Destabilization of the retinoblastoma tumor suppressor by human papillomavirus type 16 E7 is not sufficient to overcome cell cycle arrest in human keratinocytes.

The E7 oncoprotein of human papillomavirus type 16 promotes cell proliferation in the presence of antiproliferative signals. Mutagenesis of E7 has revealed that this activity requires three regions, conserved regions 1 and 2 and a C-terminal zinc finger. Binding to the retinoblastoma tumor repressor (Rb) through an LxCxE motif in conserved region 2 is necessary, but not sufficient, for E7 to induce proliferation. We tested the hypothesis that binding to Rb is not sufficient because conserved region 1 and/or the C terminus are required for E7 to functionally inactivate Rb and thus induce proliferation. One mechanism proposed for how E7 inactivates Rb is by blocking Rb-E2F binding. Either conserved region 1 or the C terminus was necessary, in combination with the LxCxE motif, for E7 to block Rb-E2F binding in vitro. While all full-length E7 proteins with mutations outside of the LxCxE motif inhibited Rb-E2F binding, some failed to abrogate cell cycle arrest, demonstrating that blocking Rb-E2F binding is not sufficient for abrogating antiproliferative signals. Another mechanism proposed for how E7 inactivates Rb is by promoting the destabilization of Rb protein. Mutations in conserved region 1 or the LxCxE motif prevented E7 from reducing the half-life of Rb. Though no specific C-terminal residues of E7 were essential for destabilizing Rb, a novel class of mutations that uncouple the destabilization of Rb from the deregulation of keratinocyte proliferation was discovered. Destabilization of Rb correlated with the abrogation of Rb-induced quiescence but was not sufficient for overriding DNA damage-induced cell cycle arrest or for increasing keratinocyte life span. Finally, the same regions of E7 required for destabilizing Rb were required for reducing p107 and p130 levels. Together, these results suggest that inactivation of all three Rb family members is not sufficient to deregulate keratinocyte cell cycle control.     

Human papillomavirus type 16 E7 oncoprotein associates with the cullin 2 ubiquitin ligase complex, which contributes to degradation of the retinoblastoma tumor suppressor

Human papillomavirus type 16 (HPV16) and other high-risk HPVs are etiologically linked to the development of cervical carcinomas and contribute to a number of other tumors of the anogenital tract, as well as oral cancers. The high-risk HPV E6 and E7 oncoproteins are consistently expressed in cervical cancer cells and are necessary for the induction and maintenance of the transformed phenotype. An important aspect of HPV16 E7's oncogenic activities is destabilization of the retinoblastoma tumor suppressor (pRB) through a ubiquitin/proteasome-dependent mechanism, although the exact molecular mechanism is unknown. Here, we report that HPV16 E7 is associated with an enzymatically active cullin 2 ubiquitin ligase complex and that the HPV16 E7/pRB complex contains cullin 2. Depletion of cullin 2 by RNA interference causes increased steady-state levels and stability of pRB in HPV16 E7-expressing cells, and ectopic expression of HPV16 E7 and the cullin 2 complex leads to pRB ubiquitination in vivo. Hence, we propose that the HPV16 E7-associated cullin 2 ubiquitin ligase complex contributes to aberrant degradation of the pRB tumor suppressor in HPV16 E7-expressing cells.     

Analysis of the p53-mediated G1 growth arrest pathway in cells expressing the human papillomavirus type 16 E7 oncoprotein.

Cells expressing human papillomavirus type 16 (HPV-16) E7, similar to those which express HPV-16 E6, are resistant to a p53-mediated G1 growth arrest. We examined the p53-mediated DNA damage response pathway in E7-expressing cells to determine the mechanism by which E7-containing cells continue to cycle. In response to DNA damage, no dramatic difference was detected in G1- or S-phase cyclin or cyclin-dependent kinase (Cdk) levels when E7-expressing cells were compared to the parental cell line, RKO. Furthermore, Cdk2 kinase activity was inhibited in both RKO cells and E7-expressing cells, while Cdk2 remained active in E6-expressing cells. However, the steady-state levels of pRB and p107 protein were substantially lower in E7-expressing cells than in the parental RKO cells or E6-expressing cells. There was no reduction in pRB mRNA levels, but the half-life of pRB in E7-expressing cells was markedly shorter. Infection of primary human foreskin keratinocytes with recombinant retroviruses expressing HPV-16 E7 resulted in a decrease in pRB protein levels, indicating this phenomenon is a consequence of E7 expression, not of immortalization or transformation. These data strongly suggest E7 interferes with the stability of pRB and p107 protein. We propose that the removal of these components of the p53-mediated G1 growth arrest pathway in E7-expressing cells contributes to the ability of E7 to overcome a p53-mediated G1 growth arrest.     

