Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/12712
標題: 假性狂犬病毒立即早期蛋白對醣蛋白X及潛伏相關轉錄體基因啟動子之調控
Regulation of the Promoters of Glycoprotein X and Latency-Associated Transcript Genes of Pseudorabies Virus by Immediate-Early Protein
作者: 歐嘉仁
Ou, Chia-Jen
關鍵字: Pseudorabies virus;假性狂犬病毒;immediate-early protein;glycoprotein X;latency-associated transcript;promoter;gel shift analysis;CAT assay;立即早期蛋白;醣蛋白X;潛伏相關轉錄體;基因啟動子;膠體移位試驗;CAT試驗
出版社: 獸醫學系
摘要: 
假性狂犬病毒(pseudorabies virus;PrV)屬於阿爾法皰疹病毒亞科的一員,是豬假性狂犬病的致病原。假性狂犬病毒在感染細胞後,病毒基因體的轉錄以層階式的調控模式進行,由立即早期、早期及晚期等三群基因依序表現,並可於宿主之神經系統建立潛伏感染。假性狂犬病毒之立即早期基因(immediate-early gene;IE gene)在感染細胞後,不需要病毒其他蛋白質的協助即可立刻表現出一分子量約為180 kDa的轉錄調控蛋白,故被稱為立即早期蛋白(immediate-early protein;IE protein,通稱為IE180)。此蛋白可活化病毒早期與晚期基因的表現,在整個病毒複製週期中扮演一起始的角色。目前假性狂犬病毒只有一種立即早期蛋白被發現。IE180與適當的病毒啟動子結合後,會吸引細胞轉錄複合物到這些結合位置,而導致有效的轉錄調控。雖然如此,假性狂犬病毒IE180是否會對病毒在潛伏感染後的復活化,病毒早期及晚期各個基因表現造成影響及其調控機制,仍有待進一步的研究。為了更清楚此基因產物的功能,遂將此病毒之國內分離株-台南株(TNL strain)的IE基因經次選殖、定序及表現,進而分析IE180的調控模式及其涉及調控機制的區域。
假性狂犬病毒TNL株之立即早期基因位於病毒基因體的倒轉重複區上,即位在BamHI酵素所切割的第8片段上,IE180的胺基酸序列與其他假性狂犬病毒株比較,結果顯示其保留性相當高且相似度高達95.8-96.5 %間。對於TNL株之胺基酸序列之分析,除了做胺基酸相似性之比對外,亦與其他的皰疹病毒科病毒進行相似產物胺基酸序列分析,結果顯示假性狂犬病毒IE180與第一型單純皰疹病毒ICP4 (infected-cell polypeptide 4;a4)的胺基酸序列具49.4 %的相似性。假性狂犬病毒IE 基因轉譯區長4,362個核苷酸,可轉譯出1,454個胺基酸。利用多重比較分析立即早期基因的胺基酸序列,發現其中有二個區域具高保留性。為了對IE180的轉錄調控機轉進行分析,於是將假性狂犬病毒的IE 基因嵌入真核表現載體,再分別將此可表現病毒立即早期基因蛋白的重組質體分別與含有假性狂犬病毒在潛伏感染時期的潛伏感染相關轉錄體(latency-associated transcripts;LATs)、病毒感染立即早期的立即早期蛋白、病毒感染早期的胸腺核苷激酵素(thymidine kinase;TK)及去氧核糖核酸分解酵素(deoxyribonuclease;DNase)、與病毒感染晚期的醣蛋白H(glycoprotein H;gH)及醣蛋白X(glycoprotein X;gX)等四大類基因啟動子(promoter);將這六種基因啟動子與CAT酵素(chloramphenicol acetyltransferase;CAT)基因所構成報導質體共同轉染(cotransfect)至mouse-L(LM)或neuro-2A細胞內,再藉由分析CAT酵素的活性證實假性狂犬病毒IE180可抑制LAT基因與本身IE基因的啟動子及活化病毒感染早期階段的TK 與DNase基因啟動子,同時也能活化病毒感染晚期的gX基因啟動子,但並不影響同為病毒感染晚期的gH基因啟動子活性。
為了進一步研究假性狂犬病毒IE180所涉及的LAT基因啟動子之調控機轉,分別製備了系列N端及C端的假性狂犬病毒IE基因缺損片段,利用pMAMneo真核表現載體表現,與一系列含CAT基因的5’端及3’端的假性狂犬病毒LAT基因啟動子缺損之報導質體,以短暫轉染分析(transient transfection assay)進行CAT酵素活性測定。由這些缺損選殖株所得的結果,發現假性狂犬病毒IE180之N端第1到1,433個胺基酸對於抑制LAT基因啟動子的活性具顯著影響。此外,利用大腸桿菌系統以pET為表現載體,進行IE180的表現,經金屬離子親和性色層分析法(metal affinity chromatography)加以純化後,由膠體移位試驗(gel shift analysis)所得的結果顯示,LAT基因啟動子TATA box上游的區域藉由與IE180及細胞蛋白質形成蛋白質-DNA複合物,而抑制LAT的轉錄活性。這種抑制現象可藉由利用缺損突變的方式將LAT基因啟動子上的5’-ATCGT-3’(預測之IE180結合位)片段去除而消失。此結果顯示LAT基因轉錄起始點上游第-43到-39個核苷酸,是執行假性狂犬病毒IE180對LAT基因啟動子進行抑制作用的關鍵區域。
