In order to ensure a productive life cycle, human papillomaviruses (HPVs) require fine regulation of their gene products. Uncontrolled activity of the viral oncoproteins E6 and E7 results in the immortalization of the infected epithelial cells and thus prevents the production of mature virions. Ectopically expressed E2 has been shown to suppress transcription of the HPV E6 and E7 region in cell lines where the viral DNA is integrated into the host genome, resulting in growth inhibition. However, it has been demonstrated that growth control of these cell lines can also occur independently of HPV E2 transcriptional activity in high-risk HPV types. In addition, E2 is unable to suppress transcription of the same region in cell lines derived from cervical tumors that harbor only episomal copies of the viral DNA. Here we show that HPV type 16 (HPV-16) E2 is capable of inhibiting HPV-16 E7 cooperation with an activated ras oncogene in the transformation of primary rodent cells. Furthermore, we demonstrate a direct interaction between the E2 and E7 proteins which requires the hinge region of E2 and the zinc-binding domain of E7. These viral proteins interact in vivo, and E2 has a marked effect upon both the stability of E7 and its cellular location, where it is responsible for recruiting E7 onto mitotic chromosomes at the later stages of mitosis. These results demonstrate a direct role for E2 in regulating the function of E7 and suggest an important role for E2 in directing E7 localization during mitosis.Human papillomaviruses (HPVs) are a large family of small, double-stranded DNA viruses. They infect cutaneous and mucosal epithelial tissue at different anatomical locations, resulting in a variety of clinical symptoms ranging from benign warts to invasive genital cancers (13,29). Infection with the high-risk types, most commonly HPV type 16 (HPV-16) and HPV-18, has been associated with the development of more than 99% of cervical cancer cases. The tumorigenicity of HPV is dependent on the activity of two virally encoded oncoproteins, E6 and E7. These bind at a high affinity and disrupt the function of p53 (46) and the retinoblastoma tumor suppressor protein pRB (6, 20), respectively. While E6 plays a role in inhibiting apoptosis and interfering with cell adhesion and polarity (30), E7 acts by driving S-phase progression, regulating gene expression, and interfering with the activities of cyclins and cyclin-dependent kinases (23). In addition to inactivating the function of pRB, other cellular targets of E7 include the TATA box-binding protein (TBP) (31), TBP-associated factors (35), members of the AP-1 transcription factor family (2), and histone deacetylases (7). E7 is a small phosphoprotein that shares some sequence homology with the adenovirus E1a protein and the simian virus 40 large T antigen (8). E7 is 98 amino acids in length and contains a zinc-binding domain in the C-terminal region whose structural integrity is necessary for the activity of E7 (2, 26). While both E6 and E7 cooperate to induce immortalization o...

Human papillomaviruses are the causative agents of cervical cancer. Previous studies have shown that loss of the viral E2 protein during malignant progression is an important feature of HPV-induced malignancy due to the resulting uncontrolled expression of the viral oncoproteins E6 and E7. We now show however that the viral E2 and E6 proteins are both capable of regulating each other's activity. When coexpressed, E2 and E6 induce marked changes in the pattern of each other's expression, with preferential accumulation in nuclear speckles. The two proteins interact directly, resulting in changes in the substrate specificities of E6 and the biochemical activities of E2. Thus, while E6 efficiently degrades its PDZ domain-containing substrates in the absence of E2, this activity is greatly diminished when E2 is present. Likewise, E2 alone drives both viral DNA replication and viral gene expression. However, in the presence of E6, viral DNA replication is inhibited while the transcriptional activity of E2 is elevated. These studies define a far more complex pattern of interaction between E2 and E6 than was previously thought and redefines the possible consequences of loss of E2 with respect to uncontrolled E6 activity and consequent malignant progression.

