The human discs large protein 1 interacts with and maintains connexin 43 at the plasma membrane in keratinocytes.

Gap junction channels, composed of connexins, allow direct cell-to-cell communication. Connexin 43 (Cx43; also known as GJA1) is widely expressed in tissues, including the epidermis. In a previous study of human papillomavirus-positive cervical epithelial tumour cells, we identified Cx43 as a binding partner of the human homologue of Drosophila Discs large (Dlg1; also known as SAP97). Dlg1 is a member of the membrane associated-guanylate kinase (MAGUK) scaffolding protein family, which is known to control cell shape and polarity. Here, we show that Cx43 also interacts with Dlg1 in uninfected keratinocytes in vitro and in keratinocytes, dermal cells and adipocytes in normal human epidermis in vivo. Depletion of Dlg1 in keratinocytes did not alter Cx43 transcription but was associated with a reduction in Cx43 protein levels. Reduced Dlg1 levels in keratinocytes resulted in a reduction in Cx43 at the plasma membrane with a concomitant reduction in gap junctional intercellular communication and relocation of Cx43 to the Golgi compartment. Our data suggest a key role for Dlg1 in maintaining Cx43 at the plasma membrane in keratinocytes.

HPV-18E6 Inhibits Interactions between TANC2 and SNX27 in a PBM-Dependent Manner and Promotes Increased Cell Proliferation.

Cancer-causing HPV E6 oncoproteins contain a PDZ-binding motif at the extreme carboxy terminus, which plays an important role in the viral life cycle and in the development of malignancy. Through this motif, HPV E6 targets a large number of cellular substrates, many of which are involved in processes related to the regulation of cell polarity. Recent studies also demonstrated E6's PDZ binding motif (PBM)-dependent association with SNX27, with a potential role in the perturbation of endocytic transport. Here, we have performed a proteomic analysis to identify SNX27-interacting partners whose binding to SNX27 is specifically perturbed in an E6-dependent manner. Extracts of HeLa cells that express GFP-tagged SNX27, transfected with control siRNA or siRNA targeting E6AP, were subject to GFP immunoprecipitation followed by mass spectroscopy, which identified TANC2 as an interacting partner of SNX27. Furthermore, we demonstrate that HPV E6 inhibits association between SNX27 and TANC2 in a PBM-dependent manner, resulting in an increase in TANC2 protein levels. In the absence of E6, SNX27 directs TANC2 toward lysosomal degradation. TANC2, in the presence of HPV-18E6, enhances cell proliferation in a PBM-dependent manner, indicating that HPV E6 targets the SNX27-mediated transport of TANC2 to promote cellular proliferation. IMPORTANCE While a great deal is known about the role of the E6 PDZ binding motif (PBM) in modulating the cellular proteins involved in regulating cell polarity, much less is known about the consequences of E6's interactions with SNX27 and the endocytic sorting machinery. We reasoned that a potential consequence of such interactions could be to affect the fate of multiple SNX27 endosomal partners, such as transmembrane proteins or soluble accessory proteins. Using a proteomic approach in HPV-18-positive cervical tumor-derived cells, we demonstrate that TANC2 is an interacting partner of SNX27, whose interaction is blocked by E6 in a PBM-dependent manner. This study therefore begins to shed new light on how E6 can regulate the endocytic transport of multiple SNX27-binding proteins, thereby expanding our understanding of the functions of the E6 PBM.

HPV-16 E7 Interacts with the Endocytic Machinery via the AP2 Adaptor µ2 Subunit.

