Effects of HCV Core Protein on HCMV MIE Promoter Activity
碩士 === 國立陽明大學 === 生物化學研究所 === 87 === Abstract HCV (Hepatitis C virus) has been proven as being the major cause of trans-fusion-associated non-A, non-B hepatitis, and the prolonged, persistent infec-tion of HCV often progress to chronic hepatitis, cirrhosis, and hepatocellular...
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碩士 === 國立陽明大學 === 生物化學研究所 === 87 === Abstract
HCV (Hepatitis C virus) has been proven as being the major cause of trans-fusion-associated non-A, non-B hepatitis, and the prolonged, persistent infec-tion of HCV often progress to chronic hepatitis, cirrhosis, and hepatocellular carcinoma by unknown molecular pathogenesis. Several studies have demon-strated that the HCV core protein has a pleiotropic function and may play a major role in the pathogenesis of HCV infection, but the molecular mechanism was not well defined. Human cytomegalovirus (HCMV) is ubiquitous with very high prevalence in population. Co-infection with HCV and HCMV is therefore possible. Since the activation of the MIE promoter-enhancer is ex-pected to play pivotal roles in HCMV infection, a detailed understanding of the regulation of its expression activity may help us to gain important insights about latency and reactivation of HCMV infection.
Analysis of the effect of HCV core protein on HCMV promoter activity has two significances. First, utilizing binding sites of various transcription factors on HCMV MIE promoter may help us to elucidate the possible molecular mechanism for the regulatory activity of HCV core protein. Second, recurrence of HCV or HCMV infection or both after allograft transplant is risk factor for transplanted patients, investigation of the interaction between these two viral factors may provide important insights for prophylaxis and treatment.
In our results, the full-length HCV core protein (c195) has biphasic effects on HCMV MIE promoter in HuH-7 cells in transient transfection experiment, while the deletion mutants of core protein have slight (c122) or no (c101) effect. With lower expression level, the full-length HCV core protein has trans-activa-tion ability on HCMV MIE promoter (containing promoter region -760~+3), while it has trans-suppression effect with the higher expression level. Additionally, the full-length HCV core protein also has trans-activation ability on four deletion mutants of HCMV MIE promoter (containing promoter region -736~+2, -541~+2, -246~+2, and -54~+2, respectively) in HuH-7 cells. Apart from the MIE promoter region spanning -54 to +2 (pMIEP-54/2), similar re-sults were found in HeLa cells, but differ from that of HuH-7 cells in activation pattern. Interestingly, an opposite, significant suppression of HCMV promoter activity was observed in HuH-7 and HeLa core protein-producing cell lines (HuH-7 /C190 and HeLa/C190, respectively) except pMIEP-54/2 in HeLa/C190 cells. Thus, the regulatory activity of the HCV core protein on HCMV MIE promoter possesses cell-type specificity.
Using electrophoretic mobility shift assay (EMSA) I found that the expres-sion of HCV core protein could enhance the DNA binding activity of YY1, Sp1, and CREB in HuH-7/C190 cells. In HeLa/C190 cells, the DNA binding activity of YY1 and CREB was reduced by core protein, but no effect were found in Sp1-binding activity. The results also suggest that these DNA-protein complex-es are large complexes composed of different transcription factors through protein-protein interaction. Furthermore, Western bolt analysis indicated that these effects on DNA binding activity did not result from the alteration of ex-pression levels of YY1, Sp1, and CREB by the HCV core protein under most conditions, except in the case of CREB in HuH-7/C190 cells.
Taken together, the results demonstrate that the HCV core protein possesses transcriptional regulatory ability on HCMV MIE promoter. Additionally, core protein may alter the expression level of some transcription factors and en-hance or suppress the transcriptional complex formation in a cell-type specific manner. Therefore, our findings imply that HCV infection may alter the HCMV life cycle. Moreover, these features of core protein may at least par-tially account for its pleiotropic effects on regulation of gene expression and the molecular mechanism of HCV pathogenesis.
