Infectious virions produced from a human papillomavirus type 18/16 genomic DNA chimera

The organotypic raft culture system has allowed the study of the differentiation-dependent aspects of the human papillomavirus (HPV) life cycle. However, genetic strategies to more completely understand the HPV life cycle are limited. The generation of chimeric viruses has been a useful tool in other virus systems to analyze infection and replication. To investigate the specificity of the interaction of nonstructural genes of one HPV type with the structural genes of another HPV type, we have replaced the L2 and L1 open reading frames (ORFs) of HPV type 18 (HPV18) with the L2 and L1 ORFs of HPV type 16 (HPV16). The resulting HPV18/16 chimeric construct was introduced into primary keratinocytes, where it was stably maintained episomally at a range of 50 to 100 copies of HPV genomic DNA, similar to that typically found in HPV-infected cells in vivo. The integrity of the HPV18/16 genomic DNA chimera was demonstrated. Upon differentiation in raft cultures, late viral functions, including viral DNA amplification, capsid gene expression, and virion morphogenesis, occurred. Chimeric HPV18/16 virions were purified from the raft cultures and were capable of infecting keratinocytes in vitro. Additionally, infection was specifically neutralized with human HPV16 virus-like particle (VLP)-specific antiserum and not with human HPV18 VLP-specific antiserum. Our data demonstrate that the nonstructural genes of HPV18 functionally interact with the structural genes of HPV16, allowing the complete HPV life cycle to occur. We believe that this is the first report of the propagation of chimeric HPV by normal life cycle pathways.     

Bacterial expression and purification of human papillomavirus type 18 L1

The human papillomavirus (HPV) 18 L1 gene, which encodes the L1 major capsid protein, was isolated from a female patient in Pusan, Korea Republic and was cloned into pGEX-4T-1 vector. The HPV-18L1 gene was expressed in Escherichia coli as a fusion protein with a glutathione-S-transferase (GST) tag. The soluble recombinant fusion protein, GST-18 L1 fusion, was isolated to high purity. HPV-18 L1 was purified from the GST-18 L1 fusant after biotinylated thrombin cleavage, and then the treated thrombin was removed serially using streptavidin conjugated resin. The purified HPV-18 L1 was confirmed by western blotting using a rabbit anti-denatured papillomavirus polyclonal antibody. The virus-like particles (VLP) from the purified full-length 18 L1 protein without any extra amino acid sequences was observed through the analysis of the electron microscope. This is the first study to report the expression and purification of HPV-18 L1 in E. coli. This expression and purification system offers a simple method of expressing and purifying HPV L1 protein, and could potentially be an effective route for the development and manufacturing of highly purified HPV-18 L1-based cervical cancer vaccines.     

Activation of human papillomavirus type 18 gene expression by herpes simplex virus type 1 viral transactivators and a phorbol ester.

Several viral trans-activators and a tumor promoter were examined for the ability to activate human papillomavirus type 18 (HPV-18) gene expression. A plasmid containing the HPV-18 noncoding region placed upstream of the chloramphenicol acetyltransferase reporter gene was cotransfected with different herpes simplex virus type 1 (HSV-1) genes into several cell lines. Both HSV-1 TIF and ICP0 activated HPV-18 expression; however, activation by TIF was observed only in epithelial cells, while ICP0 stimulated expression in a wide variety of cells. The element activated by both TIF and ICP0 was mapped to a 229-base-pair fragment which also contains an HPV-18 epithelial cell-preferred enhancer. The inclusion of a papillomavirus E2 trans-activator with TIF and ICP0 further increased HPV-18 expression. In contrast, the HSV-1 ICP4 and ICP27 genes, as well as the human T-cell lymphotropic virus type I and human immunodeficiency virus type 1 tat genes, were found to have no effect on HPV-18 expression. In transient assays, the addition of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) also activated HPV-18 expression. The region of HPV-18 activated by TPA was localized to a sequence which is homologous to other TPA-responsive elements.     

