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研究生:陳廷祺
研究生(外文):Ting-Chi Chen
論文名稱:第一型單純疱疹病毒潛伏相關轉錄體區域之新RNA產物之定性
論文名稱(外文):Characterization of Novel RNA Products in HSV-1 LAT Region
指導教授:張淑媛張淑媛引用關係
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:醫學檢驗暨生物技術學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:46
中文關鍵詞:單純皰疹病毒潛伏相關轉錄體
外文關鍵詞:HSV-1LATRNA transcript
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人類單純疱疹病毒第一型(HSV-1)為線狀雙股DNA病毒,基因體大小約為152.0KB。HSV-1在初次感染時症狀不明顯,但會潛伏於神經細胞中。待宿主遭受壓力或免疫力下降,病毒會再活化,造成疱疹甚至會引起較嚴重的疾病,例如角膜炎或腦膜炎。HSV-1的基因主要分為三種。立即早期基因(Intermediate Early;IE)在病毒感染後不需要有病毒蛋白的存在即可表現,主要功用是調節細胞和病毒基因的表現。早期基因(Early;E)必須在有IE基因產生之蛋白的存在下才能表現,主要與DNA合成和核酸代謝有關。晚期基因(Late;L)受到IE及E基因的調控,主要合成病毒顆粒的組成成分。在HSV-1潛伏時,大部分HSV-1的基因表現會停止。此時唯一會大量表現的是一群RNA分子,稱為latency-associated transcripts(LAT)。LAT依照其分子穩定度可以分為major LAT與minor LAT。Major LAT較穩定,會於細胞中大量累積,其包含三種大小的分子:2.0KB、1.5Kb與1.45Kb;minor LAT有兩種:8.3Kb與6.3Kb。其中8.3Kb會經過splicing產生6.3Kb 與2.0Kb LAT。目前已知2.0KB LAT可能與HSV-1潛伏的建立與維持、病毒的再活化與抑制神經細胞凋亡有關。在1.5Kb LAT splicing site的附近,本實驗室利用oligo dT RT-PCR與sequencing的方式發現到有不同於原先研究的RNA片段的存在R300(306bp)與R200(216bp)。接著利用306bp作為probe作Northern blot確認其來源RNA大小約為2.0KB左右。同時發現其表現與L基因表現模式相似,推測是在病毒複製晚期表現。有研究報告指出2.0KB LAT可能透過影響ICP0的splicing來調控病毒的潛伏或活化。因此我們推測此和LAT有相似序列的R300與R200片段可能藉由影響病毒基因的表現,而使病毒進入潛伏狀態。於是將R200和R300分別接入CMV promoter drived質體,將其轉染至Vero細胞後感染HSV-1。發現對於HSV-1的三個時期的基因之表現或病毒的產生均無影響。此外病毒感染神經母細胞瘤細胞株SH-SY5Y,也可偵測到R200與R300的表現。接著利用不同的引子進行不同的PCR反應,推測含有R200與R300之RNA 5端應包含至LATF,3端則不含LATR至LATR2的區域。另外根據序列與二級結構預測R300的splicing過程可能為group I intron機制,但R200的splicing過程尚無法推測。最後2006年有研究指出LAT中含有miRNA,所以我們預測在1.5Kb LAT spliced區域有一可能的microRNA存在,並預測其可能的目標基因為NFIX。
Human herpes simplex virus type 1 (HSV-1) is a double-strand DNA virus with about 152.0KB genome. It infects mucus epithelial cells primarily with no symptoms and establishes latent infection in trigeminal ganglia neuron cells. When host suffers from stress, HSV-1 would reactivation and cause cold sores, keratitis and rarely fatal encephalitis. The expression of HSV-1 gene is in a cascade initialed by expression of immediate-early genes (IE genes) which regulate other host and viral genes, followed by early genes (E genes) which are related to DNA synthesis and nucleotide metabolism. Finally, late genes (L genes) regulated by IE and E genes express and encode proteins which compose viral particle. During latent state, most of viral genes are inactive but there are a group of transcripts accumulated in latent-infected neuron cells, called latency-associated transcripts (LATs). According to molecular stability, the LATs can be divided into two groups: major LATs (2.0Kb, 1.5Kb and 1.45Kb) and minor LATs (8.3Kb and 6.3Kb). The most abundant stable LAT is a 2.0Kb LAT. 2.0Kb LAT is an intron spliced from a primary minor 8.3Kb LAT. 2.0Kb LAT expresses as viral late phase gene during lytic cycle and accumulates abundantly while virus established latent state. In previous studies, we knew the 2.0Kb LAT is correlated with establishment and maintenance of viral latency, reactivation