The retinoblastoma protein is an essential mediator that links the interferon-inducible 204 gene to cell-cycle regulation.

We have previously demonstrated that overexpression of p204, a member of the Ifi 200 gene family, inhibits growth, delays G0/G1 progression into S phase, and impairs E2F-mediated transcriptional activity. In this study, we show that p204 directly binds the retinoblastoma protein (pRb) in vivo to exert its activity. Transient p204 overexpression in Rb+/+ mouse embryo fibroblasts (MEF) inhibits cell proliferation, but does not affect cell growth in MEF derived from Rb-/- mice. Two human cell lines, Saos2 and C33A, bearing an inactive pRb, but not primary human embryo fibroblasts, are resistant to the p204 antiproliferative activity. p204 contains two 200 amino acid motifs, designated as type a or b domains, each containing a canonical Rb binding motif (LXCXE). When dominant-negative mutants at the Rb binding motif were transfected in Rb+/+ MEF, p204 lost its ability to inhibit cell growth, delay cell transition from G1 to S phase, and impair DNA synthesis. Moreover p204 overexpression in Rb+/+ MEF led to a significant decrease of both DHFR and PCNA proteins, two S phase markers. By contrast, this effect was not observed when Rb+/+ MEF were transfected with a p204 mutated at both Rb binding sites. Finally, overexpression of the LXCXE p204 mutant rendered Rb+/+ MEF resistant to the IFN-alpha antiproliferative activity, in comparison to the untransfected Rb+/+ MEF. As expected, Rb-/- cells were unsensitive to the IFN-alpha induced growth inhibition. Taken as a whole, these results suggest that (i) p204 contributes to the IFN-alpha antiproliferative activity and (ii) the primary target of p204 leading to efficient G1 arrest as well as to blockade of DNA replication from G1 phase is the pRb regulatory system.