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.     

Regulation of expression and chromosomal subunit conformation of avian retrovirus genomes.

We have investigated the copy number, chromosomal subunit conformation and regulation of expression of integrated avian retrovirus genomes. Our results indicate that there are approximately two copies of the endogenous viral genomes (RAV-O) per haploid cell genome in uninfected chick embryo fibroblasts (CEF) and red blood cells (RBC). The copy number and subunit conformation (as measured by DNAasel sensitivity) of the RAV-O genomes are independent of the level of expression of these viral DNA sequences. In cells isolated from embryos of the V+, gs-chf- and gs+chf+ phenotypes, approximately one of the two viral genomes is in a DNAase l-sensitive conformation. Upon infection with an exogenous Rous sarcoma virus (PR-RSV-C), one new viral genome is integrated per haploid CEF genome. The newly integrated RSV genome is completely sensitive to DNAase l, and the subunit conformation of the endogenous viral genomes is not altered by the integration of additional exogenous proviruses. Both the endogenous and newly integrated exogenous viral genomes are present in "nu-body" structures, and the selective sensitivity of these proviral DNA sequences to DNAase l is maintained in isolated nucleosomes. Our experiments revealing the DNAase l sensitivity of one of the two RAV-O genomes in gs-chf-CEF led us to reexamine the level of viral specific RNA in CEF of various GS genotypes. We find that GS/GS CEF contain approximately 100 copies of viral RNA per cell, gs/gs CEF contain no detectable viral RNA, and the heterozygote GS/gs CEF contain approximately 50 copies of viral specific RNA per cell. These results suggest that the GS gene controls production of RAV-O RNA sequences in CEF in a "cis" fashion. In RBCs, however, the expression of the RAV-O genome is independent of the GS gene, with both GS/GS and gs/gs RBCs containing roughly equivalent amounts of viral specific RNA. Our results suggest that the chromosomal structure of the endogenous viral genes is independent of the GS gene, and that the GS gene is cis-acting and tissue-specific.     

Chicken leukosis virus genome sequences in DNA from normal chick cells and virus-induced bursal lymphomas.

Genome sequences of two recent field isolates of avian leukosis viruses in the DNA of normal and neoplastic chicken cells were studied by DNA-RNA hybridization under conditions of DNA excess. Comparisons were made between 60-70S RNA from these viruses and that of a chicken endogenous type C virus (RAV-0), and of a series of "laboratory" leukosis and sarcoma viruses, by competitive hybridization analysis. A minimum of 18% of the genome sequences of both ALV isolates detected in DNA from lymphomas they induced were not detected in normal chicken DNA. The vast majority of the fraction of RNA sequences from ALV which do form hybrids with normal chick DNA appear to be reacting with the endogenous provirus of RAV-0. The genomic representation of a variety of avian leukosis and sarcoma viruses in normal chicken cells could not be distinguished by these methods (except that 13% of the RAV-0 genome was not shared with any of the other viruses). In contrast, the portion of the ALV genome exogenous to the normal chicken geome showed significant divergence from that of two sarcoma viruses (Pr RSV-C and B-77). The increased hybridization of ALV RNA with lymphoma DNA was used to detect the appearance of ALV specific sequences in the bursa of Fabricius following infection.increased hybridization was correlated with both the time after infection and the extent of replacement of the bursa by lymphoma. About one half of the increase in hybridization preceded histologic evidence of transformation.     

Heterogeneity of genetic loci in chickens: analysis of endogenous viral and nonviral genes by cleavage of DNA with restriction endonucleases.

Restriction endonucleases can be used to define the structure and position of genetic loci for which specific molecular hybridization reagents are available. We have used this approach to compare 18 chicken embryos with respect to several cellular genes; endogenous viral DNA related to the replicative genes of avian sarcoma virus (ASV) or to RAV-O, an endogenous virus of chickens; and sequences related to the transforming (src) gene of ASV. Each cellular gene eas remarkably homogeneous within our test population. We found little or no variation in globin and ovomucoid genes; ovalbumin and transferrin (with one exception) showed variation which is probably allelic in nature. The endogenous viral DNA which has homology with RAV-O was found at several different positions in host DNA and its structure resembled that of proviruses acquired by experimental infection, with sequences from both ends of viral RNA repeated near both ends of viral DNA. Within the population of 18 chickens, one endogenous provirus was always present, whereas the several other proviruses were each found in only a few members of this group. However, screening of additional chickens identified individuals lacking the provirus common to the initial 18 animals surveyed; in at least one embryo no RAV-O-related DNA was detected. These findings suggest that the endogenous RAV-O-related sequences have entered the germ line by relatively recent infection and are still segregating in several contemporary chicken flocks. The sequences in the chicken genome which have homology with the src gene of ASV are invariant from bird to bird and in this sense resemble a cellular gene rather than a viral sequence.     

