Activation of cap-dependent translation by mucosal human papillomavirus E6 proteins is dependent on the integrity of the LXXLL binding motif

The human papillomavirus (HPV) type 16 (HPV16) E6 protein can stimulate mechanistic target of rapamycin complex 1 (mTORC1) signaling and cap-dependent translation through activation of the PDK1 and mTORC2 kinases. Here we report that HPV18 E6 also enhances cap-dependent translation. The integrity of LXXLL and PDZ protein binding domains is important for activation of cap-dependent translation by high-risk mucosal HPV E6 proteins. Consistent with this model, low-risk mucosal HPV6b and HPV11 E6 proteins, which do not contain a PDZ protein binding motif, also activate cap-dependent translation and mTORC1, albeit at a lower efficiency than high-risk HPV E6 proteins. In contrast, cutaneous HPV5 and HPV8 E6 proteins, which lack LXXLL and PDZ motif protein binding, do not enhance cap-dependent translation. Mutagenic analyses of low-risk HPV E6 proteins revealed that association with the LXXLL motif containing ubiquitin ligase E6AP (UBE3A) correlates with activation of cap-dependent translation. Hence, activation of mTORC1 and cap-dependent translation may be important for the viral life cycle in specific epithelial tissue types and contribute to cellular transformation in cooperation with other biological activities of high-risk HPV E6-containing proteins.     

A systematic analysis of human papillomavirus (HPV) E6 PDZ substrates identifies MAGI-1 as a major target of HPV type 16 (HPV-16) and HPV-18 whose loss accompanies disruption of tight junctions

The E6 proteins from high-risk, cancer-causing types of human papillomavirus (HPV) are characterized by the presence of a PDZ (PSD95/Dlg/ZO-1) binding motif in their extreme carboxy termini, through which they interact with a number of cellular PDZ domain-containing substrates. In order to ascertain how many of these are degraded by E6 in vivo, we performed an extensive analysis of the effects of E6 ablation on the expression levels of a number of previously reported E6 PDZ substrates. Using HPV type 16 (HPV-16)-positive CaSKi cells and HPV-18-positive HeLa cells, we have found that MAGI-1 is a major degradation target of both HPV-16 and HPV-18 E6. In contrast, hDlg, hScrib, PTPN3, TIP2, FAP1, and PSD95 all exhibit various degrees of susceptibility to E6-induced degradation, and a high degree of HPV type specificity is observed for certain substrates. We also show that E6 preferentially targets MAGI-1 within the nucleus and at membrane sites. One of the direct consequences of MAGI-1 degradation is a loss of tight-junction integrity, as determined by mislocalization of the tight-junction protein ZO-1. Ablation of E6 expression restores tight junctions, and this restoration is dependent on the presence of MAGI-1. These results demonstrate that oncogenic HPV E6 proteins disrupt cellular tight junctions through the degradation of MAGI-1, and they provide further evidence of how the PDZ binding potential of E6 can contribute to HPV-induced malignancy.     

Role of the PDZ domain-binding motif of the oncoprotein E6 in the pathogenesis of human papillomavirus type 31

A number of PDZ domain-containing proteins have been identified as binding partners for the oncoprotein E6 of the high-risk type human papillomaviruses (HPVs). These include hDlg, hScrib, MAGI-1, MAGI-2, MAGI-3, and MUPP1. The PDZ domain-binding motif (-X-T-X-V) at the carboxy terminus of E6 is essential for targeting PDZ proteins for proteasomal degradation. The presence of this motif only in the high-risk HPVs suggests its possible role in HPV-induced oncogenesis. To investigate the role of the PDZ domain-binding motif of E6 in the HPV life cycle, two mutant HPV31 genomes were constructed: E6ValDelta, with a deletion of the last amino acid residue of E6 (valine), and E6ETQVDelta, with a deletion of the entire PDZ domain-binding motif of E6 (ETQV). Three human foreskin keratinocyte (HFK) cell lines were established which maintained transfected wild-type HPV31 or either of two mutant genomes. Cells containing either of two mutant genomes were significantly retarded in their growth rates and reduced in their viral copy numbers compared to those transfected with wild-type genomes. Western analysis did not reveal any significant changes in the levels of PDZ proteins following stable transfection of any HPV31 genomes into HFKs. Although the E6ETQVDelta-transfected HFKs exhibited a pattern of morphological differentiation that appeared different from the HPV31 wild-type-transfected HFKs in organotypic raft cultures, immunohistochemical analysis failed to identify substantial changes in the differentiation-dependent membrane localization of hDlg proteins. These results suggest that binding of E6 to PDZ proteins modulates the early viral functions such as proliferation and maintenance of the viral copy number in undifferentiated cells.     

