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The Gag and Gag-Pol precursors of avian sarcoma leukemia virus (ASLV) are translated from viral genomic-size mRNA at a molar ratio of about 20:1. Translation of Gag is terminated at the stop codon UAG located at the carboxyl-terminus of the viral protease (PR), whereas a ribosomal frameshift occurring at the carboxyl-terminus of Gag allows translation of the Gag-Pol precursor. To determine how PR is released from the Gag-Pol precursor, a single base (A or T) was inserted at the Gag-Pol junction in order to adjust the translation into a single reading frame. These mutations allow processing of the viral precursor when expressed in bacterial cells, but cause cessation of viral production after transfection of avian cells. The viral PR released from the large precursor is one amino acid longer than PR cleaved from the Gag polyprotein and is terminated by an Ile instead of a Leu residue.

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After budding from the plasma membrane, retrovirus particles undergo a process of maturation, which includes changes in morphology caused by several proteolytic cleavages of the precursor of the internal structural proteins, products of the gag gene. Cleavage is mediated by the viral protease, PR. The fact that in most systems cleavage appears to occur only after assembly is complete, suggests that PR may become enzymatically active as a consequence of release of the virion from the cell. Using avian leukosis virus as a model system, we tested the hypothesis that leakage of calcium ions into newly budded virions plays a role in their maturation. We found that in both quail Qt35 cells and monkey COS-1 cells, maturation occurred normally in calcium-free medium and in the presence of EGTA. A calcium ionophore also did not affect maturation. We conclude that calcium influx does not act as a trigger for PR-mediated maturation.

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Integration of retroviral DNA is a site-specific reaction involving an endonuclease encoded by the viral pol gene (pol-endo). In vitro the pol-endo from avian sarcoma and leukosis viruses (ASLVs) cleaves both DNA strands near the U5-U3 junction of tandem long terminal repeats (LTR-LTR junction) in single-stranded and replicative form (RF)-I substrates. We have reported previously that the sequences that are required for cleavage of single-stranded substrates by the alpha beta form of the pol-endo differ for the plus and minus strands (G. Duyk, M. Longiaru, D. Cobrinik, R. Kowal, P. deHaseth, A. M. Skalka, and J. Leis, J. Virol. 56:589-599, 1985). This is not the case with RF-I substrates, in which a maximum of 22 base pairs of U5 and 8 base pairs of U3 were required for alpha beta pol-endo cleavage in each strand. Insertion of a palindromic octanucleotide (CATCGATG) at the LTR-LTR junction abolished cleavage in RF-I but not in single-stranded DNA substrates. Deletion of the four nucleotides (TTAA) at the junction prevented cleavage in the plus strand of RF-I DNA, but did not affect cleavage of single-stranded DNA. Furthermore, the alpha beta form of ASLV pol-endo did not recognize heterologous LTR-LTR junction sequences from the reticuloendotheliosis virus or Moloney murine leukemia virus in either substrate form, despite their sequence and structural similarities to the ASLV junction. These results support a role for a sequence-specific interaction between the ASLV pol-endo and the LTR-LTR junction domains that are required for cleavage. By using the infectious Rous sarcoma virus clone pATV8-K, we introduced a set of deletions into the U5 region that would be incorporated into the LTR-LTR junction on viral replication. In the unintegrated provirus, the deletions started 43 base pairs from the LTR-LTR junction and extended various lengths toward the junction. Results of transfection studies with these clones indicated that the U5 sequences that are required for virus production in vivo correspond to those that are required for cleavage of RF-I DNA in vitro.

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The relationship of two early events in the establishment of infection by avian retroviruses, the inhibition of viral DNA synthesis in stationary avian cells and the secondary infection which occurs after infection of replicating cells, was investigated. When neutralizing antibody to spleen necrosis virus was used to prevent secondary infection, the amount of unintegrated linear spleen necrosis virus DNA detected was much lower in infected stationary cells than in infected replicating cells. The amount of unintegrated linear spleen necrosis virus DNA in stationary cells was less than one copy per cell even at high multiplicities of infection. Viral DNA synthesis resumed after stimulation of the cells to replicate. The time of this viral DNA synthesis was closely correlated with renewed cellular DNA synthesis. In addition, blocking secondary infection of replicating cells prevented the rate of virus production from reaching the high levels usually associated with a normal productive infection by SNV. Virus production increased if secondary infection was allowed. However, this rise in virus production was not proportional to the amounts of viral DNA integrated after secondary infection.

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Supercoiled DNA molecules were used for the molecular cloning of full-length avian sarcoma virus (ASV) DNA. Viral DNA produced by the Schmidt-Ruppin A (SR-A) strain of ASV was isolated from acutely infected transformed quail cells. Supercoiled DNA was separated from linear and open circular DNA by acid phenol extraction, opened into a full-length linear form by cleavage with the restriction endonuclease SacI, and cloned into lambda gtWES x lambda B. Four different cloned viral DNA molecules were isolated: SRA-1 contains two copies of the 330-base pair terminal redundancy normally found at each end of the linear DNA molecules, but harbors a 63-base pair deletion that spans the site at which the two copies of the terminal redundancy are joined in circular DNA molecules; SRA-2 contains two complete copies of the terminal redundancy; SRA-3 probably contains only one copy of the terminal redundancy but in all other respects appears to be similar to SRA-2; SRA-4 contains a 2,500-base pair deletion that removes all of the src gene (the gene responsible for transformation by ASVs) plus additional nucleotides adjacent to the src gene whose precise locations have not been determined. Transfection of chicken embryo fibroblasts by either SRA-1 or SRA-2 resulted both in the appearance of transformed cells and in the production of infectious virus. These results demonstrate that the cloned DNA molecules are functionally identical to viral DNA produced in vivo; therefore, molecular cloning did not cause any major alterations of the DNA. The infectivity of SRA-1 DNA indicates that the 63 base pairs missing from that molecule are not required for the initiation of viral RNA synthesis, even though the deletion is located in a copy of the terminal redundancy thought to carry a promoter for RNA synthesis. This suggests that the deletion does not remove any sequences required for the initiation of transcription.

