Jaagsiekte sheep retrovirus (JSRV) can induce rapid, multifocal lung cancer, but JSRV is a simple retrovirus having no known oncogenes. Here we show that the envelope (env) gene of JSRV has the unusual property that it can induce transformation in rat fibroblasts, and thus is likely to be responsible for oncogenesis in animals. Retrovirus entry into cells is mediated by Env interaction with particular cell-surface receptors, and we have used phenotypic screening of radiation hybrid cell lines to identify the candidate lung cancer tumor suppressor HYAL2͞LUCA2 as the receptor for JSRV. HYAL2 was previously described as a lysosomal hyaluronidase, but we show that HYAL2 is actually a glycosylphosphatidylinositol (GPI)-anchored cell-surface protein. Furthermore, we could not detect hyaluronidase activity associated with or secreted by cells expressing HYAL2, whereas we could easily detect such activity from cells expressing the related serum hyaluronidase HYAL1. Although the function of HYAL2 is currently unknown, other GPI-anchored proteins are involved in signal transduction, and some mediate mitogenic responses, suggesting a potential role of HYAL2 in JSRV Env-mediated oncogenesis. Lung cancer induced by JSRV closely resembles human bronchiolo-alveolar carcinoma, a disease that is increasing in frequency and now accounts for Ϸ25% of all lung cancer. The finding that JSRV env is oncogenic and the identification of HYAL2 as the JSRV receptor provide tools for further investigation of the mechanism of JSRV oncogenesis and its relationship to human bronchiolo-alveolar carcinoma.J aagsiekte sheep retrovirus (JSRV) is the causative agent of a contagious lung cancer of sheep called ovine pulmonary carcinoma or sheep pulmonary adenomatosis (1). Tumors originate from type II secretory alveolar and nonciliated bronchiolar epithelial cells, and late stages of the disease are accompanied by the secretion of copious lung fluid containing the virus. Purified virus induces multifocal tumors in as little as 10 days (2), suggesting the role of a viral oncogene. However, JSRV is a simple retrovirus with typical gag, pol, and env genes and no known oncogenes. The viral structural (Gag) and enzymatic (Pol) proteins are unlikely to be responsible, because they interact primarily with viral components, but there is precedent for alteration of cellular functions by the envelope (Env) protein of retroviruses, which interacts with cellular components to mediate virus entry. For example, the deleted Env protein of the Friend spleen focus-forming virus causes erythroleukemia by binding to and activating the erythropoietin receptor (3). If the JSRV Env protein was indeed oncogenic, identification of the host cell receptor for JSRV would provide key insights into the oncogenic mechanism of this highly pathogenic retrovirus. Furthermore, the contagious nature of JSRV and its ability to survive exposure to proteases and surfactants present in lung fluid suggest that vectors based on JSRV might be useful for gene therapy targeted to the lung, pr...

