Genetic control of expression of endogenous virus genes in chicken cells

Uninfected fibroblasts from chicken embryos contain avian leukosis-sarcoma virus genetic information in the form of DNA (1-3), which may exist in a latent unexpressed state or may be at least partially expressed, as shown by the presence of viral specific RNA, structural proteins, and the envelope glycoprotein (4)(5)(6)(7)(8)(9)(10)(11)(12). The last mentioned product complements a defect of the Bryan strain of Rous sarcoma virus (BH-RSV) and is thus known as chicken helper factor (chf) (8). The regulatory function which controls the expression of the endogenous virus-related genes (9, 13) appears to act at some step controlling the amount of viral RNA in the cell, either by altering the rate of transcription or by affecting the stability of the RNA (4, 5). Certain cell types, in particular those derived from line 7 or related chicken lines, spontaneously release an infectious virus, RAV-O. Virus production in these cells is genetically controlled (14,15), and appears to correlate with the presence or absence of viral RNA (unpublished results). However, most types of uninfected cells, including those used in the present study, do not produce virus particles even though viral RNA and proteins may be synthesized. This lack of virus production suggests either that certain viral genes are unexpressed in these cells, or that the endogenous viral genome is incomplete or defective.Infection of chicken cells with avian leukosis-sarcoma viruses results in substantial increases in the amounts of viral RNA and in production of progeny virus. The amounts of RNA and virus in infected cells are essentially the same in both endogenous chicken helper factor positive (chf+) and chicken helper factor negative (chf-) cells (4, 16 (13,20). Two classes of chf+ chicken embryos have been described, which contain high levels (hH) and extremely high levels (hE) of helper activity (4, 13). Where necessary, they are designated as chf+ (hH) and chf+ (hE). The Schmidt-Ruppin strain RSV, subgroup A (SR-RSV-A), SR-RSV-B (21), and SR-RSV-N8 (22) were kindly provided by S. Kawai of this laboratory; RAV-0 was spontaneously released from Line 100 chicken cells (kindly supplied by L. B. Crittenden, U.S. Dept. of Agriculture, East Lansing, Mich.). Other viruses used in this study were Rous associated virus-2 (RAV-2), RAV-7, and BH-RSV(-). Their preparation has been described before (6,21). BH-RSV-infected cell cultures were prepared by infection of chf-cells with BH-RSV(-) in the presence of ultraviolet light-inactivated Sendai virus (6, 16) and were fully transformed after several cell transfers.Nucleic Acid Extraction. Cellular and viral RNAs were extracted as described previously (4). Cell DNA was purified by the methods of Marmur (23) and Berns and Thomas (24) as modified by Schincariol and Joklik (25). For annealing experiments, the DNA was cleaved by acid hydrolysis to an average length of approximately 150 nucleotides (26).

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confidence: 99%

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confidence: 99%

Independent regulation of endogenous and exogenous avian RNA tumor virus genes.

Hayward¹,

Hanafusa²

1976

“…However, the A particles had a fine structure different from that of cores of mature C-type particles, as illustrated in Fig. 2 (18,22) were observed to be entirely devoid of the A-type particles. As will be seen belbw, the cytoplasmic A particles were elicited by several strains of avian tumor viruses.…”

Section: Resultsmentioning

confidence: 99%

“…(19,20). For most experiments, chicken cells negative for group-specific antigen (gs) and chicken helper factor (chf) (18) were used.…”

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confidence: 99%

Relationship between A-type and C-type particles in cells infected by Rous sarcoma virus.

