D R Lowy

HPV16 E6 and E7 proteins cooperate to immortalize human foreskin keratinocytes.

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)

Identification of the HPV-16 E6 protein from transformed mouse cells and human cervical carcinoma cell lines.

Human cervical carcinoma cell lines that harbor human papillomavirus (HPV) have been reported to retain selectively and express HPV sequences which could encode viral E6 and E7 proteins. The potential importance of HPV E6 to tumors is suggested further by the observation that bovine papillomavirus (BPV) E6 can induce morphologic transformation of mouse cells in vitro. To identify HPV E6 protein, a polypeptide encoded by HPV‐16 E6 was produced in a bacterial expression vector and used to raise antisera. The antisera specifically immunoprecipitated the predicted 18‐kd protein in two human carcinoma cell lines known to express HPV‐16 RNA and in mouse cells morphologically transformed by HPV‐16 DNA. The 18‐kd E6 protein was distinct from a previously identified HPV‐16 E7 protein. The HPV‐16 E6 antibodies were found to be type specific in that they did not recognize E6 protein in cells containing HPV‐18 sequences and reacted weakly, if at all, to BPV E6 protein. The results demonstrate that human tumors containing HPV‐16 DNA can express an E6 protein product. They are consistent with the hypothesis that E6 may contribute to the transformed phenotype in human cervical cancers that express this protein.

The specific DNA recognition sequence of the bovine papillomavirus E2 protein is an E2-dependent enhancer.

The upstream regulatory region (URR) of the bovine papillomavirus (BPV) genome contains an enhancer that is activated by a BPV E2 gene product. We have previously found that a bacterially derived E2 fusion protein specifically interacted with several fragments of URR DNA, suggesting that E2 may activate transcription by directly binding to the enhancer. Each of the bound fragments contains at least one copy of a conserved motif (ACCN6GGT). To determine if this motif is required and sufficient for specific E2 binding, we have now constructed a bacterial expression vector that encodes a full‐length E2 peptide and developed a refinement of the McKay DNA immunoprecipitation assay that allows the determination, to the nucleotide level, of the minimum sequence required for specific binding. The results show that the E2 recognition sequence is a single copy of this motif and that the variant ACCGN4CGGT is bound with greater affinity than the minimum ACCN6GGT motif. An oligonucleotide encoding the motif was able to inhibit E2‐dependent trans‐activation in a transient transfection assay, indicating that the virally encoded E2 also interacts with this sequence in mammalian cells. When present in two or more copies, but not in a single copy, the E2 binding element had intrinsic enhancer activity but only in cells expressing E2. The results indicate that the conserved motif alone is sufficient for E2‐mediated enhancement and that the binding of E2 to the motif is probably required for efficient enhancement. Since a single motif did not have a significant enhancer activity, it is likely that bound E2 molecules act cooperatively in activating transcription.

Prevention of cancer through immunization : Prospects and challenges for the 21st century

Persistent infection by several microbial agents is responsible for at least 15% of cancer globally, including most cancers of the liver, stomach, and cervix. The recent development of vaccines that can prevent infection and premalignant disease caused by human papillomaviruses (HPV), which cause virtually all cases of cervical cancer as well as some other cancers, has focused renewed attention on infection control as a means of reducing the global cancer burden. For vaccines to prevent cancer‐causing infection with hepatitis C virus, Helicobacter pylori, or Epstein Barr virus, new vaccine technologies to induce more effective protective responses are required. For the two available cancer control vaccines, designed to prevent infection with HPV and hepatitis B virus, the major challenge is to promote effective vaccine deployment through education programs and increased affordability/accessibility for underserved populations, particularly in the developing world, where the cancer burden attributable to infection by these two viruses is greatest.

Explanations for the high potency of HPV prophylactic vaccines

HPV L1 virus-like particle (VLP) vaccines administered in a prime/boost series of three injections over six months have demonstrated remarkable prophylactic efficacy in clinical trials and effectiveness in national immunization programs with high rates of coverage. There is mounting evidence that the vaccines have similar efficacy and effectiveness even when administered in a single dose. The unexpected potency of one dose of these VLP vaccines may largely be attributed to structural features of the particles, which lead to the efficient generation of long-lived antigen-specific antibody-producing cells and unique features of the virus life cycle that make the HPV virions highly susceptible to antibody-mediated inhibition of infection.

Remembering Irwin Freedberg

The following comments were originally published on the American Dermatological Association listserv in July 2005 and are republished here with permission from the authors and the Association.