Variations in integration site of avian oncornaviruses in different hosts.

We examined the integration site of avian oncornaviruses in the genome of different hosts with respect to the repetitive frequency of the cellular DNA sequences adjacent to the integrated proviral DNA. The following systems were studied: avian sarcoma virus (B-77) and avian leukosis virus (Rous-associated virus-61) in cultured duck embryonic cells and B-77 in cultured mouse 3T3 cells. These systems represent different host responses to viral infection, i.e., one in which both cellular transformation and viral replication occur (B-77-infected duck cells), one in which viral replication, but not transformation, occurs (Rous-associated virus-61-infected duck cells), and one in which transformation, but not viral replication, occurs (B-77-infected 3T3 cells). Two sequential hybridizations were used. First, large denatured DNA fragments (2.8 X 10(6) daltons) were reassociated to different C0t (mole-seconds per liter) values. Next, DNA remaining single stranded at different C0t values was isolated by hydroxylapatite column chromatography, immobilized on nitrocellulose filters, and hybridized with an excess of 3H-labeled 35S viral RNA to titrate the concentration of proviral DNA. Results show that B-77 sarcoma virus and Rous-associated virus-61 integrate in the unique region of duck DNA, whereas B-77 proviral DNA is associated with both repeated and unique host DNA sequences in transformed mouse 3T3 cells.     

Avian acute leukemia virus OK 10: analysis of its myc oncogene by molecular cloning.

Several DNAs representing the genome of the avian acute leukemia virus OK 10 were isolated by molecular cloning from a transformed quail cell line, 9C, which contained at least six OK 10 proviruses. Recombinant lambda phages harboring the OK 10 genome and additional flanking cellular DNA sequences were studied by restriction endonuclease mapping and hybridization to viral cDNA probes. Six of the clones represented complete proviruses with similar, if not identical, viral sequences integrated at different positions in the host DNA. The organization of the OK 10 genome was determined by electron-microscopic analysis of heteroduplexes formed between the cloned OK 10 DNA and DNAs representing the c-myc gene and the genomes of two other avian retroviruses, Rous-associated virus-1 and MC29. The results indicated that the OK 10 proviral DNA is about 7.5 kilobases in size with the following structure: 5'-LTR-gag-delta polmyc-delta env-LTR-3', where LTR indicates a long terminal repeat. The oncogene of OK 10, v-mycOK 10, forms a continuous DNA segment of around 1.7 kilobases between pol and env. It is similar in structure and length to the v-myc gene of MC29, as demonstrated by restriction endonuclease and heteroduplex analyses. Two of the OK 10 proviruses were tested in transfection experiments: both DNAs gave rise to virus with the transforming capacities of OK 10 when Rous-associated virus-1 was used to provide helper virus functions.     

Formation of Rous associated virus-60: origin of the polymerase gene.

The DNA of normal chicken embryos contains sequences related to the avian leukosis-sarcoma viruses. RNA-dependent DNA polymerase of these viruses is encoded by a genetic element known as the pol gene. The nature of the endogenous virus pol gene in chicken cells was investigated by testing its ability to participate in genetic recombination. Rous-associated virus-60-type recombinant viruses isolated after infection of chicken cells with strains tsLA337PR-B or tsNY21SR-A, both of which produce a temperature-sensitive DNA polymerase, also possessed the temperature-sensitive lesion. These results are consistent with the hypothesis that the endogenous viral information used for the generation of Rous-associated virus-60 is deficient in at least part of the pol gene and that the defect includes that portion represented by the lesions in NY21 and LA337. The frequency of polymerase-negative BH-Rous sarcoma virus alpha formation was not affected by the levels of endogenous viral expression, which suggests that the alpha defect is not derived from the endogenous pol gene.     

Helper-independent retrovirus vectors with Rous-associated virus type O long terminal repeats.