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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.

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Inoculation of avian oncoviruses into 1- to 2-month old chickens led to a rapid production of antiviral humoral antibodies. Under these conditions it was found that avian leukosis viruses are sequestered in macrophages of peripheral blood, in which they can persist for a long period of time (up to about 3 years). In contrast, avian sarcoma viruses were never found in macrophages from chickens during the progression of sarcomas or after regression of the tumors.

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We previously reported the isolation of a newly recovered avian sarcoma virus (rASV) from tumors of chickens injected with transformation-defective (td) mutants of the Schmidt-Ruppin strain of Rous sarcoma virus (SR-RSV). In this paper, we present further biological and biochemical characterization of the recovered sarcoma viruses. High titers of rASV's were generally obtained by cocultivation of tumor cells with normal chicken embryo fibroblasts or by homogenization of tumor tissues. Most rASV isolates were similar to SR-RSV, subgroup A (SR-RSV-A), in their growth characteristics and were nondefective in replication. The subgroup specificity of rASV's and the electrophoretic mobilities of their structural proteins were the same as those parental td viruses. The nondefectiveness of rASV's was further substantiated by the size of their genomic RNA, which was indistinguishable from that of SR-RSV-A and substantially larger than that of parental td RNA. Molecular hybridization using complementary DNA specific to the src gene of SR-RSV (cDNAsrc) showed that the RNAs of td mutants used in this study contained extensive deletions within the src gene (7 to 30% hybridization with cDNAsrc); the same probe hybridized up to 90% with RNA from two isolates of rASV. These data indicate that rASV has regained genetic information which had been deleted in the td mutants and strongly suggest that the generation of rASV involves a genetic interaction between td virus and host cell genetic information.

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Previous studies by Guntaka et al. have shown that the unintegrated DNA intermediates of avian RNA tumor virus replication can be readily isolated from cultures of the quail tumor line QT-6 at 1 day after infection. The intermediates include double-stranded linear and covalently closed circular DNA species. Using the analysis procedure of Southern together with previously obtained information regarding the sites of action of certain restriction endonucleases on avian sarcoma virus DNA, we have further characterized the viral DNA intermediates. Evidence is presented that, relative to the RNA genome, most of the linear species possess a direct terminal sequence redundancy equivalent to 0.5 X 10(6) +/- 0.3 X 10(6) daltons of double-stranded DNA. Some of the circular forms also possess a sequence redundancy of 0.21 X 10(6) +/- 0.03 X 10(6) daltons.

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Treatment by glucosamine of avian sarcoma virus-transformed chicken embryo fibroblast (CEF) cells completely inhibited the formation of progeny-transforming virus particles. Such cells, however, could continue to synthesize non-infectious physical particles containing both viral RNA and the enzyme RNA-dependent DNA polymerase if glucosamine exposure was performed in the presence of glucose. Glucosamine treatment was found to affect antigenic expression in transformed CEF as measured by an indirect immunofluorescence test. Inhibition to a far lesser extent was observed when a lymphocyte stimulation assay for the detection of cell-mediated immunity was used in this system.

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The OK 10 virus complex was isolated from a liver tumour of a chicken which, as an embryo, had been inoculated intravenously with a field isolate of an avian leukosis virus. The OK 10 virus complex contains at least two viruses: the interference assay and serum neutralization test indicate that the helper virus belongs to subgroup A. One of the viruses, OK 10 V, induces distinct foci in chick embryo cells under agar overlay and cells from the foci form colonies in soft agar. These properties allow in vitro assay of the virus. Injection of virus or infected cells into chicks induces acute leukaemia but no local tumours. Another virus, OK 10 AV (associated virus), comprises about 99% of the OK 10 complex. The virus does not induce foci in chick embryo cells. In chickens it causes leukosis 17 months after injection. Electron micrographs of OK 10 virus stocks show typical C type virus particles. These particles have a density of 1.16 g/ml and contain 70S RNA which, after heat denaturation, releases type b RNA subunits. The OK 10 virus complex apparently represents a strain of acute leukaemia viruses.

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Exposure of chicken embryo fibroblasts (CEF) to trypsin solubilizes cell surface components, including the initial attachment site for avian tumour viruses (ATV). The soluble attachment site activity appears to interact directly with the ATV in vitro thereby interfering with the binding of at least two ATV subgroups to both intact CEF and CEF plasma membranes. A result of this interaction in vitro is reduced ATV infectivity, demonstrated by reduced transforming capacity of RSV (RAV-I).

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