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author2 |
Yan-Hwa Wu Lee |
author_facet |
Yan-Hwa Wu Lee Ru-Tsun 麥如村 |
author |
Ru-Tsun 麥如村 |
spellingShingle |
Ru-Tsun 麥如村 Effects of HCV Core Protein on HCMV MIE Promoter Activity |
author_sort |
Ru-Tsun |
title |
Effects of HCV Core Protein on HCMV MIE Promoter Activity |
title_short |
Effects of HCV Core Protein on HCMV MIE Promoter Activity |
title_full |
Effects of HCV Core Protein on HCMV MIE Promoter Activity |
title_fullStr |
Effects of HCV Core Protein on HCMV MIE Promoter Activity |
title_full_unstemmed |
Effects of HCV Core Protein on HCMV MIE Promoter Activity |
title_sort |
effects of hcv core protein on hcmv mie promoter activity |
publishDate |
1999 |
url |
http://ndltd.ncl.edu.tw/handle/99448188118971484555 |
work_keys_str_mv |
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ndltd-TW-087YM0001070072015-10-13T11:50:26Z http://ndltd.ncl.edu.tw/handle/99448188118971484555 Effects of HCV Core Protein on HCMV MIE Promoter Activity C型肝炎病毒核心蛋白對人類巨細胞病毒MIE啟動子活性之影響 Ru-Tsun 麥如村 碩士 國立陽明大學 生物化學研究所 87 Abstract HCV (Hepatitis C virus) has been proven as being the major cause of trans-fusion-associated non-A, non-B hepatitis, and the prolonged, persistent infec-tion of HCV often progress to chronic hepatitis, cirrhosis, and hepatocellular carcinoma by unknown molecular pathogenesis. Several studies have demon-strated that the HCV core protein has a pleiotropic function and may play a major role in the pathogenesis of HCV infection, but the molecular mechanism was not well defined. Human cytomegalovirus (HCMV) is ubiquitous with very high prevalence in population. Co-infection with HCV and HCMV is therefore possible. Since the activation of the MIE promoter-enhancer is ex-pected to play pivotal roles in HCMV infection, a detailed understanding of the regulation of its expression activity may help us to gain important insights about latency and reactivation of HCMV infection. Analysis of the effect of HCV core protein on HCMV promoter activity has two significances. First, utilizing binding sites of various transcription factors on HCMV MIE promoter may help us to elucidate the possible molecular mechanism for the regulatory activity of HCV core protein. Second, recurrence of HCV or HCMV infection or both after allograft transplant is risk factor for transplanted patients, investigation of the interaction between these two viral factors may provide important insights for prophylaxis and treatment. In our results, the full-length HCV core protein (c195) has biphasic effects on HCMV MIE promoter in HuH-7 cells in transient transfection experiment, while the deletion mutants of core protein have slight (c122) or no (c101) effect. With lower expression level, the full-length HCV core protein has trans-activa-tion ability on HCMV MIE promoter (containing promoter region -760~+3), while it has trans-suppression effect with the higher expression level. Additionally, the full-length HCV core protein also has trans-activation ability on four deletion mutants of HCMV MIE promoter (containing promoter region -736~+2, -541~+2, -246~+2, and -54~+2, respectively) in HuH-7 cells. Apart from the MIE promoter region spanning -54 to +2 (pMIEP-54/2), similar re-sults were found in HeLa cells, but differ from that of HuH-7 cells in activation pattern. Interestingly, an opposite, significant suppression of HCMV promoter activity was observed in HuH-7 and HeLa core protein-producing cell lines (HuH-7 /C190 and HeLa/C190, respectively) except pMIEP-54/2 in HeLa/C190 cells. Thus, the regulatory activity of the HCV core protein on HCMV MIE promoter possesses cell-type specificity. Using electrophoretic mobility shift assay (EMSA) I found that the expres-sion of HCV core protein could enhance the DNA binding activity of YY1, Sp1, and CREB in HuH-7/C190 cells. In HeLa/C190 cells, the DNA binding activity of YY1 and CREB was reduced by core protein, but no effect were found in Sp1-binding activity. The results also suggest that these DNA-protein complex-es are large complexes composed of different transcription factors through protein-protein interaction. Furthermore, Western bolt analysis indicated that these effects on DNA binding activity did not result from the alteration of ex-pression levels of YY1, Sp1, and CREB by the HCV core protein under most conditions, except in the case of CREB in HuH-7/C190 cells. Taken together, the results demonstrate that the HCV core protein possesses transcriptional regulatory ability on HCMV MIE promoter. Additionally, core protein may alter the expression level of some transcription factors and en-hance or suppress the transcriptional complex formation in a cell-type specific manner. Therefore, our findings imply that HCV infection may alter the HCMV life cycle. Moreover, these features of core protein may at least par-tially account for its pleiotropic effects on regulation of gene expression and the molecular mechanism of HCV pathogenesis. Yan-Hwa Wu Lee 吳妍華 1999 學位論文 ; thesis 55 zh-TW |