Human papillomavirus (HPV) E7 plays a major role in HPV-induced malignancy, perturbing cell cycle regulation, and driving cell proliferation. Major targets of cancer-causing HPV E7 proteins are the pRB family of tumor suppressors, which E7 targets for proteasome-mediated degradation and whose interaction is promoted through an acidic patch, downstream of the LXCXE motif in E7, that is subject to phosphorylation by casein kinase II (CKII). In this study we show that HPV-16 E7 targets the AP2-complex, which plays a critical role in cargo recognition in clathrin-mediated endocytosis. Intriguingly, HPV-16 E7 contains a specific amino acid sequence for AP2 recognition, and this overlaps the pRb LXCXE recognition sequence but involves completely different amino acid residues. HPV-16 E7 does this by binding to the AP2-µ2 adaptor protein subunit via residues 25-YEQL-28 within the LXCXE motif. Point mutations at Y25 within 22-LYCYE-26 suggest that the interaction of E7 with AP2-µ2 is independent from pRB binding. In cells, this interaction is modulated by acidic residues downstream of LXCXE, with the binding being facilitated by CKII-phosphorylation of the serines at positions 31 and 32. Finally, we also show that association of HPV-16 E7 with the AP2 adaptor complex can contribute to cellular transformation under low-nutrient conditions, which appears to be mediated, in part, through inhibition of AP2-mediated internalization of epidermal growth factor receptor (EGFR). This indicates that E7 can modulate endocytic transport pathways, with one such component, EGFR, most likely contributing toward the ability of E7 to induce cell transformation and malignancy. These studies define a new and unexpected role for HPV-16 E7 in targeting clathrin-mediated endocytosis. IMPORTANCE Despite being a very small protein, HPV-E7 has a wide range of functions within the infected cell, many of which can lead to cell transformation. High-risk HPV-E7 deregulates the function of many cellular proteins, perturbing cellular homeostasis. We show that a novel target of HPV-E7 is the clathrin-adaptor protein 2 complex (AP2) µ2 subunit, interacting via residues within E7's pRB-binding region. Mutational studies show that an AP2 recognition motif is present in the CR2 region and is conserved in >50 HPV types, suggesting a common function for this motif in HPV biology. Mutational analysis suggests that this motif is important for cellular transformation, potentially modulating endocytosis of growth factor receptors such as EGFR, and thus being a novel activity of E7 in modulating clathrin-mediated endocytosis and cargo selection. This study has important implications for the molecular basis of E7 function in modulating protein trafficking at the cell surface.

Repression of Memo1, a Novel Target of Human Papillomavirus Type 16 E7, Increases Cell Proliferation in Cervical Cancer Cells.

Human papillomavirus (HPV)-induced carcinogenesis is associated with unregulated expression of the oncoproteins E6 and E7. HPV E7 is a viral protein that lacks enzymatic activity; however, it can target several cellular proteins to induce cell transformation and promote uncontrolled proliferation. Although several E7 targets have been described, there are still gaps in the understanding of how this oncoprotein drives cells toward malignancy. Here, using a small HPV type 16 (HPV16) E7 peptide in a proteomic approach, we report Memo1 as a new E7 binding partner, interacting through the aspartic and glutamic acid residues (E80 and D81) in the C-terminal region of HPV16 E7. Furthermore, we demonstrate that HPV16 E7 targets Memo1 for proteasomal degradation through a Cullin2-dependent mechanism. In addition, we show that overexpression of Memo1 decreases cell transformation and proliferation and that reduction of Memo1 levels correlate with activation of Akt and an increase in invasion of HPV-positive cervical cancer cell lines. Our results show a novel HPV E7 interacting partner and describe novel functions of Memo1 in the context of HPV-induced malignancy. IMPORTANCE Although numerous targets have been reported to interact with the HPV E7 oncoprotein, the mechanisms involved in HPV-induced carcinogenesis and the maintenance of cell transformation are still lacking. Here, through pulldown assays using a peptide encompassing the C-terminal region of HPV16 E7, we report Memo1 as a novel E7 interactor. High levels of Memo1 correlated with reduced cell proliferation and, concordantly, knockdown of Memo1 resulted in Akt activation in HPV-positive cell lines. These results highlight new mechanisms used by HPV oncoproteins to modulate proliferation pathways in cervical cancer cells and increase our understanding of the link between Memo1 protein and cancer.

