Human papillomavirus type 16 (HPV16) is a DNA tumour virus that can infect epithelial tissue and can cause hyperproliferative lesions that can progress to cancer. The expression of the HPV late protein, E1.;E4 is lost during malignantprogression, but in productive infections E1.;E4 is abundantly expressed inthe late stages of HPV infection. The appearance of E1.;E4 coincides with theonset of genome amplification and the upregulation of the viral replication proteins El and E2 and precedes the synthesis of the viral capsid proteins L1 and L2. In this study we show that HPV16 E1.;E4 (16E1.;E4) can interact directlywith several cellular and viral proteins including the late proteins L1 and L2 and the early proteins E7 and E2. HPV16 E2 associates with the phosphorylated, unphosphorylated and cleaved forms of 16E1.;E4, with stable association beingdependent on sequences in the central highly charged region and the extreme C-terminus of E1.;E4. We observed that co-expression of E2 and E1.;E4 in culturedepithelial cells resulted in the progressive accumulation of E2 to E1.;E4 boundstructures in the cytoplasm, and a reduction in nuclear E2 levels at late time points. This phenomenon in cells that were co-expressing E2, E1.;E4 andEl, at ratios mimicking those seen in the late stages of the virus life cycle, coincided with an increase in HPV origin-dependent replication and enhanced El/E2-mediated transcription from the wild-type HPV16 early promoter (p97). The co-expression of E1.;E4 with El and E2 at late time points was also shownto reduce p97 activity when E2 was expressed at levels that were optimised for E1/E2- mediated transactivation. We suggest that during productive infection, the ability of E1.;E4 to bind to and alter the distribution of E2 in the cellmay facilitate genome amplification and the expression of S-phase promoting HPV proteins, E6 and E7.