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.     

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.     

Differential regulation of cell-cell contact, invasion and anoikis by hScrib and hDlg in keratinocytes

The components of the Scrib/Dlg tumour suppressor complex have complementary roles in Drosophila and loss of both proteins is a common event in many different human tumours. However no studies have directly addressed the respective contributions of loss of hScrib and hDlg in the same human cell background to cellular phenotypes associated with cell transformation. In human HaCaT keratinocytes we show that removal of hScrib greatly reduces cell-cell contact and cell-matrix interactions, and promotes an invasive phenotype. Conversely, in cells lacking hDlg1 cell-cell contacts are maintained and there are decreases in both cell growth and invasion. However, hDlg-depleted cells show increased resistance to a specialized form of apoptosis known as anoikis, to which cells lacking hScrib are highly susceptible. Thus whilst it has been widely assumed that hScrib and hDlg have complementary roles, these studies in fact demonstrate that hScrib and hDlg1 have distinct and opposing functions in human keratinocytes.     

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.     

Aberrant activation of the interleukin-2 autocrine loop through the nuclear factor of activated T cells by nonleukemogenic human T-cell leukemia virus type 2 but not by leukemogenic type 1 virus

Human T-cell leukemia virus type 1 (HTLV-1) but not HTLV-2 is associated with adult T-cell leukemia. We found that HTLV-2 Tax2 protein stimulated reporter gene expression regulated by the interleukin (IL)-2 promoter through the nuclear factor of activated T cells (NFAT) in a human T-cell line (Jurkat). However, the activity of HTLV-1 Tax1 was minimal in this system. T-cell lines immortalized by HTLV-2 but not HTLV-1 constitutively exhibited activated NFAT in the nucleus and constitutively expressed IL-2 mRNA. Cyclosporine A, an inhibitor of NFAT activation, abrogated the induction of IL-2 mRNA in HTLV-2-immortalized T-cell lines and concomitantly inhibited cell growth. This growth inhibition was rescued by the addition of IL-2 to the culture. Furthermore, anti-IL-2 receptor antibodies significantly reduced the proliferation of HTLV-2-infected T-cell lines but not that of HTLV-1-infected cells. Our results suggest that Tax2 activates an IL-2 autocrine loop mediated through NFAT that supports the growth of HTLV-2-infected cells under low-IL-2 conditions. This mechanism would be especially important in vivo, where this autocrine mechanism establishes a nonleukemogenic life-long HTLV-2 infection. The results also suggest that differences in long-term cytokine production between HTLV-1 and HTLV-2 infection are another factor for the differences in pathogenesis.     

Human papillomavirus type 18 E6 protein binds the cellular PDZ protein TIP-2/GIPC, which is involved in transforming growth factor beta signaling and triggers its degradation by the proteasome

Several viral proteins expressed by DNA or RNA transforming viruses have the particular property of binding via their C-terminal end to various cellular proteins with PDZ domains. This study is focused on the PDZ protein TIP-2/GIPC, which was originally identified in two-hybrid screens performed with two different baits: the human T-cell leukemia virus type 1 Tax oncoprotein and the regulator of G signaling RGS-GAIP. Further studies have shown that TIP-2/GIPC is also able to associate with the cytoplasmic domains of various transmembrane proteins. In this report we show that TIP-2/GIPC interacts with the E6 protein of human papillomavirus type 18 (HPV-18). This event triggers polyubiquitination and proteasome-mediated degradation of the cellular protein. In agreement with this observation, silencing of E6 by RNA interference in HeLa cells causes an increase in the intracellular TIP-2/GIPC level. This PDZ protein has been previously found to be involved in transforming growth factor beta (TGF-beta) signaling by favoring expression of the TGF-beta type III receptor at the cell membrane. In line with this activity of TIP-2/GIPC, we observed that depletion of this protein in HeLa cells hampers induction of the Id3 gene by TGF-beta treatment and also diminishes the antiproliferative effect of this cytokine. Conversely, silencing of E6 increases the expression of Id3 and blocks proliferation of HeLa cells. These results support the notion that HPV-18 E6 renders cells less sensitive to the cytostatic effect of TGF-beta by lowering the intracellular amount of TIP-2/GIPC.     

