Oncoprotein E6 is essential for oncogenesis induced by human papillomaviruses (HPVs). The solution structure of HPV16-E6 C-terminal domain reveals a zinc binding fold. A model of full-length E6 is proposed and analyzed in the context of HPV evolution. E6 appears as a chameleon protein combining a conserved structural scaffold with highly variable surfaces participating in generic or specialized HPV functions. We investigated surface residues involved in two specialized activities of high-risk genital HPV E6: p53 tumor suppressor degradation and nucleic acid binding. Screening of E6 surface mutants identified an in vivo p53 degradation-defective mutant that fails to recruit p53 to ubiquitin ligase E6AP and restores high p53 levels in cervical carcinoma cells by competing with endogeneous E6. We also mapped the nucleic acid binding surface of E6, the positive potential of which correlates with genital oncogenicity. E6 structure-function analysis provides new clues for understanding and counteracting the complex pathways of HPV-mediated pathogenesis.

Antibodies are valuable tools for functional studies in vitro, but their use in living cells remains challenging because they do not naturally cross the cell membrane. Here, we present a simple and highly efficient method for the intracytoplasmic delivery of any antibody into cultured cells. By following the fate of monoclonal antibodies that bind to nuclear antigens, it was possible to image endogenous targets and to show that inhibitory antibodies are able to induce cell growth suppression or cell death. Our electrotransfer system allowed the cancer cells we studied to be transduced without loss of viability and may have applications for a variety of intracellular immuno-interventions.

Background: Intrabodies are defined as antibody molecules which are ectopically expressed inside the cell. Such intrabodies can be used to visualize or inhibit the targeted antigen in living cells. However, most antibody fragments cannot be used as intrabodies because they do not fold under the reducing conditions of the cell cytosol and nucleus.

The sequences of Saccharomyces cerevisiae mitochondrial tRNA1 Arg, tRNA2 Arg, tRNAGly, tRNA2 Lys tRNALeu and tRNAPro are reported. Special structural features were found in tRNAPro, which has A8, C21, A48 instead of the constant residues U8, A21 and pyrimidine 48, and in tRNA2 Lys, which has a U excluded from basepairing and bulging out from the TΨC stem. The tRNA1 Arg, tRNA2 Lys and tRNALeu, which belong to two‐codon families ending in a purine, have a modified uridine in the wobble position, which prevents misreading of C and U. It is likely to be 5‐carboxymethylaminomethyluridine. tRNAGly and tRNAPro have an unmodified uridine in the wobble position allowing the reading of all four codons of a four‐codon family. However, tRNA2 Arg, which is a minor species and belongs to the CGN four‐codon family, has an unmodified A in the wobble position. This very unusual feature raises the problem of the mechanism by which the codons CGA, CGG and CGC are recognized.

Cervical cancer is caused by high-risk types of human papillomaviruses (HPV) that encode the E6 and E7 oncogenes. Silencing of E6 gene expression in HPVpositive cell lines by transfection of small interfering RNA (siRNA) with cationic lipids restores the dormant p53 tumor suppressor pathway. Because cationic lipids can also be used for intracytoplasmic delivery of proteins, we tested whether the delivery of monoclonal antibodies that bind to HPV16 E6 and neutralize its biological activity in vitro could restore p53 function in tumor cells. Here, we show that the 4C6 antibody is efficiently delivered into the cell cytoplasm using a lipidic reagent used for siRNA transfection. The delivery of 4C6 resulted in the nuclear accumulation of p53 protein in CaSki and SiHa cells but not in HeLa cells. Furthermore, the antibody-mediated p53 response was dramatically increased when a peptide corresponding to the 4C6 epitope and bearing a COOHterminal cysteine residue was added to the transduction mixture. We found that a fraction of the added peptides were dimers that allowed the formation of antibody polymers adsorbed onto the lipidic matrix. With this system, the proliferation of CaSki and SiHa cells was strongly diminished, but no apoptosis was detectable. Remarkably, cell growth was almost totally suppressed by the addition of E6-specific siRNA to the transduction complex. The results indicate that the activity of E6 oncoprotein can be down-regulated in vivo by lipidmediated antibody delivery and that antibodies and siRNA act synergistically when codelivered. This novel targeting strategy is simple to implement and may find therapeutic applications.

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The E6 protein of the high-risk human papillomavirus type 16 (HPV-16) is involved in the tumorigenesis of human cervical cells by targeting numerous cellular proteins. We characterized new anti-E6 monoclonal antibodies and used them for precise localization of the E6 oncoprotein within carcinoma cells. Overexpressed E6 protein was predominantly detected in the nucleus of transiently transfected HaCaT cells. While mostly localized at the periphery of condensed chromatin, E6 was also associated with nuclear ribonucleoproteic ultrastructures and with some ribosomal areas in the cytoplasm of SiHa and CaSki cells. The chimeric b-galactosidase-E6 protein expressed in transfected HeLa cells was essentially localized in the nuclear compartment. Together, these data indicate that the E6 sequence of HPV-16 may encode a nuclear localization signal. The preferential nuclear distribution of this viral oncoprotein in HPV-transformed cells correlates with its activities at the transcriptional level.High-risk strains of human papillomaviruses (HPVs) such as HPV-16 are associated with lesions that can progress to cervical cancer (zur Hausen, 1999). The genome of these viruses encodes an oncoprotein, E6, which mediates degradation of the cellular p53 tumour suppressor protein in vitro (Scheffner et al., 1992) and in vivo (Lechner et al., 1992). Although this particular property of E6 remains the main explanation of its transforming activity, recent findings indicate that E6 is a multifunctional protein with p53-independent activities. It has been shown that E6 activates transcription of the telomerase gene (hTERT; Klingelhutz et al., 1996) and acts as a DNA-binding protein with high affinity for four-way DNA junctions (Ristriani et al., 2000(Ristriani et al., , 2001. In addition to p53, E6 interacts with a variety of cellular proteins, of which a significant number act at the transcriptional level (Mantovani & Banks, 2001).Previous studies on the localization of HPV-16 E6 within human carcinoma cells have led to contradictory results, most probably due to the low level of endogenous E6 protein and the poor reactivity of the available anti-E6 antibodies. E6 protein was localized in the cytoplasmic perinuclear region of SiHa cells (Daniels et al., 1998) and co-localized with p53 in the cytoplasm of CaSki cells (Liang et al., 1993). In transfected COS1 cells, overexpressed HPV-16 E6 protein is essentially found in the nuclear compartment (Kanda et al., 1991;Schwalbach et al., 2000;Sherman & Schlegel, 1996), while in transfected human cells, E6 of HPV-18, another malignant strain, is evenly distributed in the cytoplasm and the nucleus (Guccione et al., 2002). We generated monoclonal antibodies that specifically bind to the recombinant E6 protein of HPV-16. The aim of this study was to determine the precise intracellular localization of the E6 protein in transformed cells in order to establish whether the cellular distribution of this oncoprotein correlates with its recently described p53-independent biological functions. Here, we h...