Differences between the endogenous and exogenous DNA sequences of Rous-associated virus-O.

DNA sequences related to the endogenous retrovirus of chickens, Rous-associated virus-O (RAV-O), have been examined using site-specific DNA endonuclease analysis of cellular DNA derived from line 15 and line 100 chickens. Individual embryos from both inbred lines were used as a source of embryonic fibroblasts from which cellular DNA was isolated. Analysis of DNA containing either endogenous RAV-O sequences alone or both endogenous and exogenous RAV-O sequences produced identical patterns of RAV-O-specific DNA fragments after digestion with the endonucleases Eco RI, Hind III, BgI II, Bam HI or Xho I. Similar analysis with endonucleases Hinc II or Hha I, however, produced several RAV-O-specific DNA fragments which were derived from cellular DNA containing both endogenous and exogenous RAV-O sequences but not from cellular DNA containing only endogenous sequences. Although some differences exist between the DNA fragments specific for the endogenous viral sequences of line 15 and line 100 cellular DNA, the DNA fragments specific for the exogenous viral sequences were identical between the two inbred lines. Cleavage of an unintegrated linear RAV-O DNA molecule with Hinc II or Hha I produced DNA fragments identical to those specific for the exogenously acquired RAV-O provirus. This suggests that these characteristic fragments contain no cellular DNA. The potential DNA junction fragments containing both viral and cellular DNA, identified after analysis of DNA that contains both endogenous and exogenous viral sequences, were identical to those observed after analysis of DNA containing only endogenous viral sequences. These results support the following conclusions. First, exogenous proviral sequences are integrated into chicken cell DNA following an interaction between viral and cellular DNA that is specific with respect to the virus and nonspecific with respect to the cell. Second, both the free linear RAV-O DNA intermediate and the newly integrated exogenous provirus contain specific endonuclease sites that are not found in endogenous RAV-O DNA sequences. These results suggest that the formation of the exogenous DNA provirus involves specific alteration of the endogenous viral DNA sequences before reinsertion of the sequences as the exogenous RAV-O DNA provirus. It is possible that newly integrated exogenous RAV-O sequences are characterized by specific differences in the pattern of base methylation and a limited sequence arrangement.     

Lack of Serological Relationship Among DNA Polymerases of Avian Leukosis-Sarcoma Viruses, Reticuloendotheliosis Viruses, and Chicken Cells

Antibodies against a large and a small DNA polymerase isolated from chicken embryos and against avian myeloblastosis virus DNA polymerase were used to study the serological relationships of the DNA polymerase activities of three avian systems with RNA and a DNA polymerase-avian leukosis-sarcoma viruses, reticuloendotheliosis viruses, and a fraction from uninfected chicken cells. The DNA polymerase activity of disrupted virions of all avian leukosis-sarcoma viruses tested was neutralized to the same extent by antibody against avian myeloblastosis virus DNA polymerase and was not neutralized by the antibodies against chicken cellular DNA polymerases. The viruses tested included induced leukosis viruses and Rous-associated virus-O. The DNA polymerase activity of disrupted virions of all of the reticuloendotheliosis viruses was not neutralized by any of the antibodies. The chicken endogenous RNA-directed DNA polymerase activity was neutralized partially or completely, in different experiments, by antibody against the small DNA polymerase isolated from chicken embryos, but was not neutralized by the other two antibodies.     

Identification of an intramitochondrially synthesized proteolipid associated with the mitochondrial ATPase complex as the product of a mitochondrial gene determining oligomycin resistance in Aspergillus nidulans.

