Identification of an arginine-rich motif in human papillomavirus type 1 E1;E4 protein necessary for E4-mediated inhibition of cellular DNA synthesis in vitro and in cells.

Productive infections by human papillomaviruses (HPVs) are restricted to nondividing, differentiated keratinocytes. HPV early proteins E6 and E7 deregulate cell cycle progression and activate the host cell DNA replication machinery in these cells, changes essential for virus synthesis. Productive virus replication is accompanied by abundant expression of the HPV E4 protein. Expression of HPV1 E4 in cells is known to activate cell cycle checkpoints, inhibiting G(2)-to-M transition of the cell cycle and also suppressing entry of cells into S phase. We report here that the HPV1 E4 protein, in the presence of a soluble form of the replication-licensing factor (RLF) Cdc6, inhibits initiation of cellular DNA replication in a mammalian cell-free DNA replication system. Chromatin-binding studies show that E4 blocks replication initiation in vitro by preventing loading of the RLFs Mcm2 and Mcm7 onto chromatin. HPV1 E4-mediated replication inhibition in vitro and suppression of entry of HPV1 E4-expressing cells into S phase are both abrogated upon alanine replacement of arginine 45 in the full-length E4 protein (E1;E4), implying that these two HPV1 E4 functions are linked. We hypothesize that HPV1 E4 inhibits competing host cell DNA synthesis in replication-activated suprabasal keratinocytes by suppressing licensing of cellular replication origins, thus modifying the phenotype of the infected cell in favor of viral genome amplification.

The effect of CtBP1 binding on the structure of the C-terminal region of adenovirus 12 early region 1A.

Adenovirus early region 1A (AdE1A) binds to the C-terminal binding protein 1 (CtBP1) primarily through a highly conserved PXDLS motif located close to its C-terminus. Purified synthetic peptides equivalent to this region of AdE1A have been shown to form a series of beta-turns. In this present study the effect of CtBP1 binding on the conformation of C-terminal region of Ad12E1A has been investigated. Using one- and two-dimensional (1)H NMR spectroscopy, the conformation of 20-residue peptides equivalent to amino acids I(241)-V(260) and E(247)-N(266) of Ad12E1A were examined in the absence of CtBP1. Whilst the latter peptide forms a series of beta-turns in its C-terminal half as reported previously, the former peptide is alpha-helical over the region D(243)-Q(253). Upon interaction with CtBP1 the conformation of the backbone in the region (255)PVDLCVK(261) of the Ad12E1A E(247)-N(266) peptide reorganises from a predominately beta-turn to an alpha-helical conformation. This structural isomerisation is characterised by a shift upfield of 0.318 ppm for the delta-CH(3) proton resonance of V(256). 2-D NOESY experiments showed new signals in the amide-alpha region which correlate to transferred NOEs from the protein to the peptide residues E(251), V(256) and K(261). In further analyses the contribution of individual amino acids within the sequence (254)VPVDLS(259) was assessed for their importance in determining structure and consequently affinity of the peptide for CtBP. It has been concluded that Ad12E1A residues (255)P-V(260) serve initially as a recognition site for CtBP and then as an anchor through a beta-turns-->alpha-helix conformational rearrangement. In addition it has been predicted that regions N-terminal to the PXDLS motif in AdE1As from different virus serotypes and from mammalian proteins form alpha-helices.

Acetylation at a lysine residue adjacent to the CtBP binding motif within adenovirus 12 E1A causes structural disruption and limited reduction of CtBP binding.

C-terminal binding protein (CtBP) has been shown to bind to a highly conserved five-amino-acid motif (PXDLS) located very close to the C-terminus of adenovirus early region 1A proteins. It has also been demonstrated that amino acids C-terminal and N-terminal to this original proposed binding site contribute to the interaction. However, conflicting evidence has been presented to show that acetylation of an adjacent lysine residue in Ad5E1A may or may not influence binding. It has now been demonstrated here that acetylation of a lysine, equivalent to position 261 in Ad12 E1A and position 285 in Ad5E1A, in a synthetic peptide disrupts the binding to CtBP1 and CtBP2 and alters the K(i) of the peptide, indicative of a reduction in the affinity of the peptide for CtBP1 and CtBP2, but only to a rather limited extent (less than 2-fold). The solution structures of synthetic peptides equivalent to wild-type and acetylated forms of the Ad12 E1A peptide have been determined by proton NMR spectroscopy. The wild-type form of the peptide adopts a series of beta-turns over the region Val(254)-Arg(262). Within the acetylated isoform, the beta-turn conformation is less extensive, Val(260)-Arg(262) adopting a random confirmation. We conclude that secondary structure (beta-turns) and an appropriate series of amino acid side chains over an extended binding site (PXDLSXK) are necessary for recognition by CtBP, acetylation of lysine interfering with both of these features, but not to such an extent as to totally inhibit interaction. Moreover, it is possible that the beta-turn conformation at the C-terminus of AdE1A contributes to binding to alpha importin and nuclear import. Acetylation of lysine (261) could disrupt interaction through structural destabilization as well as charge neutralization and subsequent nuclear localization.

