Functional oligomerization of purified human papillomavirus types 16 and 6b E7 proteins expressed in Escherichia coli (original) (raw)
Journal of Virology
The L1 major capsid protein of human papillomavirus type 11 (HPV-11) was expressed in Escherichia coli, and the soluble recombinant protein was purified to near homogeneity. The recombinant L1 protein bound DNA as determined by the Southwestern assay method, and recombinant mutant L1 proteins localized the DNA-binding domain to the carboxy-terminal 11 amino acids of L1. Trypsin digestion of the full-length L1 protein yielded a discrete 42-kDa product (trpL1), determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, resulting from cleavage at R415, 86 amino acids from the L1 carboxy terminus. Sucrose gradient sedimentation analysis demonstrated that trpL1 sedimented at 11S, while L1 proteins with amino-terminal deletions of 29 and 61 residues sedimented at 4S. Electron microscopy showed that the full-length L1 protein appeared as pentameric capsomeres which self-assembled into capsid-like particles. The trpL1 protein also had a pentameric morphology but was unable to ...
Virology, 1992
Human papillomavirus type 16 (HPV 16) is often found in cervical carcinomas, while HPV 6 is frequently associated with benign genital lesions. We have compared the abilities of the E7 transforming proteins of HPV 6 and 16 to transform various established and primary rodent cells by using the same heterologous promoter system. HPV 16 E7 efficiently induced anchorage-independent growth of all the rodent cell lines tested and immortalized or cooperated with ras in transforming primary rat cells. On the other hand, the transforming activity of HPV 6 E7 was lower and was restricted. By construction of chimeras of HPV 6 and 16 E7, we found that the difference in transforming activity between the two E7 proteins was mainly determined by the difference in their 30 N-terminal amino acid residues, although some activities seem to be slightly affected by differences in their residual C-terminal portions. o 1992 Academic Press. Inc.
Journal of Virology, 2013
We describe the extensive and progressive oligomerization of human papillomavirus (HPV) genomes after transfection into the U2OS cell line. The HPV genomic oligomers are extrachromosomal concatemeric molecules containing the viral genome in a head-to-tail orientation. The process of oligomerization does not depend on the topology of the input DNA, and it does not require any other viral factors besides replication proteins E1 and E2. We provide evidence that oligomerization of the HPV18 and HPV11 genomes involves homologous recombination. We also demonstrate oligomerization of the HPV18 and HPV11 genomes in SiHa, HeLa, and C-33 A cell lines and provide examples of oligomeric HPV genomes in clinical samples obtained from HPV-infected patients.
Characterization of functional HPV-16 E7 protein produced in Escherichia coli
Journal of Biological Chemistry, 1992
Human papillomaviruses (HPVs) are the etiologic agents responsible for genital warts and are contributing factors in the pathogenesis of human cervical cancer. The HPV E7 gene is transcriptionally active in these diseases and has been shown to transform mammalian cells in vitro. We have expressed and purified the HPV-16 E7 gene product in Escherichia coli. The isolated E7 protein contains zinc in a 1:l molar ratio. X-ray absorption fine structure studies demonstrated that the zinc is coordinated by 4 sulfur ligands. We sequentially derivatized the E7 cysteines to differentiate between solvent-exposed, metal-bound, and disulfide-associated cysteines. Our results demonstrate that CysZ4 and Cyses are accessible to solvent, while cysteines in the two conserved Cys-X-X-Cys motifs are likely involved in binding zinc. We observed no evidence for the existence of disulfide bonds in recombinant E7 protein under the conditions tested. Nearly half of the known human papilloma viruses (HPVs)' can infect the genital mucosa and produce benign epithelial lesions. Epidemiological and molecular genetic studies have demonstrated that only a few of the HPV isolates that infect the genital mucosa are associated with most (75-100%) cases of cervical cancer (1). In these cases two viral genes, E6 and E7, are transcriptionally active (2). The most common HPV strain associated with cervical neoplasia is HPV-16. The HPV-16 E6 and E7 genes together have been shown to be necessary and sufficient for transformation of primary human keratinocytes (3). The HPV-16 E7 gene is able to transform established rodent fibroblasts (4), and, in conjunction with ras, primary rat epithelial cells (5). The HPV-16 E7 gene encodes a protein of 98 amino acids (6) which shares a region of homology (amino acids 2-35) with the adenovirus E1A protein and simian virus-40 large T antigen. E7, like ElA, can transactivate transcription from ~~ ~ * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Oncogene, 1998
The conserved region 3 (CR3) of the E7 protein of human papillomaviruses contains two CXXC motifs involved in zinc binding and in the homodimerization of the molecule. Studies have suggested that the intact CXXC motifs in the CR3 of HPV16 and HPV18 E7 are required for the in vitro transforming activity of these proteins. CR3 also contains a low anity pRb binding site and is involved in the disruption of the E2F/Rb1 complex. E7 is structurally and functionally related to Adenovirus E1A protein, which also has two CXXC motifs in CR3. However, the Ad E1A transforming activity appears to be independent of the presence of such domains. In fact, this viral protein exists in vivo as two dierent forms of 289 and 243 amino acids. The shorter Ad E1A form (Ad E1A243), where both CXXC motifs are deleted by internal splicing, retains its in vitro transforming activity. We have investigated if the HPV16 E7 CR3 can be functionally replaced by the Ad E1A243 CR3, which lacks both CXXC motifs. A chimeric protein (E7/E1A243) containing the CR1 and CR2 of HPV16 E7 fused to the CR3 of Ad E1A 243 was constructed. The E7/E1A243 while not able to homodimerize in the S. cerevisiae two-hybrid system retains several of the properties of the parental proteins, HPV16 E7 and Ad E1A. It associates with the`pocket' proteins, induces growth in soft agar of NIH3T3 cells and immortalizes rat embryo ®broblasts. These data suggest that the CXXC motifs in CR3 of E7 do not play a direct role in the transforming properties of this viral protein but probably are important for maintaining the correct protein con®guration.
