We have developed Mab against the Env protein of JSRV that give intense staining of lung tumors in sheep infected with JSRV and in mice exposed to an AAV6 vector encoding JSRV Env. These Mab recognized tumors in all JSRV-infected sheep examined (n = 29) from multiple countries. The antibodies did not recognize similar urethane-induced lung tumors in mice. Both urethane- and JSRV Env-induced lung tumors have the same histologic appearance, express the type II alveolar cell marker surfactant protein C and most do not express the Clara cell marker CC-10 [10, 18, 32]. The Mab did not recognize alveolar type II cell hyperplasia or other cell types in a variety of diseases in sheep that were not the result of JSRV infection. We also found that at least two of the Mab recognized the Env from ovine ENTV in tumors induced in mice by exposure to an AAV6 vector encoding ENTV Env , and in a sheep with nasal adenocarcinoma associated with ENTV infection. Together these results indicate that the Mab are highly specific for ovine betaretrovirus Env expression, and would provide a useful diagnostic test for JSRV, and possibly for ENTV as well.
The current accepted nomenclature for lung cancer resulting from JSRV infection is ovine pulmonary adenocarcinoma. The primary reason for its characterization as a malignant disease is because of the observation of metastases consisting of lung tumor epithelial cells, which occurs to a variable extent in sheep . However, the main tumor type we see in JSRV-infected sheep and in JSRV Env-expressing mice is adenoma, consistent with the previous description of the disease as an adenomatosis. Given our results in sheep and in mice, and the fact that what kills these animals is breathing difficulty, it seems the primary effect of JSRV infection, mediated through the Env protein, is to cause proliferation of lung epithelial cells. In JSRV-infected sheep, such proliferation typically increases lung fluid production and thereby facilitates aerosol transmission of the virus produced by epithelial cells. Metastasis may occur as the result of additional genetic changes resulting from virus replication and integration, or resulting directly from Env expression and stimulation of cell proliferation, but these appear not to be the primary effects of virus infection or Env expression.
It is remarkable how little Env Mab staining we observe outside of tumors in mice and sheep. Others have reported similar results in sheep by using polyclonal antibodies to detect JSRV Env or capsid proteins [31–33], but our use here of highly specific Mab that give intense staining of Env-expressing cells helps to rule out the presence of low levels of Env expression outside of tumors. In mice we know that an AAV6 vector encoding AP (ARAP4) can transduce all epithelial cell populations in the airway with relatively high efficiency , yet we see no Env staining in large or small airways or in histologically-normal alveoli in mice exposed to the AAV6 vector ARJenv, which like ARAP4 contains a strong Rous sarcoma virus promoter to drive gene expression. It is known that oncoproteins can have both growth-promoting and toxic effects in cultured cells , and perhaps only lung stem cells that are the progenitors of tumors can tolerate expression of the potent Env oncoprotein, while Env expression is toxic to the more differentiated cells.
Lack of Env expression outside of tumors in lungs of sheep infected with JSRV is particularly surprising given the presence of replicating virus in the sheep. Perhaps spread of JSRV is inhibited in sheep by an immune response, despite the finding that sheep mount a poor response against JSRV because of immune tolerance induced by proteins made by related endogenous retroviruses . Alternatively, like other simple retroviruses, JSRV may only infect dividing cells, and most potential target cells in the lung are not actively dividing. Most intriguingly, it may be that Env is toxic to most differentiated lung cell types in sheep, as proposed above for mice.
Our results provide further support for the conclusion that the JSRV cell-entry receptor Hyal2 plays no role in sheep tumorigenesis beyond its role as a receptor for virus entry. Mouse Hyal2 does not serve as a cell-entry receptor for retrovirus vectors bearing the JSRV Env protein [4, 15–17] nor does it bind the SU domain of JSRV Env , yet we have shown here that lung tumors induced in mice by Env expression alone are quite similar to lung tumors induced by JSRV in sheep having a functional Hyal2 virus receptor.
