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Human papillomaviruses (HPVs) are DNA tumor viruses that infect mucosal and cutaneous epithelial cells of more than 20 vertebrates. High-risk HPV causes about 5% of human cancers worldwide, and the viral proteins E6 and E7 promote carcinogenesis by interacting with tumor suppressors and interfering with many cellular pathways. As a consequence, they immortalize cells more efficiently in concert than individually. So far, the networks of E6 and E7 with their respective cellular targets have been studied extensively but independently. However, we hypothesized that E6 and E7 might also interact directly with each other in a novel interaction affecting HPV-related carcinogenesis. Here, we report a direct interaction between E6 and E7 proteins from carcinogenic HPV types 16 and 31. We demonstrated this interaction via cellular assays using two orthogonal methods: coimmunoprecipitation and flow cytometry–based FRET assays. Analytical ultracentrifugation of the recombinant proteins revealed that the stoichiometry of the E6/E7 complex involves two E7 molecules and two E6 molecules. In addition, fluorescence polarization showed that (I) E6 binds to E7 with a similar affinity for HPV16 and HPV31 (in the same micromolar range) and (II) that the binding interface involves the unstructured N-terminal region of E7. The direct interaction of these highly conserved papillomaviral oncoproteins may provide a new perspective for studying HPV-associated carcinogenesis and the overall viral life cycle. [ABSTRACT FROM AUTHOR]