CIGB-300 Peptide Targets the CK2 Phospho-Acceptor Domain on Human Papillomavirus E7 and Disrupts the Retinoblastoma (RB) Complex in Cervical Cancer Cells.

CIGB-300 is a clinical-grade anti-Protein Kinase CK2 peptide, binding both its substrate's phospho-acceptor site and the CK2α catalytic subunit. The cyclic p15 inhibitory domain of CIGB-300 was initially selected in a phage display library screen for its ability to bind the CK2 phospho-acceptor domain ofHPV-16 E7. However, the actual role of this targeting in CIGB-300 antitumoral mechanism remains unexplored. Here, we investigated the physical interaction of CIGB-300 with HPV-E7 and its impact on CK2-mediated phosphorylation. Hence, we studied the relevance of targeting E7 phosphorylation for the cytotoxic effect induced by CIGB-300. Finally, co-immunoprecipitation experiments followed by western blotting were performed to study the impact of the peptide on the E7-pRB interaction. Interestingly, we found a clear binding of CIGB-300 to the N terminal region of E7 proteins of the HPV-16 type. Accordingly, the in vivo physical interaction of the peptide with HPV-16 E7 reduced CK2-mediated phosphorylation of E7, as well as its binding to the tumor suppressor pRB. However, the targeting of E7 phosphorylation by CIGB-300 seemed to be dispensable for the induction of cell death in HPV-18 cervical cancer-derived C4-1 cells. These findings unveil novel molecular clues to the means by which CIGB-300 triggers cell death in cervical cancer cells.

Regulation of HPV E7 Stability by E6-Associated Protein (E6AP).

High-risk human papillomaviruses (HPVs) are responsible for most human cervical cancers, and uncontrolled expression of the two key viral oncoproteins, E6 and E7, stimulates the induction of carcinogenesis. Previous studies have shown that both E6 and E7 are closely associated with different components of the ubiquitin proteasome pathway, including several ubiquitin ligases. Most often these are utilized to target cellular substrates for proteasome-mediated degradation, but in the case of E6, the E6AP ubiquitin ligase plays a critical role in controlling E6 stability. We now show that knockdown of E6AP in HPV-positive cervical cancer-derived cells causes a marked decrease in E7 protein levels. This is due to a decrease in the E7 half-life and occurs in a proteasome-dependent manner. In an attempt to define the underlying mechanism, we show that E7 can also associate with E6AP, albeit in a manner different from that of E6. In addition, we show that E6AP-dependent stabilization of E7 also leads to an increase in the degradation of E7's cellular target substrates. Interestingly, ectopic overexpression of E6 oncoprotein results in lower levels of E7 protein through sequestration of E6AP. We also show that increased E7 stability in the presence of E6AP increases the proliferation of the cervical cancer-derived cell lines. These results demonstrate a surprising interplay between E6 and E7, in a manner which is mediated by the E6AP ubiquitin ligase. IMPORTANCE This is the first demonstration that E6AP can directly help stabilize the HPV E7 oncoprotein, in a manner similar to that observed with HPV E6. This redefines how E6 and E7 can cooperate and potentially modulate each other's activity and further highlights the essential role played by E6AP in the viral life cycle and malignancy.

Human Papillomavirus infection requires the CCT Chaperonin Complex.

