Characterization of the Human Papillomavirus 16 E8 Promoter

ABSTRACTPersistent infections with certain human papillomaviruses (HPV) such as HPV16 are a necessary risk factor for the development of anogenital and oropharyngeal cancers. HPV16 genomes replicate as low-copy-number plasmids in the nucleus of undifferentiated keratinocytes, which requires the viral E1 and E2 replication proteins. The HPV16 E8^E2C (or E8^E2) protein limits genome replication by repressing both viral transcription and the E1/E2-dependent DNA replication. How E8^E2C expression is regulated is not understood. Previous transcript analyses indicated that the spliced E8^E2C RNA is initiated at a promoter located in the E1 region upstream of the E8 gene. Deletion and mutational analyses of the E8 promoter region identify two conserved elements that are required for basal promoter activity in HPV-negative keratinocytes. In contrast, the transcriptional enhancer in the upstream regulatory region of HPV16 does not modulate basal E8 promoter activity. Cotransfection studies indicate that E8^E2C inhibits, whereas E2 weakly activates, the E8 promoter. Interestingly, the cotransfection of E1 and E2 induces the E8 promoter much more strongly than the major early promoter, and this is partially dependent upon binding of E2 to Brd4. Mutation of E8 promoter elements in the context of HPV16 genomes results in an increased genome copy number and elevated levels of viral early and late transcripts. In summary, the promoter responsible for the expression of E8^E2C is both positively and negatively regulated by viral and cellular factors, and this regulatory circuit may be crucial to maintain a low but constant copy number of HPV16 genomes in undifferentiated cells.IMPORTANCEHPV16 replicates in differentiating epithelia and can cause cancer. How HPV16 maintains its genome in undifferentiated cells at a low but constant level is not well understood but may be relevant for the immunological escape of HPV16 in the basal layers of the infected epithelium. This study demonstrates that the expression of the viral E8^E2C protein, which is a potent inhibitor of viral replication in undifferentiated cells, is driven by a separate promoter. The E8 promoter is both positively and negatively regulated by viral proteins and thus most likely acts as a sensor and modulator of viral copy number.

Construction of Expression Vector with Fruit-Specific Promoter and Genetic Transformation of Strawberry

In order to transcription and expression an exogenous gene just in transgenic strawberry fruit, the gene encoding tomato fruit-specific E8 promoter was cloned. We constructed plant expression vector with E8 promoter replace the CaMV35S of pBI121 that used for transformation of strawberry. Recombination plasmid was identified by PCR, restriction enzymes digestion and sequencing analysis, and transfered into the agrobacterium EHA105 strain. At the same time, the agrobacterium EHA105, transformation and no transformation tissue of strawberry used as the experiment materials to study the expression of reporter genegusAby the histochemical staining method. The results show that agrobacterium which containsgusAgene and strawberry organization of transformation can be dyed blue, others were not stain. So in testing the transgenic plants by agroinfiltration transient expression system can reduce or avoid false positive appearance.