Studies of ENTV and JSRV have been difficult because of the lack of an in vitro cell culture system for growing the viruses. In the case of JSRV, it has been shown that the LTR is inactive in most cell types tested (26
). Production of JSRV in culture has been accomplished by transfecting 293T cells with a recombinant JSRV genome in which the U3 region of the upstream JSRV LTR is replaced with a strong human cytomegalovirus promoter (30
). However, while virus produced in this manner can infect various ovine cell lines, it replicates poorly and little virus is produced (31
). To circumvent these difficulties, we constructed an MoMLV-based packaging line that expresses the JSRV Env protein and showed that JSRV Env is incorporated into virions with MoMLV Gag-Pol and MoMLV-based retroviral vectors (35
). We later used this to determine the host range of the JSRV Env (35
) and to identify the JSRV cell surface receptor (36
). Here we have used the same approach for ENTV and show that the ENTV Env protein can pseudotype MoMLV-based retrovirus vectors, thus allowing us to study the properties of the ENTV Env protein.
We provide strong evidence that JSRV and ENTV use the same cell surface receptor, Hyal2. Nonpermissive cells were rendered susceptible to transduction by both ENTV and JSRV vectors when Hyal2 from ovine, bovine, or human species was expressed in the cells. Furthermore, expression of JSRV Env in sheep cells blocked entry by ENTV and JSRV vectors, showing that ENTV uses a subset of the receptor(s) used by JSRV. Although we cannot entirely rule out the possibility that ENTV and JSRV use multiple receptors, previous studies indicate that at least in human cells, JSRV utilizes only one receptor. Thus, analysis of JSRV entry into radiation hybrid cells shows that only a small region of human chromosome 3 that contains the hyal2
gene can render hamster cells permissive to transduction by a JSRV vector (35
). Additionally, while two paralogs of hyal2
that might encode JSRV receptors, hyal1
, map to the same region, the proteins they encode do not function as receptors for JSRV, nor do those of the other hyal2
, that are located on human chromosome 7 (6
). The findings that both ENTV and JSRV use oHyal2 as a receptor and that Hyal2 is ubiquitously expressed in different tissues of other mammals including mice and humans (6
) argues that the tissue-specific oncogenesis by these viruses is not determined at the level of receptor-mediated virus entry. Furthermore, JSRV can enter and integrate into cell types from many tissues in culture and in animals, including nasal turbinate cells, the target for ENTV oncogenesis (14
), yet JSRV oncogenesis is limited to cells of the lower airway, again indicating that oncogenesis is not regulated at the receptor level.
The ENTV vector titers were lower for all cell types, including sheep cells, compared to the JSRV vector titers. By exchanging the TM domain of the ENTV Env with that of the JSRV Env, we were able to produce a vector that transduced sheep cells as efficiently as a JSRV vector without changing its restricted tropism for cells from other species. This particular clone of the ENTV Env may have a general defect in this region, or perhaps the ENTV Env is less able to interact with MoMLV Gag proteins and pseudotype MoMLV vectors than is the JSRV Env protein.
While both the JSRV and ENTV Env proteins can mediate efficient vector entry using the sheep Hyal2 receptor, they differ in their ability to use the Hyal2 orthologs from other species. This property has been observed previously for other viruses that utilize the same receptor; for example, both MoMLV and the closely related PVC211 retrovirus use the cationic amino acid transporter Cat1 from mouse and rat as a receptor, but only PVC211 can use hamster Cat1 as a receptor (19
). Thus, a difference in host range conferred by different Env proteins does not necessarily imply the use of unrelated receptors for Env-mediated cell entry. This apparent paradox can be explained by postulating differences in the abilities of the JSRV and ENTV Env proteins to efficiently bind specific Hyal2 orthologs from different species. Interestingly, while the ENTV vector was unable to transduce bovine cells, it was able to transduce cells transfected with a bHyal2 expression construct, presumably because overexpression of bHyal2 can offset the binding inefficiency of ENTV Env and promote virus entry. Along these lines, we found that ENTV vectors transduce 293 cells but not other human cells and hypothesize that 293 cells express more Hyal2 than do the other cell types. By exchanging a few residues within the SU domain of ENTV with equivalent residues of JSRV, we were able to create a chimeric Env that uses the endogenous human receptor, thus identifying the differences in JSRV Env that allow it to efficiently interact with Hyal2 orthologs.
Mechanisms of retroviral oncogenesis have previously been divided into two major categories: insertional mutagenesis, which typically involves proviral integration in or near a cellular proto-oncogene, such as c-myc
), leading to increased expression of an oncogenic protein, and virus expression of oncogenes derived from cellular genes, as in the case of v-src
or v -sis
). ENTV provides a new example of a third mechanism of retroviral oncogenesis that involves the presence of oncogenic viral proteins that are not derived from cellular genetic material. JSRV and avian hemangioma virus are recent additions to the latter category, and as we have shown for ENTV, the Env proteins from these viruses all exhibit transforming activity (1
). Spleen focus-forming virus is an early example of a retrovirus with an oncogene derived from viral env
sequences, but in this case the Env protein is nonfunctional, and spleen focus-forming virus is replication defective (25
). Last, human T-cell leukemia virus type 1 and bovine leukemia virus are complex retroviruses that exhibit characteristics of the latter category due to the oncogenic properties of Tax, a viral protein that normally transactivates the viral LTR and which can also interact with cellular transcription factors, leading to the activation of many cellular genes, including those that stimulate growth (5
). These results provide increasing evidence that retroviral proteins can evolve oncogenic properties, as have the proteins of viruses in many other virus families.
ENTV and JSRV use the same receptor for cell entry, and both express Env proteins with transforming activity, indicating that some other factor governs their tissue-specific tumorigenesis. JSRV and ENTV have markedly different enhancer regions within their LTRs, suggesting that tissue specificity of disease might be controlled at the transcriptional level. Indeed, the JSRV LTR is specifically active in cells from the lower airway, including type II pneumocytes and Clara cells (26
), the targets for JSRV oncogenesis. The sequence of the U3 region of the JSRV LTR reveals potential binding sites for transcription factors hepatocyte nuclear factor 3 (HNF-3), NF-1, and SP-1 (26
), which have been shown to function synergistically in regulating expression of lung-specific genes, such as the surfactant genes (18
). Biochemical analysis shows that HNF-3 strongly transactivates the JSRV LTR when overexpressed in NIH 3T3 cells, whereas other transcription factors that are expressed in the epithelial cells of the lung did not cause transactivation (26
). The U3 region of the ENTV LTR (4
) is dramatically different from the JSRV LTR in that it contains only the SP-1 site and lacks many of the other regulatory sequences that are found in JSRV, including the NF-1 site and the two HNF-3 sites. The ENTV LTR contains a binding site for the nuclear transcription factor Y which is not found in the JSRV LTR. Identification of the factors involved in ENTV transcriptional regulation will help to clarify how ENTV and JSRV cause tissue-specific transformation. However, such studies are complicated because it has been difficult to identify candidate transcription factors that transactivate the ENTV LTR and cells which comprise the nasal epithelium are quite diverse, making it difficult to identify the correct lineage of cells for such studies. A good approach to addressing these questions would be to study induction of disease in animals infected with hybrid viruses that contain the structural genes of ENTV or JSRV under the control of the LTR from the other virus. Direct evidence for the transcriptional regulation of ENTV will help to better define the mechanism by which ENTV and JSRV induce tissue-specific cancers.