The purification of an RNA-dependent DNA polymerase from the allantoic fluid of uninfected, embryonated chicken eggs is described in detail. Comparison to the polymerase of avian myeloblastosis virus shows that the two enzymes are different with respect to ion concentrations for optimal reaction, response to increasing concentrations of substrate, thermal stability and protection from thermal inactivation by viral RNA. It is concluded that the enzymes compared to each other are different proteins, which must have been coded by different genes. The RNA-dependent DNA polymerase in the allantoic fluid, therefore, does not derive from the partial or complete expression of the endogenous virus genome of the normal chicken cell or from infection by exogenous viruses.     

Organization of shared and unshared sequences in the genomes of chicken endogenous and sarcoma viruses.

The genome of the genetically transmitted endogenous C type virus of chickens, RAV-O, is closely related to that of Rous sarcoma virus (RSV). Nevertheless, these viruses differ widely in oncogenicity and regulation by the host cell. Competitive hybridization analysis of ¹²⁵I-labeled genomic RNA demonstrated that the genome of RAV-O lacks about 35% of the sequences of nondefective RSV which formed hybrids with proviral DNA from RSV-infected cells, and that the genome of transformation-defective deletion mutants of RSV (td RSV) lacks about 15% of these sequences. Conversely, about 12% of the RAV-O sequences forming hybrids with normal chicken cell DNA were not detected in the sarcoma virus. A technique was developed to map the location of these unshared sequences by competitive hybridization. The deletion in the genome of td RSV was seen to begin at about 0.2 and to end at about 0.05 of the genome length from the 3′ end of sarcoma virus RNA, confirming the results of other laboratories using the method of mapping RNAase TI resistance of oligonucleotides. The 35% of RSV sequences missing and/or diverged in the genome of RAV-O were concentrated within 40% of the sarcoma virus genome from the 3′ end, and most of this large section did not appear to form hybrids with chicken DNA under the conditions of these experiments. A low level of hybrid formation was, however, detected between uninfected chicken cellular DNA and a small fraction of the nucleotides in the region of the td deletion. Analysis of RAV-O 3′ end fragments demonstrated that the genomic sequences of RAV-O missing in RSV were concentrated at the 3′ end of the endogenous viral genome. We conclude that the sequence differences between endogenous and sarcoma viruses are largely concentrated in specific regions of the viral genome.     

The distribution of endogenous chicken retrovirus sequences in the DNA of galliform birds does not coincide with avian phylogenetic relationships.

The chicken is a domesticated form of Red Jungle-fowl (Gallus gallus), which belongs to the Pheasant family (Phasianidae) within the order Galliformes. Domestic chickens carry the genome of the endogenous retrovirus RAV-O as DNA sequences integrated into host chromosomes transmitted through the germ line. We have examined the presence and distribution of RAV-O-related sequences in the DNA of Red Junglefowl and other closely related species of Junglefowl, as well as more distantly related Pheasants and Quail. DNA sequences homologous to RAV-O were analyzed by molecular hybridization in liquid and after electrophoresis of restriction endonuclease fragments. The presence of RAV-O-related sequences in avian DNA does not correlate with phylogenetic relationships. Under stringent conditions of hybridization in liquid, DNA sequences homologous to RAV-O cDNA were detected at high levels (greater than 80% homology( only in the genomes of the domestic chicken and its phylogenetic ancestor, the Red Junglefowl (Gallus gallus). The DNA of two other species of Gallus (G. sonnerati, Sonnerat's Junglefowl and G. varius, Green Junglefowl), of Ring-necked Pheasant and of Japanese Quail contained sequences with less than 10% homology to RAV-O cDNA. Under conditions permitting mismatching, however, Ring-necked Pheasant DNA hybridized up to 50% of the RAV-O cDNA, and Quail DNA 24%, whereas the extent of hybridization to Sonnerat's and Green Junglefowl DNA was not markedly increased. Analysis of restriction enzyme digests revealed several distinct fragments of DNA hybridizing to chick retrovirus cDNA in both Red Junglefowl and domestic chicken, and multiple fragments in DNA from two species of Phasianus. No fragments with sequences related to chicken retroviruses were found, however, in digests of DNA prepared from Sonnerat's, Ceylonese and Green Junglefowl, from two other Pheasant genera (Chrysolophus and Lophura), or from one Quail genus (Coturnix). Thus the DNA of three Junglefowl species closely related to Gallus gallus lacked RAV-O sequences while the DNA of more distantly related Phasianus species showed significant homology. These results show that RAV-O-related sequences have not diverged together with the normal host genes during the evolution of the Phasianidae. Although RAV-O sequences are endogenous in all domestic chickens and Red Junglefowl studied thus far, it appears that the RAV-O genome has been introduced relatively recently into the germ line of Gallus gallus, following speciation but before domestication, and independently of the related sequences found in members of the genus Phasianus.     