Human papillomavirus (HPV) infection is a multi-step process that implies complex interactions of the viral particles with cellular proteins. The HPV capsid includes the two structural proteins L1 and L2, that play crucial roles on infectious viral entry. L2 is particularly relevant for the intracellular trafficking of the viral DNA towards the nucleus. Here, using proteomic studies we identified CCT proteins as novel interaction partners of HPV-16 L2. The CCT multimeric complex is an essential chaperonin which interacts with a large number of protein targets. We analysed the binding of different components of the CCT complex to L2. We confirmed the interaction of this structural viral protein with the CCT subunit 3 (CCT3) and we found that this interaction requires the N-terminal region of L2. Defects in HPV-16 pseudoviral particle (PsVs) infection were revealed by siRNA-mediated knockdown of some CCT subunits. While a substantial drop in the viral infection was associated with the ablation of CCT component 2, even more pronounced effects on infectivity were observed upon depletion of CCT component 3. Using confocal immunofluorescence assays, CCT3 co-localised with HPV PsVs at early times after infection, with L2 being required for this to occur. Further analysis showed the colocalization of several other subunits of CCT with the PsVs. Moreover, we observed a defect in capsid uncoating and a change in PsVs intracellular normal processing when ablating CCT3. Taken together, these studies demonstrate the importance of CCT chaperonin during HPV infectious entry.ImportanceSeveral of the mechanisms that function during the infection of target cells by HPV particles have been previously described. However, many aspects of this process remain unknown. In particular, the role of cellular proteins functioning as molecular chaperones during HPV infections has been only partially investigated. To the best of our knowledge, we describe here for the first time, a requirement of the CCT chaperonin for HPV infection. The role of this cellular complex seems to be determined by the binding of its component 3 to the viral structural protein L2. However, CCT's effect on HPV infection most probably comprises the whole chaperonin complex. Altogether, these studies define an important role for the CCT chaperonin in the processing and intracellular trafficking of HPV particles and in subsequent viral infectious entry.

Human Papillomavirus 16 L2 Recruits both Retromer and Retriever Complexes during Retrograde Trafficking of the Viral Genome to the Cell Nucleus.

Previous studies have identified an interaction between the human papillomavirus (HPV) L2 minor capsid protein and sorting nexins 17 and 27 (SNX17 and SNX27) during virus infection. Further studies show the involvement of both retromer and retriever complexes in this process since knockdown of proteins from either complex impairs infection. In this study, we show that HPV L2 and 5-ethynyl-2'-deoxyuridine (EdU)-labeled pseudovirions colocalize with both retromer and retriever, with components of each complex being bound by L2 during infection. We also show that both sorting nexins may interact with either of the recycling complexes but that the interaction between SNX17 and HPV16 L2 is not responsible for retriever recruitment during infection, instead being required for retromer recruitment. Furthermore, we show that retriever recruitment most likely involves a direct interaction between L2 and the C16orf62 subunit of the retriever, in a manner similar to that of its interaction with the VPS35 subunit of retromer.IMPORTANCE Previous studies identified sorting nexins 17 and 27, as well as the retromer complex, as playing a role in HPV infection. This study shows that the newly identified retriever complex also plays an important role and begins to shed light on how both sorting nexins contribute to retromer and retriever recruitment during the infection process.

SGEF forms a complex with Scribble and Dlg1 and regulates epithelial junctions and contractility.

The canonical Scribble polarity complex is implicated in regulation of epithelial junctions and apical polarity. Here, we show that SGEF, a RhoG-specific GEF, forms a ternary complex with Scribble and Dlg1, two members of the Scribble complex. SGEF targets to apical junctions in a Scribble-dependent fashion and functions in the regulation of actomyosin-based contractility and barrier function at tight junctions as well as E-cadherin-mediated formation of adherens junctions. Surprisingly, SGEF does not control the establishment of polarity. However, in 3D cysts, SGEF regulates the formation of a single open lumen. Interestingly, SGEF's nucleotide exchange activity regulates the formation and maintenance of adherens junctions, and in cysts the number of lumens formed, whereas SGEF's scaffolding activity is critical for regulation of actomyosin contractility and lumen opening. We propose that SGEF plays a key role in coordinating junctional assembly and actomyosin contractility by bringing together Scribble and Dlg1 and targeting RhoG activation to cell-cell junctions.

Diverse Papillomavirus Types Induce Endosomal Tubulation.