Surface plasmon resonance analysis of the binding of high‐risk mucosal HPV E6 oncoproteins to the PDZ1 domain of the tight junction protein MAGI‐1

The E6 oncoproteins from high‐risk mucosal human papillomavirus (HPV) induce cervical cancer via two major activities, the binding and the degradation of the p53 protein and PDZ domain‐containing proteins. Human MAGI‐1 is a multi‐PDZ domain protein implicated into protein complex assembly at cell–cell contacts. High‐risk mucosal HPV E6 proteins interact with the PDZ1 domain of MAGI‐1 via a C‐terminal consensus binding motif. Here, we developed a medium throughput protocol to accurately measure by surface plasmon resonance affinity constants of protein domains binding to peptidic sequences produced as recombinant fusions to the glutathione‐S‐transferase (GST). This approach was applied to measure the binding of MAGI‐1 PDZ1 to the C‐termini of viral or cellular proteins. Both high‐risk mucosal HPV E6 C‐terminal peptides and cellular partners of MAGI‐1 PDZ1 bind to MAGI‐1 PDZ1 with comparable dissociation constants in the micromolar range. MAGI‐1 PDZ1 shows a preference for C‐termini with a valine at position 0 and a negative charge at position ?3, confirming previous studies performed with HPV18 E6. A detailed combined analysis via site‐directed mutagenesis of the HPV16 C‐terminal peptide and PDZ1 indicated that interactions mediated by charged residues upstream the PDZ‐binding motif strongly contribute to binding selectivity of this interaction. In addition, our work highlighted the K₄₉₉ residue of MAGI‐1 as a novel determinant of binding specificity. Finally, we showed that MAGI‐1 PDZ1 also binds to the C‐termini of LPP and Tax proteins, which were already known to bind to PDZ proteins but not to MAGI‐1. Copyright © 2010 John Wiley & Sons, Ltd.     

Structures of a human papillomavirus (HPV) E6 polypeptide bound to MAGUK proteins: mechanisms of targeting tumor suppressors by a high-risk HPV oncoprotein

Human papillomavirus (HPV) E6 oncoprotein targets certain tumor suppressors such as MAGI-1 and SAP97/hDlg for degradation. A short peptide at the C terminus of E6 interacts specifically with the PDZ domains of these tumor suppressors, which is a property unique to high-risk HPVs that are associated with cervical cancer. The detailed recognition mechanisms between HPV E6 and PDZ proteins are unclear. To understand the specific binding of cellular PDZ substrates by HPV E6, we have solved the crystal structures of the complexes containing a peptide from HPV18 E6 bound to three PDZ domains from MAGI-1 and SAP97/Dlg. The complex crystal structures reveal novel features of PDZ peptide recognition that explain why high-risk HPV E6 can specifically target these cellular tumor suppressors for destruction. Moreover, a new peptide-binding loop on these PDZs is identified as interacting with the E6 peptide. Furthermore, we have identified an arginine residue, unique to high-risk HPV E6 but outside the canonical core PDZ recognition motif, that plays an important role in the binding of the PDZs of both MAGI-I and SAP97/Dlg, the mutation of which abolishes E6's ability to degrade the two proteins. Finally, we have identified a dimer form of MAGI-1 PDZ domain 1 in the cocrystal structure with E6 peptide, which may have functional relevance for MAGI-1 activity. In addition to its novel insights into the biochemistry of PDZ interactions, this study is important for understanding HPV-induced oncogenesis; this could provide a basis for developing antiviral and anticancer compounds.     

