Lyman B. Crittenden

Activation of the cellular oncogene c-erbB by ltr insertion: Molecular basis for induction of erythroblastosis by avian leukosis virus

Avian leukosis virus (ALV), a slowly oncogenic retrovirus, induces in chickens a variety of neoplasms, including lymphoid leukosis and erythroblastosis. In lymphoid leukosis, a cellular oncogene, c-myc, is activated by the insertion of ALV LTR. We provide evidence that ALV utilizes a similar mechanism in erythroblastosis induction by activating a different cellular oncogene, c-erbB. We report the isolation, from leukemic erythroblast DNA, of a clone that represents the viral-cell junction fragment and carried the ALV LTR and part of the c-erbB locus. Restriction and sequence analyses reveal that the LTR is located upstream from the erbB coding region and is oriented in the same transcriptional direction; such a structure would be compatible with the promoter-insertion type of activation. Our findings provide a molecular explanation for the multipotency of slowly oncogenic retroviruses.

Transgenic chickens: Insertion of retroviral genes into the chicken germ line

We infected early chicken embryos by injection of wild-type and recombinant avian leukosis viruses into the yolk of unincubated, fertile eggs. The viremic males (designated generation 0 (G-0] were tested for transmission of proviral DNA to their G-1 progeny. Nine of 37 G-0 viremic males were mosiac and proviral DNA was transmitted to their progeny at frequencies varying from 1 to 11%. All of the G-1 progeny examined by restriction enzyme analysis for clonality of proviral junction fragments had one to three simple but different fragments. The proviral DNA was transmitted from G-1 to the G-2 progeny in a Mendelian fashion thus proving that retroviral genes have been inserted into the chicken germ line. One of the viruses is a candidate vector for insertion of foreign genes into the chicken germ line.

Exogenous and endogenous leukosis virus genes--a review.

Lymphoid leukosis is induced by exogenous retroviruses (LLV) that replicate via a DNA intermediate that is integrated into host somatic cell DNA. Variation in endogenous LLV expression is largely controlled by location of DNA proviruses integrated in the host germ-line and inherited as Mendelian genes. Present evidence suggests that endogenous viral genes (ev loci) arose from germ-line integration of exogenous LLVs and that those integrated ev loci expressing high levels of virus are at a selective disadvantage. Exogenous LLV infection leads not only to development of LL in some chickens but also to reduced productivity of layers. Results of preliminary experiments suggest that variation in ev locus expression influences non-oncogenic pathology and immune response after infection with an LLV known to be structurally related to ev loci. The effect of these loci on response to infection by unrelated micro organisms and on productivity should now be studied to determine whether the breeder should be concerned with these genes.

Interaction of genes controlling resistance to RSV(RAV-O).

Abstract Inbred lines 6, 7, 15, and 100 and some of their F 1 and backcross chick embryos were studied for resistance to subgroups A, B, C, and E sarcoma viruses, and the occurrence of the R 1 erythrocyte and viral group-specific (gs) antigens in cell culture. Embryos from the same matings were assayed for resistance to subgroup B and E viruses on the chorioallantoic membrane and for the occurrence of the R 1 antigen. The data suggest that (1) RSV(RAV-O) cannot readily infect subgroup B virus resistant cells; (2) the dominant gene, I e , probably exists in line 6 and is associated with the occurrence of the gs antigen; (3) the occurrence of the R 1 erythrocyte antigen is associated with enhancement of susceptibility to RSV(RAV-O) in the presence of the dominant inhibitor ( I e ); (4) the R 1 antigen is associated with the occurrence of gs antigen in the absence of I e , (5) genes exist which cause resistance to RSV(RAV-O) on the chorioallantoic membrane, but not in culture. These results suggest that the tumor virus b locus controls the existence of a receptor for subgroup B and E viruses, but that other genes, associated with the occurrence of viral antigens (gs and R 1 ), interact to inhibit or enhance susceptibility to RSV(RAV-O).

Lymphoid leukosis viruses and GS antigen in unincubated chicken eggs.

Lymphoid leukosis viruses and viral group-specific antigen were found in albumen of unincubated chicken eggs stored at 8 degrees C. Infectious virus was detected for up to 22 days and antigen was stable for 63 days. Tests for virus were conducted on albumen withdrawn from eggs prior to incubation and on extracts of embryos from the same eggs. When albumen was from eggs stored no longer than 6 days, virus was isolated from 20% more albumen samples than embryo extracts. The techniques described should be useful in programmes to eradicate lymphoid leukosis viruses from commercial poultry.