We have constructed nonpermuted replication-competent avian retrovirus vectors that derive from Rous sarcoma virus (S. H. Hughes, J. J. Greenhouse, C. J. Petropoulos, and P. Sutrave, J. Virol. 61:3004-3012, 1987). We describe here the construction and properties of corresponding vectors in which the long terminal repeats (LTRs) of the parental virus have been replaced by the LTRs of the endogenous chicken virus Rous-associated virus type O. The Rous-associated virus type O LTR vectors replicated approximately 1/10 as well as the parental vectors and expressed a test gene, chloramphenicol acetyltransferase, approximately 1/30 to 1/50 as well.     

Intracellular precursors to the major glycoprotein of avian oncoviruses in chicken embryo fibroblasts.

A 96,000-dalton glycoprotein, p(96), was present in cell extracts obtained from gs-chf- chicken embryo fibroblasts infected with the avian RNA tumor viruses Rous-associated virus-2 subgroup B (RAV-2) and the Schmidt-Ruppin strain of Rous sarcoma virus subgroup A (SR-RSV-A), as well as from uninfected gsLchf+ (HE) cell extracts. It was not found in cell extracts from uninfected gs-chf- or gs+chf+ (HH) cells, nor from gs-chf- cells infected with envelope-deficient Bryan high-titer Rous sarcoma virus. Immunoprecipitation, kinetic, and biochemical data indicate the this polyprotein contains information that gives rise to the major virion glycoprotein gp85. A second polyprotein of 80,000 daltons, p/80), is also present in the RAV-2- and SR-RSV-A-infected gs-chf- cells. This second polyprotein contains less carbohydrate than p(96), and kinetic and biochemical data indicate that p(80) may be an immature form of p(96).     

Single-step conversion of cells to retrovirus vector producers with herpes simplex virus-Epstein-Barr virus hybrid amplicons

We report here on the development and characterization of a novel herpes simplex virus type 1 (HSV-1) amplicon-based vector system which takes advantage of the host range and retention properties of HSV-Epstein-Barr virus (EBV) hybrid amplicons to efficiently convert cells to retrovirus vector producer cells after single-step transduction. The retrovirus genes gag-pol and env (GPE) and retroviral vector sequences were modified to minimize sequence overlap and cloned into an HSV-EBV hybrid amplicon. Retrovirus expression cassettes were used to generate the HSV-EBV-retrovirus hybrid vectors, HERE and HERA, which code for the ecotropic and the amphotropic envelopes, respectively. Retrovirus vector sequences encoding lacZ were cloned downstream from the GPE expression unit. Transfection of 293T/17 cells with amplicon plasmids yielded retrovirus titers between 10(6) and 10(7) transducing units/ml, while infection of the same cells with amplicon vectors generated maximum titers 1 order of magnitude lower. Retrovirus titers were dependent on the extent of transduction by amplicon vectors for the same cell line, but different cell lines displayed varying capacities to produce retrovirus vectors even at the same transduction efficiencies. Infection of human and dog primary gliomas with this system resulted in the production of retrovirus vectors for more than 1 week and the long-term retention and increase in transgene activity over time in these cell populations. Although the efficiency of this system still has to be determined in vivo, many applications are foreseeable for this approach to gene delivery.     

A new avian leukosis virus-based packaging cell line that uses two separate transcomplementing helper genomes.

An avian leukosis virus-based packaging cell line was constructed from the genome of the Rous-associated virus type 1. The gag, pol, and env genes were separated on two different plasmids; the packaging signal and the 3' long terminal repeat were removed. On a plasmid expressing the gag and pol genes, the env gene was replaced by the hygromycin resistance gene. The phleomycin resistance gene was inserted in the place of the gag-pol genes on a plasmid expressing the env gene. The plasmid containing the gag, pol, and Hygror genes was transfected into QT6 cells. Clones that produced high levels of p27gag were transfected with the plasmid containing the Phleor and env genes. Clones that produced high levels of env protein (as measured by an interference assay) were tested for their ability to package NeoR-expressing replication-defective vectors (TXN3'). One of the clones (Isolde) was able to transfer the Neo+ phenotype to recipient cells at a titer of 10(5) resistance focus-forming units per ml. Titers of supernatants of cells infected with Rous-associated virus type 1 prior to transfection by Neor vectors were similar. Tests for recombination events that might result in intact helper virus showed no evidence for the generation of replication-competent virus. The use of selectable genes inserted next to the viral genes to generate high-producer packaging cell lines is discussed.     