E6 and E7 from human papillomavirus type 16 cooperate to target the PDZ protein Na/H exchange regulatory factor 1

Previous studies have shown that the PDZ-binding motif of the E6 oncoprotein from the mucosal high-risk (HR) human papillomavirus (HPV) types plays a key role in HPV-mediated cellular transformation in in vitro and in vivo experimental models. HR HPV E6 oncoproteins have the ability to efficiently degrade members of the PDZ motif-containing membrane-associated guanylate kinase (MAGUK) family; however, it is possible that other PDZ proteins are also targeted by E6. Here, we describe a novel interaction of HPV type 16 (HPV16) E6 with a PDZ protein, Na(+)/H(+) exchange regulatory factor 1 (NHERF-1), which is involved in a number of cellular processes, including signaling and transformation. HPV16 E6 associates with and promotes the degradation of NHERF-1, and this property is dependent on the C-terminal PDZ-binding motif of E6. Interestingly, HPV16 E7, via the activation of the cyclin-dependent kinase complexes, promoted the accumulation of a phosphorylated form of NHERF-1, which is preferentially targeted by E6. Thus, both oncoproteins appear to cooperate in targeting NHERF-1. Notably, HPV18 E6 is not able to induce NHERF-1 degradation, indicating that this property is not shared with E6 from all HR HPV types. Downregulation of NHERF-1 protein levels was also observed in HPV16-positive cervical cancer-derived cell lines, such as SiHa and CaSki, as well as HPV16-positive cervical intraepithelial neoplasia (CIN). Finally, our data show that HPV16-mediated NHERF-1 degradation correlates with the activation of the phosphatidylinositol-3'-OH kinase (PI3K)/AKT signaling pathway, which is known to play a key role in carcinogenesis.     

Interaction of the Human Papillomavirus E6 Oncoprotein with Sorting Nexin 27 Modulates Endocytic Cargo Transport Pathways

A subset of high-risk Human Papillomaviruses (HPVs) are the causative agents of a large number of human cancers, of which cervical is the most common. Two viral oncoproteins, E6 and E7, contribute directly towards the development and maintenance of malignancy. A characteristic feature of the E6 oncoproteins from cancer-causing HPV types is the presence of a PDZ binding motif (PBM) at its C-terminus, which confers interaction with cellular proteins harbouring PDZ domains. Here we show that this motif allows E6 interaction with Sorting Nexin 27 (SNX27), an essential component of endosomal recycling pathways. This interaction is highly conserved across E6 proteins from multiple high-risk HPV types and is mediated by a classical PBM-PDZ interaction but unlike many E6 targets, SNX27 is not targeted for degradation by E6. Rather, in HPV-18 positive cell lines the association of SNX27 with components of the retromer complex and the endocytic transport machinery is altered in an E6 PBM-dependent manner. Analysis of a SNX27 cargo, the glucose transporter GLUT1, reveals an E6-dependent maintenance of GLUT1 expression and alteration in its association with components of the endocytic transport machinery. Furthermore, knockdown of E6 in HPV-18 positive cervical cancer cells phenocopies the loss of SNX27, both in terms of GLUT1 expression levels and its vesicular localization, with a concomitant marked reduction in glucose uptake, whilst loss of SNX27 results in slower cell proliferation in low nutrient conditions. These results demonstrate that E6 interaction with SNX27 can alter the recycling of cargo molecules, one consequence of which is modulation of nutrient availability in HPV transformed tumour cells.     

PDZRN3/LNX3 Is a Novel Target of Human Papillomavirus Type 16 (HPV-16) and HPV-18 E6

High-risk human papillomavirus (HPV) E6 proteins have a C-terminal PDZ binding motif through which they bind, and target for proteasome-mediated degradation, a number of PDZ-containing cellular targets. Recent studies have suggested that the RING-containing ubiquitin-protein ligase PDZRN3 might also be an HPV E6 target. In this analysis, we show that HPV-16 and HPV-18 E6 can target PDZRN3 in a PDZ- and proteasome-dependent manner and provide a connection between the HPV life cycle and differentiation-related STAT signaling.     

Multi-PDZ domain protein MUPP1 is a cellular target for both adenovirus E4-ORF1 and high-risk papillomavirus type 18 E6 oncoproteins

