH2: molecular cloning of proviral DNA and structural analysis of viral RNA and protein.

Viral RNA, molecularly cloned proviral DNA, and virus-specific protein of avian retrovirus MH2 were analyzed. The complexity and sequence conservation of the transformation-specific v-myc sequences of MH2 RNA were compared with those of the other members of the MC29 subgroup of acute leukemia viruses, MC29, CMII, and OK10, and with chicken cellular c-myc sequences. All T1 oligonucleotides mapping within the 1.3-kilobase coding region of MC29 v-myc have homologous counterparts in the RNAs of all MC29 subgroup viruses and in c-myc. These counterparts are either identical in composition or altered by single point mutations. Hence, the 47,000-dalton carboxy-terminal sequences of the transforming proteins of these viruses and of the cellular gene product are probably highly conserved but may contain single amino acid substitutions. T1 oligonucleotide mapping of MH2 RNA indicated that the MH2 v-myc sequences map close to the 3' end of viral RNA. A genomic library of an MH2-transformed quail cell line was prepared by using the Charon 4A vector system. By screening with an myc-specific probe, a clone containing the entire MH2 provirus (lambda MH2-1) was isolated. Digestion of cloned DNA with KpnI yielded a 5.1-kilobase fragment hybridizing to both gag- and myc-specific probes. Further restriction mapping of lambda MH2-1 DNA showed that about 1.6 kilobases of the gag gene are present near the 5' end of proviral DNA, and the conserved part of v-myc, i.e., 1.3 kilobases, is present near the 3' end of proviral DNA. These two domains are separated by a segment of at least 1 kilobase of different genetic origin, including additional unique sequences unrelated to virion genes. Tryptic peptide analysis of the gag-related protein of MH2, p100, revealed gag-specific peptides and several unique methionine-containing peptides. One of the latter is possibly shared with the polymerase precursor protein Pr180gag-pol, but no myc-specific peptides, defined for the MC29 protein p110gag-myc, appear to be present in MH2 p100. The data on viral RNA, proviral DNA, and protein of MH2 reveal a unique genetic structure for this virus of the MC29 subgroup and suggest that its v-myc gene is not expressed as a gag-related protein.

Host Antigens on Avian Oncoviruses: Presence on Viruses Produced by Quail, Duck and Rat Cells of Antigens Related to Membrane Antigens of Chick Embryo Fibroblasts and Chicken Erythrocytes

Avian sarcoma viruses (ASV) produced by Japanese quail embryo fibroblasts (QEF) were inactivated to the same degree as ASV produced by chick embryo fibroblasts (CEF), with or without complement, by rabbit antisera to CEF and to membrane antigens of chick embryo and adult chicken erythrocytes, in particular their unique age-specific antigens. ASV produced by duck embryo fibroblasts (DEF) were only inactivated by the antisera to the chicken erythrocyte antigens. Hence, viruses produced by QEF and DEF bear on their envelope antigens related to the chicken antigens picked up by ASV in CEF. These antigens are presumably coded by the quail and duck cells, since antigens related to the chicken antigens were found on QEF and on quail and duck erythrocytes. Antigens related to the chicken antigens have also been found on ASV shed in low amounts by semi-permissive rat sarcoma cells (line 17RBI77) and on the surface of these cells, as well as on that of another semi-permissive cell line (RBH) originating from a hamster sarcoma produced by inoculation of the rat cells. The origin of these antigens, which may play a role in semi-permissiveness, remains to be explained since they do not appear to be normally expressed by rat or hamster cells. It was also found that, contrary to an earlier conclusion and in agreement with a recent report, uninfected CEF bear on their membrane an antigen that is related or identical to the specific antigen of chick embryo erythrocytes. Therefore, only the antigen related to the adult-specific chicken erythrocyte antigen does not pre-exist on CEF.

Characterization of a 105,000 molecular weight gag-related phosphoprotein from cells transformed by the defective avian sarcoma virus PRCII

In cells infected with the replication-defective avian sarcoma virus PRCII a single virus-specific product is detectable, a polyprotein of 105,000 molecular weight (p105). P105 can be precipitated with antisera to gag proteins of avian leukosis and sarcoma viruses. By two-dimensional tryptic peptide analysis of [ 35S]methionine-labeled proteins we have shown that p105 contains peptides of helper virion gag proteins p19 and p27, but not of p15. In addition a number of peptides are present in p105 that are not found in any of the helper virus gene products including gPr95 env and Pr180 gag-pol . These p105-specific peptides are not detectable in the p60 src protein of Rous sarcoma virus (RSV) nor in the gag-related polyproteins encoded by avian myelocytoma and carcinoma viruses MC29 and MH2 or avian erythroblastosis virus AEV. P105 is not detectably glycosylated, but is heavily phosphorylated. In this respect it resembles p60 src of RSV rather than the polyproteins of avian leukemia viruses. Since p105 is the only viral gene product detectable in nonproducing cells transformed by PRCII, this protein may be important in the initiation and maintenance of oncogenic transformation. The nonstructural sequences in p105 would then represent a new class of transforming gene in avian oncoviruses.

