Induction of autoantibodies to mouse CCR5 with recombinant papillomavirus particles.
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The vertebrate immune system has evolved to respond vigorously to microbial infection but to ignore self-antigens. Evidence has emerged that B cell responses to viruses are initiated by immune recognition of ordered arrays of antigen on the viral surface. To test whether autoantibodies against a self-antigen can be induced by placing it in a context that mimics the ordered surface of a viral particle, a peptide representing an extracellular loop of the mouse chemokine receptor CCR5 was incorporated into an immunodominant site of the bovine papillomavirus virus L1 coat protein, which self-assembles into virus-like particles. Mice inoculated with chimeric L1-CCR5 particles generated autoantibodies that bound to native mouse CCR5, inhibited binding of its ligand RANTES, and blocked HIV-1 infection of an indicator cell line expressing a human-mouse CCR5 chimera. These results suggest a general method for inducing autoantibodies against self-antigens, with diverse potential basic research and clinical applications. (+info)
A mutational analysis of the transforming functions of the E8 protein of bovine papillomavirus type 4.
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The E8 protein of BPV-4 contributes to transformation of primary bovine cells (PalFs) by inducing anchorage-independent growth and by down-regulating gap junction intercellular communication, likely due to its binding to 16K ductin. We show here that, in addition, E8 confers on PalF cells the ability to grow in low serum and to escape from contact inhibition (focus formation). E8 also transactivates an exogenous human cyclin A gene promoter, suggesting that overexpression of cyclin A is responsible for the transformed phenotype. Mutant forms of E8 were generated to establish whether the transforming functions of the protein could be segregated. Mutations were introduced both in the hydrophobic domain and in the hydrophilic C-terminal "tail", and chimeras with BPV-1 E5 were constructed. Cells expressing either wild-type E8 or mutant forms were analyzed for their ability to grow in low serum and in suspension and to form foci. Wild-type E8 and its mutants were also analyzed for their ability to transactivate the cyclin A promoter. We show here that the transforming functions of E8 can be segregated and that both the hydrophilic C-terminal tail and the residue at position 17 in the hydrophobic domain are crucial for E8 functions and for the transactivation of the cyclin A promoter. These results support the hypothesis that the different aspects of cellular transformation brought about by E8 might be due to interaction with different cellular targets. They suggest that E8 might function differently from BPV-1 E5 and demonstrate that the separate domains of E5 and E8 are not functionally interchangeable. (+info)
Long-term episomal maintenance of bovine papillomavirus type 1 plasmids is determined by attachment to host chromosomes, which Is mediated by the viral E2 protein and its binding sites.
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Papillomavirus genomes are stably maintained as extrachromosomal nuclear plasmids in dividing host cells. To address the mechanisms responsible for stable maintenance of virus, we examined nuclear compartmentalization of plasmids containing the full-length upstream regulatory region (URR) from the bovine papillomavirus type 1 (BPV1) genome. We found that these plasmids are tightly associated with the nuclear chromatin both in the stable cell lines that maintain episomal copies of the plasmids and in transiently transfected cells expressing the viral E1 and E2 proteins. Further analysis of viral factors revealed that the E2 protein in trans and its multiple binding sites in cis are both necessary and sufficient for the chromatin attachment of the plasmids. On the other hand, the BPV1 URR-dependent plasmid replication and chromatin attachment processes are clearly independent of each other. The ability of the plasmids to stably maintain episomes correlates clearly with their chromatin association function. These data suggest that viral E2 protein-mediated attachment of BPV1 genomes to the host cell chromatin could provide a mechanism for the coupling of viral genome multiplication and partitioning to the host cell cycle during viral latent infection. (+info)
Effect of bovine papillomavirus E2 protein-specific monoclonal antibodies on papillomavirus DNA replication.
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The bovine papillomavirus type 1 (BPV-1) E2 protein is the master regulator of papillomavirus replication and transcription. We have raised a panel of monoclonal antibodies (MAbs) against the BPV-1 E2 protein and used them to probe the structure and function of the protein. Five MAbs reacted with linear epitopes, and four MAbs recognized conformation-dependent epitopes which mapped within the C-terminal DNA-binding and dimerization domain. MAb 1E2 was able to recognize the replication- and transactivation-defective but not the competent conformation of the transactivation domain of the E2 protein. MAb 5H4 prevented efficiently the formation of E2-DNA as well as E2-dependent E1-E2-origin complexes and also dissociated preformed complexes in a concentration-dependent manner. Cotransfection of several MAbs with the BPV-1 minimal origin plasmid pUCAlu into CHO4.15 cells resulted in a dose-dependent inhibition of replication. Inhibition of replication by MAb 5H4 and the Fab' fragment of 5H4 correlated with their ability to dissociate the E2 protein from the DNA. MAb 3F12 and MAbs 1H10 and 1E4, directed against the hinge region, were also capable of inhibiting BPV-1 origin replication in CHO4.15 cells. However, the Fab' fragments of 1H10 and 3F12 had no effect in the transient replication assay. These data suggest that MAbs directed against the hinge region sterically hinder the inter- or intramolecular interactions required for the replication activity of the E2 protein. (+info)
Papillomavirus capsid protein expression level depends on the match between codon usage and tRNA availability.