Previous studies have shown that the endoplasmic reticulum (ER)-anchored protein VAP is strictly required by human papillomavirus type 16 (HPV-16) for successful infectious entry. Entry appeared to be mediated in part through the induction of endosomal tubulation and subsequent transport of the virion to the trans-Golgi network (TGN). In this study, we were interested in investigating whether this mechanism of infectious entry is conserved across multiple Papillomavirus types. To do this, we analyzed the role of VAP and endosomal tubulation following infection with Pseudovirions (PsVs) derived from the alpha, beta, delta, kappa, and pi papillomavirus genera, reflecting viruses that are important human and animal pathogens. We demonstrate that VAP is essential for infection with all PV types analyzed. Furthermore, we find that VAP and EGFR-dependent endosomal tubulation is also induced by all these different Papillomaviruses. These results indicate an evolutionarily conserved requirement for VAP-induced endocytic tubulation during Papillomavirus infectious entry.

The HPV-18 E7 CKII phospho acceptor site is required for maintaining the transformed phenotype of cervical tumour-derived cells.

The Human Papillomavirus E7 oncoprotein plays an essential role in the development and maintenance of malignancy, which it achieves through targeting a number of critical cell control pathways. An important element in the ability of E7 to contribute towards cell transformation is the presence of a Casein Kinase II phospho-acceptor site within the CR2 domain of the protein. Phosphorylation is believed to enhance E7 interaction with a number of different cellular target proteins, and thereby increase the ability of E7 to enhance cell proliferation and induce malignancy. However, there is little information on how important this site in E7 is, once the tumour cells have become fully transformed. In this study, we have performed genome editing of the HPV-18 E7 CKII recognition site in C4-1 cervical tumour-derived cells. We first show that mutation of HPV18 E7 S32/S34 to A32/A34 abolishes CKII phosphorylation of E7, and subsequently we have isolated C4-1 clones containing these mutations in E7. The cells continue to proliferate, but are somewhat more slow-growing than wild type cells, reach lower saturation densities, and are also more susceptible to low nutrient conditions. These cells are severely defective in matrigel invasion assays, partly due to downregulation of matrix metalloproteases (MMPs). Mechanistically, we find that phosphorylation of E7 plays a direct role in the ability of E7 to activate AKT signaling, which in turn is required for optimal levels of MMP secretion. These results demonstrate that the E7 CKII phospho-acceptor site thus continues to play an important role for E7's activity in cells derived from cervical cancers, and suggests that blocking this activity of E7 could be expected to have therapeutic potential.

Phosphorylation of Human Papillomavirus Type 16 L2 Contributes to Efficient Virus Infectious Entry.

The human papillomavirus (HPV) capsid comprises two viral proteins, L1 and L2, with the L2 component being essential to ensure efficient endocytic transport of incoming viral genomes. Several studies have previously reported that L1 and L2 are posttranslationally modified, but it is uncertain whether these modifications affect HPV infectious entry. Using a proteomic screen, we identified a highly conserved phospho-acceptor site on the HPV-16 and bovine papillomavirus 1 (BPV-1) L2 proteins. The phospho-modification of L2 and its presence in HPV pseudovirions (PsVs) were confirmed using anti-phospho-L2-specific antibodies. Mutation of the phospho-acceptor sites of both HPV-16 and BPV-1 L2 resulted in the production of infectious virus particles, with no differences in efficiencies of packaging the reporter DNA. However, these mutated PsVs showed marked defects in infectious entry. Further analysis revealed a defect in uncoating, characterized by a delay in the exposure of a conformational epitope on L1 that indicates capsid uncoating. This uncoating defect was accompanied by a delay in the proteolysis of both L1 and L2 in mutated HPV-16 PsVs. Taken together, these studies indicate that phosphorylation of L2 during virus assembly plays an important role in optimal uncoating of virions during infection, suggesting that phosphorylation of the viral capsid proteins contributes to infectious entry.IMPORTANCE The papillomavirus L2 capsid protein plays an essential role in infectious entry, where it directs the successful trafficking of incoming viral genomes to the nucleus. However, nothing is known about how potential posttranslational modifications may affect different aspects of capsid assembly or infectious entry. In this study, we report the first phospho-specific modification of the BPV-1 and HPV-16 L2 capsid proteins. The phospho-acceptor site is very highly conserved across multiple papillomavirus types, indicating a highly conserved function within the L2 protein and the viral capsid. We show that this modification plays an essential role in infectious entry, where it modulates susceptibility of the incoming virus to capsid disassembly. These studies therefore define a completely new means of regulating the papillomavirus L2 proteins, a regulation that optimizes endocytic processing and subsequent completion of the infectious entry pathway.

