The E2 proteins from oncogenic (high-risk) human papillomaviruses (HPVs) can induce apoptotic cell death in both HPV-transformed and non-HPV-transformed cells. Here we show that the E2 proteins from HPV type 6 (HPV6) and HPV11, two nononcogenic (low-risk) HPV types, fail to induce apoptosis. Unlike the high-risk HPV16 E2 protein, these low-risk E2 proteins fail to bind p53 and fail to induce p53-dependent transcription activation. Interestingly, neither the ability of p53 to activate transcription nor the ability of p53 to bind DNA, are required for HPV16 E2-induced apoptosis in non-HPV-transformed cells. However, mutations that reduce the binding of the HPV16 E2 protein to p53 inhibit E2-induced apoptosis in non-HPV-transformed cells. In contrast, the interaction between HPV16 E2 and p53 is not required for this E2 protein to induce apoptosis in HPV-transformed cells. Thus, our data suggest that this high-risk HPV E2 protein induces apoptosis via two pathways. One pathway involves the binding of E2 to p53 and can operate in both HPV-transformed and non-HPV-transformed cells. The second pathway requires the binding of E2 to the viral genome and can only operate in HPV-transformed cells.Human papillomaviruses (HPVs) that infect the genital tract can be divided into two groups. High-risk (HR) or oncogenic viral types, such as HR-HPV type 16 (HR-HPV16) and HR-HPV18, are associated with cervical cancer and some other human tumors (7). In contrast, low-risk (LR) or nononcogenic types, such as LR-HPV6 and LR-HPV11, cause genital warts and are not associated with cancer. An appreciation of the differences between the HR-and LR-HPV types is central to an understanding of the origins of cervical cancer and other HPV-induced diseases. The E6 and E7 proteins from HR-HPV have long been recognized to be oncoproteins (33), and significant differences in the properties of E6 and E7 proteins from HR and LR viral types have been identified. For example, the E6 proteins from HR-HPV16 and HR-HPV18 bind to the p53 tumor suppressor protein, resulting in increased degradation of p53 (22,41). Although the E6 proteins from LR-HPV6 and LR-HPV11 can also bind to p53, these interactions do not increase p53 degradation (6,28,41). The E7 proteins from HR-and LR-HPV types also show significant differences. Although the E7 proteins from HR-HPV16 and HR-HPV18 bind to the retinoblastoma tumor suppressor protein p105Rb in vitro, the E7 proteins from LR-HPV6 and LR-HPV11 bind this protein poorly (16,34). Interesting though these differences in the E6 and E7 proteins are, it is important to note that the HR-HPV infections are relatively common and the presence of an HR-HPV type does not therefore always result in oncogenesis (36). Thus, although these differences in the E6 and E7 proteins from different HPV types are undoubtedly important in the establishment and/or progression of cervical cancer, they cannot in themselves completely explain why some HPV types are oncogenic while others are not.In cervical warts the HPV genome exists as an extrach...

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, many proteins are present as a freely diffusing population in free exchange with a sub-population that is tightly associated with a particular subcellular domain or structure. In situ subcellular fractionation allows the visualization of protein compartmentalization and can also reveal protein sub-populations that localize to specific structures. For example, removal of soluble cytoplasmic proteins and loosely held nuclear proteins can reveal the stable association of some transcription factors with chromatin. Subsequent digestion of DNA can in some cases reveal association with the network of proteins and RNAs that is collectively termed the nuclear scaffold or nuclear matrix.Here we describe the steps required during the in situ fractionation of adherent and non-adherent mammalian cells on microscope coverslips. Protein visualization can be achieved using specific antibodies or fluorescent fusion proteins and fluorescence microscopy. Antibodies and/or fluorescent dyes that act as markers for specific compartments or structures allow protein localization to be mapped in detail. In situ fractionation can also be combined with western blotting to compare the amounts of protein present in each fraction. This simple biochemical approach can reveal associations that would otherwise remain undetected. Protocol I. Preparation for fractionationThis section describes the preparation of poly-L-Lysine coated microscope coverslips and the attachment of cells prior to fractionation. If required the cells can be transiently transfected with protein expression vectors either before or after attachment. 2. Coat clean coverslips with poly-L-Lysine by incubating them in the solution for at least 1 hour on a rocking platform at 22°C. 3. Wash the coated coverslips with sterile distilled water twice and follow with a single wash with 96% ethanol. 4. Air dry the coated coverslips on a piece of filter paper and keep them in a dry container for future use (once dry they can be stacked). A. Preparation of poly-L-1. Place a poly-L-Lysine coated coverslip in a well in a 6-well plate with the coated surface facing up. 2. Seed K562 cells at a density of 7x10 5 cells/well in Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), and PS (penicillin 100units/ml, streptomycin 100mg/ml). In the case of K562 cells transient transfection can be performed using electroporation (0.4cm electroporation cuvettes with 1x10 7 cells in 200μl of media at 250V / 975μF) before seeding the cells. 3. Incubate the cells for 24 hours at 37°C in 5% CO2. 4. Pour off the medium and wash the cells twice with ice cold phosphate buffered saline (PBS). 5. Follow with the subcellular fractionation protocol.1. Place an uncoated coverslip in a well in a 6-well plate. 2. Wash twice in PBS prewarmed at 37°C. 3. Detach the cells by digesting with trypsin (0.03% EDTA, 0.25% trypsin) ...

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