E1 empty set E4 protein of human papillomavirus type 16 associates with mitochondria

The human papillomavirus (HPV) E1 empty set E4 protein is the most abundantly expressed viral protein in HPV-infected epithelia. It possesses diverse activities, including the ability to bind to the cytokeratin network and to DEAD-box proteins, and in some cases induces the collapse of the former. E1 empty set E4 is also able to prevent the progression of cells into mitosis by arresting them in the G(2) phase of the cell cycle. In spite of these intriguing properties, the role of this protein in the life cycle of the virus is not clear. Here we report that after binding to and collapsing the cytokeratin network, the HPV type 16 E1 empty set E4 protein binds to mitochondria. When cytokeratin is not present in the cell, E1 empty set E4 appears associated with mitochondria soon after its synthesis. The leucine cluster within the N-terminal portion of the E1 empty set E4 protein is pivotal in mediating this association. After the initial binding to mitochondria, the E1 empty set E4 protein induces the detachment of mitochondria from microtubules, causing the organelles to form a single large cluster adjacent to the nucleus. This is followed by a severe reduction in the mitochondrial membrane potential and an induction of apoptosis. HPV DNA replication and virion production occur in terminally differentiating cells which are keratin-rich, rigid squamae that exfoliate after completion of the differentiation process. Perturbation of the cytokeratin network and the eventual induction of apoptotic properties are processes that could render these unyielding cells more fragile and ease the exit of newly synthesized HPVs for subsequent rounds of infection.     

Human papillomavirus type 16 E1 E4-induced G2 arrest is associated with cytoplasmic retention of active Cdk1/cyclin B1 complexes

Human papillomavirus type 16 (HPV16) can cause cervical cancer. Expression of the viral E1 E4 protein is lost during malignant progression, but in premalignant lesions, E1 E4 is abundant in cells supporting viral DNA amplification. Expression of 16E1 E4 in cell culture causes G2 cell cycle arrest. Here we show that unlike many other G2 arrest mechanisms, 16E1 E4 does not inhibit the kinase activity of the Cdk1/cyclin B1 complex. Instead, 16E1 E4 uses a novel mechanism in which it sequesters Cdk1/cyclin B1 onto the cytokeratin network. This prevents the accumulation of active Cdk1/cyclin B1 complexes in the nucleus and hence prevents mitosis. A mutant 16E1 E4 (T22A, T23A) which does not bind cyclin B1 or alter its intracellular location fails to induce G2 arrest. The significance of these results is highlighted by the observation that in lesions induced by HPV16, there is evidence for Cdk1/cyclin B1 activity on the keratins of 16E1 E4-expressing cells. We hypothesize that E1 E4-induced G2 arrest may play a role in creating an environment optimal for viral DNA replication and that loss of E1 E4 expression may contribute to malignant progression.     

Modelling of the human papillomavirus type 16 E5 protein

Cytochrome (cyt) c forms complexes, undergoes a conformational change and becomes partly reduced at interaction with membrane anchored alkaline phosphatase (AP), a glycoprotein which is released into the body fluid in forms differing in hydrophobicity. The proportion of products formed in the mixtures depends on pH, ionic strength, temperature and the buffer composition. The reaction terminates in an equilibrium between cyt c(FeII) and other cyt c conformers. Optimal conditions for the rate of the reaction are 100 mM glycine/NaOH, pH 9.7-9.9, at which 68-74% of cyt c is found in the reduced state. The interaction affects compactness of the haem cleft as shown by changes induced in CD spectra of the Soret region and changes in optical characteristics of phenylalanine, tyrosine and tryptophan residues. Differential scanning calorimetry of AP+cyt c mixtures revealed a creation of at least two types of complexes. A complex formed by non-coulombic binding prevails at substoichiometric AP/cyt c ratios, at higher ratios more electrostatic attraction is involved and at 1:1 molar ratio an apparent complexity of binding forces occurs. The rapid phase of the cyt c(FeII) formation depends on the presence of the hydrophobic alkylacylphosphoinositol (glycosylphosphatidylinositol) moiety, the protein part of the enzyme participates in an electrostatic and much slower phase of cyt c(FeII) creation. The results show that non-coulombic interaction may participate at interaction of cyt c with cellular proteins.     

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.     

人乳头瘤病毒16型L1蛋白的克隆及表达

采用PCR技术从宫颈癌组织中扩增人乳头瘤病毒16型(Human papillomavirus type16,HPV16)L1全长基因片段,目的片段克隆到pMD18T载体后经酶切鉴定及测序确认。构建重组原核表达质粒pGEX4T1-L1,转化大肠杆菌E.coliBL21,IPTG诱导表达出以非可溶性蛋白形式存在的表达蛋白,该重组蛋白的表达量占菌体总蛋白的17%,免疫印迹检测表明,表达蛋白与宫颈癌病人血清出现特异性反应。成功构建了重组原核表达质粒pGEX4T1-L1,并且在原核细胞中得到表达,为进一步研究L1蛋白的免疫学活性及疫苗的研发奠定了基础。     

Identification of T- and B-cell epitopes of the E7 protein of human papillomavirus type 16.