Jaagsiekte sheep retrovirus (JSRV) is a type D retrovirus associated with a contagious lung tumor of sheep, ovine pulmonary carcinoma. Other than sheep, JSRV is known to infect goats, but there is no evidence of human infection. Until now it has not been possible to study the host range for JSRV because of the inability to grow this virus in culture. Here we show that the JSRV envelope protein (Env) can be used to pseudotype Moloney murine leukemia virus (MoMLV)-based retrovirus vectors and that such vectors can transduce human cells in culture. We constructed hybrid retrovirus packaging cells that express the JSRV Env and the MoMLV Gag-Pol proteins and can produce JSRV-pseudotype vectors at titers of up to 10 6 alkaline phosphatase-positive focus-forming units/ml. Using this high-titer virus, we have studied the host range for JSRV, which includes sheep, human, monkey, bovine, dog, and rabbit cells but not mouse, rat, or hamster cells. Jaagsiekte sheep retrovirus (JSRV) is the causative agent of a contagious lung cancer of sheep known as ovine pulmonary carcinoma (OPC), also known as sheep pulmonary adenomatosis or jaagsiekte. OPC is a veterinary problem with significant economic impact in several countries. In addition, OPC shares characteristics with human bronchioalveolar carcinoma (BAC) (12,32,49), and BAC represents about 25% of human lung cancer cases (6). Lung cancer being the most common fatal form of cancer in humans (10), recent interest in JSRV stems from the hypothesis that OPC could be useful as a naturally occurring animal model for understanding the mechanism of pulmonary carcinogenesis (26,49).JSRV has been classified as a type D retrovirus, based on genomic organization, but has a type B-like Env protein (61). The sheep genome carries multiple copies of JSRV-like endogenous sheep retrovirus (ESRV) sequences (25, 27, 61), but subsequent studies have shown that JSRV is an exogenous virus distinct from ESRV sequences (4, 5, 44) and is specifically associated with OPC. Recent studies by Palmarini et al. (48) using an infectious molecular clone of JSRV have confirmed that JSRV is the causative agent of OPC. The mechanism of oncogenesis by JSRV is not known. JSRV has the genomic organization of a simple replication-competent retrovirus with no known oncogenes. The incubation period in naturally acquired OPC seems to range from months to years, suggesting insertional mutagenesis. However, OPC can be induced experimentally in 3 to 4 weeks, suggesting a mechanism of action more similar to that of a transforming retrovirus.The main sites of JSRV replication and assembly are the transformed epithelial cells of the lung, especially the alveolar type II cells (45). The lung fluid and tumor extracts of infected sheep can be used for virus isolation and also for experimentally transmitting the disease to lambs by intratracheal inoculation (14,37,59), suggesting that the virus is stable in lung fluid. The stability of the virus in lung fluid as well as the ability to infect the epithelial cells of the lung i...

Enzootic nasal tumor virus (ENTV) induces nasal epithelial cancer in infected sheep, but it is a simple retrovirus lacking a known oncogene. ENTV is closely related to jaagsiekte sheep retrovirus (JSRV), which also causes cancer in sheep but in the epithelial cells of the lower airways and alveoli. Here we show that as with JSRV, the envelope (Env) protein of ENTV can transform cultured cells and thus is likely to be responsible for oncogenesis in animals. In addition, the ENTV Env protein mediates virus entry using the same receptor as does JSRV Env, the candidate tumor suppressor Hyal2. However, ENTV Env mediates entry into cells from a more restricted range of species than does JSRV, and based on this finding we have identified amino acid regions in the Env proteins that are important for virus entry. Also, because ENTV does not efficiently use human Hyal2 as a receptor, we cloned the ovine Hyal2 cDNA and show that the encoded protein functions as an efficient receptor for both ENTV and JSRV. In summary, although ENTV and JSRV use the same cell surface receptor for cell entry and apparently transform cells by the same mechanism, they induce cancer in different tissues of infected sheep, indicating that oncogenesis is regulated at some other level. The transcriptional regulatory elements in these viruses are quite different, indicating that tissue-specific oncogenesis is likely regulated at the level of viral gene expression. Enzootic nasal tumor virus (ENTV) is a simple retrovirusthat is transmitted horizontally and induces nasal adenocarcinoma in sheep (4, 10) and goats (8, 9). ENTV can be found in the nasal fluid of animals with intranasal tumors, which eventually progress and cause severe cranial deformations and respiratory blockage, resulting in death (41). ENTV is closely related to jaagsiekte sheep retrovirus (JSRV) (Ͼ95% overall amino acid similarity) (4), which is the causative agent of ovine pulmonary adenocarcinoma (also called sheep pulmonary adenomatosis or jaagsiekte) (30). Unlike tumors caused by ENTV, which arise from nasal epithelial cells, JSRV-induced tumors arise from epithelial cells in the lower airway, including type II alveolar and bronchiolar epithelial cells (27). The mechanism of oncogenesis by ENTV and its relationship to that caused by JSRV are unknown. Both of these viruses are present in many countries worldwide and have a significant economic and animal health impact. In addition, the disease induced by JSRV exhibits histological features similar to those of many human pulmonary adenocarcinomas, including bronchioloalveolar carcinoma (28, 33). Thus, study of adenocarcinoma induced by JSRV and ENTV may provide insights into the etiology of human lung cancer. While ENTV and JSRV do not appear to cause lung cancer in humans having occupational exposure to these viruses, a recent study shows that antiserum directed against the JSRV capsid protein cross-reacts with 30% of human pulmonary adenocarcinoma samples but not with nontumorous lung lesions, normal lung tissue, or many adenoc...

