Transfection by DNAs of avian erythroblastosis virus and avian myelocytomatosis virus strain MC29.

Chicken embryo fibroblasts and NIH 3T3 mouse cells were transformable by DNAs of chicken cells infected with avian myelocytomatosis virus strain MC29 or with avian erythroblastosis virus. Transfection of chicken cells appeared to require replication of MC29 or avian erythroblastosis virus in the presence of a nontransforming helper virus. In contrast, NIH 3T3 cells transformed by MC29 or avian erythroblastosis virus DNA contained only replication-defective transforming virus genomes.     

Neoplastic response of turkey liver to MC29 leukosis virus.

Twelve to fourteen days after intravenous inoculation of MC29 virus (1 x 10(5)LD50) into 1-day-old turkeys, liver tumour developed in 100% of the infected animals and led to death of birds. The tumour proved to be hepatomas histologically and electron microscopically. Numerous C-type particles were detectable among the tumour cells, as well as budding from the cell membrane. C-type particles were also observed in the tumour-free liver tissue in the spaces between the cells, budding was not detectable. Large number of virus particles were found in spleen extracellularly and intracellular vacuoles. Kidney tumours did not develop, but a few extracellular virus particles were located in the tissue. The MC29 virus-induced primary liver tumour in the turkey seems to be a suitable model for the morphological study of the relationship between oncogenic viruses and eukaryotic cells.     

Intranuclear degradation of the transformation-inducing protein encoded by avian MC29 virus

The nuclear protein, p110, encoded by the avian MC29 virus degrades with a half-life of 30 to 40 min in virus-transformed cells. Inhibitors of lysosomal proteolysis had no effect on this degradation. When inhibitors of RNA or protein synthesis were added immediately after pulse-labeling the p110 with [35S]methionine, degradation was impeded. Treatment of cells with cycloheximide prior to, and after, the pulse extended the half-life of p110 further than post-treatment alone, and addition of both actinomycin D and cycloheximide to cells pretreated with cycloheximide extended the half-life even further. In cells depleted of cellular ATP using a glucose-deficient medium containing oligomycin, degradation of p110 was only partially inhibited, indicating no direct involvement of ATP in degradation. Isolation of nuclei or nuclear matrices containing labeled p110, with subsequent incubation, resulted in minimal loss of p110 during several hours. These results suggest that p110 is degraded by a protease which is itself labile and freely diffusible from the nucleus, and, in addition, degradation may involve interaction of p110 with newly synthesized RNA.     

Genetic determinant of rapid-onset B-cell lymphoma by avian leukosis virus.

Infection of 10 day-old chicken embryos with the recombinant avian leukosis virus (ALV) EU-8 induces a high incidence of rapid-onset B-cell lymphoma by insertional activation of the c-myb gene. LR-9, a related ALV with differences from EU-8 in the gag and pol genes, induces rapid-onset lymphoma at only a low incidence. To localize the viral determinant(s) responsible for this biologic difference, we constructed and tested a series of reciprocal chimeras between EU-8 and LR-9 ALVs. The ability to induce rapid-onset lymphoma efficiently was localized to a 925-nucleotide (nt) region of the EU-8 gag gene. Sequence analysis of the region revealed a 42-nt deletion in EU-8 relative to LR-9, as well as some single-nucleotide changes. A mutant virus, delta LR-9, constructed by deleting these 42 nt from LR-9, also induced rapid-onset lymphoma at a high frequency, confirming the biologic significance of this deletion. This deletion removed nt 735 to 776, which lies within a cis-acting RNA element that negatively regulates splicing (NRS). The deletion was shown to cause an increase in splicing efficiency, which may lead to increased production of a truncated myb gene product from an ALV-myb readthrough RNA.     

Rate of virus-specific RNA synthesis in synchronized chicken embryo fibroblasts infected with avian leukosis virus.

