The human papillomavirus types (HPVs) most often associated with cancer of the cervix, such as HPV16, have been reported previously to immortalize normal human foreskin keratinocytes in vitro, while the types that are primarily associated with benign cervical lesions failed to do so. In this study we have determined the HPV16 genes that are responsible for the immortalizing activity of the viral genome. Transfection with a plasmid in which E6 and E7 were the only intact open reading frames (ORFs) induced an indefinite life‐span in the keratinocytes with an efficiency similar to that of the entire early region of the viral DNA. Mutants in the E6E7 clone with inactivating lesions in E6 or E7 failed to induce immortalization. When transfected alone, E7 could induce hyperproliferation, but these cells eventually senesced. By itself, E6 exhibited no activity, Co‐transfection of a plasmid with an intact E6 ORF and a second plasmid with an intact E7 ORF generated keratinocyte lines with indefinite growth potential. The E6 and E7 proteins were detected in the lines induced by the E6E7 DNA and by co‐transfection of the E6 and E7 plasmids. Therefore, we conclude that HPV16 E6 and E7 cooperative to immortalize human keratinocytes in vitro. Changes in cellular gene expression are probably also required for immortalization since all of the keratinocyte lines examined were aneuploid. Serum and calcium resistant sublines were isolated from the E6E7 induced lines, indicating that other HPV genes do not play an obligatory role in the generation of resistance to differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)

The Harvey murine sarcoma virus (Ha-MuSV) transforming gene, v-rasH, encodes a 21,000 molecular weight protein (p21) that is closely related to the p21 proteins encoded by the cellular transforming genes of the ras gene family. The primary translation product (prop21), which is found in the cytosol, undergoes posttranslational modification and the mature protein subsequently becomes associated with the inner surface of the plasma membrane and binds lipid tightly. The p21 proteins have the capacity to bind guanine nucleotides non-covalently in vitro. To assess the biological relevance of these biochemical features of the protein, we have now studied a series of deletion mutants located at or near the C-terminus of the viral p21 protein. Our tissue culture studies indicate that amino acids located at or near the C-terminus are required for cellular transformation, membrane association and lipid binding.

Background-Previous studies have demonstrated that genital infection with high-risk types of human papillomavirus (HPV), most often HPV16, is the most significant risk factor for the development of cervical cancer. However, serologic assays that have been developed to identify high-risk HPV infection have either failed to associate serum reactivity with other indicators of HPV infection or have identified only a minority of HPV-infected individuals.

Previous studies of premature chain termination mutants and in frame deletion mutants of the p21 ras transforming protein encoded by the transforming gene of Harvey murine sarcoma virus (Ha‐MuSV) have suggested that the C terminus is required for cellular transformation, lipid binding, and membrane localization. We have now further characterized the post‐translational processing of these mutants and have also studied two C‐terminal v‐rasH point mutants: one encodes serine in place of cysteine‐186, the other threonine for valine‐187. The Thr‐187 mutant was transformation‐competent, and its p21 protein was processed normally, as was the p21 encoded by a transformation‐competent deletion mutant from which amino acids 166‐175 had been deleted. The Ser‐186 mutant was defective for transformation. The p21s encoded by the Ser‐186 mutant and by the previously described transformation‐defective mutants did not undergo the posttranslational processing common to biologically active ras proteins: their electrophoretic migration rate did not change, they remained in the cytosol, and they failed to bind lipid. Since the cell‐encoded ras proteins also contain this cysteine, we conclude that this amino acid residue is required for all ras proteins.

We tested the ability of vaccination with virus-like particles (VLPs) to protect domestic rabbits against papillomas induced by the cottontail rabbit papillomavirus (CRPV). A recombinant baculovirus system that expressed only the L1 major papillomavirus structural protein or L1 plus the minor L2 protein was used in insect cells as the source of VLPs. Groups of 10 rabbits were immunized with native or denatured VLPs from CRPV or type 1 bovine papillomavirus by using Freund's adjuvant. Alum was used as the adjuvant for an additional group immunized with CRPV L1-L2 VLPs. Animals were challenged with 5 ؋ 10 10 and 2 ؋ 10 11 particles on opposing flanks. No protection was seen in rabbits immunized with native or denatured bovine papillomavirus L1-L2 or with denatured CRPV L1-L2. In these groups, the lower and higher challenge doses resulted in 27 of 30 animals with extensive papillomas, with each of the remaining animals having a smaller number of persistent papillomas. Progression to carcinoma developed in 20 rabbits. Animals inoculated with native CRPV VLPs composed of L1 alone or L1-L2 developed many fewer lesions; the lower and higher challenge doses resulted in 17 of 29 and 5 of 29 rabbits, respectively, with no lesions, and the remainder developed only one to eight papillomas, which all regressed except for those on 1 rabbit. None developed cancer within 1 year of infection. Rabbits vaccinated with native CRPV VLPs developed high-titer antibodies in an enzyme-linked immunosorbent assay based on native VLPs, and passive transfer of serum or immunoglobulin G from rabbits immunized with CRPV VLPs protected against CRPV challenge. We conclude that native VLPs can induce antibody-mediated, type-specific protection against experimental papillomavirus infection.

Human cervical carcinoma cell lines that harbor human papillomavirus (HPV)

An ELISA to detect serum IgG antibody response to human papillomavirus (HPV) type 16 virus-like particles (VLPs) was evaluated in a case-control study of cervical neoplasia, nested within a prospective cohort study. Subjects included 688 controls with continued normal cytology and 152 cases with confirmed incident squamous intraepithelial lesions who were tested for DNA for a broad spectrum of HPV types at cohort and follow-up of controls, 16.6% were seropositive compared with 30.8% and 52.4% of cases with low- and high-grade lesions, respectively. Of HPV-16 DNA-negative subjects, 16.5% were seropositive. Seropositivity increased from 22.2% in subjects who were HPV-16 DNA-positive by polymerase chain reaction once only (enrollment or follow-up) to 83.3% in those who were HPV-16 DNA-positive at both time points. These data imply that serum antibody to HPV-16 VLPs is a relatively sensitive indicator of persisting cervical HPV-16 infection.

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.