Induction of insulin release in fibroblasts transfected with genomic rat islet DNA

L cells (tk-) were cotransfected with total genomic rat islet DNA and a plasmid containing thymidine kinase gene (ptk). Transfectants were tested for their ability to release insulin into the medium. At least 10% of the colonies contained immunoreactive insulin (IRI) during the initial two weeks. The insulin secreted competed linearly with rat insulin in RIA, the majority of the insulin antigenicity comigrating with rat insulin on G-50 Sephadex chromatography. With continuing propagation the IRI activity diminished; however 3 selected cultures demonstrated increased secretion of IRI following stimulation with glucose. These findings indicate that glucose-induced insulin secretion can be obtained in non-beta cells; however the frequency of success was below one stable transfection for every 5 X 10(8) Ltk- cells exposed to the transfection procedure.     

Differentiation-specific expression from the bovine papillomavirus type 1 P2443 and late promoters.

The papillomavirus life cycle is tightly linked with keratinocyte differentiation in squamous epithelia. Vegetative viral DNA replication begins in the spinous layer, while synthesis of capsid proteins and virus maturation is restricted to the most differentiated or granular layer of the epithelium. In this study, in situ hybridization of bovine fibropapillomas was used to demonstrate that the activity of two promoters of bovine papillomavirus type 1 (BPV-1) is regulated in a differentiation-specific manner. In situ hybridization with a late promoter (PL)-specific oligonucleotide probe suggested that PL is dramatically upregulated in the granular layer of the fibropapilloma. Northern (RNA) blot analysis of RNA from BPV-1-infected fibropapillomas indicated that the three major BPV-1 late-region mRNAs were transcribed from PL. These RNAs include the previously described L1 (major capsid) mRNA as well as two larger mRNAs. The two larger mRNAs were characterized and shown to contain the L2 (minor capsid protein) open reading frame as well as the L1 open reading frame. In contrast to PL, the P2443 promoter was maximally active in basal keratinocytes and the fibroma. The major mRNA transcribed from P2443 is the putative E5 oncoprotein mRNA which is spliced between nucleotides 2505 and 3225. No signal was detected above the basal layer with use of a probe specific for this mRNA. The E5 oncoprotein has previously been localized by immunoperoxidase staining to the granular cell layer as well as the basal cell layer of the fibropapilloma (S. Burnett, N. Jareborg, and D. DiMaio, Proc. Natl. Acad. Sci. USA 89:5665-5669, 1992). These data suggest that E5 proteins in the basal cell and granular cell layers are not translated from the same mRNA.     

Induction of cell surface insulin antigenicity in fibroblasts transfected with islet genomic DNA

Intact platelets were stimulated with thrombin and the amount of GTP-binding protein (G-protein) oligomers was assessed by measuring ADP ribosylation of 40-41 kDa protein by pertussis toxin in isolated membranes. The toxin substrate fell by 57-62% in 10-60 s, but then returned towards normal over 5 min. Recovery was greatly enhanced by removal of thrombin from receptors with hirudin. Phorbol myristate acetate increased ADP-ribosylatable protein, but only back to initial levels prior to PMA. In contrast prostaglandin D2 plus theophylline (which increase cyclic AMP) did not increase ADP ribosylation, but could completely block the fall of the toxin substrate caused by thrombin. These results indicate that activation of thrombin receptors promotes the dissociation of G-protein oligomers to release free alpha-subunits, and this effect can be modulated by protein kinase C and cyclic AMP-dependent protein kinase. The possible relationships of these findings to the regulation of stimulus-response coupling in platelets is discussed.     

Characterization of primary human keratinocytes transformed by human papillomavirus type 18.

Primary human epithelial cells were cotransfected with pHPV-18 and pSV2neo, and cell strains were generated by selecting in G418. One cell strain (FE-A), which exhibits an extended life span, is currently in its 30th passage. In comparison, control cultures can only be maintained up to the seventh passage. Southern blot analysis revealed the presence of at least one intact, integrated viral genome in these cells. FE-A cells showed altered growth properties, characterized by a change in morphology, and clonal density. Differentiation markers analyzed by Western blotting (immunoblotting), such as cytokeratins and involucrin, indicated that the cells resembled a partially differentiated epithelial population. Increased expression of the 40-kilodalton cytokeratin was observed in FE-A cells, similar to that observed in simian virus 40-immortalized human keratinocytes (M. Steinberg and V. Defendi, J. Cell Physiol. 123:117-125, 1985). FE-A cells were also found to be defective in their response to terminal differentiation stimuli. Calcium and 12-O-tetradecanoyl-phorbol-13-acetate treatment induced normal epithelial cells to differentiate, whereas the human papillomavirus 18 (HPV-18)-containing keratinocytes were resistant to these signals, indicating their partially transformed nature. These cells were not able to induce tumors in nude mice over a period of up to 8 months. A second cell strain, FE-H18L, also generated by transfecting HPV-18, also exhibited an extended life span and similar alterations in morphology. Viral RNA transcribed from the early region of HPV-18 was detected in both cell strains by Northern (RNA) blot analysis. These cell strains should provide a useful model for determining the role of HPV in carcinogenesis.     