There is strong evidence implicating human papillomavirus type 16 (HPV16) in the genesis of human genital cancer. Viral DNA has been identified in invasive carcinoma of the uterine cervix and in cell lines derived from cervical carcinomas. These sequences are actively transcribed, and translation products corresponding to the early (E)-region genes have been identified. The most abundant viral protein is the E7 protein, which has been shown to possess transforming activity for both established and primary cells. In addition, it has been shown to bind to a cellular tumor suppressor, the retinoblastoma gene product (pRb-105). In view of these properties, we have undertaken the immunological analysis of this protein and have identified four T-cell epitopes and three B-cell epitopes by using a series of overlapping peptides spanning the entire HPV16 E7 sequence. Two of the B-cell epitopes were recognized by antisera from mice with three different murine (H-2) haplotypes (k, d, and s) immunized with two different E7 fusion proteins and from Fischer rats seeded with baby rat kidney cells transformed by HPV16 E7 and ras. A third B-cell epitope was recognized by antisera from CBA mice seeded with HPV16 E7-expressing L cells. Two regions of the protein contain common B- and T-cell epitopes, one of which appears to be particularly immunodominant.     

Production of human papillomavirus type 16 E7 protein in Lactococcus lactis

The E7 protein of human papillomavirus type 16 was produced in Lactococcus lactis. Secretion allowed higher production yields than cytoplasmic production. In stationary phase, amounts of cytoplasmic E7 were reduced, while amounts of secreted E7 increased, suggesting a phase-dependent intracellular proteolysis. Fusion of E7 to the staphylococcal nuclease, a stable protein, resulted in a highly stable cytoplasmic protein. This work provides new candidates for development of viral screening systems and for oral vaccine against cervical cancer.     

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.     

Identification of the E5 open reading frame of human papillomavirus type 16.

Sequencing of the E5 open reading frame (ORF) of human papillomavirus type 16 revealed an additional nucleotide, a thymidine residue, at position 3903 compared with the original sequence (Seedorf et al., Virology 145:181-185, 1985). The additional T had two effects; first, in reading frame 2, in which the original E5 ORF was predicted, the additional T changed the reading frame downstream of position 3903 to create an ORF, which we designated E5, that terminated at position 4018 and potentially encoded a 52-amino-acid polypeptide. Secondly, in reading frame 3, a new ORF was created (positions 3807 to 4097), which we propose is the authentic papillomavirus type 16 E5 ORF. It contained a methionine residue and encoded an additional 82 amino acids. Both ORFs have been cloned into bacterial expression vectors (pATH), and the fusion proteins have been used to generate polyclonal antibodies in rabbits.     

Functional analysis of the human papillomavirus type 16 E1=E4 protein provides a mechanism for in vivo and in vitro keratin filament reorganization

High-risk human papillomaviruses, such as human papillomavirus type 16 (HPV16), are the primary cause of cervical cancer. The HPV16 E1=E4 protein associates with keratin intermediate filaments and causes network collapse when expressed in epithelial cells in vitro. Here, we show that keratin association and network reorganization also occur in vivo in low-grade cervical neoplasia caused by HPV16. The 16E1=E4 protein binds to keratins directly and interacts strongly with keratin 18, a member of the type I intermediate-filament family. By contrast, 16E1=E4 bound only weakly to keratin 8, a type II intermediate-filament protein, and showed no detectable affinity for the type III protein, vimentin. The N-terminal 16 amino acids of the 16E1=E4 protein, which contains the YPLLXLL motif that is conserved among supergroup A viruses, were sufficient to target green fluorescent protein to the keratin network. When expressed in the SiHa cervical epithelial cell line, the full-length 16E1=E4 protein caused an almost total inhibition of keratin dynamics, despite the phosphorylation of keratin 18 at serine 33, which normally leads to 14-3-3-mediated keratin solubilization. Mutant 16E1=E4 proteins which lack the LLKLL motif, or which have lost amino acids from their C termini, and which were compromised in the ability to associate with keratins did not disturb normal keratin dynamics. 16E1=E4 was found to exist as dimers and hexamers, whereas a C-terminal deletion mutant (16E1=E4Delta87-92) existed as monomers and formed multimeric structures only poorly. Considered together, our results suggest that by associating with keratins through its N terminus, and by associating with itself through its C terminus, 16E1=E4 may act as a keratin cross-linker and prevent the movement of keratins between the soluble and insoluble compartments. The increase in avidity associated with multimeric binding may contribute to the ability of 16E1=E4 to sequester its cellular targets in the cytoplasm.     

Crystal structure of the E2 transactivation domain of human papillomavirus type 11 bound to a protein interaction inhibitor

Interaction between the E2 protein and E1 helicase of human papillomaviruses (HPVs) is essential for the initiation of viral DNA replication. We recently described a series of small molecules that bind to the N-terminal transactivation domain (TAD) of HPV type 11 E2 and inhibits its interaction with E1 in vitro and in cellular assays. Here we report the crystal structures of both the HPV11 TAD and of a complex between this domain and an inhibitor, at 2.5- and 2.4-A resolution, respectively. The HPV11 TAD structure is very similar to that of the analogous domain of HPV16. Inhibitor binding caused no significant alteration of the protein backbone, but movements of several amino acid side chains at the binding site, in particular those of Tyr-19, His-32, Leu-94, and Glu-100, resulted in the formation of a deep hydrophobic pocket that accommodates the indandione moiety of the inhibitor. Mutational analysis provides functional evidence for specific interactions between Tyr-19 and E1 and between His-32 and the inhibitor. A second inhibitor molecule is also present at the binding pocket. Although evidence is presented that this second molecule makes only weak interactions with the protein and is likely an artifact of crystallization, its presence defines additional regions of the binding pocket that could be exploited to design more potent inhibitors.