Cyclin-dependent kinase inhibitors (CDKIs) have been shown to block human immunodeficiency virus and herpes simplex virus. It is hypothesized that CDKIs block viral replication by inhibiting transcription of specific cellular genes. Here we find that three CDKIs, flavopiridol, purvalanol A, and methoxy-roscovitine, block Moloney murine leukemia virus (MLV) transcription events. Using gene expression microarray technology to examine the inhibitory effects of CDKIs, we observed a cellular gene, the pre-B-cell leukemia transcription factor 1 (Pbx1) gene, down-regulated by CDKI treatment. The PBX consensus element (PCE), TGATTGAC, is conserved in the long terminal repeats of several murine retroviruses, including Moloney MLV. Mutations in the PCE completely inhibited viral transcription whereas overexpression of PBX1 and a PBX1-associated protein, PREP1, enhanced viral transcription. The interaction between the PCE and PBX1-PREP1 proteins was confirmed by gel shift experiments. Blocking PBX1 protein synthesis resulted in a significant decrease in viral transcription. Collectively, our results represent the first work demonstrating that the homeodomain proteins PBX1 and PREP1 are cellular factors involved in Moloney MLV transcription regulation.Cyclin-dependent kinases (CDKs) are key regulators of cell cycle control and transcription. Nine CDKs have been identified thus far. CDK1 to CDK7 are required for cell cycle regulation, and CDK7 to CDK9 are involved in RNA polymerase II-dependent transcription (33). In fact, CDK2, CDK4, and CDK6 also regulate transcription by activating E2F via specific phosphorylation of the retinoblastoma gene product (Rb protein) in complex with E2F (13, 49). It has been shown elsewhere that approximately 90% of all neoplasias are associated with CDK hyperactivation and subsequent Rb pathway inactivation (21). Therefore, CDKs represent attractive targets for cancer therapy, and several CDK inhibitors (CDKIs) have been developed (4,16,18,48). Two CDKIs, flavopiridol (Flavo) and UCN-1, are already in clinical trials as potential anticancer therapeutics (44,49).The antiviral activities of CDKIs were first observed in studies describing their interaction with positive transcription elongation factor b (P-TEFb), a factor required for human immunodeficiency virus (HIV) replication (32, 54). P-TEFb, composed of CDK9 and a cyclin subunit derived from one of three different genes (cyclin T1, T2, or K), controls RNA polymerase II-dependent transcription elongation by phosphorylating the carboxyl-terminal domain of the large subunit of RNA polymerase II (41).