No abstract

No abstract

Bovine papillomavirus type 1 (BPV-1) and its cloned full-length DNA can transform rodent cells in vitro, and the viral DNA persists as an extrachromosomal multicopy plasmid in these transformed cells. Previous studies have identified at least five discrete viral RNAs that are expressed in BPV-1 transformed cells and have shown that these transcripts share a 3' coterminus. To further define the structure of these RNAs and to characterize the functions of individual viral transcripts, we constructed a cDNA library with mRNA from BPV-1-transformed mouse C127 cells using an Okayama and Berg plasmid. From a library of 105 independent clones, 200 BPV-1 specific clones were isolated and characterized. Sequence analysis has revealed differential splicing patterns for the mRNA species in BPV-1 transformed cells. In conjunction with the open reading frames (ORFs) deduced from the BPV-1 DNA sequence, it is possible to predict the structure of the potential encoded proteins. The vector used to generate these cDNA clones contains mammalian cell transcriptional regulatory elements, facilitating their functional characterization. We have identified two distinct classes of cDNA clones that can each independently transform mouse C127 cells. One class of cDNA clones contains the-E2 ORF intact and the second contains the E6 ORF intact. These two putative viral functions appear to act synergistically in transforming mouse C127 cells in vitro.fied several regions of BPV-1 genome that influence the expression of the viral transforming functions (9-12). Deletion mutants affecting the E2 ORF are significantly impaired in their ability to transform mouse C127 cells, suggesting that the putative E2 protein is an important transforming protein (9). The expression of E2 alone, however, is not sufficient for the fully transformed phenotype. Deletion mutagenesis studies map an additional function that is critical for the fully transformed phenotype to the region between the Hpa I site (base 1) and the Sma I site (base 945) (9). These previous studies did not distinguish whether these two distinct regions of the viral genome encode separate proteins or whether they contain exons that are spliced together at the level of mRNA to generate a single transforming protein.To further define the structure of the mRNAs in transformed cells and to characterize the functions encoded by individual viral transcripts, we constructed a cDNA library using mRNA from BPV-1-transformed mouse cells. The vector used for cDNA cloning contains the simian virus 40 (SV40) early promoter, the SV40 late region introns, and the SV40 late region polyadenylylation site, allowing expression of cDNA clones in mouse cells for functional analysis (17). In this study, expression vectors that carried individual cDNA inserts were tested for their ability to transform mouse C127 cells.

Bovine papillomavirus (BPV-1) contains two independent transforming genes that have been mapped to the E5 and E6 open reading frames (ORF's). The E5 transforming protein was identified by means of an antiserum against a synthetic peptide corresponding to the 20 COOH-terminal amino acids of the E5 ORF. The E5 polypeptide is the smallest viral transforming protein yet characterized; it had an apparent size of 7 kilodaltons. The transforming polypeptide is encoded entirely within the second half of the E5 ORF and its predicted amino acid composition is very unusual; 68% of the amino acids are strongly hydrophobic and 34% are leucine. Cell fractionation studies localized this polypeptide predominantly to cellular membranes.

Infection of the lung epithelial cell line A549 by respiratory syncytial virus (RSV) resulted in the elevated synthesis of multiple cellular cytokines, including a number of interleukins (ILs). Detailed studies of IL-11 induction revealed that it required infection by viable virus and involved a net increase in the steady state level of IL-11 mRNA. Nuclear run-on assays showed a direct effect of RSV on IL-11 gene transcription. Mutational analysis of the IL-11 promoter fused to a reporter luciferase gene demonstrated the requirement of a region 720 nucleotides upstream of the mRNA start site in the transcriptional induction of IL-11 by RSV. Two nearly identical 10-nucleotide-long sequences GGGGTCTCCC and GGGTCTCCCC in this region resembled the NF-kappa B consensus motif. Mutation of either sequence greatly reduced RSV-mediated induction of IL-11 promoter activity. NF-kappa B sites in IL-1 alpha, IL-6, and IL-8 promoters were also required for RSV-mediated induction of transcription of these promoters. Immunological studies and use of reporter gene constructs provided direct evidence for the activation and nuclear translocation of NF-kappa B by RSV. Sodium salicylate and aspirin, inhibitors of NF-kappa B activation, abolished transcriptional induction of all these cytokines by RSV. Together, these studies demonstrated an essential role of NF-kappa B in RSV-mediated transcription of multiple cytokines genes and suggested a possible use of salicylates in managing airway inflammation and viral pathogenesis during RSV infection.