HTLV-2 Tax Immortalizes Human CD4+ Memory T Lymphocytes by Oncogenic Activation and Dysregulation of Autophagy

Human T cell leukemia virus type 1 and type 2 (HTLV-1 and -2) are two closely related retroviruses with the former causing adult T cell leukemia. HTLV-2 infection is prevalent among intravenous drug users, and the viral genome encodes the viral transactivator Tax, which is highly homologous to the transforming protein Tax from HTLV-1. However, the link between HTLV-2 infection and leukemia has not been established. In the present study, we evaluated the activity of HTLV-2 Tax in promoting aberrant proliferation of human CD4 T lymphocytes. Tax2 efficiently immortalized CD4⁺ memory T lymphocytes with a CD3/TCRαβ/CD4/CD25/CD45RO/CD69 immunophenotype, promoted constitutive activation of PI3K/Akt, IκB kinase/NF-κB, mitogen-activated protein kinase, and STAT3, and it also increased the level of Mcl-1. Disruption of these oncogenic pathways led to growth retardation and apoptotic cell death of the Tax2-established T cell lines. We further found that Tax2 induced autophagy by interacting with the autophagy molecule complex containing Beclin1 and PI3K class III to form the LC3⁺ autophagosome. Tax2-mediated autophagy promoted survival and proliferation of the immortalized T cells. The present study demonstrated the oncogenic properties of Tax2 in human T cells and also implicated Tax2 in serving as a molecular tool to generate distinct T cell subtype lines.     

Accelerated G(1) phase progression induced by the human T cell leukemia virus type I (HTLV-I) Tax oncoprotein.

Tax, the human T cell leukemia virus type I oncoprotein, plays a crucial role in viral transformation and the development of the virally associated disease adult T cell leukemia. Because oncogenesis involves alterations in cell growth, it is important to examine the effects of Tax on cell cycle progression. Using a synchronized cell system, we have found that Tax expression accelerates G(1) phase progression and S phase entry with concomitant DNA replication. This accelerated progression is accompanied by an earlier onset of cdk2 kinase activity. In contrast to the shortening of G(1) phase, the length of S phase is unaffected by Tax expression. As a result of a more rapid cell cycle progression, cells expressing Tax exhibit faster growth kinetics and display an altered cell cycle distribution. Additionally, the decreased time allowed for growth in the presence of Tax results in a decreased cell size. Tax-associated acceleration of cell cycle progression may play a role in the ability of this viral oncoprotein to mediate cellular transformation and promote the development of human T cell leukemia virus type I-associated diseases.     

Effect of human T-cell leukemia virus type I tax protein on activation of the human vimentin gene.

We report that the expression of the vimentin gene, a cytoskeletal growth-regulated gene, is activated in trans by the Tax (p40x) transactivator protein encoded by the human T-cell leukemia virus type I. Expression of the Tax protein activates a number of cellular genes, such as those coding for the alpha chain of the high-affinity interleukin-2 receptor and interleukin-2. These findings indicate that the Tax protein is involved in the unregulated T-cell growth associated with human T-cell leukemia virus type I infection. Higher levels of vimentin mRNA were expressed in two human T-cell leukemia virus type I-transformed T cell lines, C91/PL and C81-66/45, when compared with that in Jurkat T cells. We demonstrate that this activation is conferred by the vimentin upstream flanking sequences. Indeed, enhanced activity was detected when constructs with the vimentin promoter linked to the chloramphenicol acetyltransferase gene were transfected in HeLa cells and in two cell lines of hematopoietic origin (Jurkat T lymphoblastoid cells and U937 promonocytic cells) together with a Tax expression plasmid. By introducing a series of deletions in the vimentin promoter, we further restrict these sequences to 30 base pairs, located between 241 and 210 base pairs upstream of the mRNA cap site. A 40-base-pair oligonucleotide containing this regulatory region proved sufficient to confer Tax inducibility upon a heterologous promoter linked to chloramphenicol acetyltransferase. Importantly, this segment includes an 11-base-pair promoter segment that has homology with the binding site for the NF-kappa B transactivating factor. Our findings indicate that constitutive expression of the vimentin gene under the control of the Tax protein may be relevant in understanding the progression of the lymphoproliferative process associated with human T-cell leukemia virus type I infection.     

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.     

Two independent domains of hDlg are sufficient for subcellular targeting: the PDZ1-2 conformational unit and an alternatively spliced domain

hDlg, a human homologue of the Drosophila Dig tumor suppressor, contains two binding sites for protein 4.1, one within a domain containing three PSD-95/Dlg/ZO-1 (PDZ) repeats and another within the alternatively spliced I3 domain. Here, we further define the PDZ- protein 4.1 interaction in vitro and show the functional role of both 4.1 binding sites in situ. A single protease-resistant structure formed by the entirety of both PDZ repeats 1 and 2 (PDZ1-2) contains the protein 4.1-binding site. Both this PDZ1-2 site and the I3 domain associate with a 30-kD NH2-terminal domain of protein 4.1 that is conserved in ezrin/radixin/moesin (ERM) proteins. We show that both protein 4.1 and the ezrin ERM protein interact with the murine form of hDlg in a coprecipitating immune complex. In permeabilized cells and tissues, either the PDZ1-2 domain or the I3 domain alone are sufficient for proper subcellular targeting of exogenous hDlg. In situ, PDZ1-2- mediated targeting involves interactions with both 4.1/ERM proteins and proteins containing the COOH-terminal T/SXV motif. I3-mediated targeting depends exclusively on interactions with 4.1/ERM proteins. Our data elucidates the multivalent nature of membrane-associated guanylate kinase homologue (MAGUK) targeting, thus beginning to define those protein interactions that are critical in MAGUK function.     