Roles for APIS and the 20S proteasome in adenovirus E1A-dependent transcription.

We have determined distinct roles for different proteasome complexes in adenovirus (Ad) E1A-dependent transcription. We show that the 19S ATPase, S8, as a component of 19S ATPase proteins independent of 20S (APIS), binds specifically to the E1A transactivation domain, conserved region 3 (CR3). Recruitment of APIS to CR3 enhances the ability of E1A to stimulate transcription from viral early gene promoters during Ad infection of human cells. The ability of CR3 to stimulate transcription in yeast is similarly dependent on the functional integrity of yeast APIS components, Sug1 and Sug2. The 20S proteasome is also recruited to CR3 independently of APIS and the 26S proteasome. Chromatin immunoprecipitation reveals that E1A, S8 and the 20S proteasome are recruited to both Ad early region gene promoters and early region gene sequences during Ad infection, suggesting their requirement in both transcriptional initiation and elongation. We also demonstrate that E1A CR3 transactivation and degradation sequences functionally overlap and that proteasome inhibitors repress E1A transcription. Taken together, these data demonstrate distinct roles for APIS and the 20S proteasome in E1A-dependent transactivation.

The APC/C and CBP/p300 cooperate to regulate transcription and cell-cycle progression.

The anaphase-promoting complex/cyclosome (APC/C) is a multicomponent E3 ubiquitin ligase that, by targeting protein substrates for 26S proteasome-mediated degradation through ubiquitination, coordinates the temporal progression of eukaryotic cells through mitosis and the subsequent G1 phase of the cell cycle. Other functions of the APC/C are, however, less well defined. Here we show that two APC/C components, APC5 and APC7, interact directly with the coactivators CBP and p300 through protein-protein interaction domains that are evolutionarily conserved in adenovirus E1A. This interaction stimulates intrinsic CBP/p300 acetyltransferase activity and potentiates CBP/p300-dependent transcription. We also show that APC5 and APC7 suppress E1A-mediated transformation in a CBP/p300-dependent manner, indicating that these components of the APC/C may be targeted during cellular transformation. Furthermore, we establish that CBP is required in APC/C function; specifically, gene ablation of CBP by RNA-mediated interference markedly reduces the E3 ubiquitin ligase activity of the APC/C and the progression of cells through mitosis. Taken together, our results define discrete roles for the APC/C-CBP/p300 complexes in growth regulation.

Accumulation of p53 in response to adenovirus early region 1A sensitizes human cells to tumor necrosis factor alpha-induced apoptosis.

Many tumor cells are resistant to tumor necrosis factor alpha (TNFalpha)-induced apoptosis. Adenovirus early region 1A (AdE1A) sensitizes the otherwise resistant cells to TNFalpha. AdE1A also stabilizes the p53 protein. The present study demonstrates a correlation between AdE1A-induced sensitization and stabilization of p53 in TNFalpha-induced apoptosis since the N-terminal and CR2 regions, the binding sites for CBP/p300, Rb and 26S proteasome regulatory components, are required for both these actions of AdE1A. TNFalpha does not induce apoptosis and AdE1A fails to sensitize TNFalpha cytotoxicity in p53-negative cells. However, introduction of exogenous p53 overcomes the cellular resistance to TNFalpha toxicity and enhances AdE1A sensitization, demonstrating that AdE1A sensitizes TNFalpha-induced apoptosis by its stabilization of p53. A proteasome inhibitor, lactacystin, enhances TNFalpha cytotoxicity in p53-positive and -negative cells, suggesting that accumulation of cellular proteins other than p53 might also regulate the cellular response to TNFalpha signaling.

The interaction of the hnRNP family member E1B-AP5 with p53.