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.     

Antigenic determinants specific to the envelope glycoprotein of exogenous avian tumor viruses.

Antibody in the sera of 15B X 7(2) chickens was measured for reactivity to determinants of the envelope glycoprotein of endogenous and exogenous avian tumor viruses. The pattern of reactivity of animals infected with exogenous sarcoma virus served to operationally define a class of determinants present on the envelope glycoprotein of exogenous viruses and absent from that of the endogenous virus Rous-associated virus-0.     

Avian retroviral long terminal repeats bind CCAAT/enhancer-binding protein.

DNA-protein interactions involving enhancer and promoter sequences within the U3 regions of several avian retroviral long terminal repeats (LTRs) were studied by DNase I footprinting. The rat CCAAT/enhancer-binding protein, C/EBP, bound to all four viral LTRs examined. The Rous sarcoma virus binding site corresponded closely to the 5' limit of the LTR enhancer; nucleotides -225 to -188 were protected as a pair of adjacent binding domains. The Fujinami sarcoma virus LTR bound C/EBP at a single site at nucleotides -213 to -195. C/EBP also bound to the promoter region of the enhancerless Rous-associated virus-0 LTR at nucleotides -77 to -57. The avian myeloblastosis virus LTR bound C/EBP at three sites: nucleotides -262 to -246, -154 to -134, and -55 to -39. We have previously observed binding of C/EBP to an enhancer in the gag gene of avian retroviruses. A heat-treated nuclear extract from chicken liver bound to all of the same retroviral sequences as did C/EBP. Alignment of the avian retroviral binding sequences with the published binding sites for C/EBP in two CCAAT boxes and in the simian virus 40, polyoma, and murine sarcoma virus enhancers suggested TTGNNGCTAATG as a consensus sequence for binding of C/EBP. When two bases of this consensus sequence were altered by site-specific mutagenesis of the Rous sarcoma virus LTR, binding of the heat-stable chicken protein was eliminated.     

Nucleotide Sequences Derived from Pheasant DNA in the Genome of Recombinant Avian Leukosis Viruses with Subgroup F Specificity

Recombination between viral and cellular genes can give rise to new strains of retroviruses. For example, Rous-associated virus 61 (RAV-61) is a recombinant between the Bryan high-titer strain of Rous sarcoma virus (RSV) and normal pheasant DNA. Nucleic acid hybridization techniques were used to study the genome of RAV-61 and another RAV with subgroup F specificity (RAV-F) obtained by passage of RSV-RAV-0 in cells from a ring-necked pheasant embryo. The nucleotide sequences acquired by these two independent isolates of RAV-F that were not shared with the parental virus comprised 20 to 25% of the RAV-F genomes and were indistinguishable by nucleic acid hybridization. (In addition, RAV-F genomes had another set of nucleotide sequences that were homologous to some pheasant nucleotide sequences and also were present in the parental viruses.) A specific complementary DNA, containing only nucleotide sequences complementary to those acquired by RAV-61 through recombination, was prepared. These nucleotide sequences were pheasant derived and were not present in the genomes of reticuloendotheliosis viruses, pheasant viruses, and avian leukosis-sarcoma viruses of subgroups A, B, C, D, and E. They were partially endogenous, however, to avian DNA other than pheasant. The fraction of these nucleotide sequences present in other avian DNAs generally paralleled the genetic relatedness of these avian species to pheasants. However, there was a high degree of homology between these pheasant nucleotide sequences and related nucleotide sequences in the DNA of normal chickens as indicated by the identical melting profiles of the respective hybrids.     

