Activation of the human epsilon- and beta-globin promoters by SV40 T antigen.

We have studied the effect of the SV40 T antigen on expression from human globin promoters fused to the bacterial chloramphenicol acetyltransferase (CAT) gene and compared its effect with the SV40 enhancer and the adenovirus E1A protein. We have observed that expression of p epsilon GLCAT and p beta GLCAT (the epsilon-globin or beta-globin promoter linked to the CAT gene) was significantly stimulated when cotransfected with a cloned T antigen plasmid into CV-1 cells, indicating that trans-activation of the globin promoters was mediated by SV40 T antigen. Transfection of the p beta GLCAT-SV (p beta GLCAT containing the SV40 enhancer element) into CV-1 cells resulted in a 50-60-fold increase in CAT activity as compared to p beta GLCAT (no enhancer). However, cotransfection of the p beta GLCAT-SV with the cloned T antigen resulted in an additional increase of CAT expression, which suggests that T antigen and the SV40 enhancer activate globin gene expression independently. We found that T antigen but not E1A could further stimulate the expression of an enhancer-containing plasmid in CV-1 cells; whereas E1A but not T antigen could further stimulate p epsilon GLCAT expression in COS-1 cells which constitutively express the SV40 T antigen. These results suggest that T antigen and E1A also act independently. Deletion analysis showed that the minimum sequence required for a detectable level of stimulation of the epsilon-globin promoter by T antigen is 177 bp 5' to the cap site, suggesting that the target sequences for response to T antigen do not reside in the canonical 100 bp promoter region, but rather reside in sequences further upstream, and therefore the cellular factors interacting with T antigen are not the TATA or CAT box binding proteins, but the proteins interacting with upstream regulatory sequences.     

Genetic definition of a new bovine papillomavirus type 1 open reading frame, E5B, that encodes a hydrophobic protein involved in altering host-cell protein processing.

We have previously observed that bovine papillomavirus type 1 (BPV-1) induces the appearance of five cellular proteins in C127 mouse fibroblasts, four of which appear to arise by altered processing of resident endoplasmic reticulum proteins. Studies of various cell lines revealed that expression of the 3' end of the BPV early region was sufficient for induction of these changes. To identify the BPV gene responsible, we have utilized the simian virus 40 (SV40)/BPV-1 recombinant virus Pava-1, which expresses the 3' end of the BPV early region behind an SV40 early promoter. C127 cells infected with Pava-1 for 48 h show the expected BPV-associated alterations, as do cells infected with Pava constructs mutated in the E5 or E2 genes. However, a mutation in the start codon of a previously ignored open reading frame extending from nucleotides 4013 to 4170 (E5B) eliminated the BPV-associated changes. Similar results were obtained with COS cells infected with the Pava mutants and C127 cells transformed by full-length mutated BPV. Despite its influence on the processing of cellular endoplasmic reticulum proteins, this mutation in E5B did not alter BPV-transforming efficiency or the ability of transformants to form colonies in soft agar. The E5B open reading frame encodes a hydrophobic 52-amino-acid polypeptide that shares structural similarities with HPV6 E5A and HPV16 E5. Speculations on a role for E5B in the viral life cycle are discussed.     

Effects of Methylation Status of CpG Sites within the HPV16 Long Control Region on HPV16-Positive Head and Neck Cancer Cells

ObjectiveTo map comprehensively the methylation status of the CpG sites within the HPV16 long control region (LCR) in HPV-positive cancer cells, and to explore further the effects of methylation status of HPV16 LCR on cell bioactivity and E6 and E7 expression. In addition, to analyze the methylation status of the LCR in HPV-positive oropharyngeal squamous cell carcinoma (OPSCC) patients.ResultsHypermethylation status of the LCR in UM-SCC47 (79.8%) and CaSki cells (90.0%) and unmethylation status of the LCR in SiHa cells (0%) were observed. Upon demethylation, the cells with different methylation levels responded differently during growth, apoptosis, and cell cycle arrest, as well as in terms of their E6 and E7 expression. In HPV16-positive OPSCC patients, the methylation rates were 9.5% in the entire LCR region, 13.9% in the 5′-LCR, 6.0% in the E6 enhancer, and 9.5% in the p97 promoter, and hypermethylation of p97 promoter was found in a subset of cases (20.0%, 2/10).ConclusionsOur study revealed two different methylation levels of the LCR in HPV16-positive cancer cells and OPSCC patients, which may represent different carcinogenesis mechanisms of HPV-positive cancers cells. Demethylating the meCpGs in HPV16 LCR might be a potential target for a subgroup of HPV16-positive patients with head and neck squamous cell carcinoma.