In this study, we investigated the structural basis of human papillomavirus type 11 (HPV-11) E5a transforming activity at the amino acid level. The effects of insertion, deletion, and substitution mutations on the E5a transforming activity were determined by the assay of anchorage-independent growth. In the conserved Cys-X-Cys structure, substitution of Ser for Cys-73 resulted in indistinguishable transforming activity, whereas substitution of Ser for Cys-75 or Ser for both Cys-73 and Cys-75 retained 50 and 42% transformation, respectively. This suggests that Cys at position 75 may be important for transformation. Charge and structural changes at the COOH termini of several mutants impaired transformation significantly, but those at the middle region did so only mildly. In addition, the 16,000-molecular-weight pore-forming protein (16K protein) is known to associate with BPV-1, HPV-6, and HPV-16 E5 proteins. In this study, we investigated the correlation between E5a-16K binding affinity and the transforming activity of E5a by the use of 11 E5a mutants. Results show that E5a and these 11 E5a mutants could bind to the 16K protein when these proteins were coexpressed in COS cells, suggesting that simple binding of the 16K protein by E5a may not be sufficient for cell transformation.
Previously, we found that the E5 protein can be expressed in HPV-16 infected precancerous lesions and cervical cancer. In this study, we investigated the presence of sequence variants of E5 in HPV-16 infected tissues. Toward this end, we amplified the E5 gene by polymerase chain reaction from 29 HPV-16 infected tissues including eight normal tissues, seven high grade neoplastic tissues (high grade squamous intraepithelial lesions (HSIL) and 14 cervical cancer tissues. Sequence analysis demonstrated that there were three mutational hot spots at positions 3979, 4042, and 4077 of the HPV-16 DNA; these and other mutations resulted in six variants in the E5 sequence. This resulted in four E5 protein mutants, named WTE5 [wild type E5 protein], 14E5, 21E5 and 56E5. Functional analysis of these four mutant proteins revealed that the transforming activities of 14E5, 21E5 and 56E5 were 0.95, 0.59, and 0.89 fold of WTE5, respectively. Although E5 was expressed in all of the HSIL and cervical cancer tissues, but in only one of the eight normal tissues tested, only WT E5 protein was found in HSIL while in cervical cancer tissues both WT and mutant E5 proteins were detected. Since these E5 proteins exhibited the same in vitro transforming activity, these data suggest that expression of E5 is important in development and progression toward malignancy but mutation of E5 does not affect the transformation process.
We previously established a human fibroblast cell line, HFL 6-2, that contains a temperature sensitive simian virus 40 (SV40) T antigen, permitting cell growth at 35 degrees C but restricting growth at 39 degrees C. p21 (Waf1/Cip1) was significantly induced by temperature shifts in HFL 6-2 cells. Here we show that all-trans-retinoic acid (RA) treatment prevented the growth restriction of HFL 6-2 cells at 39 degrees C. In the presence of RA, HFL 6-2 cells proliferated into sizeable colonies even at 39 degrees C. [3H]Thymidine incorporation and flow cytometry analysis revealed that cells exposed to RA maintained DNA synthesis at 39 degrees C. Prevention of growth restriction by RA was correlated with a lack of induction of p21 at the transcription level. These observations suggest that RA may prevent the senescence process by repressing p21 gene expression, and perturb the growth regulation of somatic cells.
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