Our results also argue against a role for insertional oncogene activation or insertional mutagenesis in sheep tumorigenesis. An AAV6 vector was used to transfer and express JSRV Env in the mice analyzed here, and tumor induction followed single-hit kinetics , a result that is inconsistent with a requirement for insertional events in addition to Env expression for tumorigenesis. In addition, inclusion of an excess of a non-oncogenic AAV6 vector during transduction by the JSRV Env-expressing AAV6 vector reduced the number of tumors , again indicating that additional genetic changes that might be caused by the AAV6 vector are not important for tumorigenesis. Together with results shown here that tumors induced by JSRV Env in mice are quite similar to tumors induced by JSRV in sheep, these results indicate that JSRV tumorigenesis is primarily dependent on the oncogenic activity of the JSRV Env protein and does not require genetic changes resulting from JSRV integration.
The main tumor type induced by systemic administration of the AAV6 vector encoding JSRV Env was hepatocellular adenoma. Generation of this non-malignant proliferative tumor is consistent with the activity of JSRV Env in the lung to generate adenomas arising from lung epithelial cells. Given the low frequency of hepatocellular adenocarcinomas following JSRV Env vector administration, it is likely that additional events are required for adenocarcinoma formation, as they appear to be following expression of other oncoproteins such as Myc .
Systemic administration of the AAV6 vector encoding JSRV Env to mice induced multiple hemangiomas and some hemangiosarcomas, tumors that arise from uncontrolled and disorganized proliferation of endothelial cells. Endothelial cells in these tumors were uniformly and uniquely stained by the Env Mab, indicating a direct effect of Env on endothelial cells in these tumors.
Oncogenes from other viruses can also induce hemangiomas and have helped elucidate a common pathway for hemangiogenesis that involves phosphatidylinositol 3-kinase (PI3K) activation, downstream activation of Akt, and increased vascular endothelial growth factor production; the latter being a key stimulus for hemangiogenesis. For example, avian sarcoma virus 16 induces hemangiomas and was found to express a viral oncogene derived from the gene encoding the catalytic subunit of PI3K . Viral vectors expressing the viral or cellular forms of the PI3K catalytic subunit could induce hemangiosarcomas in chickens and could transform chicken embryo fibroblasts in culture . Transformation in culture was accompanied by Akt activation and VEGF production, and overexpression of a myristylated form of Akt or VEGF itself could induce hemangiosarcoma formation in chicken embryos . Interestingly, JSRV Env has been shown to transform cultured fibroblasts from mice, rats, and chickens [4–6], and transformation is accompanied by activation of PI3K and Akt in these cells [8, 40, 41], suggesting that JSRV Env may induce hemangioma formation by activation of the PI3K-Akt-VEGF pathway in mouse endothelial cells.
Another retrovirus that induces hemangiomas is avian hemangioma virus, and like JSRV, this appears to be due to expression of the viral Env protein [42, 43]. However, the avian hemangioma virus Env protein shows no similarity to that of JSRV, so it is difficult to predict if the mechanisms of hemangiogenesis are similar. It will be interesting to see if AHV also activates members of the PI3K-Akt-VEGF pathway.
In 10% of JSRV-infected sheep studied we observed masses of Env+ fibroblastic cells that appear to be separate neoplasms. The ability of JSRV Env to transform fibroblasts from several species in tissue culture [4–6], and the uniform Env+ staining of the fibroblastic cell masses in sheep, make it tempting to speculate that these masses represent a novel tumor type. However, the frequent observation of non-neoplastic fibroblast or mesenchymal cell proliferation in response to a number of tissue insults complicates this interpretation. Others have observed similar proliferation of connective tissue in association with epithelial tumors in JSRV-infected sheep , but immunohistochemical analysis for Env expression was not performed. We did not see such Env+ fibroblastic masses in mice, but this could simply be due to a lower frequency of the parental cell type in mouse lung. Regardless, these fibroblastic masses were an infrequent occurrence in sheep and thus do not account for the typical disease observed in JSRV-infected sheep.
Nasal administration of the AAV6-Jenv vector to normal C57BL/6 mice results in strong immune responses against Env that limit tumor formation, therefore we have used immunodeficient C57BL/6 Rag-2 mice to model tumor formation by JSRV Env. The question arises whether an immunodeficient mouse is an appropriate model for a disease that occurs in immunocompetent sheep. In fact, expression of multiple endogenous retroviruses related to JSRV in sheep results in immunotolerance toward JSRV infection , thus immunodeficient mice appear to be a good model in which to study this intriguing viral disease.