Oncogenic comparison of human papillomavirus type 58 E7 variants.

Human papillomavirus 58 (HPV58) ranks the second or third in East Asian cervical cancers. Current studies on HPV58 are scarce and focus on the prototype. Previously, we identified the three most common circulating HPV58 E7 strains contained amino acid alterations: G41R/G63D (51%), T20I/G63S (22%) and T74A/D76E (14%) respectively. Among them, the T20I/G63S variant (V1) had a stronger epidemiological association with cervical cancer. We therefore suggested that V1 possessed stronger oncogenicity than the other two variants. Here, we performed phenotypic assays to characterize and compare their oncogenicities with HPV58 E7 prototype. Our results showed that overexpression of V1 conferred a higher colony-forming ability to primary murine epithelial cells than prototype (P < 0.05) and other variants, implicating its higher immortalising potential. Further experiments showed that both V1 and prototype enhanced the anchorage-independent growth of NIH/3T3 cells (P < 0.001), implicating their stronger transforming power than the two other variants. Moreover, they possessed an increased ability to degrade pRb (P < 0.001), which is a major effector pathway of E7-driven oncogenesis. Our work represents the first study to compare the oncogenicities of HPV58 E7 prototype and variants. These findings deepened our understanding of HPV58 and might inform clinical screening and follow-up strategy.

The Human Papillomavirus E6 PDZ Binding Motif Links DNA Damage Response Signaling to E6 Inhibition of p53 Transcriptional Activity.

The presence of a PDZ binding motif (PBM) in the human papillomavirus (HPV) E6 oncoprotein appears to be a characteristic marker of high oncogenic potential and confers interaction with a number of different cellular PDZ domain-containing substrates. The E6 PBM is also subject to phosphorylation, resulting in inhibition of E6 PDZ binding activity and instead allowing E6 to associate with 14-3-3 proteins. In this study, we analyzed the conditions under which the E6 PBM is phosphorylated. We demonstrate that in normal cycling cells, the levels of E6 phosphorylation are very low. However, following exposure of cells to oxidative stress or the induction of DNA damage, there is a striking increase in the levels of E6 phosphorylation. Depending on the specific stimulus, this phosphorylation of E6 can involve the ATM/ATR pathway and is performed primarily through Chk1, although the Chk2 pathway is also involved indirectly through activation of protein kinase A (PKA). To understand the biological relevance of these phospho-modifications of E6, we analyzed their effects upon the ability of E6 to inhibit p53 transcriptional activity. We show that an intact E6 phospho-acceptor site plays an essential role in the ability of E6 to inhibit p53 transcriptional activity on a subset of p53-responsive promoters in a manner that is independent of E6's ability to direct p53 degradation. These results are, to our knowledge, the first example of a DNA damage response controlling PBM-PDZ recognition. This study also provides links between the DNA damage response, the regulation of E6 PBM function, and the inhibition of p53 activity and begins to explain how HPV-infected cells remain within the cell cycle, despite activation of DNA damage response pathways during productive virus infections.IMPORTANCE The cancer-causing HPV E6 oncoproteins all possess a PDZ binding motif at their extreme carboxy termini. Depending upon whether this motif is phosphorylated, E6 can recognize PDZ domain-containing proteins or members of the 14-3-3 family of proteins. We show here that DNA damage response pathways directly signal to the E6 PBM, resulting in Chk1- and Chk2-driven phosphorylation. This phosphorylation is particularly pronounced following treatment of cells with a variety of different chemotherapeutic drugs. A direct functional consequence of this signaling is to confer an enhanced ability upon E6 to inhibit p53 transcriptional activity in a proteasome-independent but phosphorylation-dependent manner. These results are the first example of DNA damage signaling pathways regulating PBM-PDZ interactions and provide the mechanistic link between E6 PBM function and perturbation of p53 activity.