Adenovirus early region 1B-associated protein 5, E1B-AP5, a member of the heterogeneous nuclear ribonucleoprotein (hnRNP) family, was originally isolated on the basis of its ability to bind to the adenovirus 5 early region1B55K protein. Here, it has been demonstrated that E1B-AP5 interacts with mutant and wild-type p53 from human cells in pull-down assays using GST-E1B-AP5. This interaction has been confirmed by co-immunoprecipitation studies and pull-down experiments with in vitro translated E1B-AP5 and GST-p53. The binding site for E1B-AP5 has been mapped to the C-terminal region of p53. In reciprocal experiments, it has been shown that several regions of E1B-AP5 bound to p53 although it is probable that a major site of interaction is located between amino acids 395 and 732 of E1B-AP5. In reporter assays, E1B-AP5 inhibited p53 transcriptional activity although not as efficiently as the Ad5E1B55K protein. Transfection of E1B-AP5 into human tumour cells affected the cellular response to UV radiation, such that, although p53 expression was induced, little change in the level of p53-inducible genes could be observed.

Recruitment of CBP/p300, TATA-binding protein, and S8 to distinct regions at the N terminus of adenovirus E1A.

The N-terminal region of the adenovirus (Ad) 12S E1A gene product targets several cellular proteins that are essential for the induction of S phase, cellular immortalization, cellular transformation, transcriptional repression, and transcriptional activation. The precise binding sites for these proteins, however, remain to be resolved. We therefore undertook an extensive site-directed mutagenesis approach to generate specific point mutants and to precisely map the binding sites for CBP, p300, TATA-binding protein (TBP), S4, S8, hGcn5, P/CAF, and Ran within the first 30 amino acids of the Ad5 12S E1A protein. We determined that although common residues within the N-terminal region can form partial binding sites for these proteins, point mutants were also generated that could discriminate between binding sites. These data indicate that AdE1A can target each of these proteins individually through distinct binding sites. It was evident, however, that the mutation of specific hydrophobic residues typically had the greatest effect upon AdE1A's ability to bind individual partners. Indeed, the mutation of L at positions 19 and 20 eliminated the ability of AdE1A to interact with any of the N-terminal binding proteins studied here. Interestingly, although TBP and S8 or CBP/p300 can exist as functional complexes, RNA interference revealed that the recruitment of either TBP, S8, or CBP/p300 to AdE1A was not dependent upon the expression of the other proteins. These data further indicate that AdE1A can target individual partner proteins in vivo and that it does not necessarily recruit these proteins indirectly as components of larger macromolecular complexes. Finally, we took advantage of the fine-mapping data to ascertain which proteins were targeted during the transformation process. Consistent with previous studies, CBP/p300 was found to be targeted by AdE1A during this process, although our data suggest that binding to other N-terminal proteins is also important for transformation.

Cooperation between different forms of the human papillomavirus type 1 E4 protein to block cell cycle progression and cellular DNA synthesis.

Posttranslational modification-oligomerization, phosphorylation, and proteolytic cleavage-of the human papillomavirus (HPV) E4 protein occurs as the infected keratinocytes migrate up through the suprabasal wart layers. It has been postulated that these events modify E4 function during the virus life cycle. In HPV type 1 (HPV1)-induced warts, N-terminal sequences are progressively cleaved from the full-length E4 protein (E1(wedge)E4) of 17 kDa to produce a series of polypeptides of 16, 11 and 10 kDa. Here, we have shown that in human keratinocytes, a truncated protein (E4-16K), equivalent to the 16-kDa species, mediated a G(2) arrest in the cell cycle that was dependent on a threonine amino acid in a proline-rich domain of the protein. Reconstitution of cyclin B1 expression in E4-16K cells reversed the G(2) arrest. Expression of E4-16K also induced chromosomal rereplication, and this was associated with aberrant nuclear morphology. Perturbation of the mitotic cell cycle was a biological activity specific to the truncated protein. However, coexpression of the full-length E1(wedge)E4 protein and the truncated E4-16K protein inhibited normal cellular proliferation and cellular DNA rereplication but did not prevent cells from arresting in G(2). Our findings provide the first evidence to support the hypothesis that proteolytic cleavage of the E1(wedge)E4 protein modifies its function. Also, different forms of the HPV1 E4 protein cooperate to negatively influence keratinocyte proliferation. We predict that these distinct biological activities of E4 act to support efficient amplification of the viral genome in suprabasal keratinocytes.

The targeting of the proteasomal regulatory subunit S2 by adenovirus E1A causes inhibition of proteasomal activity and increased p53 expression.

