Genome structure and expression of the ev/J family of avian endogenous viruses.

We recently reported the identification of sequences in the chicken genome that show over 95% identity to the novel envelope gene of the subgroup J avian leukosis virus (S. J. Benson, B. L. Ruis, A. M. Fadly, and K. F. Conklin, J. Virol. 72:10157-10164, 1998). Based on the fact that the endogenous subgroup J-related env genes were associated with long terminal repeats (LTRs), we concluded that these LTR-env sequences defined a new family of avian endogenous viruses that we designated the ev/J family. In this report, we have further characterized the content and expression of the ev/J proviruses. The data obtained indicate that there are between 6 and 11 copies of ev/J proviruses in all chicken cells examined and that these proviruses fall into six classes. Of the 18 proviruses examined, all share a high degree of sequence identity and all contain an internal deletion that removes all of the pol gene and various amounts of gag and env gene sequences. Sequencing of the gag genes, LTRs, and untranslated regions of several ev/J proviruses revealed a high level of identity between isolates, indicating that they have not undergone significant sequence variation since their introduction into the avian germ line. Although the ev/J gag gene showed a relatively weak relationship (46% identity and 61% similarity at the amino acid level) to that of the avian leukosis-sarcoma virus family, it retains several sequences of demonstrated importance for virus assembly, budding, and/or infectivity. Finally, evidence was obtained that at least some members of the ev/J family are expressed and, if translated, could encode Gag- and Env-related polypeptides.     

Immunological phenotype of lymphomas induced by avian leukosis viruses.

The production of immunoglobulin by six cell lines derived from bursal tumors induced by avian leukosis virus follows two general patterns: (i) three cell lines that have been extensively passaged in culture synthesize and secrete light chains only; (ii) three cell lines that are recently isolated produce and secrete monomeric immunoglobulin M in addition to free light chains. All six cell lines synthesize and secrete both glycosylated and unglycosylated forms of light chain. We conclude that the cell lines established from lymphomas induced by avian leukosis virus represent relatively mature, but possibly abnormal, stages in the development of chicken B-lymphocytes. The immunoglobulin M produced by the cell lines failed to form detectable immune complexes with avian leukosis virus. It therefore appears that the immunoglobulin M is not directed against viral antigens and that autogenous antigenic stimulus cannot account for the sustained growth of the neoplastic B-lymphocytes.     

Absence of missense mutations in activated c-myc genes in avian leukosis virus-induced B-cell lymphomas.

We have determined the nucleotide sequences of two independent DNA clones which contained the activated c-myc genes from avian leukosis virus-induced B-cell lymphomas. Neither of these c-myc genes contained missense mutations. This strongly supports the notion that the c-myc proto-oncogene in avian leukosis virus-induced B-cell lymphomas can be oncogenically activated by altered expression of the gene without a change in the primary structure of the gene product.     

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.     

Detection and Assay of Avian Tumor Virus Group-Specific Antigen and Antibody by the Paired Radioiodine-Labeled Antibody Technique

The paired radioiodine-labeled antibody technique (PRILAT) was applied to the detection and quantitation of avian tumor virus group-specific (gs) antigens and antibody. The technique proved to be specific, repeatable, and appreciably more sensitive than the microcomplement-fixation test for avian leukosis (COFAL). The PRILAT facilitated direct measurement of comparative antigen content of several types of transformed, neoplastic, or virus-infected cells and the magnitude of nonspecific antibody binding by appropriate control cells. The versatility of the technique was illustrated by application to the detection and quantitation of gs antibody content of chicken, turkey, pigeon, and hamster sera. Antibodies were detected in COFAL-negative sera from hamsters bearing tumors induced by the Schmidt-Ruppin strain of Rous sarcoma virus. Sera from chickens bearing similar tumors were not positive for gs antibodies, although sera from turkeys and chickens immunized with avian leukosis virus did contain gs antibodies.     

Artificial insertion of a dominant gene for resistance to avian leukosis virus into the germ line of the chicken

Summary This report describes the unique biological properties of a transgenic chicken line that contains a defective avian leukosis virus (ALV) proviral insert that we call alv6. Chick embryo fibroblasts (CEF) containing this insert express subgroup A envelope glycoprotein since they yield focus-forming pseudotype virus when co-cultivated with transformed quail cells expressing envelope-defective Bryan high-liter Rous sarcoma virus (RSV). In addition, these cells display high interference to subgroup A RSV but not to subgroup B RSV infection. Chickens containing this insert are highly resistant to pathogenic subgroup A ALV infection, but show little immunological tolerance to subgroup B ALV infection. Thus we have artificially inserted a dominant gene for resistance to avian leukosis infection into the chicken germ line.     

Acutely transforming avian leukosis virus subgroup J strain 966: defective genome encodes a 72-kilodalton Gag-Myc fusion protein

Avian leukosis virus subgroup J (ALV-J), the most recent member of the avian retroviruses, is predominantly associated with myeloid leukosis in meat-type chickens. We have previously demonstrated that the acutely transforming virus strain 966, isolated from an ALV-J-induced tumor, transformed peripheral blood monocyte and bone marrow cells in vitro and induced rapid-onset tumors, suggesting transduction of oncogenes (L. N. Payne, A. M. Gillespie, and K. Howes, Avian Dis. 37:438-450, 1993). In order to understand the molecular basis for the rapid transformation and tumor induction, we have determined the complete genomic structure of the provirus of the 966 strain. The sequence of the 966 provirus clone revealed that its genome is closely related to that of HPRS-103 but is defective, with the entire pol and parts of the gag and env genes replaced by a 1,491-bp sequence representing exons 2 and 3 of the c-myc gene. LSTC-IAH30, a stable cell line derived from turkey monocyte cultures transformed by the 966 strain of ALV-J, expressed a 72-kDa Gag-Myc fusion protein. The identification of the myc gene in 966 virus as well as in several other ALV-J-induced tumors suggested that the induction of myeloid tumors by this new subgroup of ALV occurs through mechanisms involving the activation of the c-myc oncogene.