Human Papillomavirus 16 Infection Induces VAP-Dependent Endosomal Tubulation.

Human papillomavirus (HPV) infection involves complex interactions with the endocytic transport machinery, which ultimately facilitates the entry of the incoming viral genomes into the trans-Golgi network (TGN) and their subsequent nuclear entry during mitosis. The endosomal pathway is a highly dynamic intracellular transport system, which consists of vesicular compartments and tubular extensions, although it is currently unclear whether incoming viruses specifically alter the endocytic machinery. In this study, using MICAL-L1 as a marker for tubulating endosomes, we show that incoming HPV-16 virions induce a profound alteration in global levels of endocytic tubulation. In addition, we also show a critical requirement for the endoplasmic reticulum (ER)-anchored protein VAP in this process. VAP plays an essential role in actin nucleation and endosome-to-Golgi transport. Indeed, the loss of VAP results in a dramatic decrease in the level of endosomal tubulation induced by incoming HPV-16 virions. This is also accompanied by a marked reduction in virus infectivity. In VAP knockdown cells, we see that the defect in virus trafficking occurs after capsid disassembly but prior to localization at the trans-Golgi network, with the incoming virion-transduced DNA accumulating in Vps29/TGN46-positive hybrid vesicles. Taken together, these studies demonstrate that infection with HPV-16 virions induces marked alterations of endocytic transport pathways, some of which are VAP dependent and required for the endosome-to-Golgi transport of the incoming viral L2/DNA complex.IMPORTANCE Human papillomavirus infectious entry involves multiple interactions with the endocytic transport machinery. In this study, we show that incoming HPV-16 virions induce a dramatic increase in endocytic tubulation. This tubulation requires ER-associated VAP, which plays a critical role in ensuring the delivery of cargoes from the endocytic compartments to the trans-Golgi network. Indeed, the loss of VAP blocks HPV infectious entry at a step after capsid uncoating but prior to localization at the trans-Golgi network. These results define a critical role for ER-associated VAP in endocytic tubulation and in HPV-16 infectious entry.

HPV-16 virions can remain infectious for 2 weeks on senescent cells but require cell cycle re-activation to allow virus entry.

Successful infection with Human Papillomaviruses requires mitosis, when incoming viral genomes gain access to nuclear components. However, very little is known about how long HPV particles can remain infectious in non-dividing cells or in which cellular compartments these viruses may reside. To investigate these questions we have used BJ cells as a reversible model of senescence and show that HPV-16 can only infect early-passage proliferating cells. Late-passage senescent cells are resistant to HPV infection, but this can be reversed by inducing cell cycle re-entry with a p53 siRNA. In senescent cells we find that efficient virus entry can be attained upon cell cycle re-entry 16 days after infection, demonstrating that HPV can persist for 2 weeks prior to induction of mitosis. However, exposing cells to anti-HPV-16 L1 neutralising antibody blocks infection at these late time points, suggesting that the virions reside near the cell surface. Indeed, immunofluorescence analysis shows that virions accumulate on the cell surface of senescent cells and only enter endocytic vesicles upon stimulation with p53 siRNA. These results demonstrate that HPV-16 virions can remain viable on a non-dividing cell for extended periods of time, but are nonetheless vulnerable to antibody-induced neutralisation throughout.

The VPS4 component of the ESCRT machinery plays an essential role in HPV infectious entry and capsid disassembly.