from latent state, neuron survival and antiapoptosis. In study of 1.5Kb splicing, we used reverse-transcription polymerase chain reaction (RT-PCR) and sequencing. We discovered two novel products called R200 (216bp) and R300 (306bp). Further, we found R300 may come from 2.0Kb RNA. The expression pattern of R200 and R300 is the same with L gene and suggested that R200 and R300 should express at late phase in viral lytic cycle. It has been reported that 2.0Kb LAT might influence an IE gene, ICP0, expression by blocking its splicing and this might lead virus into latent phase. We proposed that R300 and R200 which are similar to LAT may influence viral replication and promote virus into latent state. We expressed two fragments in Vero cells and infected with HSV-1. We discovered there were no difference between transfercted and wild type Vero cells and suggested that R300 and R200 have no effect on viral replication. Besides we used neuron cell line, SH-SY5Y, as target and can detect R300 and R200. We also used different primers for PCR to find the 3’ and 5’ end of RNA contained R300 and R200. We suggested that 3’ end of them should be up to LATF, but 5’ end of them didn’t include the region of LATR to LATR2. According to the sequence and RNA structure, we supposed that the mechanism of R300 splicing is group I intronic splicing. Howerer, we couldn’t predict the possible one of R200. Finally, we predicted a microRNA in 1.5Kb LAT splicing region, and its possible human gene is NFIX.
目錄 1
圖表目錄 2
中文摘要 3
英文摘要 4
第一章 前言 6
1.1 人類單純疱疹病毒
1.2 人類單純疱疹病毒的基因結構與表現
1.3 潛伏相關轉錄體
1.4 潛伏相關轉錄體之功能
1.5 研究目的

第二章 實驗材料與方法 11
2.1 實驗材料
2.2 實驗方法

第三章 實驗結果 22
3.1 經由反轉錄-聚合酵素連鎖反應發現不同分子大小的LAT
3.2 R200與R300之特性
3.3 R200與R300於神經細胞株SH-SY5Y之研究
3.4 尋找含有R200與R300之RNA可能之位置

第四章 討論 25

第五章 參考文獻 29

第六章 附圖表格 35



圖表目錄
Fig. 1 HSV-1 LAT示意圖 35
Fig. 2 RT-PCR發現不同之RNA分子,R300與R200 36
Fig. 3 北方墨點法 37
Fig. 4 R300與R200之序列比對圖 38
Fig. 5 R300與R200於HSV-1感染後2、12與16小時之表現 39
Fig. 6 R300與R200對HSV-1基因表現之影響 40
Fig. 7 R300與R200對HSV-1複製之影響 41
Fig. 8 R300與R200於神經細胞株SH-SY5Y之表現 42
Fig. 9  R300與R200來源RNA可能之3端與5端位置 43
Fig. 10 RNA二級結構圖 44
Fig. 11 LAT區域之預測miRNA示意圖 45
1.Nishiyama, Y., Herpes simplex virus gene products: the accessories reflect her lifestyle well. Rev Med Virol, 2004. 14(1): p. 33-46.
2.Spivack, J.G. and N.W. Fraser, Detection of herpes simplex virus type 1 transcripts during latent infection in mice. J Virol, 1987. 61(12): p. 3841-7.
3.Stroop, W.G., D.L. Rock, and N.W. Fraser, Localization of herpes simplex virus in the trigeminal and olfactory systems of the mouse central nervous system during acute and latent infections by in situ hybridization. Lab Invest, 1984. 51(1): p. 27-38.
4.Farrell, M.J., A.T. Dobson, and L.T. Feldman, Herpes simplex virus latency-associated transcript is a stable intron. Proc Natl Acad Sci U S A, 1991. 88(3): p. 790-4.
5.Rodahl, E. and J.G. Stevens, Differential accumulation of herpes simplex virus type 1 latency-associated transcripts in sensory and autonomic ganglia. Virology, 1992. 189(1): p. 385-8.
