Structure and function of the human papillomavirus E1 and E2 replication proteins

The papillomavirus is a group of small DNA tumor viruses with a double-stranded circular DNA genome of about 8 kb. Papillomavirus DNA replication requires the viral E1 and E2 proteins, the replication origin sequences, and the host replication machinery. The E1 protein of papillomavirus is an ATP-dependent DNA helicase. The E2 protein is an important viral transcription factor and an origin recognition protein. The E2 protein consists of three structural and functional domains: the amino-terminal trans-acting domain, the carboxyl-terminal DNA binding and protein dimerization domain, and the connecting hinge region. Domains of the E1 protein are not clearly defined. in our first study, we used the transient replication assay as an indicator for E1/E2 protein interaction. We found that the human papillomavirus type 16 E1 C-terminal domain, which overlaps the putative ATPase activity region, determined the E1 specificity toward the E2 protein, suggesting that this domain is involved in the E1-E2 interaction. In our second study, we fused the green fluorescence protein (gfp) to the N-terminus of HPV-11 E2 protein and E2 subdomains. By examining the cellular distribution of each gfp fusion protein, we showed that the N-terminal trans-acting domain and the hinge region are independently associated with the nuclear matrix. The amino acid patch RKRAR in the hinge region plays an important role in the nuclear localization of the E2 protein. Our data suggest that the hinge region is the major determinant for nuclear retention of the E2 protein. In our third study, initiated to develop vectors for gene transfer experiment and ultimately genetic therapy, we cloned the viral DNA sequences including HPV origin of DNA replication, E1 and E2 open reading frames into the mammalian expression vector pMT2. The resulting pMT2 vectors demonstrated self-replicating activity when transiently transfected into human cell lines. We demonstrated that HPV E1 and E2 proteins can be expressed from the same viral genomic DNA fragment in a single expression cassette, that a trans-gene cassette can be inserted without affecting the replication of the replicon plasmid, and that the replication level can be manipulated by modulating E1 and E2 expression.