Regulation of the hDlg/hScrib/Hugl-1 tumour suppressor complex

The proper function of the Scribble tumour suppressor complex is dependent upon the correct localisation of its components. Previously we observed dynamic relocalisation of the hDlg component under conditions of osmotic stress. We now show that the other two components of the complex, hScrib and Hugl-1 display similar patterns of expression. We demonstrate, by shRNA ablation of hScrib expression, that hDlg and Hugl-1 are in part dependent upon hScrib for their correct localization. However under conditions of osmotic stress this apparent dependency no longer exists: hDlg and Hugl-1 localise to cell membranes independently of hScrib. We also demonstrate an interaction between the three components of the hScrib complex and the tSNARE syntaxin 4, and show that correct localization of the Scrib complex is in part tSNARE dependent. This is the first detailed analysis of the co-localisation and function of the hScrib complex in mammalian cells and demonstrates a direct link between the control of the hScrib complex and vesicle transport pathways.     

Human T cell leukemia virus type 1 Tax inhibits innate antiviral signaling via NF-kappaB-dependent induction of SOCS1

Human T cell leukemia virus type 1 (HTLV-1) inhibits host antiviral signaling pathways although the underlying mechanisms are unclear. Here we found that the HTLV-1 Tax oncoprotein induced the expression of SOCS1, an inhibitor of interferon signaling. Tax required NF-κB, but not CREB, to induce the expression of SOCS1 in T cells. Furthermore, Tax interacted with SOCS1 in both transfected cells and in HTLV-1-transformed cell lines. Although SOCS1 is normally a short-lived protein, in the presence of Tax, the stability of SOCS1 was greatly increased. Accordingly, Tax enhanced the replication of a heterologous virus, vesicular stomatitis virus (VSV), in a SOCS1-dependent manner. Surprisingly, Tax required SOCS1 to inhibit RIG-I-dependent antiviral signaling, but not the interferon-induced JAK/STAT pathway. Inhibition of SOCS1 by RNA-mediated interference in the HTLV-1-transformed cell line MT-2 resulted in increased IFN-β expression accompanied by reduced HTLV-1 replication and p19(Gag) levels. Taken together, our results reveal that Tax inhibits antiviral signaling, in part, by hijacking an interferon regulatory protein.     

Cell-type-specific trans-activation of herpes simplex virus thymidine kinase promoter by the human T-cell leukemia virus type I Tax protein.

The human T-cell leukemia virus type I Tax protein (HTLV-I Tax) is known as a trans-activating factor for a variety of genes, including those of cytokines. Here, we show that Tax is capable of activating the herpes simplex virus thymidine kinase (HSV-TK) promoter in certain mammalian cell lines. In murine NIH 3T3 fibroblasts and human HeLa cells, trans-activation by Tax was remarkably strong, whereas in human chondrocytic HCS-2/8 and monkey kidney Cos-7 cells, the responsiveness of the TK promoter to Tax was poor. Deletion analysis revealed that one of the two previously described Sp1 sites is required for the Tax responsiveness, whereas the CTF binding site is not. The results suggest possible interactions between the oncogenic Tax protein and the viral TK in coinfected cells in vivo. Care should be taken in the context of HTLV-I research, as the HSV-TK promoter has been widely used in molecular biology and gene therapeutics.     

Distinct IkappaB kinase regulation in adult T cell leukemia and HTLV-I-transformed cells

We have recently shown constitutive IkappaB kinase (IKK) activation and aberrant p52 expression in adult T cell leukemia (ATL) cells that do not express human T cell leukemia virus type I (HTLV-I) Tax, but the mechanism of IKK activation in these cells has remained unknown. Here, we demonstrate distinct regulation of IKK activity in ATL and HTLV-I-transformed T cells in response to protein synthesis inhibition or arsenite treatment. Protein synthesis inhibition for 4 h by cycloheximide (CHX) barely affects IKK activity in Tax-positive HTLV-I-transformed cells, while it diminishes IKK activity in Tax-negative ATL cells. Treatment of ATL cells with a proteasome inhibitor MG132 prior to protein synthesis inhibition reverses the inhibitory effect of CHX, and MG132 alone greatly enhances IKK activity. In addition, treatment of HTLV-I-transformed cells with arsenite for 1 h results in down-regulation of IKK activity without affecting Tax expression, while 8 h of arsenite treatment does not impair IKK activity in ATL cells. These results indicate that a labile protein sensitive to proteasome-dependent degradation governs IKK activation in ATL cells, and suggest a molecular mechanism of IKK activation in ATL cells distinct from that in HTLV-I-transformed T cells.