A general theme that has emerged from studies of DNA tumor viruses is that otherwise unrelated oncoproteins encoded by these viruses often target the same important cellular factors. Major oncogenic determinants for human adenovirus type 9 (Ad9) and high-risk human papillomaviruses (HPV) are the E4-ORF1 and E6 oncoproteins, respectively, and although otherwise unrelated, both of these viral proteins possess a functional PDZ domain-binding motif that is essential for their transforming activity and for binding to the PDZ domain-containing and putative tumor suppressor protein DLG. We report here that the PDZ domain-binding motifs of Ad9 E4-ORF1 and high-risk HPV-18 E6 also mediate binding to the widely expressed cellular factor MUPP1, a large multi-PDZ domain protein predicted to function as an adapter in signal transduction. With regard to the consequences of these interactions in cells, we showed that Ad9 E4-ORF1 aberrantly sequesters MUPP1 within the cytoplasm of cells whereas HPV-18 E6 targets this cellular protein for degradation. These effects were specific because mutant viral proteins unable to bind MUPP1 lack these activities. From these results, we propose that the multi-PDZ domain protein MUPP1 is involved in negatively regulating cellular proliferation and that the transforming activities of two different viral oncoproteins depend, in part, on their ability to inactivate this cellular factor.     

The structural and dynamic response of MAGI-1 PDZ1 with noncanonical domain boundaries to the binding of human papillomavirus E6

PDZ domains are protein interaction domains that are found in cytoplasmic proteins involved in signaling pathways and subcellular transport. Their roles in the control of cell growth, cell polarity, and cell adhesion in response to cell contact render this family of proteins targets during the development of cancer. Targeting of these network hubs by the oncoprotein E6 of “high-risk” human papillomaviruses (HPVs) serves to effect the efficient disruption of cellular processes. Using NMR, we have solved the three-dimensional solution structure of an extended construct of the second PDZ domain of MAGI-1 (MAGI-1 PDZ1) alone and bound to a peptide derived from the C-terminus of HPV16 E6, and we have characterized the changes in backbone dynamics and hydrogen bonding that occur upon binding. The binding event induces quenching of high-frequency motions in the C-terminal tail of the PDZ domain, which contacts the peptide upstream of the canonical X-[T/S]-X-[L/V] binding motif. Mutations designed in the C-terminal flanking region of the PDZ domain resulted in a significant decrease in binding affinity for E6 peptides. This detailed analysis supports the notion of a global response of the PDZ domain to the binding event, with effects propagated to distal sites, and reveals unexpected roles for the sequences flanking the canonical PDZ domain boundaries.     

E6AP-dependent degradation of DLG4/PSD95 by high-risk human papillomavirus type 18 E6 protein

In most cervical cancers, DNAs of high-risk mucosotropic human papillomaviruses (HPVs), such as types 16 and 18, are maintained so as to express two viral proteins, E6 and E7, suggesting that they play important roles in carcinogenesis. The carboxy-terminal PDZ domain-binding motif of the E6 proteins is in fact essential for transformation of rodent cells and induction of hyperplasia in E6-transgenic mouse skin. To date, seven PDZ domain-containing proteins, including DLG1/hDLG, which is a human homologue of the Drosophila discs large tumor suppressor (Dlg), have been identified as targets of high-risk HPV E6 proteins. Here, we describe DLG4/PSD95, another human homologue of Dlg, as a novel E6 target. DLG4 was found to be expressed in normal human cells, including cervical keratinocytes, but only to a limited extent in both HPV-positive and HPV-negative cervical cancer cell lines. Expression of HPV18 E6 in HCK1T decreased DLG4 levels more strongly than did HPV16 E6, the carboxy-terminal motif of the proteins being critical for binding and degradation of DLG4 in vitro. DLG4 levels were restored by expression of either E6AP-specific short hairpin RNA or bovine papillomavirus type 1 E2 in HeLa but not CaSki or SiHa cells, reflecting downregulation of DLG4 mRNA as opposed to protein by an HPV-independent mechanism in HPV16-positive cancer lines. The tumorigenicity of CaSki cells was strongly inhibited by forced expression of DLG4, while growth in culture was not inhibited at all. These results suggest that DLG4 may function as a tumor suppressor in the development of HPV-associated cancers.     

