Envelope-induced cell transformation by ovine betaretroviruses

Abstract

Ovine betaretroviruses include Jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV). JSRV and ENTV represent a unique class of oncogenic retroviruses that induce tumors of the respiratory tract. JSRV and ENTV are highly related but induce different diseases. Expression of the JSRV envelope (Env) induces transformation of rodent fibroblasts in vitro and phosphorylation of Akt, a central player in the phosphatidylinositol 3-kinase (PI-3K)/Akt signal transduction pathway. However, little information is available on the molecular biology of ENTV. In this study, we initially assessed whether the ENTV Env has the same properties as the homologous JSRV protein. We performed entry and interference assays using retroviral vectors pseudotyped with either the JSRV or the ENTV Env and sheep choroid plexus cells, choroid plexus cells stably expressing the JSRV Env protein, human 293T cells, mouse NIH 3T3 cells, or NIH 3T3 cells expressing human hyaluronidase 2 (HYAL2), the cellular receptor for JSRV. The results obtained indicated that ENTV and JSRV share the same receptor in sheep cells and that they can use human HYAL2 as a cellular receptor in mouse cells. The ENTV Env induces transformation of rodent fibroblasts in vitro. As with the JSRV Env, the tyrosine at position 590 is critical for ENTV Env-induced cell transformation, and Akt is phosphorylated in ENTV Env-transformed cells but not in the parental cell lines. Thus, ovine betaretroviruses share a common mechanism of cell transformation. We further investigated the relevance of Akt activation in cells transformed by ovine betaretroviruses. A PI-3K inhibitor blocked Akt phosphorylation in JSRV Env-transformed cells, suggesting a possible involvement of PI-3K in JSRV and ENTV Env-induced cell transformation. In addition, phosphorylated Akt was detected in a cell line derived from a lung tumor of a sheep with naturally occurring ovine pulmonary adenocarcinoma.

FIG. 1.

Amino acid alignment of the JSRV and ENTV Env proteins (GenBank accession no. AF105220 and Y16627). The identity of the JSRV and ENTV Env proteins at the amino acid level is 84%, but in the VR3 region they are only approximately 50% identical. The Y-X-X-M motif (positions 590 to 593) required for JSRV Env transformation is conserved in the ENTV Env.

FIG. 2.

Entry assays with MoMLV-luciferase vectors pseudotyped with the JSRV or ENTV Env protein. Values are expressed as relative light units (RLU) per milliliter; threefold dilutions were used in this experiment. Both JSRV and ENTV vectors enter SCP cells (CP), but their entry is inhibited in CP-ENVchim cells, which stably express JSRV Env, indicating that the two viruses use the same cellular receptor. Likewise, JSRV Env and ENTV Env can enter human (293) cells but cannot enter mouse (NIH 3T3) cells unless they express human HYAL2. Values are averages from duplicate experiments for each dilution. The experiments were repeated at least once with a different DNA preparation and gave essentially the same results.

FIG. 3.

Transformation assays in NIH 3T3 and 208F cells. NIH 3T3 and 208F cells were transfected with pCMV3JS21ΔGP or pCMV3ENTVΔGP as described in the text. Micrographs show the typical appearance of transformed foci after 3 weeks (NIH 3T3 cells) and 12 days (208F cells).

FIG. 4.

Akt activation in ENTV Env-transformed cells. Serum-starved cells transformed by ENTV or JSRV Env protein and the parental cell lines were photographed (A) and analyzed by SDS-PAGE and Western blotting (B) as described in Materials and Methods. Akt phosphorylated in serine is visible in the transformed cells but not in the parental cell lines. The same lysates were analyzed by using an antiserum against Akt as a loading control.

FIG. 4.

Akt activation in ENTV Env-transformed cells. Serum-starved cells transformed by ENTV or JSRV Env protein and the parental cell lines were photographed (A) and analyzed by SDS-PAGE and Western blotting (B) as described in Materials and Methods. Akt phosphorylated in serine is visible in the transformed cells but not in the parental cell lines. The same lysates were analyzed by using an antiserum against Akt as a loading control.

FIG. 5.

Akt activation in JSRV Env-transformed cells is PI-3K dependent. (A) Lysates of serum-starved NIH 3T3.JSRV cells (NIH 3T3 cells transformed by JSRV Env) were obtained after 30 min of incubation with different amounts of LY294002, a specific PI-3K inhibitor, and were analyzed by SDS-PAGE and Western blotting for the presence of phosphorylated Akt. (Top) Activation of Akt is inversely related to the quantity of LY294002 added to the medium. A 12.5 μM concentration of LY294002 is sufficient to inhibit Akt phosphorylation in JSRV Env-transformed cells. (Bottom) The same lysates were analyzed by using an antiserum against Akt as a loading control. (B) Akt activation is shown in NIH 3T3-JSENV cells, which stably express JSRV Env, but not in NIH 3T3-JSENVY590D cells, which express JSRV Env with a mutation of Y590. As above, the lysates were also analyzed for the presence of Akt as a loading control. (C) Lysates of serum-starved JS-8 cells were analyzed by SDS-PAGE and Western blotting for the presence of phosphorylated Akt. (Top) JS-8 shows constitutive activation of Akt, as does the JSRV-transformed cell line NIH 3T3.JSRV. (Bottom) Presence of Akt in the cell lysates as a loading control.