Human Papillomavirus (HPV) infection involves multiple steps, from cell attachment, through endocytic trafficking towards the trans-Golgi network, and, ultimately, the entry into the nucleus during mitosis. An essential viral protein in infectious entry is the minor capsid protein L2, which engages different components of the endocytic sorting machinery during this process. The ESCRT machinery is one such component that seems to play an important role in the early stages of infection. Here we have analysed the role of specific ESCRT components in HPV infection, and we find an essential role for VPS4. Loss of VPS4 blocks infection with multiple PV types, suggesting an evolutionarily conserved critical step in infectious entry. Intriguingly, both L1 and L2 can interact with VPS4, and appear to be in complex with VPS4 during the early stages of virus infection. By using cell lines stably expressing a dominant-negative mutant form of VPS4, we also show that loss of VPS4 ATPase activity results in a marked delay in capsid uncoating, resulting in a defect in the endocytic transport of incoming PsVs. These results demonstrate that the ESCRT machinery, and in particular VPS4, plays a critical role in the early stages of PV infection.

The PTPN14 Tumor Suppressor Is a Degradation Target of Human Papillomavirus E7.

Activation of signaling pathways ensuring cell growth is essential for the proliferative competence of human papillomavirus (HPV)-infected cells. Tyrosine kinases and phosphatases are key regulators of cellular growth control pathways. A recently identified potential cellular target of HPV E7 is the cytoplasmic protein tyrosine phosphatase PTPN14, which is a potential tumor suppressor and is linked to the control of the Hippo and Wnt/beta-catenin signaling pathways. In this study, we show that the E7 proteins of both high-risk and low-risk mucosal HPV types can interact with PTPN14. This interaction is independent of retinoblastoma protein (pRb) and involves residues in the carboxy-terminal region of E7. We also show that high-risk E7 induces proteasome-mediated degradation of PTPN14 in cells derived from cervical tumors. This degradation appears to be independent of cullin-1 or cullin-2 but most likely involves the UBR4/p600 ubiquitin ligase. The degree to which E7 downregulates PTPN14 would suggest that this interaction is important for the viral life cycle and potentially also for the development of malignancy. In support of this we find that overexpression of PTPN14 decreases the ability of HPV-16 E7 to cooperate with activated EJ-ras in primary cell transformation assays.IMPORTANCE This study links HPV E7 to the deregulation of protein tyrosine phosphatase signaling pathways. PTPN14 is classified as a potential tumor suppressor protein, and here we show that it is very susceptible to HPV E7-induced proteasome-mediated degradation. Intriguingly, this appears to use a mechanism that is different from that employed by E7 to target pRb. Therefore, this study has important implications for our understanding of the molecular basis for E7 function and also sheds important light on the potential role of PTPN14 as a tumor suppressor.

Monitoring HPV-16 E7 phosphorylation events.

HPV-16 E7 is one of the key proteins that, by interfering with the host metabolism through many protein-protein interactions, hijacks cell regulation and contributes to malignancy. Here we report the high resolution investigation of the CR3 region of HPV-16 E7, both as an isolated domain and in the full-length protein. This opens the way to the atomic level study of the many interactions in which HPV-16 E7 is involved. Along these lines we show here the effect of one of the key post-translational modifications of HPV-16 E7, the phosphorylation by casein kinase II.

A naturally occurring variant of HPV-16 E7 exerts increased transforming activity through acquisition of an additional phospho-acceptor site.

Human Papillomavirus E6 and E7 play critical roles in cancer development, although not all isolates of the viral oncoproteins are identical. A common E7 variant encodes an amino acid change at N29S. We show that this change increases the levels of phosphorylation by CKII by creating an additional phospho-acceptor site at S29. This confers increased phospho-dependent interaction with a number of cellular targets, including TATA Box Binding Protein (TBP) and pRb. A further consequence is an increased ability to target pRb and p130 for degradation. Biologically, these biochemical differences are reflected in an increased ability of the N29S variant to transform primary rodent cells. This is the first study to demonstrate an important biochemical change in E7 function caused by a naturally occurring variation, and we suggest that the N29S variant merits further assessment to determine whether it has an increased association with the development of HPV-associated malignancies.