The human papillomavirus (HPV) minor capsid protein L2 plays important roles in the generation of infectious viral particles and in the initial steps of infection. Here we show that HPV-16 L2 protein is sumoylated at lysine 35 and that sumoylation affects its stability. Interestingly, the sumoylated form of L2 cannot bind to the major capsid protein L1, suggesting a mechanism by which capsid assembly may be modulated in an infected cell. Additionally, L2 appears to modulate the overall sumoylation status of the host cell. These observations indicate a complex interplay between the HPV L2 protein and the host sumoylation machinery.Posttranslational modification is a central method of diversifying protein function. Ubiquitin-like proteins such as SUMO (small ubiquitin-related modifier) are known to be key regulators of several biological functions (31). In humans, at least three SUMO forms (SUMO1, -2, and -3) are expressed. SUMO2 and SUMO3 (here called SUMO2/3) are closely related, sharing 97% identity, whereas SUMO1 shares 43% identity with SUMO2/3. SUMO modification exerts a variety of effects on its targets, altering a target's cellular localization, its stability, its ability to interact with other proteins, and its activity (31). Most known sumoylation targets are transcription factors or other proteins involved in chromatin structure, regulation, and expression (12), supporting a fundamental role for this modification system in regulating cellular homeostasis. Hence, it is no surprise that viral proteins can exploit the host sumoylation system; proteins from both RNA and DNA viruses have been shown to be sumoylated and/or to interact with the sumoylation machinery (8). The viruses that exhibit interplay between sumoylation and viral proteins can be divided into two groups: viruses that have their proteins sumoylated and viruses whose proteins directly modify host sumoylation. In both cases, the outcome is a cellular environment more favorable for viral replication (5, 8).Human papillomavirus (HPV) infects both mucosal and cutaneous epithelia, and certain high-risk HPV types are the causative agents of cervical cancer (11). Two structural proteins, L1 and L2, form the papillomavirus capsid (16). The minor capsid protein L2 plays a critical role in the generation of infectious viral particles and in early events of HPV infection (13), although its precise functions in HPV entry, intracellular trafficking, endosomal escape, and the nuclear import of the HPV genome have not been fully elucidated (21).Previous studies have shown sumoylation to be important in the HPV life cycle. The functions of two early proteins, E1 and E2, are modified directly by sumoylation (17,19,26,28), and three others, E2 (27), E6 (3), and E7 (14), affect host sumoylation pathways.HPV type 16 (HPV-16) L2's potential involvement with the sumoylation machinery has not been documented, although a consensus sequence, ⌿KXE, where ⌿ is a large hydrophobic amino acid, corresponding to the SUMO acceptor site in most known SUMO substrate proteins...

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An important characteristic of the E6 proteins derived from cancer-associated human papillomaviruses (HPVs) is their ability to target cellular proteins for ubiquitin-mediated degradation. Degradation of the p53 tumour suppressor protein by E6 is known to involve the cellular ubiquitin ligase, E6-AP; however, it is presently not known how E6 targets the Drosophila discs large (Dlg) tumour suppressor and the membrane-associated guanylate kinase inverted (MAGI) family of proteins for degradation. By using an in vitro E6-AP immunodepletion assay, these targets were tested for degradation in a E6-AP-dependent manner. The data showed clearly that E6 can direct the degradation of Dlg and the MAGI family of proteins in the absence of E6-AP in this in vitro system. These results provide compelling evidence for the role of E6-associated ubiquitin ligases other than E6-AP in the degradation of certain E6 targets.The ubiquitin/26S proteasome pathway is a major route for the selective degradation of eukaryotic proteins. Through the removal of key regulatory components, the pathway helps to control many aspects of cell homeostasis, growth and development. High specificity within the ubiquitin system is achieved by the ubiquitin protein ligase family of E3 enzymes. These enzymes bind to the target substrates, either directly or indirectly, and catalyse the last step in the conjugation process [covalent attachment of ubiquitin to the substrate (Ciechanover, 1998;Ciechanover et al., 2000)]. The first example of a viral protein that uses the proteasome machinery to direct the degradation of a cellular protein was provided by the human papillomavirus (HPV) E6 protein and its stimulation of the ubiquitination and degradation of the cellular tumour suppressor protein p53 (Scheffner et al., 1990;Werness et al., 1990). In vitro studies in reticulocyte lysates have shown that the HPV E6 oncoprotein binds to a cellular protein of 100 kDa, termed E6-AP (Huibregtse et al., 1991, which is the prototype of the HECT (homologous to E6-AP carboxyl terminus) domain-containing ubiquitin ligases . Under normal circumstances, p53 is regulated via the Mdm2 ubiquitin ligase and E6-AP has no role in this pathway (Haupt et al., 1997;Kubbutat et al., 1997). However, in HPV-containing cells, the Mdm2 pathway is bypassed and p53 is degraded constitutively via the E6/E6-AP ubiquitin ligase complex (Scheffner et al., , 1994Rolfe et al., 1995;Hengstermann et al., 2001). . The discovery that high-risk, but not low-risk, HPV E6 proteins can bind to the PDZ domain-containing proteins and target them for ubiquitin-mediated degradation was particularly interesting, considering that high-risk types cause lesions that can progress to cervical carcinoma, whereas the low-risk types are very rarely associated with malignancies (zur Hausen & Schneider, 1987). PDZ domains are motifs of 80-90 aa, which are present in a variety of proteins that are involved in clustering of ion channels, signalling enzymes and adhesion molecules to specific structures at the membranecyt...