為了研究假性狂犬病毒IE180對病毒晚期基因之一的gX基因之啟動子的活化機制,本實驗亦構築一系列含有gX基因啟動子缺損片段的CAT報導質體進行測試。結果顯示IE180的N端從第136到第732個胺基酸的區域為其激活gX啟動子所必須,而IE180對gX基因啟動子主要調控區域乃位於gX啟動子之transcriptional enhancer factor-1(TEF-1)結合位。為了進一步定義IE180對gX基因啟動子激活的特性,經由試管內表現IE蛋白,經由西方墨點法鑑定後,與含有TEF-1結合位且經定點突變所得的一系列gX基因啟動子片段之探針進行膠體移位分析及Sounthwestern墨點法等試驗。結果得知,IE180藉由與gX啟動子上的TEF-1結合區形成複合物的模式對gX啟動子進行轉錄活化。
本實驗以選殖的假性狂犬病毒IE基因及其基因產物IE180進行IE180對病毒基因調控之研究。筆者利用CAT試驗分析IE180在病毒感染細胞時,對各個階段啟動子的調控影響。接著,分析IE180對gX基因啟動子的激活作用,以及它對LAT基因啟動子的抑制現象之作用機轉,進而定位出負責執行調控機制的重要功能區。本試驗的結果顯示假性狂犬病毒IE180可藉由細胞內的轉錄因子的參與,形成DNA-蛋白質複合物,進而激活或抑制病毒各階段基因的表現。

Pseudorabies virus (PrV), a member of Alphaherpesvirinae, is the causative agent of Aujeszky's disease in pigs. PrV replicates lytically in the periphery and can establish a latent infection in neurons. PrV genes are generally classified into three kinetic classes, defined as immediate-early, early, and late, on the basis of their temporal expression during the viral lytic cycle. PrV has only one major immediate-early (IE) gene, and is the first viral genome transcribed during a production infection. This gene codes for the transcriptional regulatory protein of molecular weight 180 kDa (IE180). The IE180, a promiscuous regulator of gene expression, is considered to be required for efficient initiation of lytic infection and reactivation from latency. In addition, IE180 appears to function as a transcription factor by interacting with components of the basal transcription initiation complex to allow transcription of early and late genes. A combination of biochemical and genetic analyses has successfully defined multiple functional domains of the protein and provided clues to some of the function mechanisms of the actions of IE180. However, the mechanism of activation or suppression in IE180 is not completely understood. IE180 is known to be essential for the activation of later classes of promoters and the repression of some viral genes, including its own gene. Nevertheless, the regulatory mechanism of IE180 remained to be investigated. In this study, we identified the PrV (TNL strain) IE gene and gene product. The mechanism responsible for transactivation or repression of IE180 were examined, and the structure-function relationships of PrV IE180 and promoters of other PrV genes were studied.