Giuli¹,

Hanafusa²,

Kawai³

et al. 1975

Chicken cells infected with avian RNA tumor virus often contain small cytoplasmic A-type particles which commonly exist as clusters of 50-100 particles when viewed in thin sections. These particles were found more consistently in Rous sarcoma virus-infected than Rous-associated virus-infected cultures, but were generally present in only a small fraction of the total infected cells. The results of the survey of cells infected with various strains of leukosissarcoma viruses led to the hypothesis that the A particles develop in cells undergoing cytopathological degeneration. The hypothesis explains also the evanescent nature of the appearance of these particles in infected cells. The application of immunoelectron microscopic methods using monospecific antisera against viral internal proteins revealed that the A particles contain components immunologically related to the proteins of C-type virus.The process of assembly, release by budding, and maturation of the oncogenic RNA agents, including particles classified morphologically as C-type viruses (1, 2), occurs at the plasma membrane of infected cells (3-5). In avian systems one generally does not observe any recognizable precursor internal structures at the site of egress equivalent to the "A"-type particles found in murine and other infections. However, commencing with the report by Bernhard et al. (6), presence of aggregates of cytoplasmic A particles 60-70 nm in diameter has been described (7-10). Since such particles are not an invariable component of virus-producing avian cells they were considered to be not essential for replication. By contrast, in the case of murine agents A particles are present consistently, notably in tissue of mouse mammary carcinoma (2, 11), plasma cell tumors, and lymphomas (12)(13)(14) (19,20). For most experiments, chicken cells negative for group-specific antigen (gs) and chicken helper factor (chf) (18) were used.Antisera and Immunoelectron Microscopy. Monospecific antisera against purified RAV-2 proteins P27, P19, and P15 were obtained from immunized rats as described previously (21, 22). The specificity of each serum was determined by means of radioimmunoassay employing as antigen each respective polypeptide purified on guanidine-hydrochloride agarose columns and indicated less than 1% cross reactivity with any other antigen (22). Since antibody does not penetrate the plasma membrane of living cells, prior to immunological tagging the cells were released from monolayer by trypsin, washed repeatedly in cold Dulbecco's phosphate-buffered saline, made permeable by exposure for 2 min to 0.005% Nonidet P40 (NP40) to rupture the membrane, centrifuged at 800 X g for 10 min into a pellet, resuspended in Eagle's MEM nutrient medium (23), and incubated with monospecific antiserum at 370 for 30 min with continuous agitation. Unattached antibody was removed by centrifuging the cells and resuspending in nutrient medium twice. In some cases when the indirect immunoferritin technique (24) was employed the ruptured cells were exposed fi...

“…In other lines, partial expression of the endogenous provirus results in production of viral core proteins [group-specific antigens (gs)] and envelope proteins [chicken helper factors (chf)] (10,11). In addition to the gs-chf-phenotype, which does not produce either viral product, two other phenotypes have been described: gs+ chf+, in which both products are made (12,13), and gsL chfhE in which gs antigens are produced at low levels and helper is produced at high levels (14,15). By nucleic acid hybridization, the amount of viral specific DNA appears to be the same in all three phenotypes (1,2,16).…”

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confidence: 99%

Structural studies on oncornavirus-related sequences in chicken genomic DNA: two-step analyses of EcoRI and Bgl I restriction digests and tentative mapping of a ubiquitous endogenous provirus digests and tentative mapping of a ubiquitous endogenous provirus.

McClements¹,

Hanafusa²,

Tilghman³

et al. 1979

DNA from a variety of uninfected chicken cell types has been analyzed by using restriction endonuclease digestion and RPC-5 ion-exchange chromatography followed by agarose gel electrophoresis. Endogenous retrovirus sequences were detected by using a 32P-labeled avian leukosis viral RNA probe. One simple pattern was identified in an individual containing unexpressed endogenous proviral genes (gs-chf-phenotype for group-specific antigens and chicken helper factor) that was common to all individuals studied. A tentative restriction map has been derived for this and one other gs-chfendogenous provirus. Other gs-chf-individuals and individuals with other phenotypes (e.g., gs+ chf+ and gsL chfhE) showed more complicated patterns that often included additional bands and thus probably additional proviruses. RNA from an avian sarcoma virus was used to detect cellular sequences (sarc) homologous to the viral transforming gene (src). Results have revealed that a single restriction endonuclease EcoRI fragment of 13 X 106 daltons contains the majority of these sequences and confirm that they are not adjacent to the endogenous provirus.Uninfected chicken cells possess DNA sequences homologous to at least part of the RNA genomes of the avian retroviruses (1-5). Nucleic acid hybridization has shown these endogenous proviral sequences to be present in a few copies per haploid genome; however, a precise determination of the copy number is not possible by this technique (1, 4). Cells from certain chicken lines spontaneously release an endogenous virus, Rous-associated virus type 0 (RAV-0), (6, 7). These lines and other normally nonproducing lines that can be chemically induced to release virus (8, 9) must contain a complete viral genome that is capable of expression. In other lines, partial expression of the endogenous provirus results in production of viral core proteins [group-specific antigens (gs)] and envelope proteins [chicken helper factors (chf)] (10, 11). In addition to the gs-chf-phenotype, which does not produce either viral product, two other phenotypes have been described: gs+ chf+, in which both products are made (12, 13), and gsL chfhE in which gs antigens are produced at low levels and helper is produced at high levels (14, 15). By nucleic acid hybridization, the amount of viral specific DNA appears to be the same in all three phenotypes (1, 2, 16). However, Hayward and Hanafusa (17,18) have shown quantitative and qualitative differences in the virus-specific RNAs. Thus, differences in expression may be regulated at the transcriptional level.In addition to the endogenous provirus, all chicken cells contain DNA homologous to the transformation-specific (src) gene of avian sarcoma viruses (19). These cellular sequences (called "sarc") are not linked to the provirus (20) and are independently transcribed into poly(A)-RNA that is found in polysomes (21,22). The function of sarc in normal cells is ullknown.To investigate the organization of the provirus in normal cellular DNA and its possible relationship to vi...