6.Spivack, J.G. and N.W. Fraser, Expression of herpes simplex virus type 1 latency-associated transcripts in the trigeminal ganglia of mice during acute infection and reactivation of latent infection. J Virol, 1988. 62(5): p. 1479-85.
7.Thomas, D.L., et al., The 2-kilobase intron of the herpes simplex virus type 1 latency-associated transcript has a half-life of approximately 24 hours in SY5Y and COS-1 cells. J Virol, 2002. 76(2): p. 532-40.
8.Wu, T.T., et al., Evidence that two latency-associated transcripts of herpes simplex virus type 1 are nonlinear. J Virol, 1996. 70(9): p. 5962-7.
9.Krummenacher, C., J.M. Zabolotny, and N.W. Fraser, Selection of a nonconsensus branch point is influenced by an RNA stem-loop structure and is important to confer stability to the herpes simplex virus 2-kilobase latency-associated transcript. J Virol, 1997. 71(8): p. 5849-60.
10.Spivack, J.G., G.M. Woods, and N.W. Fraser, Identification of a novel latency-specific splice donor signal within the herpes simplex virus type 1 2.0-kilobase latency-associated transcript (LAT): translation inhibition of LAT open reading frames by the intron within the 2.0-kilobase LAT. J Virol, 1991. 65(12): p. 6800-10.
11.Alvira, M.R., et al., Genetic studies exposing the splicing events involved in herpes simplex virus type 1 latency-associated transcript production during lytic and latent infection. J Virol, 1999. 73(5): p. 3866-76.
12.Thompson, R.L. and N.M. Sawtell, Evidence that the herpes simplex virus type 1 ICP0 protein does not initiate reactivation from latency in vivo. J Virol, 2006. 80(22): p. 10919-30.
13.Cai, W., et al., The herpes simplex virus type 1 regulatory protein ICP0 enhances virus replication during acute infection and reactivation from latency. J Virol, 1993. 67(12): p. 7501-12.
14.Halford, W.P. and P.A. Schaffer, ICP0 is required for efficient reactivation of herpes simplex virus type 1 from neuronal latency. J Virol, 2001. 75(7): p. 3240-9.
15.Leib, D.A., et al., Immediate-early regulatory gene mutants define different stages in the establishment and reactivation of herpes simplex virus latency. J Virol, 1989. 63(2): p. 759-68.
16.Perng, G.C., et al., The latency-associated transcript gene enhances establishment of herpes simplex virus type 1 latency in rabbits. J Virol, 2000. 74(4): p. 1885-91.
17.Thompson, R.L. and N.M. Sawtell, The herpes simplex virus type 1 latency-associated transcript gene regulates the establishment of latency. J Virol, 1997. 71(7): p. 5432-40.
18.Sawtell, N.M. and R.L. Thompson, Herpes simplex virus type 1 latency-associated transcription unit promotes anatomical site-dependent establishment and reactivation from latency. J Virol, 1992. 66(4): p. 2157-69.
19.Maillet, S., et al., Herpes simplex virus type 1 latently infected neurons differentially express latency-associated and ICP0 transcripts. J Virol, 2006. 80(18): p. 9310-21.
20.Amelio, A.L., P.K. McAnany, and D.C. Bloom, A chromatin insulator-like element in the herpes simplex virus type 1 latency-associated transcript region binds CCCTC-binding factor and displays enhancer-blocking and silencing activities. J Virol, 2006. 80(5): p. 2358-68.
21.Feldman, L.T., et al., Spontaneous molecular reactivation of herpes simplex virus type 1 latency in mice. Proc Natl Acad Sci U S A, 2002. 99(2): p. 978-83.
22.Chen, S.H., et al., A viral function represses accumulation of transcripts from productive-cycle genes in mouse ganglia latently infected with herpes simplex virus. J Virol, 1997. 71(8): p. 5878-84.
23.Kubat, N.J., et al., Specific histone tail modification and not DNA methylation is a determinant of herpes simplex virus type 1 latent gene expression. J Virol, 2004. 78(3): p. 1139-49.