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Translation of mRNA encoding the L1 and L2 capsid proteins of papillomavirus (PV) is restricted in vivo to differentiated epithelial cells, although transcription of the L1 and L2 late genes occurs more widely. The codon composition of PV late genes is quite different from that of most mammalian genes. To test the possibility that PV late gene codon composition determines the efficiency of PV late gene expression in some cell types, synthetic bovine papillomavirus type 1 (BPV1) late genes were constructed with codon composition modified to resemble the typical mammalian gene. Expression of these genes from a strong promoter in Cos-1 cells was compared with expression of wild-type BPV1 late genes from the same promoter. Both unmodified and modified PV late genes were transcribed in Cos-1 cells, but only the codon-modified genes were translated. In vitro translation of wild-type but not synthetic BPV1 L1 mRNA was markedly enhanced by addition of aminoacyl-tRNAs. Codon composition thus limits BPV1 late gene translation in Cos-1 cells, and this limitation can be overcome by modification of the codon composition of the genes or by provision of excess tRNA. Replacement of codons in the green fluorescent protein (gfp) gene with those frequently used in PV late genes did not alter gfp transcription in Cos-1 cells but almost abolished translation, supporting the hypothesis that the observed differences in efficiency of translation of modified and unmodified PV capsid genes were related to codon usage rather than mRNA structure. As tRNA populations vary within and between tissues in the same eukaryotic organism, we speculate that matching of tRNA availability to codon usage may be one determinant of the restriction of expression of PV late genes to differentiated epithelium. (+info)
Nucleotides 1506-1625 of bovine papillomavirus type 1 genome can enhance DNA packaging by L1/L2 capsids.
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We have previously described a DNA-packaging assay using bovine papillomavirus type 1 (BPV-1) virus-like particles (VLPs) and have identified a region of the BPV genome that assists in packaging. In this study, we identify a specific BPV sequence involved in DNA packaging by BPV-1 VLPs. In the initial screening of BPV-1 genomic sequences essential for DNA packaging, we observed that a plasmid with deletions between nucleotides (nt) 948 and 2113 failed to be packaged into BPV-1 VLPs. However, plasmids containing nt 948 to 2113 were efficiently packaged, suggesting that this 1.2-kb fragment contains a packaging enhancement sequence (PES). Further mapping of the BPV-1 genome showed that this packaging sequence lies between nt 1506 and 1625. Furthermore, this packaging sequence is also recognized by HPV6b VLPs, suggesting that a common packaging mechanism may be used by the two papillomavirus types. Given the phylogenetic difference between these two viral types, it is likely that other papillomavirus types may also use the same packaging mechanism. Identification of the PES has allowed a minimal viral genome sequence to be used in the packaging assay, improving the usefulness of the assay in studying the process of papillomavirus DNA encapsidation. (+info)
Papillomavirus E2 induces p53-independent apoptosis in HeLa cells.
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We have previously shown that expression of the papillomavirus E2 protein in HeLa cells induces p53 accumulation and causes both cell cycle arrest and apoptosis. In contrast to growth arrest, onset of apoptosis was not correlated with an increase of p53 transcriptional activity. In the present study, we conducted biochemical and genetic experiments in order to determine whether E2-induced apoptosis was independent of p53 induction. We showed that E2 did not alter the transcription of Bax, a known p53-activated cell death inducer. The time course of apoptotic cell death preceded p53 induction by several hours. Overexpression of the HPV18 E6 oncogene prevented E2-mediated p53 accumulation, but did not alter the rate of cell death. Finally, point mutants of the HPV18 E2 transactivation domain induced apoptosis, although they were unable to induce high p53 accumulation or cell cycle arrest. In addition, the results obtained with these mutants indicated that both transcriptional activation and replication functions of E2 were dispensable for the induction of cell death. These observations show that E2-induced apoptosis is an early event, independent of p53 accumulation and unrelated to downstream p53-dependent transcriptional events. (+info)
An enhanced epithelial response of a papillomavirus promoter to transcriptional activators.
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Mucosal epitheliotropic papillomaviruses have a similar long control region (LCR) organization: a promoter region, an enhancer region, and a highly conserved distribution of E2 DNA binding sites. The enhancer of these viruses is epithelial-specific, as it fails to activate transcription from heterologous promoters in nonepithelial cell types (Gloss, B., Bernard, H. U., Seedorf, K., and Klock, G. (1987) EMBO J. 6, 3735-3743; Morgan, I. M., Grindlay, G. J., and Campo, M. S. (1999) J. Gen. Virol. 80, 23-27). Studies on E2 transcriptional regulation of the human mucosal epitheliotropic papillomaviruses have been hindered by poor access to the natural target cell type and by the observation that some of the human papillomavirus promoters, including human papillomavirus-16, are repressed in immortalized epithelial cells. Here we present results using the bovine papillomavirus-4 (BPV-4) LCR and a bovine primary cell system as a model to study the mechanism of E2 transcriptional regulation of mucosal epitheliotropic papillomaviruses and the cell type specificity of this regulation. E2 up-regulates transcription from the BPV-4 LCR preferentially in epithelial cells (Morgan, I. M., Grindlay, G. J., and Campo, M. S. (1998) J. Gen. Virol. 79, 501-508). We demonstrate that the epithelial-specific enhancer element of the BPV-4 LCR is not required for the enhanced activity of E2 in epithelial cells and that the BPV-4 promoter is more responsive, not only to E2, but to other transcriptional activators in epithelial cells. This is the first time a level of epithelial specificity has been shown to reside in a papillomavirus promoter region. (+info)