Correlation of RNA binding affinity of avian oncornavirus p19 proteins with the extent of processing of virus genome RNA in cells.

We purified the p19 proteins from the Prague C strain of Rous sarcoma virus, avian myeloblastosis virus, B77 sarcoma virus, myeloblastosis-associated virus-2(0), and PR-E 95-C virus and measured their binding affinities for 60S viral RNA by the nitrocellulose filter binding technique. The apparent association constants of the p19 proteins from Rous sarcoma virus Prague C, avian myeloblastosis virus, and B77 sarcoma virus for homologous and heterologous 60S RNAs were similar (1.5 x 10(11) to 2.6 x 10(11) liters/mol), whereas those of myeloblastosis-associated virus-2(0) and PR-E 95-C virus were 10-fold lower. The sizes and relative amounts of the virus-specific polyadenylic acid-containing RNAs in the cytoplasms of cells infected with Rous sarcoma virus Prague C, myeloblastosis-associated virus-2(0), and PR-E 95-C virus were determined by fractionating the RNAs on agarose gels containing methylmercury hydroxide, transferring them to diazobenzyloxymethyl paper and hybridizing them to a 70-nucleotide complementary DNA probe. In cells infected with Rous sarcoma virus Prague C we detected 3.4 x 10(6)-, 1.9 x 10(6)-, and 1.1 x 10(6)-dalton RNAs, in PR-E 95-C virus-infected cells we detected 3.4 x 10(6)-, 1.9 x 10(6)- and 0.7 x 10(6)-dalton RNAs, and in cells infected with myeloblastosis-associated virus-2(0) we detected 3 x 10(6)- and 1.3 x 10(6)-dalton RNAs. Each of these RNA species contained RNA sequences derived from the 5' terminus of genome-length RNA, as evidenced by hybridization with the 5' 70-nucleotide complementary DNA. The ratios of subgenomic mRNA's to genome-length RNAs in cells infected with myeloblastosis-associated virus-2(0) and PR-E 95-C virus were three- to five-fold higher than the ratio in cells infected with Rous sarcoma virus Prague C. These results suggest that more processing of viral RNA in infected cells is correlated with lower binding affinities of the p19 protein for viral RNA, and they are consistent with the hypothesis that the p19 protein controls processing of viral RNA in cells.     

In vivo analysis of a new lacZ retrovirus vector suitable for cell lineage marking in avian and other species

To obtain a replication-defective retrovirus vector well suited for cell lineage marking in early avian embryos, we have constructed and tested a derivative of the avian spleen necrosis virus (SNV) carrying the marker gene lacZ. Consistently high titers of this virus, designated CXL, were produced from retroviral packaging cells with no evidence of contaminating helper virus even after 12 months of continuous culture. CXL expresses lacZ strongly and stably in avian cells and has a host range that extends to other avian and some mammalian species. We show that CXL has the potential to mark a wide variety of chick embryo cell types by infection in ovo. The high titers obtainable with this virus can provide a significant advantage over alternative lacZ vectors, especially in lineage marking of early stage embryos. As an example of this, we show that CXL can be used to mark cells of the precardiac mesoderm in stage 4-5 chick embryos.     

Embryonic infection with the endogenous avian leukosis virus Rous-associated virus-0 alters responses to exogenous avian leukosis virus infection.

We inoculated susceptible chicken embryos with the endogenous avian leukosis virus Rous-associated virus-0 (RAV-0) on day 6 of incubation. At 1 week after hatching, RAV-0-infected and control chickens were inoculated with either RAV-1 or RAV-2, exogenous viruses belonging to subgroups A and B, respectively. The chickens injected with RAV-0 as embryos remained viremic with exogenous virus longer and either failed to develop type-specific humoral immunity to exogenous virus or developed it later than the control chickens not inoculated with RAV-0. The RAV-0-injected chickens also developed neoplasms at a much higher frequency than did the control chickens. We suggest that the lower immune responses of the RAV-0-injected chickens were due to an immunological tolerance to envelope group-specific glycoproteins shared among endogenous and exogenous viruses.     