Although adenovirus early region 1A (AdE1A) can modulate protein expression through its interaction with transcriptional regulators it can also influence the ability of the cell to degrade proteins by binding to components of the 26 S proteasome. We demonstrate here that AdE1A interacts with the S2 subunit of the 19 S regulatory complex in addition to the ATPase subunits S4 and S8 previously identified. S2 forms complexes with both the 13 and 12 S AdE1A proteins both in vivo and in vitro. Mutational analysis has shown direct binding through a short sequence toward the N terminus of conserved region 2 of AdE1A, which encompasses the LXCXE motif, involved in interaction with the pRb family of proteins. In vivo, additional contacts are made between AdE1A and proteasomal components, as well as within the proteasome, such that deletion of the N-terminal region of E1A as well as part of conserved region 2 is required to completely disrupt S2 binding. Mutation of AdE1A, which disrupts complex formation with S2, results in the loss of its ability to stabilize the p53 protein. Similarly down-regulation of S2 expression using small interfering RNAs leads to the inhibition of p53 degradation. These effects were observed in normally growing cells and those subjected to UV irradiation. Furthermore, AdE1A had no effect on the Mdm2-mediated ubiquitination of p53. We suggest therefore that interaction of AdE1A with S2, as well as with the ATPases S4 and S8, directly causes inhibition of proteasomal activity and consequent increase in the protein levels of p53.

Human papillomavirus (HPV) type 16-specific CD8+ T cell responses in women with high grade vulvar intraepithelial neoplasia.

Human papillomavirus (HPV)-associated vulvar intraepithelial neoplasia (VIN) has serious sequelae for the sufferer. Current treatments are associated with poor response and high relapse rates. The development of HPV-specific T cell immunotherapies offers a new approach to treatment. This will require a detailed understanding of the spectrum of T cell responses induced by HPV antigens, and how effectively viral antigens can be accessed by the immune system. We have investigated the frequency and spectrum of HPV16-specific CD8+ T cell responses to three HPV16 antigens in 9 women with high grade VIN (VIN3). CD4-depleted populations of responder cells were screened against overlapping 30-35mer peptides covering the sequences of HPV16 E6, E7 and E4 using ELISPOT assays of IFN-gamma release. We demonstrated CD8+ T cell reactivity to one or more of the proteins in 6 of 9 patient samples. All 6 of these responders recognised peptides covering the E7 protein, 3 of 9 women responded to E6 peptides, but no reactivity was seen to E4. Our results suggest that HPV16-specific cytotoxic T cells (CTLs) are relatively common in women with persistent VIN3. The HPV-specific CTL response, however, seems to be ineffective. There is some evidence that there are problems associated with the processing and presentation of HPV antigens by the infected vulvar epithelium. It will be crucial to address this in the design of any T cell based therapy for HPV-associated VIN and vulval cancer.

The ND10 component promyelocytic leukemia protein relocates to human papillomavirus type 1 E4 intranuclear inclusion bodies in cultured keratinocytes and in warts.

Human papillomavirus type 1 (HPV1) E4 protein is associated with cytoplasmic and nuclear inclusions in productively infected keratinocytes. Here we have used transient expression of HPV1 E4 (also known as E1E4) protein in keratinocytes to reproduce formation of E4 inclusions. Immunofluorescence analysis showed that progressive formation of inclusions correlated with diminished colocalization between E4 and keratin intermediate filaments (IFs). Our results support a model in which the HPV1 E4-keratin IF association is transient, occurring only at an early stage of inclusion formation. We also demonstrate that E4 induces relocation of the promyelocytic leukemia protein (PML) from multiple intranuclear speckles (ND10 bodies) to the periphery of nuclear E4 inclusions and that this activity is specific to full-length E4 protein. Analysis of HPV1-induced warts demonstrated that nuclear PML-E4 inclusions were present in productively infected keratinocytes, indicating that reorganization of PML occurs during the virus's replication cycle. It has been suggested that ND10 bodies are the sites for papillomavirus genome replication and virion assembly. Our finding that E4 induces reorganization of ND10 bodies in vitro and in vivo is further strong evidence that these domains play an important role in the papillomavirus life cycle. This study indicates that HPV1 is analogous to other DNA viruses that disrupt or reorganize ND10 domains, possibly to increase efficiency of virus infection. We hypothesize that HPV1 E4-induced reorganization of PML is necessary for efficient replication of the virus during the virus-producing phase.

Caspase-mediated cleavage of adenovirus early region 1A proteins.