Cell killing by avian leukosis viruses.

Infection of chicken cells with a cytopathic avian leukosis virus resulted in the detachment of killed cells from the culture dish. The detached, dead cells contained more unintegrated viral DNA than the attached cells. These results confirm the hypothesis that cell killing after infection with a cytopathic avian leukosis virus is associated with accumulation of large amounts of unintegrated viral DNA. No accumulation of large amounts of integrated viral DNA was found in cells infected with cytopathic avian leukosis viruses.     

Recombination between endogenous and exogenous RNA tumor virus genes as analyzed by nucleic acid hybridization.

Certain chicken cells that do not spontaneously release virus particles have been shown to produce a subgroup E avian RNA tumor virus, Rous-associated virus 60 (RAV-60), after infection with viruses of other subgroups. The nucleic acids of RAV-60 were analyzed for sequence homologies with the viral nucleic acids contained in the uninfected cell and with those of RAV-2, the exogenous virus used for the preparation of this particular RAV-60 isolate. In addition, these nucleic acids were compared with those of RAV-0, an endogenous virus spontaneously released from line 100 chicken cells. RAV-60 appears to be intermediate between RAV-0 and RAV-2 in its genetic composition, based on the pattern of hybridization obtained with the nucleic acids of these viruses and on the melting profiles of the various hybrid combinations. Of the three viruses tested, RAV-0 appears to have the greatest sequence homology with the viral nucleic acids of the uninfected cell. Hybridization between RAV-60 3-H-labeled complementary DNA and either DNA or RNA from the uninfected cell indicates that RAV-60 contains some nucleic acid sequences which are not present in the cell. In addition, some RAV-60 sequences which hybridize with the cell nucleic acid contain significant amounts of mismatching, as indicated by the lower thermal stability of these hybrid duplexes. Hybrid formation between these partially homologous sequences was excluded under stringent annealing conditions. The data indicate that RAV-60 is a recombinant between exogenous and endogenous viral genes.     

Gs, an allele of chickens for endogenous avian leukosis viral antigens, segregates with ev 3, a genetic locus that contains structural genes for virus.

Gs is an allele of chickens for the expression of endogenous avian leukosis virus-related core (gs) and envelope (chf) antigens. Progeny of a genetic cross in which Gs was segregating were analyzed for endogenous viral DNA as well as for the expression of endogenous viral antigens. Viral genetic information was identified by cleavage of embryo DNA with restriction endonucleases, electrophoretic separation of the resulting fragments, and identification of bands containing viral sequences by hybridization of the DNA to 32P-labeled viral RNA. Four different chromosomal sites of residence of endogenous viral sequences were identified by this method. These sites were the same as those previously assigned to the endogenous viral loci ev 1, ev 3, ev 4, and ev 5. ev 1 was present in all of the progeny of the cross. ev 3, ev 4, and ev5 were present in various combinations with ev 1. ev 3 cosegregated with the gs+chf+ phenotpye. Cells which did not contain ev 3 but contained ev 1, ev 4, and/or ev 5, did not express detectable levels of viral antigens. We suggest that Gs contains the structural genes for endogenous virus which reside at ev 3 and that these structural genes code for gs and chf in gs+chf+ cells.     

