SUMMARYWe recently reported the expression of human papillomavirus type 18 (HPV-18) E6 protein in bacteria and the production of anti-E6 polyclonal antibodies. This work has now been extended with the production of a panel ofmonoclonal antibodies against the HPV-18 E6 protein. These antibodies demonstrate that there is little antigenic conservation in the E6 protein between HPV-16 and HPV-18, with only one antibody recognizing a cross-reactive epitope. We have used both the monoclonal and the polyclonal antibodies to look for E6 expression in a number of HPV DNA-containing cell lines. These reagents specifically detected a 16.5K tool. wt. polypeptide in cells derived from a human cervical carcinoma. INTRODUCTIONHuman papillomaviruses (HPVs) induce epithelial or fibroepithelial proliferations of skin or mucosa (zur Hausen, 1977). Infection with specific HPV types correlates with specific diseases such as common warts, epidermodysplasia verruciformis and genital warts (Pfister, 1984). In addition infection with some HPV types has been closely linked with specific human cancers. In particular, HPV-16 and HPV-18 DNAs have been found in a large proportion of malignant lesions of the cervix (Durst et al., 1983 ;Boshart et al., 1984), as well as in a number of cell lines derived from human cervical carcinomas. In some cases these DNAs are transcribed (Schwarz et al., 1985;Yee et al., 1985) and HPV-coded proteins are expressed (Smotkin & Wettstein, 1986).Until recently, bovine papillomavirus (BPV-1) served as the prototype for the study of the molecular biology and transforming genes of papillomaviruses. These studies have shown that the BPV-1 E6 open reading frame (ORF) is necessary for efficient transformation of mouse C 127 cells (Schiller et aL, 1984;Yang et al., 1985; Androphy eta[., 1985), and in one case using a BPV-1 E6-specific antiserum, E6 protein has been demonstrated (Androphy et al., 1985). More recently, the direct transforming ability of HPV-16 D NA has been demonstrated in vitro, both in the form of molecularly cloned HPV-16 DNA (Yasumoto et al., 1986) and also HPV-16 DNA derived directly from a cervical tumour biopsy (Tsunokawa et al., 1986). In both cases the HPV-16 DNA was transcribed.We recently reported the synthesis of HPV-18 E6 polypeptide in bacteria (Matlashewski et al., 1986a). The present paper reports the isolation of a panel of monoclonal antibodies to the HPV-18 E6 polypeptide, and their use in the immunological analysis of the E6 protein derived from different HPVs. The use of these reagents for analysis of E6 expression in a variety of HPV DNA-containing transformed cells is also described.

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SUMMARYHuman papiUomavirus type 18 (HPV-18) has recently been closely linked with human malignant cervical lesions. The early region of the genome of the related bovine papillomavirus (BPV) has been shown to be important for the production of the transformed phenotype. BPV E6 has been shown to be a transforming protein. We report the primary structure of the HPV-18 E6 open reading frame and its predicted amino acid sequence. Both E6 protein and E6-fl-galactosidase fusion protein were synthesized in bacteria. Antisera were raised against the E6-fl-galactosidase fusion protein and against an E6 N-terminal peptide which was 14 amino acids long. We show that these antisera reacted on Western blots against E6 synthesized in bacteria. The HPV E6 antigen and antibodies described here will be useful in understanding HPV expression and its association with human malignancies and may also be diagnostically useful.

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Certain ␤-subunits exert profound effects on the kinetics of voltage-gated (K v ) potassium channel inactivation through an interaction between the amino-terminal "inactivation domain" of the ␤-subunit and a "receptor" located at or near the cytoplasmic mouth of the channel pore. Here we used a bacterial random peptide library to examine the structural requirements for this interaction. To identify peptides that bind K v 1.1 we screened the library against a synthetic peptide corresponding to the predicted S4-S5 cytoplasmic loop of the K v 1.1 ␣-subunit (residues 313-328). Among the highest affinity interactors were peptides with significant homology to the amino terminus of K v ␤1. We performed a second screen using a peptide from the amino terminus of K v ␤1 (residues 2-31) as "bait" and identified peptide sequences with significant homology to the S4-S5 loop of K v 1.1.