Structural Insights into a Wildtype Domain of the Oncoprotein E6 and Its Interaction with a PDZ Domain

The high-risk human papilloma virus (HPV) oncoproteins E6 and E7 interact with key cellular regulators and are etiological agents for tumorigenesis and tumor maintenance in cervical cancer and other malignant conditions. E6 induces degradation of the tumor suppressor p53, activates telomerase and deregulates cell polarity. Analysis of E6 derived from a number of high risk HPV finally yielded the first structure of a wild-type HPV E6 domain (PDB 2M3L) representing the second zinc-binding domain of HPV 51 E6 (termed 51Z2) determined by NMR spectroscopy. The 51Z2 structure provides clues about HPV-type specific structural differences between E6 proteins. The observed temperature sensitivity of the well-folded wild-type E6 domain implies a significant malleability of the oncoprotein in vivo. Hence, the structural differences between individual E6 and their malleability appear, together with HPV type-specific surface exposed side-chains, to provide the structural basis for the different interaction networks reported for individual E6 proteins. Furthermore, the interaction of 51Z2 with a PDZ domain of hDlg was analyzed. Human Dlg constitutes a prototypic representative of the large family of PDZ proteins regulating cell polarity, which are common targets of high-risk HPV E6. Nine C-terminal residues of 51Z2 interact with the second PDZ domain of hDlg2. Surface plasmon resonance in conjunction with the NMR spectroscopy derived complex structure (PDB 2M3M) indicate that E6 residues N-terminal to the canonical PDZ-BM of E6 significantly contribute to this interaction and increase affinity. The structure of the complex reveals how residues outside of the classical PDZ-BM enhance the affinity of E6 towards PDZ domains. Such mechanism facilitates successful competition of E6 with cellular PDZ-binding proteins and may apply to PDZ-binding proteins of other viruses as well.     

Comprehensive Analysis of Host Cellular Interactions with Human Papillomavirus E6 Proteins Identifies New E6 Binding Partners and Reflects Viral Diversity

We have begun to define the human papillomavirus (HPV)-associated proteome for a subset of the more than 120 HPV types that have been identified to date. Our approach uses a mass spectrometry-based platform for the systematic identification of interactions between human papillomavirus and host cellular proteins, and here we report a proteomic analysis of the E6 proteins from 16 different HPV types. The viruses included represent high-risk, low-risk, and non-cancer-associated types from genus alpha as well as viruses from four different species in genus beta. The E6 interaction data set consists of 153 cellular proteins, including several previously reported HPV E6 interactors such as p53, E6AP, MAML1, and p300/CBP and proteins containing PDZ domains. We report the genus-specific binding of E6s to either E6AP or MAML1, define the specific HPV E6s that bind to p300, and demonstrate several new features of interactions involving beta HPV E6s. In particular, we report that several beta HPV E6s bind to proteins containing PDZ domains and that at least two beta HPV E6s bind to p53. Finally, we report the newly discovered interaction of proteins of E6 of beta genus, species 2, with the Ccr4-Not complex, the first report of a viral protein binding to this complex. This data set represents a comprehensive survey of E6 binding partners that provides a resource for the HPV field and will allow continued studies on the diverse biology of the human papillomaviruses.     

Emerging theme: cellular PDZ proteins as common targets of pathogenic viruses

More than a decade ago, three viral oncoproteins, adenovirus type 9 E4-ORF1, human T-lymphotropic virus type 1 Tax, and high-risk human papillomavirus E6, were found to encode a related carboxyl-terminal PDZ domain-binding motif (PBM) that mediates interactions with a select group of cellular PDZ proteins. Recent studies have shown that many other viruses also encode PBM-containing proteins that bind to cellular PDZ proteins. Interestingly, these recently recognized viruses include not only some with oncogenic potential (hepatitis B virus, rhesus papillomavirus, cottontail rabbit papillomavirus) but also many without this potential (influenza virus, Dengue virus, tick-borne encephalitis virus, rabies virus, severe acute respiratory syndrome coronavirus, human immunodeficiency virus). Examination of the cellular PDZ proteins that are targets of viral PBMs reveals that the viral proteins often interact with the same or similar types of PDZ proteins, most notably Dlg1 and other members of the membrane-associated guanylate kinase protein family, as well as Scribble. In addition, cellular PDZ protein targets of viral PBMs commonly control tight junction formation, cell polarity establishment, and apoptosis. These findings reveal a new theme in virology wherein many different virus families encode proteins that bind and perturb the function of cellular PDZ proteins. The inhibition or perturbation of the function of cellular PDZ proteins appears to be a widely used strategy for viruses to enhance their replication, disseminate in the host, and transmit to new hosts.     