The rate of avian leukosis virus (ALV)-specific RNA synthesis has been examined in bot- uninfected and ALV-infected synchronized chicken embryo fibroblasts. RNA from cells labeled for 2h with [3H]uridine was hybridized with avian myeloblastosis virus poly(dC)-DNA, and the hybridized RNA was analyzed with poly(I)-spephadex chromatography. Approximately 0.5% of the RNA synthesized in ALV-infected cells was detected as virus specific, and no more than a twofold variation in the rate of synthesis was detected at different times in the cell cycle. In synchronized uninfected chicken embryo fibroblasts, approximately 0.03% of the RNA synthesized was detected as virus specific, and no significant variation in the rate of synthesis was observed during the cell cycle. Treatment of ALV-infected chicken embryo fibroblasts with cytosine arabinoside or colchicine was used to block cells at different stages in the cell cycle. The rates of virus-specific RNA synthesis in cells so treated did not differ significantly from the rates in either stationary or unsynchronized virus-infected chicken embryo fibroblasts. These findings support the conclusion that after the initial division of an ALV-infected chicken embryo fibroblast and the initiation of virus RNA synthesis, the rate of virus-specific RNA synthesis is independent of the cell cycle.     

Transformation of avian myogenic cultures with myelocytomatosis virus strain 29

Summary Primary cultures of proliferating chick presumptive myoblasts were exposed of either to two RNA tumor viruses and shortly thereafter treated with 5-bromodeoxyuridine (BUdR) to suppress differentiation. The effect of a Rous sarcoma virus which was temperature-sensitive for transformation (tsRSV) has been characterized previously and was used as a reference for evaluating the effect of a myelocytomatosis virus (MC29) and its helper. Two subcultures following exposure, both infected cultures were extensively transformed as indicated by cell morphology. Relaxation of the BUdR block at this time resulted in cultures which still appeared transformed and did not contain myoblast or myotube-like cells or two of their molecular markers. In contrast, uninfected controls and tsRSV-infected cultures which were shifted-up to the nonpermissive temperature produced numerous spontaneously contracting myotubes. The results confirm previous evidence that infection of presumptive myoblasts by tsRSV at the premissive temperature preserves the extant state of differentiation of presumptive myoblasts and suggest, by analogy, that MC29-infection renders a similar effect.     

Proteins specified by Sindbis virus in chick embryo fibroblast cells

Large amounts of high molecular weight polypeptides were detected in “aged” chick embryo fibroblast cells infected with Sindbis virus. These polypeptides were shown to be virus specific by several criteria. At least some of the above polypeptides were shown to be precursors to smaller viral structural proteins by a pulse-chase experiment.     

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.     

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.     

In situ expression of helper-free avian leukosis virus (ALV)-based retrovirus vectors in early chick embryos.

Defective avian leukosis-based vectors expressing the bacterial lacZ gene were used as helper-free preparations to infect early stage Brown-Leghorn embryos. Both in toto X-gal staining and DNA analysis using Southern blot technique were applied to detect virus integration and expression. Our results demonstrate a low efficiency of in vitro infection in early stages of embryonic development. Southern blot analysis reveals that only 1% of embryonic cells integrate the vector genome after infection using 2 to 12 virus particle per embryonic cell. In situ expression of the lacZ marker gene was detected in only 0.06% of embryonic cells. These results lead us to conclude that only 6% of infected cells express efficiently the lacZ marker gene. This low level of expression could result from avian leukosis virus LTRs inhibition in chicken embryonic cells at an early stage of development. In spite of the low efficiency of infection, no evidence for tissue restrictive expression was observed. However, vector containing LTRs from RAV-2 virus allows preferential expression of provirus vector in neural tube tissue, whereas cardiac localization of the preferential expression was observed using vector containing the RAV-1 LTRs. The chronological analysis of the marker gene expression in terms of location of expression foci and sizes of these foci, lead us to hypothesize the putative regulation of retrovirus expression linked to embryonic development.