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 th e Ifi 200 gene family, inhibits growth, delays G0/G1 progression into S pha se, and impairs E2F-mediated transcriptional activity. In this study, we sh ow that p204 directly binds the retinoblastoma protein (pRb) in vivo to exe rt its activity. Transient p204 overexpression in Rb+/+ mouse embryo fibrob lasts (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 t o the p204 antiproliferative activity. p204 contains two 200 amino acid mot ifs, designated as type a or b domains, each containing a canonical Rb bind ing motif (LXCXE). When dominant-negative mutants at the Rb binding motif w ere transfected in Rb+/+ MEF, p204 lost its ability to inhibit cell growth, delay cell transition from G1 to S phase, and impair DNA synthesis. Moreov er p204 overexpression in Rb+/+ MEF led to a significant decrease of both D HFR and PCNA proteins, two S phase markers. BS contrast, this effect was no t observed when Rb+/+ MEF were transfected with a p204 mutated at both Rb b inding 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 activ ity and (ii) the primary target of p204 leading to efficient G1 arrest as w eb as to blockade of DNA replication from G1 phase is the pRb regulatory sy stem.