Adenovirus 2 and 12 early region 1A (Ad2 and Ad12 E1A) proteins were cleaved during cisplatin-induced apoptosis of Ad-transformed rat and human cells. Cleavage was inhibited in the presence of caspase inhibitors such as Z-VAD-FMK. In Ad12 transformants both 13S and 12S E1A proteins were cleaved at a similar rate. In Ad2 transformants the E1A 13S component was appreciably less stable than the 12S component. In in vitro studies Ad2 and Ad12 E1A 13S and Ad2 12S proteins were rapidly cleaved by caspase 3 whereas Ad12 12S E1A and Ad12 13S E1A were rapidly degraded by caspase 7. Cleavage sites in Ad12 13S proteins for caspase 3 have been determined. Initial cleavage occurred at D24 and D150; this was followed by cleavage at D204 and D242. Caspase-3-mediated cleavage of Ad12 13S E1A destroyed its ability to bind to CBP and TBP but interaction between C terminal E1A polypeptides and CtBP was observed. During viral infection Ad5 and Ad12 E1A 12S proteins were markedly more stable than 13S proteins but no difference was observed in Ad E1A levels in the absence or presence of the caspase inhibitors Z-VAD-FMK or Z-D(OMe)-E(OMe)-V-D(OMe)-CH(2)F. Limited caspase 3 and 10 activation occurred during infection with the E1B 19K(-) virus Ad2 pm1722 but little or no activation of caspase 3 was observed during wt virus infection. Examination of protein cleavage during viral infection of A549 cells showed proteolysis of lamin B and PARP in response to Ad5 wt and Ad2 pm1722. Protein degradation in response to both viruses was partially inhibited by Z-VAD-FMK. Following infection of human skin fibroblasts lamin B was degraded, although only limited changes in PARP levels were observed. We have concluded that Ad E1A is cleaved by caspases during apoptosis but not during viral infection. However, some of the processes commonly associated with apoptosis occur during viral infection, particularly with E1B 19K(-) mutants, although apoptosis per se is not evident.

Transcriptional regulation of the human glycoprotein hormone common alpha subunit gene by cAMP-response-element-binding protein (CREB)-binding protein (CBP)/p300 and p53.

We have investigated the functional interactions between adenovirus early region 1A (AdE1A) protein, the co-activators cAMP-response-element-binding protein (CREB)-binding protein (CBP)/p300 and SUG1, and the transcriptional repressor retinoblastoma (Rb) in mediating T3-dependent repression. Utilizing the human glycoprotein hormone common alpha-subunit (alpha-subunit) promoter and AdE1A mutants with selective binding capacity to these molecules we have determined an essential role for CBP/p300. In normal circumstances, wild-type 12 S AdE1A inhibited alpha-subunit activity. In contrast, adenovirus mutants that retain both the SUG1- and Rb-binding sites, but lack the CBP/p300-binding site, were unable to repress promoter activity. We have also identified a role for the tumour-suppressor gene product p53 in regulation of the alpha-subunit promoter. Akin to 12 S AdE1A, exogenous p53 expression repressed alpha-subunit activity. This function resided in the ability of p53 to interact with CBP/p300; an N-terminal mutant incapable of interacting with CBP/p300 did not inhibit alpha-subunit activity. Stabilization of endogenous p53 by UV irradiation also correlated positively with reduced alpha-subunit activity. Intriguingly, T3 stimulated endogenous p53 transcriptional activity, implicating p53 in T3-dependent signalling pathways. These data indicate that CBP/p300 and p53 are key regulators of alpha-subunit activity.

Detection of CD4(+)- and CD8(+)-T-cell responses to human papillomavirus type 1 antigens expressed at various stages of the virus life cycle by using an enzyme-linked immunospot assay of gamma interferon release.

Human papillomavirus (HPV) antigens are expressed in epithelial cells at different stages of differentiation, and this may affect how they are handled by the immune system. We assessed the relative immunogenicities of four different HPV type 1 proteins: E6 and E7, which are made early in basal or parabasal cells; E4, which is made suprabasally in differentiating cells; and L1, a late protein which appears in the highly differentiated upper spinous layers. Pools of 15-mer peptides covering the primary sequences of all four proteins were used to screen 15 normal donors in enzyme-linked immunospot assays of gamma interferon release for both CD4(+)- and CD8(+)-T-cell reactivities. CD8(+)-T-cell responses were detected to the L1 protein in 7 of the 15 samples examined. No responses to E6, E7, or E4 were detected. CD4(+)-T-cell reactivities were again detected in 7 of the 15 donors. A broader spectrum of responses to E6 (three of seven), E4 (six of seven), and L1 (three of seven) was apparent, but there was no reactivity to E7. The predominant CD4(+) response was to E4. Reactivities were seen in some cases to corresponding regions on other common HPV types but were probably due to a multiple infection rather than to a cross-reaction. Antibodies to HPV1 virus-like particles were detected in 12 of the 15 (80%) donors, but antibody status did not correlate with T-cell reactivity. The differences in the relative immunogenicities of the four proteins revealed in this study are discussed in relation to how they may be processed and presented to the immune system by differentiating epithelial cells.