Localisation of human papillomavirus 16 E7 oncoprotein changes with cell confluence

E7 is one of the best studied proteins of human papillomavirus type 16, largely because of its oncogenic potential linked to cervical cancer. Yet the sub-cellular location of E7 remains confounding, even though it has been shown to be able to shuttle between the nucleus and the cytoplasm. Here we show with immunocytochemistry that E7 proteins are located in the nucleus and cytoplasm in sub-confluent cells, but becomes cytoplasmic in confluent cells. The change in E7's location is independent of time in culture, cell division, cell cycle phase or cellular differentiation. Levels of E7 are also increased in confluent cells as determined by Western blotting. Our investigations have also uncovered how different analytical techniques influence the observation of where E7 is localised, highlighting the importance of technical choice in such analysis. Understanding the localisation of E7 will help us to better comprehend the function of E7 on its target proteins.     

The E7 gene of human papillomavirus type 16 is sufficient for immortalization of human epithelial cells.

The contribution of the E6 and E7 open reading frames of human papillomavirus type 6b (HPV6b) and HPV16 to immortalization of human keratinocytes was evaluated by using amphotropic recombinant retroviruses. The HPV16 E7 gene could immortalize primary human keratinocytes without the cooperation of the viral E6 gene; however, E6 was able to contribute significantly to the efficiency of the E7 immortalizing function. Infection of HFE cells with retroviruses carrying the 16E6, 6bE6, or 6bE6E7 open reading frame did not result in immortalization.     

Induction of pRb degradation by the human papillomavirus type 16 E7 protein is essential to efficiently overcome p16INK4a-imposed G1 cell cycle Arrest

It has previously been shown that the E7 protein from the cutaneous human papillomavirus type 1 (HPV1), which is associated with benign skin lesions, binds the product of the tumor suppressor gene retinoblastoma (pRb) with an efficiency similar to that of the E7 protein from the oncogenic HPV type 16. Despite this ability, HPV1 E7 does not display any activity in transforming primary cells. In addition, the two viral proteins differ in their mechanisms of targeting pRb. HPV16 E7 promotes pRb destabilization, while cells expressing HPV1 E7 do not show any decrease in pRb levels. In this study, we show that HPV1 E7, in contrast to HPV16 E7, has only a weak activity to neutralize the effect of cyclin-dependent kinase inhibitor p16INK4a. By generation of HPV1/16 E7 chimeric proteins, we have identified a central motif in the two E7 proteins, which determines their different abilities to overcome the p16INK4a-mediated cell cycle arrest. This motif is located downstream of the pRb-binding domain and comprises only three amino acids in HPV16 E7. Swapping this central motif in the two viral proteins causes an exchange of their activities involved in circumventing the inhibitory function of p16INK4a. Most importantly, our data show that the efficiency of the E7 proteins in neutralizing the inhibitory effect of p16INK4a correlates with their ability to promote pRb degradation.     

NK cells inhibit T cell proliferation via p21-mediated cell cycle arrest

NK cells have been shown to influence immune responses via direct interaction with cells of the adaptive immune system, such as dendritic cells, B cells, and T cells. A role for NK cells in down-regulation of T cell responses has been implicated in several studies; however, the underlying mechanism of this suppression has remained elusive. In this study we show that dark Agouti rat NK cells inhibit syngeneic T cell proliferation via up-regulation of the cell cycle inhibitor, p21, resulting in a G0/G1 stage cell cycle arrest. The inhibition is cell-cell contact dependent, reversible, and Ag nonspecific. Interestingly, NK cells do not inhibit IL-2 secretion or IL-2R up-regulation and do not induce T cell death. Thus, our results show that NK cells do not affect early T cell activation events, but specifically inhibit T cell proliferation by direct interaction with T cells. Our findings suggest that NK cells may play an important role in maintaining immune homeostasis by directly regulating clonal expansion of activated T cells. This novel mechanism of T cell regulation by NK cells provides insight into NK cell-mediated regulation of adaptive immunity and provides a mechanistic link between NK cell function and suppression of T cell responses.     

Human papillomavirus type 16 E6 promotes retinoblastoma protein phosphorylation and cell cycle progression

