Domains of the E1 protein of human papillomavirus type 33 involved in binding to the E2 protein.
Papillomavirus E1 and E2 proteins are essential for the initiation of viral DNA replication. We have now analyzed the interaction of E1 and E2 of human papillomavirus type 33, which is associated with cervical carcinoma. When synthesized in insect cells using the baculovirus expression system, the E1 and E2 proteins interacted efficiently at 4 degree. A monoclonal antibody recognizing E1 amino acids 584--600 inhibited the binding of E2 and vice versa, indicating that these amino acids are involved in E2 binding. To confirm this result, a mutational analysis of E1 was performed. The E2 binding activity of E1 deletion and point mutant proteins was assayed using glutathione S-transferase E1 fusion proteins and in vitro translated proteins. At 4 degree, the C-terminal portion of E1 including amino acids 312--644 was sufficient for E2 binding. Introduction of C-terminal deletions or a point mutation at position 586 (Pro --> Glu) resulted in the loss of the E2 binding activity. A second more N-terminally located binding domain (E1 amino acids 312--450) became active when the assays were performed at 22 degrees. The monoclonal antibody still inhibited E2 binding at this temperature, indicating that both E2 binding domains are engaged in the context of the full-length protein.
Müller
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F
Institut für Medizinische Mikrobiologie und Hygiene, Universität Mainz, Germany.
Sapp
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M
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000914:Antibodies, Viral / Q000276:immunology; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D018604:Epitope Mapping; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009856:Oncogene Proteins, Viral / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D027383:Papillomaviridae / Q000737:chemistry / Q000378:metabolism*; D017354:Point Mutation; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D018411:Spodoptera / Q000166:cytology; D013696:Temperature; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D017931:DNA Primers; D009856:Oncogene Proteins, Viral; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase