Walleye retroviral cyclins phosphorylate pRb tumor suppressor and the walleye dermal sarcoma retrovirus cyclin and G2/M cyclins repress transcription of p14ARF tumor suppressor through interaction with TBX2, possibly contributing to tumorigenesis.

摘要

Walleye dermal sarcoma virus (WDSV) and walleye epidermal hyperplasia viruses 1 and 2 (WEHV 1 and 2) are associated with a benign skin tumor and skin hyperplasias on walleye fish that develop and regress seasonally. Viral transcript Orf-A is the only transcript consistently detected in developing tumors and hyperplasias and the predicted orf-A proteins have limited homology with cellular cyclins D1, C, and A implying that they may contribute to these diseases. Here, we show that the orf-A proteins (rv-cyclins) from WDSV, WEHV1 and 2 induce foci in NIH3T3 cells. Mechanistically, we demonstrate that the rv-cyclin-CDK4 complexes phosphorylate the pRb tumor suppressor in in vitro kinase assays, potentially contributing to cell proliferation and tumorigenesis. Interestingly, it has been previously shown that the WDSV rv-cyclin interacts with CDK8 to variably affect gene expression. Previously, in the yeast two-hybrid screening, two clones of mouse TBX2 were retrieved using the WDSV rv-cyclin as bait. TBX2 activates or represses expression of many genes that are rationally linked to cell proliferation, including Cdc2, MEK kinase, p21Cip1 CKI, and tumor suppressors MAD2 and p14 ARF. We investigated the effect of the rv-cyclin interaction with TBX2 on gene expression using a well-established mammalian system, i.e. repression assays of the human p14ARF promoter. We demonstrate that the WDSV rv-cyclin, but not the WEHV cyclins, increases TBX2 repression of transcription from the Arf promoter. The repression of the Arf promoter by the WDSV rv-cyclin is dependent on TBX2, the TBX2-binding site, and WDSV rv-cyclin carboxy terminal coiled-coil domain that interacts with CDK8. We propose that WDSV and WEHV rv-cyclins phosphorylate and inactivate pRb tumor suppressor and/or activate or repress expression of many genes to regulate cell cycle progression and cell proliferation, thereby contributing to the development of WDS and WEH. Additionally, we demonstrate that G2/M, but not G1/S, cyclins repress the transcription of p14ARF in reporter assays, suggesting that the rv-cyclins and G2/M cyclins evolved to impair the expression of ARF. We also show several genes, including p53, that have potential TBX2-binding sites. This implies that TBX2 may coordinate the expression of different genes, contributing to tumor formation.