We show that E6 proteins from benign human papillomavirus type 1 (HPV1) and oncogenic HPV16 have the ability to alter the regulation of the G(1)/S transition of the cell cycle in primary human fibroblasts. Overexpression of both viral proteins induces cellular proliferation, retinoblastoma (pRb) phosphorylation, and accumulation of products of genes that are negatively regulated by pRb, such as p16(INK4a), CDC2, E2F-1, and cyclin A. Hyperphosphorylated forms of pRb are present in E6-expressing cells even in the presence of ectopic levels of p16(INK4a). The E6 proteins strongly increased the cyclin A/cyclin-dependent kinase 2 (CDK2) activity, which is involved in pRb phosphorylation. In addition, mRNA and protein levels of the CDK2 inhibitor p21(WAF1/CIP1) were strongly down-regulated in cells expressing E6 proteins. The down-regulation of the p21(WAF1/CIP1) gene appears to be independent of p53 inactivation, since HPV1 E6 and an HPV16 E6 mutant unable to target p53 were fully competent in decreasing p21(WAF1/CIP1) levels. E6 from HPV1 and HPV16 also enabled cells to overcome the G(1) arrest imposed by oncogenic ras. Immunofluorescence staining of cells coexpressing ras and E6 from either HPV16 or HPV1 revealed that antiproliferative (p16(INK4a)) and proliferative (Ki67) markers were coexpressed in the same cells. Together, these data underline a novel activity of E6 that is not mediated by inactivation of p53.     

Perturbation of the p53 response by human papillomavirus type 16 E7.

The p53 tumor suppressor protein can induce both cell cycle arrest and apoptosis in DNA-damaged cells. In human carcinoma cell lines expressing wild-type p53, expression of E7 allowed the continuation of full cell cycle progression following DNA damage, indicating that E7 can overcome both G1 and G2 blocks imposed by p53. E7 does not interfere with the initial steps of the p53 response, however, and E7 expressing cells showed enhanced expression of p21(waf1/cip1) and reductions in cyclin E- and A-associated kinase activities following DNA damage. One function of cyclin-dependent kinases is to phosphorylate pRB and activate E2F, thus allowing entry into DNA synthesis. Although E7 may substitute for this activity during cell division by directly targeting pRB, continued cell cycle progression in E7-expressing cells was associated with phosphorylation of pRB, suggesting that E7 permits the retention of some cyclin-dependent kinase activity. One source of this activity may be the E7-associated kinase, which was not inhibited following DNA damage. Despite allowing cell cycle progression, E7 was unable to protect cells from p53-induced apoptosis, and the elevated apoptotic response seen in these cells correlated with the reduction of cyclin A-associated kinase activity. It is possible that inefficient cyclin A-dependent inactivation of E2F at the end of DNA synthesis contributes to the enhanced apoptosis displayed by E7-expressing cells.     

Complex formation of human papillomavirus E7 proteins with the retinoblastoma tumor suppressor gene product.

The E7 proteins encoded by the human papillomaviruses (HPVs) associated with anogenital lesions share significant amino acid sequence homology. The E7 proteins of these different HPVs were assessed for their ability to form complexes with the retinoblastoma tumor suppressor gene product (p105-RB). Similar to the E7 protein of HPV-16, the E7 proteins of HPV-18, HBV-6b and HPV-11 were found to associate with p105-RB in vitro. The E7 proteins of HPV types associated with a high risk of malignant progression (HPV-16 and HPV-18) formed complexes with p105-RB with equal affinities. The E7 proteins encoded by HPV types 6b and 11, which are associated with clinical lesions with a lower risk for progression, bound to p105-RB with lower affinities. The E7 protein of the bovine papillomavirus type 1 (BPV-1), which does not share structural similarity in the amino terminal region with the HPV E7 proteins, was unable to form a detectable complex with p105-RB. The amino acid sequences of the HPV-16 E7 protein involved in complex formation with p105-RB in vitro have been mapped. Only a portion of the sequences that are conserved between the HPV E7 proteins and AdE1A were necessary for association with p105-RB. Furthermore, the HPV-16 E7-p105-RB complex was detected in an HPV-16-transformed human keratinocyte cell line.     

Leaky scanning is the predominant mechanism for translation of human papillomavirus type 16 E7 oncoprotein from E6/E7 bicistronic mRNA

Human papillomaviruses (HPV) are unique in that they generate mRNAs that apparently can express multiple proteins from tandemly arranged open reading frames. The mechanisms by which this is achieved are uncertain and are at odds with the basic predictions of the scanning model for translation initiation. We investigated the unorthodox mechanism by which the E6 and E7 oncoproteins from human papillomavirus type 16 (HPV-16) can be translated from a single, bicistronic mRNA. The short E6 5' untranslated region (UTR) was shown to promote translation as efficiently as a UTR from Xenopus beta-globin. Insertion of a secondary structural element into the UTR inhibited both E6 and E7 expression, suggesting that E7 expression depends on ribosomal scanning from the 5' end of the mRNA. E7 translation was found to be cap dependent, but E6 was more dependent on capping and eIF4F activity than E7. Insertion of secondary structural elements at various points in the region upstream of E7 profoundly inhibited translation, indicating that scanning was probably continuous. Insertion of the E6 region between Renilla and firefly luciferase genes revealed little or no internal ribosomal entry site activity. However when E6 was located at the 5' end of the mRNA, it permitted over 100-fold-higher levels of downstream cistron translation than did the Renilla open reading frame. Internal AUGs in the E6 region with strong or intermediate Kozak sequence contexts were unable to inhibit E7 translation, but initiation at the E7 AUG was efficient and accurate. These data support a model in which E7 translation is facilitated by an extreme degree of leaky scanning, requiring the negotiation of 13 upstream AUGs. Ribosomal initiation complexes which fail to initiate at the E6 start codon can scan through to the E7 AUG without initiating translation, but competence to initiate is achieved once the E7 AUG is reached. These findings suggest that the E6 region of HPV-16 comprises features that sponsor both translation of the E6 protein and enhancement of translation at a downstream site.     

