Role of virus receptor Hyal2 in oncogenic transformation of rodent fibroblasts by sheep betaretrovirus env proteins

Abstract

The ovine betaretroviruses jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV) cause contagious cancers in the lungs and upper airways of sheep and goats. Oncogenic transformation assays using mouse and rat fibroblasts have localized the transforming activity to the Env proteins encoded by these viruses, which require the putative lung and breast cancer tumor suppressor hyaluronidase 2 (Hyal2) to promote virus entry into cells. These results suggested the hypothesis that the JSRV and ENTV Env proteins cause cancer by inhibiting the tumor suppressor activity of Hyal2. Consistent with this hypothesis, we show that human Hyal2 and other Hyal2 orthologs that can promote virus entry, including rat Hyal2, can suppress transformation by the Env proteins of JSRV and ENTV. Furthermore, we provide direct evidence for binding of the surface (SU) region of JSRV Env to human and rat Hyal2. However, mouse Hyal2 did not mediate entry of virions bearing JSRV or ENTV Env proteins, bound JSRV SU poorly if at all, and did not suppress transformation by the JSRV or ENTV Env proteins, indicating that mouse Hyal2 plays no role in transformation of mouse fibroblasts and that the Env proteins can transform at least some cells by a Hyal2-independent mechanism. Expression of human Hyal2 in mouse cells expressing JSRV Env caused a marked reduction in Env protein levels, indicating that human Hyal2 suppresses Env-mediated transformation in mouse cells by increasing Env degradation rather than by exerting a more general Env-independent tumor suppressor activity.

FIG. 1.

Human Hyal2 can reverse the transformed phenotype induced by JSRV Env in NIH 3T3 cells. NIH 3T3 cells transformed by JSRV Env protein were exposed to LXSN-based retroviral vectors encoding human Hyal1, human Hyal2, or alkaline phosphatase, or to no vector, and were selected in a medium containing G418 starting 1 day after vector exposure. Pictures were taken after 5 days of selection, when untransduced control cells were all dead.

FIG. 2.

Flow cytometric analysis of JSRV SU-IgG binding to cells expressing Hyal2 orthologs. Cells transduced by retroviral vectors expressing rat, mouse, or human Hyal2 or human Hyal1 or by the empty retroviral vector LXSN were incubated with the JSRV SU-IgG fusion protein (1 ml of unpurified culture medium containing ∼30 ng of fusion protein for NIH 3T3 cells or 0.1 ml of PBS containing ∼60 ng of purified fusion protein and 2% FBS for 208F cells) on ice for 3.5 h. The cells were washed, incubated with an FITC-conjugated rabbit anti-human Fc antibody, washed again, and analyzed by flow cytometry. Histograms for cells expressing human Hyal1 were virtually identical to those for cells expressing the empty vector LXSN and are not shown in order to simplify the figure. Results shown are representative of multiple experiments.

FIG. 3.

Scatchard analysis of JSRV SU-IgG fusion protein binding to Hyal2 orthologs. (A) 208F cells transduced by vectors encoding rat Hyal2, mouse Hyal2, or human Hyal2 or by the empty retroviral vector LXSN were incubated with increasing amounts of purified JSRV SU-IgG fusion protein, washed, incubated with an FITC-conjugated rabbit anti-human antibody, washed again, and analyzed by flow cytometry. The geometric means (log₁₀) of fluorescence (y axis) were plotted against the concentration of fusion protein used (x axis). The experiment was repeated once with similar results. (B) Scatchard analysis of JSRV SU-IgG binding to 208F cells expressing rat or human Hyal2. The bound/free ratio is plotted against the geometric means of bound fluorescence measured by flow cytometric analysis and expressed as the number of bound JSRV SU-IgG molecules per cell. Results are from a representative experiment.

FIG. 4.

208F cell transformation by JSRV and ENTV Env proteins is suppressed by human Hyal2 and rat Hyal2 but not by mouse Hyal2. 208F cells were transduced by LXSN-based retroviral vectors encoding rat Hyal2 (rHyal2), mouse Hyal2 (mHyal2), human Hyal2 (hHyal2), or human Hyal1 (hHyal1) or by an empty LXSN vector (none) and were selected for the presence of the vectors in G418. The transduced cells were then cotransfected with 10 μg of plasmid DNA encoding JSRV Env (pSX2.Jenv), ENTV Env (pSX2.Eenv), a Fos oncoprotein (pFBJ/R), or 10A1 MLV Env (pSX2) plus 1 μg of plasmid pLAPSN, which expresses AP. Transformed foci were counted approximately 2 weeks posttransfection, and the cells were fixed and stained for AP. Results are presented as the ratios of transformed foci to AP⁺ foci and are means from two independent experiments. In this experiment, the numbers of foci induced by pSX2.Jenv, pSX2.Eenv, and pFBJ/R in 208F/LXSN cells were similar and the means ranged from 120 to 160 foci per μg of plasmid DNA. Asterisks indicate values statistically different from those obtained by using cells expressing the control vector LXSN (*, P < 0.05; **, P < 0.01).

FIG. 5.

Hyal2 expression reduces JSRV Env protein levels. NIH 3T3 cells transformed by pSX2-Jenv-FLAG were transduced with retroviral vectors encoding human Hyal2 or AP. After 10 days of selection in G418, these cells and untreated NIH 3T3 cells were immunostained using anti-FLAG antibodies. The same conditions of illumination and photography were used for all panels.