Mechanism of cell entry and transformation by enzootic nasal tumor virus

Abstract

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.

FIG. 1.

JSRV Env expression blocks transduction by ENTV and JSRV vectors in SSF cells. SSF cells and two clonal SSF cell lines (SSF/LJeSN c8 and c9) which contain the JSRV Env expression vector LJeSN were exposed to LAPSN vectors made with the indicated envelope proteins. Transduction was measured 3 days after vector exposure by staining the cells for AP⁺ foci. Results are means of two to three independent experiments with duplicate determinations performed in each experiment.

FIG. 2.

Evaluation of hHyal2, oHyal2, or bHyal2 proteins as receptors for ENTV and JSRV. HT-1080 and NIH 3T3 cells were seeded at a density of 5 × 10⁵/dish in 60-mm-diameter dishes. After 24 h, the cells were transfected with expression plasmids encoding hHyal2, oHyal2, or bHyal2 or an empty expression plasmid (none). The following day, the transfected cells were trypsinized (1.5 ml of trypsin), and 100-μl aliquots of each preparation were seeded in the wells of six-well dishes. SSF cells were also seeded at a density of 10⁵/well in six-well dishes. After 24 h, the cells were exposed to the ENTV or JSRV vectors. Two days after vector exposure, the cells were stained for AP⁺ foci. Results are means of data from two to four experiments, each done in duplicate. nd, not done.

FIG. 3.

Comparison of Hyal2 proteins from different species. (A) Comparison of human (U09577), mouse (AF302843), rat (AF034218), ovine (AF411974), and bovine (AF411973) Hyal2 protein sequences (GenBank accession numbers in parentheses). Amino acid identity (*), strong similarity (:), and weaker similarity (.) and amino acid differences between oHyal2 and bHyal2 (↓) are indicated. (B) Dendrogram of receptor similarity plotted using ClustalW. Scale bar indicates 5% sequence divergence.

FIG. 3.

Comparison of Hyal2 proteins from different species. (A) Comparison of human (U09577), mouse (AF302843), rat (AF034218), ovine (AF411974), and bovine (AF411973) Hyal2 protein sequences (GenBank accession numbers in parentheses). Amino acid identity (*), strong similarity (:), and weaker similarity (.) and amino acid differences between oHyal2 and bHyal2 (↓) are indicated. (B) Dendrogram of receptor similarity plotted using ClustalW. Scale bar indicates 5% sequence divergence.

FIG. 4.

Transduction of human and sheep cells by vectors bearing chimeric ENTV/JSRV Env proteins. (A) LAPSN vectors bearing the indicated Env proteins were made by transient transfection as described in Materials and Methods. SSF and HT-1080 cells were seeded at 10⁵ cells per well of six-well plates, exposed to the vectors 1 day later, and stained to detect AP⁺ foci 2 days after vector exposure. Results are means of data from two independent experiments with duplicate determinations performed in each experiment. (B) Sequence alignment of JSRV and ENTV Env proteins. Dots indicate identical amino acids, and the dash indicates a gap. The cleavage sites for restriction enzymes are indicated by vertical lines. The predicted locations of the endoplasmic reticulum signal sequence, the cleavage site between the Env protein surface (SU) and TM subunits, the membrane-spanning domain, and the cytoplasmic tail are shown.

FIG. 4.

Transduction of human and sheep cells by vectors bearing chimeric ENTV/JSRV Env proteins. (A) LAPSN vectors bearing the indicated Env proteins were made by transient transfection as described in Materials and Methods. SSF and HT-1080 cells were seeded at 10⁵ cells per well of six-well plates, exposed to the vectors 1 day later, and stained to detect AP⁺ foci 2 days after vector exposure. Results are means of data from two independent experiments with duplicate determinations performed in each experiment. (B) Sequence alignment of JSRV and ENTV Env proteins. Dots indicate identical amino acids, and the dash indicates a gap. The cleavage sites for restriction enzymes are indicated by vertical lines. The predicted locations of the endoplasmic reticulum signal sequence, the cleavage site between the Env protein surface (SU) and TM subunits, the membrane-spanning domain, and the cytoplasmic tail are shown.

FIG. 5.

The ENTV and JSRV env genes induce transformed foci in cultured mouse cells. A morphologically flat subline of NIH 3T3 mouse cells (originally from Doug Lowy) was seeded at 5 × 10⁵ cells per 6-cm-diameter dish. One day later, the cells were transfected with 10 μg each of the plasmids pSX2.Eenv (ENTV env), pSX2.Jenv (JSRV env), pSX2 (10A1 env) or pFBJ/R (viral fos oncogene) (23) plus 1 μg of pLAPSN (to measure transfection efficiency) by using the CaPO₄ coprecipitation method. One day after transfection, the cells were trypsinized and divided in a 1:5 ratio into new dishes. Four days after transfection, the medium was changed to medium containing 5% fetal bovine serum, and the cells were refed with this medium every 3 days thereafter. Eleven days after transfection, the cells were fixed and stained for AP. Representative foci are shown; no foci were observed in the 10A1 env dish. Two AP⁺ cells are visible at the lower right in the 10A1 env panel.