Viral oncoproteins from tumorigenic viruses play crucial roles in the induction of tumors by affecting cell cycle regulation. The Marek's disease EcoRI-Q (Meq) oncoprotein of Marek's disease virus (MDV) is consistently detected in all tumor samples from birds infected with MDV and in cell lines induced by MDV. Meq interacts with cyclin-dependent kinase 2 (Cdk2) and deregulates the cell cycle. Although Meq is the most well-studied protein in MDV, there are still many questions as to its functions during the cell cycle. To better understand the functions of Meq during the cell cycle in the chicken, we have identified and characterized two cell cycle regulatory proteins and evaluated their interaction with Meq.
First, we investigated chicken cyclin-dependent kinase inhibitor 1B (p27 Kip1), which is a major cell cycle regulatory protein. It functions in arresting the cell cycle in the G1 phase and thus, inhibits cell growth. p27Kip1 plays a role in the apoptotic process; overexpression of p27Kip1 induces apoptosis in some cancer cell lines. We identified and characterized chicken p27Kip1, a 198-amino acid protein containing a cyclin-dependent kinase inhibitor (CDI) domain. Transfection studies indicated that chicken p27Kip1 localized to the nucleus and that it inhibited cell growth. Unlike its expression in tissues, the expression of p27Kip1 was decreased in MDV induced spleen tumor cells. In co-transfection and immunoprecipitation studies, chicken p27Kip1 co-localized with Meq in the nucleus. Similarly, it co-localized with a Meq mutant that had a deletion of the transactivation domain showing, that this co-localization was due to amino terminal domain of Meq. Furthermore, based on immunoprecipitation studies, Meq physically interacted with chicken p27Kip1 in constitutively expressing Meq, DF-1 cells as well as MDV-induced transformed cell lines. These results strongly suggest that the N-terminus of Meq interacts with chicken p27Kip1, either directly or indirectly, during the cell cycle and thereby influences the cell cycle, especially at the G1/S boundary and early S phase.
The second cell cycle regulatory protein that was investigated in this study was S-phase kinase associated protein 2 (Skp2), which functions in the transition from G1 to S phase and helps in DNA replication during the S phase. Skp2 facilitates ubiquitin-mediated p27Kip1 degradation in G1/S and S phase. Chicken Skp2 has an F-box motif in the N-terminus and leucine-rich repeats in the C-terminus. Transfection studies with chicken Skp2 demonstrated that it localized to the nucleus of the cell. It was also found that overexpression of chicken Skp2 induced cell growth, even under culture conditions using low serum media. Cell cycle analysis indicated that overexpression of chicken Skp2 shifted the cell population from G1 to the S and G2 phases in low serum conditions, thereby suggesting that chicken Skp2 can force the transition from G1 to S phase and induce cell proliferation, even in unfavorable conditions. Co-transfection studies of chicken Skp2 and Meq demonstrated that Skp2 also co-localizes with Meq in the nucleus; however, further studies are required to determine the interaction of these proteins.
In summary, this research identified and characterized chicken p27 Kip1 and Skp2. These studies also found that chicken p27Kip1 and Meq physically interact, and that p27Kip1 overexpression decreased the effect of Meq. In addition, these studies demonstrate the oncogenic potential of chicken Skp2 and although not confirmed with direct binding studies, our results suggest that chicken Skp2 may also interact with Meq during the cell cycle and may therefore participate in cell transformation during MDV infection.