臺灣博碩士論文加值系統

牛第四型疱疹病毒能感染血管內皮細胞並大量複製，而且兔子感染這種病毒後其血管動脈硬化的生成速度會加快。然而至今尚未有文獻探討牛第四型疱疹病毒在血管內皮細胞中基因的表現、調控與功能。牛第四型疱疹病毒在上皮細胞表現二種前早期基因(immediate-early gene, IE gene)。表現量較少的IE RNA 2所轉錄的IE2蛋白具有調控早期與晚期基因的功能，而表現量較多的IE RNA 1其功能尚未被發現。本研究的目的即在探討牛第四型疱疹病毒感染血管內皮細胞後其IE1基因表現、調控與功能。我們發現在病毒感染細胞之前早期階段IE RNA 1表現量明顯增加，而且IE1基因區域在病毒感染後期階段尚表現一些經不同剪接方式生成的轉錄產物(alternative transcripts)。這些經由不同剪接方式所生成的轉錄產物都是使用一對隱藏在第四個外顯子(exon 4)內的剪接提供(splice donor)與接受(acceptor)序列來移除一段378-bp的插入子(intron)。IE1基因也使用多個轉錄起始點與聚腺苷酸化(polyadenylation)位置來增添調控的選擇性。報告基因分析(reporter assay)與膠體電泳位移分析(electrophoretic mobility shift assay)結果可知IE2蛋白經由辨識IE1基因啟動子上一段37-bp (-549/-513)的片段調控著IE1基因的表現，且這個調控現象能隨著IE2蛋白的表現量增加而更加明顯。由於IE1基因能產生這麼多種轉錄產物且它的表現受到IE2蛋白的調控，所以我們運用共同轉染(co-transfection)與反轉錄聚合酶連鎖反應(RT-PCR)分析哪一種轉錄產物受到IE2蛋白的調控。有趣的是，IE2蛋白的確能活化IE1基因的轉錄並產生大量的IE RNA 1，而其它以不同剪接方式生成的轉錄產物則是必須要牛第四型疱疹病毒的ORF57蛋白幫助它們移除隱藏在第四個外顯子的插入子才能大量生成。兩種經不同剪接方式生成的轉錄產物的全長cDNA序列已定序完成，它們分別被命名為IE RNA 1-SV1與IE RNA 1-SV2，而它們可能轉譯出的蛋白則分別命名為IE1_174蛋白與IE1_165蛋白。IE RNA 1所轉譯的IE1蛋白與IE1_165蛋白具有RING E3泛素連接酶(RING E3 ubiquitin ligase)的二種特微：一個C4HC3 RING-CH motif與二個穿膜區域。然而，IE1_174蛋白只有一個不完整的C4HC3 RING-CH motif，而且不具有穿膜區域。我們並發現IE1蛋白具有抑制第一型主要組織相容性複合體(MHC class I)分子於細胞表面分布的功能，推測可能幫助病毒逃避免疫系統攻擊。IE1_174蛋白則無此功能。以上的研究結果顯示，IE1基因在內皮細胞的表現有著極複雜的調控機轉，而IE2蛋白與ORF57蛋白分別在轉錄時與轉錄後進行調控。

Bovine herpesvirus type 4 (BHV-4), an endothelial-tropic virus, is known to accelerate atherosclerosis process in a rabbit model. However, the expression and regulation of BHV-4 gene in infected endothelial cells remains unclear. BHV-4 expresses two immediate-early genes in epithelial cells. The minor transcript, IE RNA 2, encodes a transcription factor IE2 protein, which regulates several BHV-4 early and late genes. The function of the major transcript IE RNA 1 remains unknown. The purposes of this study were to investigate the IE1 gene regulation and function in BHV-4-infected endothelial cells. We found that IE RNA 1 expressed prominently in the immediate-early stage of infection. Several alternative transcripts using a pair of cryptic internal splice donor and acceptor sites within exon 4 as well as different initiation and polyadenylation sites were produced at later stages of infection. Analytic results of reporter assays and electrophoretic mobility shift assays revealed that IE2 protein, via a 37-bp fragment (-549/-513), dose-dependently regulated IE1 gene. IE2 protein activated the IE1 gene transcription and produced a large amount of IE RNA 1 as evidenced by co-transfection and RT-PCR assay. Interestingly, alternative transcripts were increased in the presence of ORF57 protein of BHV-4, which participated in splicing of viral transcripts via promoting the removal of the cryptic intron located in exon 4. The full-length cDNA sequences of two alternative transcripts, IE RNA 1-SV1 and IE RNA 1-SV2, were obtained from RACE experiments. IE RNA 1-SV1 and IE RNA 1-SV2 were predicted to encode IE1_174 protein and IE1_165 protein, respectively. The IE1 protein, encoded by IE RNA 1, and IE1_165 protein contained two characteristics of RING E3 ubiquitin ligase, a C4HC3 RING-CH motif and two transmembrane regions. However, IE1_174 protein contained an imperfect RING-CH motif and was lack of two transmembrane regions. Furthermore, we showed that IE1 protein, but not IE1_174 protein, participated in immune evasion by down-regulation of MHC class I molecules on the cell surface. Altogether, these data suggest that regulation of IE1 gene is extreme complex in endothelial cells, and IE2 protein and ORF57 protein regulate IE1 gene at the transcription level and the post-transcription level, respectively.

Contents
Abstract in Chinese 摘要 1
Abstract 3
Acknowledgment致謝 5
Abbreviations 7
Contents 9
Table list 11
Figure list 12
Introduction 13
Infection and atherosclerosis 13
BHV-4 14
BHV-4 and atherosclerosis 15
BHV-4 genes and regulation 16
Viruses exploit ubiquitin system 18
Specific aims 20
Materials and methods 21
Cell culture and virus propagation 21
Reverse transcription-polymerase chain reaction (RT-PCR) 21
Detection of viral DNA replication 22
Western blot analysis 23
Plasmid construction 24
Transient transfection and reporter assay 24
5’- and 3’-rapid amplification of cDNA ends (RACE) 25
Electrophoretic mobility shift assay (EMSA) 25
Immunofluorescence assay 26
Evaluation of data and statistics 27
Results 28
Analysis of IE1 gene transcription in BHV-4-infected endothelial cells 28
Identification of the 5’- and 3’-ends of IE 1 gene transcripts expressed late in infection 29
Basal promoter activity of IE1 gene promoter 30
IE2 protein regulates IE1 gene promoter in a dose-dependent manner 31
IE2 protein binds a 37-bp fragment of IE1 promoter 32
Putative consensus binding sequence of the IE2 protein 33
IE2 protein enhances IE1 gene transcription whereas ORF57 protein promotes the production of alternative transcripts 34
The expression of IE1 gene products in BHV-4-infected endothelial cells 36
IE1 gene products containing a RING-CH motif 36
IE1 protein down-regulates cell surface MHC class I molecules 37
Discussion 39
Differential regulation of BHV-4 IE1 gene in endothelial cells and in epithelial cells 39
Alternative transcripts of BHV-4 IE1 gene 40
BHV-4 IE2 protein response elements 41
Putative strategy used by IE1 protein to interfere surface distribution of MHC class I molecules 42
References 45
Tables 56
Figures 58
Part II. The effects of Chlamydia pneumoniae infection on endothelial hemostasis 73
Author’s curriculum vitae 92

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