Efficient Intracellular Assembly of Papillomaviral Vectors
Journal of Virology, 2004
Although the papillomavirus structural proteins, L1 and L2, can spontaneously coassemble to form viruslike particles, currently available methods for production of L1/L2 particles capable of transducing reporter plasmids into mammalian cells are technically demanding and relatively low-yield. In this report, we describe a simple 293 cell transfection method for efficient intracellular production of papillomaviral-based gene transfer vectors carrying reporter plasmids. Using bovine papillomavirus type 1 (BPV1) and human papillomavirus type 16 as model papillomaviruses, we have developed a system for producing papillomaviral vector stocks with titers of several billion transducing units per milliliter. Production of these vectors requires both L1 and L2, and transduction can be prevented by papillomavirus-neutralizing antibodies. The stocks can be purified by an iodixanol (OptiPrep) gradient centrifugation procedure that is substantially more effective than standard cesium chloride gradient purification. Although earlier data had suggested a potential role for the viral early protein E2, we found that E2 protein expression did not enhance the intracellular production of BPV1 vectors. It was also possible to encapsidate reporter plasmids devoid of BPV1 DNA sequences. BPV1 vector production efficiency was significantly influenced by the size of the target plasmid being packaged. Use of 6-kb target plasmids resulted in BPV1 vector yields that were higher than those with target plasmids closer to the native 7.9-kb size of papillomavirus genomes. The results suggest that the intracellular assembly of papillomavirus structural proteins around heterologous reporter plasmids is surprisingly promiscuous and may be driven primarily by a size discrimination mechanism.
SpringerPlus, 2013
A fusion protein comprising a cell penetrating and immunostimulatory peptide corresponding to residues 32 to 51 of the Limulus polyphemus protein linked to human papillomavirus (HPV)-16 E7 antigen (LALF32-51-E7) was expressed in E. coli BL21 (DE3) cells. The recombinant protein in E. coli accounted for approximately 18% of the total cellular protein and purified with a single affinity chromatographic step. Yields of approximately 38 mg purified LALF32-51-E7 per liter of induced culture was obtained with an overall 52% recovery and constitutes a promising setting for the future production and scaling-up. Purified protein was characterized as soluble aggregates with molecular weight larger than 670 kDa, which is considered an important property to increase the immunogenicity of an antigen preparation. The recombinant fusion protein LALF32-51-E7 will be a promising vaccine candidate for the treatment of HPV-16 related malignancies.
Journal of Translational Medicine, 2016
Background: High-risk human papillomaviruses (HR-HPVs) types 16 and 18 are the main etiological agents of cervical cancer, with more than 550,000 new cases each year worldwide. HPVs are also associated with other ano-genital and head-and-neck tumors. The HR-HPV E6 and E7 oncoproteins are responsible for onset and maintenance of the cell transformation state, and they represent appropriate targets for development of diagnostic and therapeutic tools. Methods: The unmutated E6 gene from HPV16 and HPV18 and from low-risk HPV11 was cloned in a prokaryotic expression vector for expression of the Histidine-tagged E6 protein (His 6-E6), according to a novel procedure. The structural properties were determined using circular dichroism and fluorescence spectroscopy. His 6-E6 oncoprotein immunogenicity was assessed in a mouse model, and its functionality was determined using in vitro GST pull-down and protein degradation assays. Results: The His 6-tagged E6 proteins from HPV16, HPV18, and HPV11 E6 genes, without any further modification in the amino-acid sequence, were produced in bacteria as soluble and stable molecules. Structural analyses of HPV16 His 6-E6 suggests that it maintains correct folding and conformational properties. C57BL/6 mice immunized with HPV16 His 6-E6 developed significant humoral immune responses. The E6 proteins from HPV16, HPV18, and HPV11 were purified according to a new procedure, and investigated for protein-protein interactions. HR-HPV His 6-E6 bound p53, the PDZ1 motif from MAGI-1 proteins, the human discs large tumor suppressor, and the human ubiquitin ligase E6-associated protein, thus suggesting that it is biologically active. The purified HR-HPV E6 proteins also targeted the MAGI-3 and p53 proteins for degradation. Conclusions: This new procedure generates a stable, unmutated HPV16 E6 protein, which maintains the E6 properties in in vitro binding assays. This will be useful for basic studies, and for development of diagnostic kits and immunotherapies in preclinical mouse models of HPV-related tumorigenesis.