Comparative peptide analysis of avian oncoviral structural protein markers

The internal structural proteins of certain avian oncoviruses show strain-specific variation in their electrophoretic mobilities. The primary structures of these gag protein markers were compared by proteolytic digestion and two-dimensional peptide analysis. Specific differences in the peptide fingerprints of homologous p27, p19, and p15 proteins were detected. These differences were consistent with the observed electrophoretic properties of strain-specific structural protein markers and segregated with the markers in genetic recombinants. It is concluded that the altered electrophoretic properties of avian oncoviral gag :markers are due to small differences in amino acid sequences of corresponding proteins.

Mutants of avian myelocytomatosis virus with smaller gag gene-related proteins have an altered transforming ability.

Avian myelocytomatosis virus strain MC29 is a replication-defective avian oncovirus which in newborn chickens causes myelocytomatosis and liver and kidney tumours. In vitro infection of bone marrow cells gives rise to colonies of transformed macrophage-like cells, and cloned viruses is also capable of transforming fibroblasts. The genome of MC29 contains cellular sequences which are closely related to those in other defective leukaemia viruses with similar transforming spectra. Consequently, these cellular sequences have been postulated to represent a new oncogene which has been designated mac, for macrophage transformation. MC29-transformed cells contain a gag gene-related protein of a 110,000 molecular weight (MW) (p110), which by tryptic peptide analysis has been shown to be a fusion product comprised of a gag gene-derived sequences and sequences which are presumed to be coded by the adjacent mac gene. These findings suggest that this protein may be implicated in transformation by MC29. We now describe three mutants of MC29 and synthesize smaller gag gene-related proteins. These mutants have an altered ability to transform bone marrow cells but not fibroblasts. This demonstrates for the first time a direct involvement of the p110 protein of MC29 in transformation.

Avian oncovirus mutant (SE21Q1b) deficient in genomic RNA: characterization of a deletion in the provirus.

We have previously described a nonconditional mutant of avian sarcoma virus (SE21Q1b) which fails to package viral RNA (Gallis et al., Virology 94:146-161, 1979; Linial et al., Cell 15:1371-1381, 1978). Quail cells transformed by SE21Q1b contain normal amounts of intracellular viral mRNA's for src, env, and gag-pol and release particles with the density of normal virus containing a typical complement of virion proteins, including reverse transcriptase. These virions are noninfectious for both chicken and quail cells and contain primarily cellular rather than viral RNA. Analysis by gel electrophoresis of the cellular DNA of quail cells transformed by SE21Q1b after restriction endonuclease digestion indicated the presence of a single provirus. The provirus was located at one site in the genome of the host cell and was flanked by the characteristic terminally repeated sequences derived from the 3' and 5' ends of viral RNA. The only defect detected in the SE21Q1b provirus was a deletion of ca. 150 base pairs of DNA somewhere between 300 and 600 bases from the left (gag-pol) end of the provirus. Analyses of the proviral DNA of cells transformed by wild-type recombinants between SE21Q1b and leukosis viruses reveal that the recombinants no longer contain this deletion. The deletion, therefore, defines a region on the viral RNA which is required for correct packaging of the virion RNA.

Concanavalin A agglutinability of some enveloped RNA viruses modified by host cell transformation.

The dependence on concanavalin A (Con A) concentration of agglutinability of some enveloped RNA viruses grown in transformed cells was compared with that of those grown in nontransformed cells. The avian oncoviruses were purified by centrifuging to equilibrium in a combination equilibrium: viscosity gradient of potassium tartrate and glycerol after conventional isopycnic sucrose density gradient centrifugation. Avian oncoviruses were more agglutinable with Con A when grown in transformed cells than when grown in nontransformed cells. Vesicular stomatitis virus grown in transformed cells was also more agglutinable than the virus grown in nontransformed cells. These results agree with the concept that the envelopes are modified by host cell transformation and that, therefore, viruses grown in transformed cells are expected to be more agglutinable with Con A than those grown in nontransformed cell.

Avian oncovirus proteins expressed on the surface of infected cells

Lactoperoxidase-catalyzed iodination and anti-AMV immunoprecipitation showed that both chicken and duck fibroblasts infected with a sarcoma virus (Rous sarcoma virus PrC) or a leukemia virus (avian myeloblastosis virus; AMV) had on their surface a protein of approximately 120 kilodaltons molecular weight (120K), as well as envelope glycoprotein precursors of 90–92 kd. Uninfected chicken fibroblasts of the gs −, chf − phenotype had much lower, but detectable amounts of surface 120K, whereas uninfected duck fibroblasts did not have any, suggesting a relationship between surface 120K and expression of chicken virus information in the cell. 120K is a glycoprotein, since it could be labeled with [ 3H]mannose and contained a component that bound to a concanavalin A affinity column. The 120K protein was characterized by tryptic fingerprinting after reiodination with chloramine-T. Total and Con A-selected 120K from infected chicken cells and total 120K from infected duck cells had essentially identical fingerprints. Moreover, they were extensively related to the iodinated fingerprint of Pr76 gag, the intracellular precursor of viral core proteins. These results indicate that expression on the cell surface of glycosylated forms of gag polyproteins occurs also in avian oncornavirus infections, similarly to findings in the murine leukemia virus system.