Production of unique C-type viruses by chicken cells grown in bromodeoxyuridine

Abstract Growth in bromodeoxyuridine (BrdU) increased the level of expression of endogenous avian C-type virus in certain 100 × 7 and line 15 chick embryo fibroblasts (CEF). The virus produced by BrdU treated 100 × 7 CEF is a subgroup E avian leukosis virus (ALV) with a more restricted host range than found for other subgroup E ALVs. The virus produced by BrdU-treated line 15 cells did not grow on cells which supported the growth of subgroups A, B, C, D, and E ALV and appears to be either a new or defective avian C-type virus. CEF from nine commercial strains and one other inbred line of chickens did not produce avian C-type virus either before or after growth in BrdU.

Genetic control of RAV-0 production in chickens

Abstract Progeny of backcross and second backcross matings of a RAV-0-producing line to a line producing RAV-0 at a low frequency were assayed for RAV-0 production. The data agreed with the hypothesis that the producing line was homozygous for an autosomaldominant gene controlling RAV-0 production. A second line was crossed and backcrossed to the low-producing line, and the frequency of RAV-0 production by the progeny also agreed with the single-dominant gene hypothesis. However, when the two producing lines were crossed and their F 1 progeny were mated to the low-producing line, 80% of the progeny produced RAV-0. This deviation from the expected of 100%, assuming that the two lines were homozygous at the same locus, suggested that the RAV-0-controlling genes were at different loci in the two lines. An alternative hypothesis, which supposes that the negative tester line carries a defective provirus which is complemented by recessive, alleles from either positive line also is suggested.

The role of the tvb locus in genetic resistance to RSV(RAV-0)

Abstract Genetic susceptibility to RSV(RAV-0), an avian sarcoma virus belonging to subgroup E. has been studied in two sets of inbred chicken lines. Data from the Reaseheath lines suggested that a recessive gene, independent of susceptibility to subgroup B, controlled resistance to subgroup E virus. This gene was assumed to be a new tve locus. Studies of the Regional Poultry Research Laboratory (RPRL) lines suggested that a recessive gene for resistance was located at the tvb locus and thus genetically linked to resistance to subgroup B virus. Crosses and backcrosses of Reaseheath and RPRL lines were assayed for susceptibility to RSV(RAV-0) and RSV(RAV-2) on the chorioallantoic membrane. The data completely support the hypothesis that a single locus controls recessive resistance to subgroup B and E viruses. Therefore, there was no evidence for an independent tve locus.

Expression of avian reticuloendotheliosis virus envelope confers host resistance.

We constructed two reticuloendotheliosis virus (REV) envelope gene expression plasmids, one containing the REV-A envelope gene, the other the spleen necrosis virus (SNV) envelope gene. Cell lines were generated by transfecting each of the REV envelope plasmids into D17 cells, a canine cell line. The levels of REV envelope glycoprotein in the cell lines were assayed by immunoprecipitating the envelope glycoproteins from lysates of cells that were labeled with [35S]methionine. Virological challenge assays determined the degree of resistance of each of the cell lines to REV-A or SNV infection. The expression of either envelope gene protected the cells from infection by either REV-A or SNV virus. Several cell lines were significantly more resistant to REV infection than the parental D17 cells, and two lines were 25,000-fold more resistant, approaching the resistance of REV-infected D17 cells to reinfection. The resistant cell lines were not able to confer resistance to susceptible cells by cocultivation. The level of resistance was correlated with the uniformity of expression of the REV envelope glycoproteins by the individual cells in a cell line and not with the absolute level of expression by the population of cells.

Endogenous virus expression in chicken lines maintained at the Regional Poultry Research Laboratory

Abstract Lines of chickens maintained at the Regional Poultry Research Laboratory (RPRL) and free of exogenous lymphoid leukosis virus (LLV) infection were assayed for expression of endogenous LLV. Embryos were assayed for the expression of infectious subgroup E LLVs, particulate RNA-directed DNA polymerase, antigens which could serve as envelope antigens for the envelope defective Bryan Rous sarcoma virus (chf), and the group-specific antigens of the leukosis-sarcoma virus group (gs). RPRL inbred lines 61, 63, 72, 100, and 15B were similar in endogenous virus expression to the previously characterized Beltsville Agricultural Research Center inbred lines 6, 72, 100, and 15. The expression of endogenous LLV was characterized for the first time in three sublines of RPRL line 15 chickens. Line 151, was gs+ chf+; line 15I4, gs− chf+; and line 15I5, gs− chf+. Some 151, and 15I5 embryos produced infectious subgroup E, but all 15I4 embryos produced infectious subgroup E virus. Two RPRL noninbred lines were also characterized Line P was segregating for subgroup E virus production and the gs+ chf+ phenotype. Line N was segregating for subgroup E virus production, the gs+ chf+ and gs− chf+ phenotypes.