Detection of Avian Tumor Virus RNA in Uninfected Chicken Embryo Cells

Uninfected chicken embryo cells were analyzed for the presence of viral ribonucleic acid (RNA) by molecular hybridization with the single-stranded deoxyribonucleic acid (DNA) product of the RNA-dependent DNA polymerase contained in avian sarcoma-leukosis virions. Viral RNA was detected in all cells which contained the avian tumor virus group-specific antigen and the virus-related helper factor. The amounts of viral RNA in these cells ranged from approximately 3 to 40 copies of viral-specific sequences per cell. In general, the viral RNA content correlated with the level of helper activity in the cells. Cells infected with Rous-associated virus 2 contained 3,000 to 4,000 copies of viral RNA per cell. RNA from these infected cells hybridized with nearly 100% of the viral (3)H-DNA. By contrast, a maximum of less than 50% hybridization was obtained with RNA from the uninfected helper-positive cells, suggesting that not all of the viral RNA sequences were present in these cells. No viral RNA was detected in cells which lacked group-specific antigen and helper activity. Under the conditions used in these studies, less than 0.3 viral genome equivalents of RNA per cell would have been detected.     

Selective shedding and congenital transmission of endogenous avian leukosis viruses.

Shedding and congenital transmission of endogenous avian leukosis viruses were studied in viremic White Leghorn hens exogenously infected with viruses with endogenous long terminal repeats (LTRs) and in four semicongenic lines of hens that naturally express infectious endogenous viruses (EVs). Relatively high titers of infectious virus EV7 (encoded at locus ev7), Rous-associated virus-0 (RAV-0), and recombinant 882/-16 RAV-0 were detected in blood cells and sera from exogenously infected hens, but marked differences were noted in the incidence of congenitally infected progeny. In enzyme immunoassays that detect viral group-specific antigen, little or no p27 was detected in albumens from dams infected with RAV-0. However, hatchmates infected with either EV7 or recombinant 882/-16 RAV-0, which was constructed with an RAV-0 LTR, shed high titers of p27. Similarly, semicongenic hens that expressed RAV-0 (EV2) (encoded at locus ev2) shed little or no p27 into albumens, but hens that harbored ev10, ev11, and ev12 shed high titers of p27. A slower electrophoretic mobility of p27, considered to be characteristic of EVs that are restricted in congenital transmission, was not associated with low levels of shedding or congenital transmission; p27 from other EVs and p27 from an avian leukosis virus field strain, all of which are shed at high levels, had mobilities identical to that of p27 from RAV-0. Although shedding and congenital transmission appear to be controlled by the viral genome, there was no correlation between low efficiency of shedding or congenital transmission and endogenous LTR or p27 sequences.     

Reticuloendotheliosis Virus Nucleic Acid Sequences in Cellular DNA

Reticuloendotheliosis virus 60S RNA labeled with (125)I, or reticuloendotheliosis virus complementary DNA labeled with (3)H, were hybridized to DNAs from infected chicken and pheasant cells. Most of the sequences of the viral RNA were found in the infected cell DNAs. The reticuloendotheliosis viruses, therefore, replicate through a DNA intermediate. The same labeled nucleic acids were hybridized to DNA of uninfected chicken, pheasant, quail, turkey, and duck. About 10% of the sequences of reticuloendotheliosis virus RNA were present in the DNA of uninfected chicken, pheasant, quail, and turkey. None were detected in DNA of duck. The specificity of the hybridization was shown by competition between unlabeled and (125)I-labeled viral RNAs and by determination of melting temperatures. In contrast, (125)I-labeled RNA of Rous-associated virus-O, an avian leukosis-sarcoma virus, hybridized 55% to DNA of uninfected chicken, 20% to DNA of uninfected pheasant, 15% to DNA of uninfected quail, 10% to DNA of uninfected turkey, and less than 1% to DNA of uninfected duck.