Maximal activation of murine infection-A4 (IFNA4) gene transcription following viral infection requires the presence of four cooperating DNA sequences (denoted A to D), which make up the virus responsive element VRE-A4. The B, C, and D modules, when tandemized, form binding sites for the virus-induced factor (VIF), a multiprotein complex that is detected early after viral infection in the nuclei of mouse L929 cells. We now demonstrate that IFN regulatory factor-3 (IRF-3) is a component of VIF and that VIF is different from the previously identified virus-activated complexes containing IRF-3 and coactivators of transcription, such as CREB binding protein (CBP) or p300. We also show that the C module is critical for both IRF-3-mediated and virus-induced transcription of the murine IFNA4 gene. Consistently, DNase I footprinting experiments and EMSA performed with increasing amounts of recombinant GST-IRF-3(DBD) fusion proteins demonstrate that cooperativity between the modules facilitate the binding of IRF-3 and recruitment of transcription coactivators on the IFNA4 promoter. These results indicate that VIF differentially recognizes the virus-responsive modules of VRE-A4 and further actualize our previous model concerning the differential expression of murine IFNA genes.