Detection of avian oncovirus group-specific antigens by the enzyme-linked immunosorbent assay.

A three-step sandwich enzyme-linked immunosorbent assay was developed for the detection of avian oncovirus group-specific (gs) antigens. The assay procedure was to coat the wells of microtitre plates with hamster anti-gs IgG, react with crude or purified antigen and finally with hamster anti-gs IgG linked to horseradish peroxidase. The sensitivity was 8 picograms (pg) of input avian myeloblastosis virus (AMV) protein, with negligible background. As the ELISA takes less than 2 h to perform, large-scale screening for infected birds is feasible. A blocking assay was also developed for detecting anti-gs antibodies by adding unlabelled antiserrum after the antigen step.

Avian retroviruses that cause carcinoma and leukemia: identification of nucleotide sequences associated with pathogenicity.

Avian myelocytomatosis virus (MC29V) is a retrovirus that transforms both fibroblasts and macrophages in culture and induces myelocytomatosis, carcinomas, and sarcomas in birds. Previous work identified a sequence of about 1,500 nucleotides (here denoted onc(MCV)) that apparently derived from a normal cellular sequence and that may encode the oncogenic capacity of MC29V. In an effort to further implicate onc(MCV) in tumorigenesis, we used molecular hybridization to examine the distribution of nucleotide sequences related to onc(MCV) among the genomes of various avian retroviruses. In addition, we characterized further the genetic composition of the remainder of the MC29V genome. Our work exploited the availability of radioactive DNAs (cDNA's) complementary to onc(MCV) (cDNA(MCV)) or to specific portions of the genome of avian sarcoma virus (ASV). We showed that genomic RNAs of avian erythroblastosis virus (AEV) and avian myeloblastosis virus (AMV) could not hybridize appreciably with cDNA(MCV). By contrast, cDNA(MCV) hybridized extensively (about 75%) and with essentially complete fidelity to the genome of Mill Hill 2 virus (MH2V), whose pathogenicity is very similar to that of MC29V, but different from that of AEV or AMV. Hybridization with the ASV cDNA's demonstrated that the MC29V genome includes about half of the ASV envelope protein gene and that the remainder of the MC29V genome is closely related to nucleotide sequences that are shared among the genomes of many avian leukosis and sarcoma viruses. We conclude that onc(MCV) probably specifies the unique set of pathogenicities displayed by MC29V and MH2V, whereas the oncogenic potentials of AEV and AMV are presumably encoded by a distinct nucleotide sequence unrelated to onc(MCV). The genomes of ASV, MC29V, and other avian oncoviruses thus share a set of common sequences, but apparently owe their various oncogenic potentials to unrelated transforming genes.

Tryptic Peptide Analysis of Avian Oncovirus gag and pol Gene Products

Tryptic Peptide Analysis of Avian Oncovirus <i>gag</i> and <i>pol</i> Gene Products

Host antigens on avian oncoviruses: evidence for virus envelope antigens related to specific chicken erythrocyte membrane antigens.

Avian sarcoma viruses (ASV) of subgroups A to D, produced by chick embryo fibroblasts (CEF), are inactivated to a high degree by rabbit antisera to the membrane antigens of adult chicken and chick embryo erythrocytes, notably by antisera to an antigen of embryo erythrocytes, which is lost by adult erythrocytes and to another antigen specific to the latter erythrocytes. Contrary to virus inactivation by anti-CEF serum reported earlier, virus inactivation by the antisera to these two age-specific antigens does not require complement and is not paralleled by virolysis but by aggregation of virions. The two antigens related, or identical, to the age-specific erythrocyte membrane antigens thus shown to be present on the virus envelope do not pre-exist, or pre-exist only in a low amount, on the CEF membrane, since the virus-inactivating capacity of their antisera is not removed by absorption with CEF. Their appearance on the virus does not depend on cell transformation but only on infection, since both antigens are found on a ts ASV mutant produced at restrictive temperature by untransformed CEF and the virus-inactivating capacity of their antisera is removed by absorption with CEF infected with Rous-associated virus (RAV-1). These findings suggest that infection of CEF by avian oncoviruses may elicit the appearance, or enhance the expression at the cell surface of antigens characteristic of another cell type which may contribute to the formation of specific virus budding sites.

Recombination in the avian oncoviruses as a model for the generation of defective transforming viruses.

Recombination in the avian oncoviruses as a model for the generation of defective transforming viruses.