Regions of human papillomavirus type 16 E7 oncoprotein required for immortalization of human keratinocytes.

Binding of the retinoblastoma gene product (pRB) by viral oncoproteins, including the E7 of human papillomavirus type 16 (HPV 16), is thought to be important in transformation of cells. One of the steps in transformation is the immortalization process. Here we show that mutations in E7 within the full-length genome which inhibit binding of pRB do not abrogate the ability of the HPV 16 DNA to immortalize primary human epithelial (keratinocyte) cells. A mutation in one of the cysteines of a Cys-X-X-Cys motif which is contained in the carboxy half of the E7 and is part of a zinc finger arrangement completely eliminates the ability of HPV 16 DNA to immortalize cells. The results indicate the importance of E7 in the immortalization of primary keratinocytes but suggest that the binding of pRB is not essential.     

Human papillomavirus type 16 E7 oncoprotein associates with the centrosomal component gamma-tubulin

Expression of a high-risk human papillomavirus (HPV) E7 oncoprotein is sufficient to induce aberrant centrosome duplication in primary human cells. The resulting centrosome-associated mitotic abnormalities have been linked to the development of aneuploidy. HPV type 16 (HPV16) E7 induces supernumerary centrosomes through a mechanism that is at least in part independent of the inactivation of the retinoblastoma tumor suppressor pRb and is dependent on cyclin-dependent kinase 2 activity. Here, we show that HPV16 E7 can concentrate around mitotic spindle poles and that a small pool of HPV16 E7 is associated with centrosome fractions isolated by sucrose density gradient centrifugation. The targeting of HPV16 E7 to the centrosome, however, was not sufficient for centrosome overduplication. Nonetheless, we found that HPV16 E7 can associate with the centrosomal regulator γ-tubulin and that the recruitment of γ-tubulin to the centrosome is altered in HPV16 E7-expressing cells. Since the association of HPV16 E7 with γ-tubulin is independent of pRb, p107, and p130, our results suggest that the association with γ-tubulin contributes to the pRb/p107/p130-independent ability of HPV16 E7 to subvert centrosome homeostasis.     

Abrogation of growth arrest signals by human papillomavirus type 16 E7 is mediated by sequences required for transformation.

Cells arrest in the G1 or G0 phase of the cell cycle in response to a variety of negative growth signals that induce arrest by different molecular pathways. The ability of human papillomavirus (HPV) oncogenes to bypass these signals and allow cells to progress into the S phase probably contributes to the neoplastic potential of the virus. The E7 protein of HPV-16 was able to disrupt the response of epithelial cells to three different negative growth arrest signals: quiescence imposed upon suprabasal epithelial cells, G1 arrest induced by DNA damage, and inhibition of DNA synthesis caused by treatment with transforming growth factor beta. The same set of mutated E7 proteins was able to abrogate all three growth arrest signals. Mutant proteins that failed to abrogate growth arrest signals were transformation deficient and included E7 proteins that bound retinoblastoma protein in vitro. In contrast, HPV-16 E6 was able to bypass only DNA damage-induced G1 arrest, not suprabasal quiescence or transforming growth factor beta-induced arrest. The E6 and E7 proteins from the low-risk virus HPV-6 were not able to bypass any of the growth arrest signals.     

Loss of p53 protein in human papillomavirus type 16 E6-immortalized human mammary epithelial cells.

We have shown previously that introduction of the human papillomavirus type 16 (HPV16) or HPV18 genome into human mammary epithelial cells induces their immortalization. These immortalized cells have reduced growth factor requirements. We report here that transfection with a single HPV16 gene E6 is sufficient to immortalize these cells and reduce their growth factor requirements. The RB protein is normal in these cells, but the p53 protein is sharply reduced, as shown by immunoprecipitation with anti-p53 antibody (pAB 421). We infer that the E6 protein reduces the p53 protein perhaps by signalling its destruction by the ubiquitin system. The HPV-transforming gene E7 was unable to immortalize human mammary epithelial cells. Thus, cell-specific factors may determine which viral oncogene plays a major role in oncogenesis.