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Jaagsiekte sheep retrovirus (JSRV) replicates in the lungs of sheep and causes the secretion of copious lung fluid containing the virus. Adaptation of JSRV to infection and replication in the lung and its apparent resistance to the denaturing activity of lung fluid suggest that vectors based on JSRV would be useful for gene therapy targeted to the lung. We show here that a retrovirus vector bearing the JSRV Env is stable during treatment with lung surfactant while an otherwise identical vector bearing an amphotropic Env is inactivated. Furthermore, the JSRV vector was stable during centrifugation, allowing facile vector concentration, and showed no loss of activity after six freeze-thaw cycles. However, the JSRV vector was inactivated by standard disinfectants, indicating that JSRV vectors pose no unusual safety risk related to their improved stability under other conditions.

Jaagsiekte sheep retrovirus (JSRV) is a type D retrovirus associated with a contagious lung tumor of sheep, ovine pulmonary carcinoma. Other than sheep, JSRV is known to infect goats, but there is no evidence of human infection. Until now it has not been possible to study the host range for JSRV because of the inability to grow this virus in culture. Here we show that the JSRV envelope protein (Env) can be used to pseudotype Moloney murine leukemia virus (MoMLV)-based retrovirus vectors and that such vectors can transduce human cells in culture. We constructed hybrid retrovirus packaging cells that express the JSRV Env and the MoMLV Gag-Pol proteins and can produce JSRV-pseudotype vectors at titers of up to 10 6 alkaline phosphatase-positive focus-forming units/ml. Using this high-titer virus, we have studied the host range for JSRV, which includes sheep, human, monkey, bovine, dog, and rabbit cells but not mouse, rat, or hamster cells. Considering the inability of the JSRV-pseudotype vector to transduce hamster cells, we used the hamster cell line-based Stanford G3 panel of whole human genome radiation hybrids to phenotypically map the JSRV receptor ( JVR) gene within the p21.3 region of human chromosome 3. JVR is likely a new retrovirus receptor, as none of the previously identified retrovirus receptors localizes to the same position. Several chemokine receptors that have been shown to serve as coreceptors for lentivirus infection are clustered in the same region of chromosome 3; however, careful examination shows that the JSRV receptor does not colocalize with any of these genes.

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Local production of cytokines by genetically engineered tumor cells decreases their tumorigenicity and elicits protective immune responses against the parental tumor cells. An alternative approach to elicit a therapeutic immune response is to use fusion proteins that can target tumor cells and simultaneously activate effector cells. Fusion proteins between human IL-2, murine or human GM-CSF, and sFv of antihuman carcinoma antibody L6 have been constructed, expressed in both COS and Chinese hamster ovary (CHO) cells, and purified by affinity chromatography. The biologic activity of L6 sFV-hIL-2, L6 sFv-mGM-CSF, and L6 sFv-hGM-CSF was tested on human T cell blasts, factor-dependent FDCP-1, and TF-1 cells, respectively. The ability of soluble L6 sFv-hIL-2, L6 sFv-mGM-CSF, and L6 sFv-hGM-CSF to stimulate the proliferation of the indicator cells was found to be comparable to that of recombinant hIL-2, mGM-CSF, or hGM-CSF. Tumor cells coated with L6 sFV-mGM-CSF or L6 sFv-hGM-CSF were also tested in this way and were found to be potent stimulators, indicating that the cytokines were functionally active when bound to the tumor cell surface. This work demonstrates the feasibility of targeting sFv-cytokine fusion proteins for the activation of effector cells as an alternative to cytokine gene therapy.