This approximately 180 kDa protein is encoded by the gene within the 8th BamHI fragment which is present in two copies in the viral genome. The complete DNA sequence coding for the IE180 of PrV TNL strain was determined. The coding region of IE180 is 4,362 nucleotides for 1,454 amino acid residues. The G + C content of the gene is 79.4 %. We identified this gene encoding the sequences within the PrV homologous to the herpes simplex virus-1 (HSV-1) Rs 1 gene that encodes ICP4. Clusters of amino acid homologies are observed among IE180 of PrV, ICP4 of HSV-1, and IE protein of simian varicella virus (SVV), and they exhibited two conserved regions among herpesviruses. In order to characterize the role of IE180 in the regulation of PrV gene expression, the IE gene of PrV was cloned into an eukaryotic expression vector and a recombinant expression plasmid was constructed. The regulatory mechanism of IE180 was analyzed by cotransfection with chloramphenicol acetyltransferase (CAT) reporter plasmids containing the promoters of PrV genes of latency-associated transcript (LAT), immediate-early protein (IE180), thymidine kinase (TK), deoxyribonuclease (DNase), glycoprotein H (gH) and glycoprotein X (gX). The results of CAT assay showed that IE180 could significantly increase the expression of CAT gene when it was under the control of promoters of TK, DNase and gX. However, the results showed that the activity of LAT or IE promoter was dramatically repressed by the IE180. Moreover, our result of CAT assay demonstrated that IE180 had no influence on the promoter of PrV glycoprotein H gene.
To further analyze the regulatory model between IE180 and the LAT promoter, we constructed a set of IE180 variants with a deletion in the N-termini or C-termini of IE gene. Cotransfection of these truncated mutants of IE gene individually with the deletion variants of the LAT promoter-CAT reporter plasmid and then assayed the CAT production by CAT assay. The results showed that the N-terminal amino acid residues 1 to 1433 of IE180 retained its significant function of repression to viral LAT promoter. To explore the possible mechanism of repression, an electrophoretic mobility shift assay (EMSA) using nuclear extracts from neuronal cells was performed. The formation of protein-DNA complexes between IE180 and the oligonucleotide probe, from -46 to -19 relative to the start site of LAT transcription, was demonstrated. The association of IE180 with the region encompassing the putative IE180 binding site and the TATA box upstream of PrV LAT gene was further confirmed by supershift of EMSA complexes using IE180 specific antibody. The results suggested that IE180 repressed the LAT promoter via an interaction between IE180, LAT promoter, and cellular protein(s).
To further identify the activation domains of IE180 protein that interact with the gX promoter sequences, various truncated mutants of IE180 gene and gX promoter gene were constructed and analyzed by CAT and gel retardation assay. Results revealed that the N-terminal amino acid residues 133 to 736 of IE180 could interact with the binding site of transcriptional enhancer factor-1 (TEF-1) that resides in the gX promoter. Formation of protein-DNA complexes between the IE180 protein and the TEF-1 element of the gX promoter was demonstrated using electrophoretic mobility shift assay (EMSA) as well as Southwestern blot analysis. These results indicated that a direct interaction occurred between IE180 and the TEF-1 element; and this interaction was abolished if the TEF-1 element was mutated. The association of IE180 with the TEF-1 element was further confirmed by supershift phenomenon of EMSA using IE180 specific antibody. Taken together, our results suggested that formation of a complex between the IE180 protein and TEF-1 element in the gX promoter region was involved in the transcriptional activation of the gX gene.
In this study, we first identified the DNA sequences coding for the IE180 of PrV TNL strain. Transient transfection assays performed with the mutants of IE180 and CAT genes linked to the promoter of PrV LAT, IE, TK, Dnase, gH, or gX gene revealed that certain IE180 residues are essential for repression of the LAT promoter and some other IE180 residues are essential for the induction of the gX promoter. The results presented here provide evidence for these conclusions: (i) regulation of the PrV genes is initiated by the formation of multi-protein complex containing the viral protein(s) and cellular factor(s); (ii) the complex binds to the elements for eukaryotic transcription factors or promoter of each viral gene; (iii) multi-functional IE180 is necessary for the activation/repression of viral gene expression.
URI: http://hdl.handle.net/11455/12712
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