Suppression in vivo of human papillomavirus type 18 E6-E7 gene expression in nontumorigenic HeLa X fibroblast hybrid cells.

The E6 and E7 genes of the cancer-associated human papillomavirus (HPV) types 16 (HPV16) and 18 (HPV18) can induce cell immortalization in vitro in normal human keratinocytes. This, however, is not associated with tumorigenicity in vivo. On the other hand, tumorigenicity of HPV18-positive HeLa cervical carcinoma cells can be suppressed by fusion of HeLa cells with normal human keratinocytes or fibroblasts. We have addressed the question of whether suppression of tumorigenicity in HeLa x fibroblast hybrid cells might be due to a reduced ability of these cells to express the HPV18 E6-E7 genes in vivo. Nontumorigenic hybrid cells and tumorigenic hybrid segregants were transplanted as organotypical cultures or injected subcutaneously into immunocompromised mice and were analyzed for HPV18 E6-E7 gene expression by RNA-RNA in situ hybridization. The tumorigenic hybrid cells showed a continuous and invasive growth that was associated with high levels of HPV18 E6-E7 mRNAs at all time points examined. In contrast, the nontumorigenic hybrid cells stopped cell proliferation approximately 3 days after transplantation. At this time they expressed the E6-E7 genes at low levels, whereas at day 2 high expression levels were observed. However, the mRNA levels of the cytoskeletal genes beta-actin and vimentin remained high for at least 14 days, demonstrating that inhibition of growth and of HPV18 E6-E7 gene expression was not due to cell death. These results suggest that growth inhibition of the nontumorigenic HeLa x fibroblast hybrid cells in vivo might be caused by suppression of HPV18 E6-E7 gene expression and are compatible with the idea of an intracellular surveillance mechanism for HPV gene expression existing in nontumorigenic cells.     

Enzymatic amplification and characterization of large DNA fragments from genomic DNA

Conditions for DNA amplification in vitro using modified T7 DNA polymerase have been devised to obtain 2000-bp DNA fragments of the HGPRT gene directly from human genomic DNA. The DNA obtained from a 1.2 x 10(5)-fold amplification has been used for direct sequencing.     

DNA sequence templates adjacent nucleosome and ORC sites at gene amplification origins in Drosophila

Eukaryotic origins of DNA replication are bound by the origin recognition complex (ORC), which scaffolds assembly of a pre-replicative complex (pre-RC) that is then activated to initiate replication. Both pre-RC assembly and activation are strongly influenced by developmental changes to the epigenome, but molecular mechanisms remain incompletely defined. We have been examining the activation of origins responsible for developmental gene amplification in Drosophila. At a specific time in oogenesis, somatic follicle cells transition from genomic replication to a locus-specific replication from six amplicon origins. Previous evidence indicated that these amplicon origins are activated by nucleosome acetylation, but how this affects origin chromatin is unknown. Here, we examine nucleosome position in follicle cells using micrococcal nuclease digestion with Ilumina sequencing. The results indicate that ORC binding sites and other essential origin sequences are nucleosome-depleted regions (NDRs). Nucleosome position at the amplicons was highly similar among developmental stages during which ORC is or is not bound, indicating that being an NDR is not sufficient to specify ORC binding. Importantly, the data suggest that nucleosomes and ORC have opposite preferences for DNA sequence and structure. We propose that nucleosome hyperacetylation promotes pre-RC assembly onto adjacent DNA sequences that are disfavored by nucleosomes but favored by ORC.