Human scribble (Vartul) is targeted for ubiquitin-mediated degradation by the high-risk papillomavirus E6 proteins and the E6AP ubiquitin-protein ligase

The high-risk human papillomavirus (HPV) E6 proteins stimulate the ubiquitination and degradation of p53, dependent on the E6AP ubiquitin-protein ligase. Other proteins have also been shown to be targeted for degradation by E6, including hDlg, the human homolog of the Drosophila melanogaster Discs large (Dlg) tumor suppressor. We show here that the human homolog of the Drosophila Scribble (Vartul) (hScrib) tumor suppressor protein is also targeted for ubiquitination by the E6-E6AP complex in vitro and that expression of E6 induces degradation of hScrib in vivo. Characterization of the E6AP-E6-hScrib complex indicated that hScrib binds directly to E6 and that the binding is mediated by the PDZ domains of hScrib and a carboxyl-terminal epitope conserved among the high-risk HPV E6 proteins. Green fluorescent protein-hScrib was localized to the periphery of MDCK cells, where it colocalized with ZO-1, a component of tight junctions. E6 expression resulted in loss of integrity of tight junctions, as measured by ZO-1 localization, and this effect was dependent on the PDZ binding epitope of E6. Thus, the high-risk HPV E6 proteins induce the degradation of the human homologs of two Drosophila PDZ domain-containing tumor suppressor proteins, hDlg and hScrib, both of which are associated with cell junction complexes. The fact that Scrib/Vart and Dlg appear to cooperate in a pathway that controls Drosophila epithelial cell growth suggests that the combined targeting of hScrib and hDlg is an important component of the biologic activity of high-risk HPV E6 proteins.     

The Role of Protein Kinase A Regulation of the E6 PDZ-Binding Domain during the Differentiation-Dependent Life Cycle of Human Papillomavirus Type 18

Human papillomavirus (HPV) E6 proteins of high-risk alpha types target a select group of PSD95/DLG1/ZO1 (PDZ) domain-containing proteins by using a C-terminal PDZ-binding motif (PBM), an interaction that can be negatively regulated by phosphorylation of the E6 PBM by protein kinase A (PKA). Here, we have mutated the canonical PKA recognition motif that partially overlaps with the E6 PBM in the HPV18 genome (E6153PKA) and compared the effect of this mutation on the HPVl8 life cycle in primary keratinocytes with the wild-type genome and with a second mutant genome that lacks the E6 PBM (E6ΔPDZ). Loss of PKA recognition of E6 was associated with increased growth of the genome-containing cells relative to cells carrying the wild-type genome, and upon stratification, a more hyperplastic phenotype, with an increase in the number of S-phase competent cells in the upper suprabasal layers, while the opposite was seen with the E6ΔPDZ genome. Moreover, the growth of wild-type genome-containing cells was sensitive to changes in PKA activity, and these changes were associated with increased phosphorylation of the E6 PBM. In marked contrast to E6ΔPDZ genomes, the E6153PKA mutation exhibited no deleterious effects on viral genome amplification or expression of late proteins. Our data suggest that the E6 PBM function is differentially regulated by phosphorylation in the HPV18 life cycle. We speculate that perturbation of protein kinase signaling pathways could lead to changes in E6 PBM function, which in turn could have a bearing on tumor promotion and progression.     

The Invasive Capacity of HPV Transformed Cells Requires the hDlg-Dependent Enhancement of SGEF/RhoG Activity

A major target of the HPV E6 oncoprotein is the human Discs Large (hDlg) tumour suppressor, although how this interaction contributes to HPV-induced malignancy is still unclear. Using a proteomic approach we show that a strong interacting partner of hDlg is the RhoG-specific guanine nucleotide exchange factor SGEF. The interaction between hDlg1 and SGEF involves both PDZ and SH3 domain recognition, and directly contributes to the regulation of SGEF''s cellular localization and activity. Consistent with this, hDlg is a strong enhancer of RhoG activity, which occurs in an SGEF-dependent manner. We also show that HPV-18 E6 can interact indirectly with SGEF in a manner that is dependent upon the presence of hDlg and PDZ binding capacity. In HPV transformed cells, E6 maintains a high level of RhoG activity, and this is dependent upon the presence of hDlg and SGEF, which are found in complex with E6. Furthermore, we show that E6, hDlg and SGEF each directly contributes to the invasive capacity of HPV-16 and HPV-18 transformed tumour cells. These studies demonstrate that hDlg has a distinct oncogenic function in the context of HPV induced malignancy, one of the outcomes of which is increased RhoG activity and increased invasive capacity.