Avian leukosis virus infection: analysis of viremia and DNA integration in susceptible and resistant chicken lines

Avian leukosis viruses induce lymphoid leukosis, a lymphoma which develops within the bursa of Fabricius several months after virus infection. Chickens from the Hyline SC and FP lines are, respectively, susceptible and resistant to avian leukosis virus-induced lymphoid leukosis. We examined plasma and cellular DNA obtained from avian leukosis virus-infected chickens for the presence of viremia and integrated viral sequences to determine whether the extent of virus infection is comparable in individuals of both lines. A less than twofold difference in the frequency of viremia was detected between chickens of the two different lines. Although the analysis of plasma samples, which were obtained at different times postinfection, demonstrated that the duration of viremia was comparable in both susceptible and resistant chickens, the onset of the viremia observed in susceptible chickens generally preceded by 1 week that observed in resistant chickens. Moreover, integrated viral sequences were detected in approximately 90% of the SC and 40% of the FP chickens. The appearance of infectious virus in the plasma was, in general, associated with the presence of integrated viral sequences in both the bursal cells and the erythrocytes obtained from the same chicken. The presence of both the viremia and the integrated viral DNA sequences was transient, suggesting a mechanism for the elimination of virus-infected cells in both susceptible and resistant chickens. Furthermore, at 5 weeks postinfection no integrated exogenous viral sequences were detected in splenic lymphocytes obtained from either chicken line, regardless of whether these chickens were viremic or had integrated viral sequences detectable in other tissues. Our results indicate that extensive avian leukosis virus replication occurs in approximately 50% of the FP and 100% of the SC chickens. Although it appears that the viral infection spreads more quickly in the SC chickens, our results afford no obvious explanation of the resistance to the development of lymphoma exhibited by FP chickens.     

Molecular cloning and characterization of gag-, pol-, and env-related gene sequences in the ev- chicken.

Using less stringent hybridization conditions and cloned viral DNA probes representing the avian sarcoma virus gag, pol, env, and long terminal repeat (LTR) gene sequences, we detected related sequences in two avian species purportedly lacking all endogenous avian leukosis viruses, the ev- chicken and the Japanese quail. The blot hybridization patterns obtained with the various probes suggest the presence of between 40 and 100 copies of retrovirus-related sequences in the genomes of these two species. An ev- chicken genomic DNA library was prepared and screened with gag-specific and pol-specific DNA probes. Several different clones were obtained from this library and characterized. Analysis of these clones revealed that the retrovirus-related gene sequences are linked in the order LTR-gag-pol-env-LTR, a structure indicative of a complete provirus. These data indicate the presence of previously unidentified endogenous retrovirus species in avian cells, suggesting that under the appropriate conditions of hybridization additional, more distantly evolved families of endogenous retrovirus genes may be identified in vertebrate species.     

Avian leukosis viruses of different subgroups and types isolated after passage of Rous sarcoma virus-Rous-associated virus-0 in cells from different ring-necked pheasant embryos.

Avian leukosis viruses of subgroups A and F (RAV-A and RAV-F) arose at a low rate after passage of Rous sarcoma virus-Rous-associated virus-0, which is subgroup E, in cells from ring-necked pheasant embryos. In cells of two embryos, all of the viruses isolated after virus passage were RAV-F. However, in cells of a third embryo, both RAV-A and RAV-F were isolated. In addition, there sometimes were type-specific differences among the different isolates of RAV-A and RAV-F from the cells of single embryos. These results indicate that the RAV-A and RAV-F probably arose by recombination of viral and cellular genes, that different ring-necked pheasant embryo may have different endogenous avian leukosis virus-related nucleotide sequences, and that recombination at different sites in these endogenous sequences might give rise to type-specific differences among the RAV-A and RAV-F.     

Electron Microscopy of Chick Fibroblasts Infected by Defective Rous Sarcoma Virus and Its Helper

Nonproducing Rous sarcoma cells of the chicken and their virus-producing as well as uninfected counterparts were studied with an electron microscope. The structural peculiarities of transformed cells included cytoplasmic annulate lamellae, aggregates of membrane-bound, glycogen-like granules, and empty, virus-like shells. Of 69 individual lines of nonproducing Rous sarcoma cells, 64 contained small numbers of viral particles. These particles were morphologically indistinguishable from mature avian tumor virus but lacked demonstrable infectivity. In sessile normal and leukosis virus-infected fibroblasts, microtubules and fibrils occurred in parallel arrays at the periphery of the cytoplasm. This cortical organization was absent from rounded Rous sarcoma cells. The characteristics of microtubular arrangement seemed to reflect differences in the locomotory activity of normal and transformed cells.