24.Wang, Q.Y., et al., Herpesviral latency-associated transcript gene promotes assembly of heterochromatin on viral lytic-gene promoters in latent infection. Proc Natl Acad Sci U S A, 2005. 102(44): p. 16055-9.
25.Kubat, N.J., et al., The herpes simplex virus type 1 latency-associated transcript (LAT) enhancer/rcr is hyperacetylated during latency independently of LAT transcription. J Virol, 2004. 78(22): p. 12508-18.
26.Amelio, A.L., et al., Deacetylation of the herpes simplex virus type 1 latency-associated transcript (LAT) enhancer and a decrease in LAT abundance precede an increase in ICP0 transcriptional permissiveness at early times postexplant. J Virol, 2006. 80(4): p. 2063-8.
27.Giordani, N.V., et al., During herpes simplex virus type 1 infection of rabbits, the ability to express the latency-associated transcript increases latent-phase transcription of lytic genes. J Virol, 2008. 82(12): p. 6056-60.
28.Leib, D.A., et al., A deletion mutant of the latency-associated transcript of herpes simplex virus type 1 reactivates from the latent state with reduced frequency. J Virol, 1989. 63(7): p. 2893-900.
29.Jin, L., et al., Reactivation phenotype in rabbits of a herpes simplex virus type 1 mutant containing an unrelated antiapoptosis gene in place of latency-associated transcript. J Neurovirol, 2007. 13(1): p. 78-84.
30.Jin, L., et al., A herpes simplex virus type 1 mutant expressing a baculovirus inhibitor of apoptosis gene in place of latency-associated transcript has a wild-type reactivation phenotype in the mouse. J Virol, 2005. 79(19): p. 12286-95.
31.Thomas, S.K., et al., Herpes simplex virus latency-associated transcript encodes a protein which greatly enhances virus growth, can compensate for deficiencies in immediate-early gene expression, and is likely to function during reactivation from virus latency. J Virol, 1999. 73(8): p. 6618-25.
32.Thomas, S.K., et al., A protein encoded by the herpes simplex virus (HSV) type 1 2-kilobase latency-associated transcript is phosphorylated, localized to the nucleus, and overcomes the repression of expression from exogenous promoters when inserted into the quiescent HSV genome. J Virol, 2002. 76(8): p. 4056-67.
33.Perng, G.C., et al., A herpes simplex virus type 1 latency-associated transcript mutant with increased virulence and reduced spontaneous reactivation. J Virol, 1999. 73(2): p. 920-9.
34.Perng, G.C., et al., Virus-induced neuronal apoptosis blocked by the herpes simplex virus latency-associated transcript. Science, 2000. 287(5457): p. 1500-3.
35.Ahmed, M., et al., Regions of the herpes simplex virus type 1 latency-associated transcript that protect cells from apoptosis in vitro and protect neuronal cells in vivo. J Virol, 2002. 76(2): p. 717-29.
36.Garber, D.A., P.A. Schaffer, and D.M. Knipe, A LAT-associated function reduces productive-cycle gene expression during acute infection of murine sensory neurons with herpes simplex virus type 1. J Virol, 1997. 71(8): p. 5885-93.
37.Gupta, A., et al., Anti-apoptotic function of a microRNA encoded by the HSV-1 latency-associated transcript. Nature, 2006. 442(7098): p. 82-5.
38.Kleymann, G., et al., New helicase-primase inhibitors as drug candidates for the treatment of herpes simplex disease. Nat Med, 2002. 8(4): p. 392-8.
39.Devi-Rao, G.B., et al., Relationship between polyadenylated and nonpolyadenylated herpes simplex virus type 1 latency-associated transcripts. J Virol, 1991. 65(5): p. 2179-90.
40.Rodahl, E. and L. Haarr, Analysis of the 2-kilobase latency-associated transcript expressed in PC12 cells productively infected with herpes simplex virus type 1: evidence for a stable, nonlinear structure. J Virol, 1997. 71(2): p. 1703-7.
41.Shamovsky, I. and E. Nudler, Gene control by large noncoding RNAs. Sci STKE, 2006. 2006(355): p. pe40.
42.Prasanth, K.V. and D.L. Spector, Eukaryotic regulatory RNAs: an answer to the ''genome complexity'' conundrum. Genes Dev, 2007. 21(1): p. 11-42.
43.Mattick, J.S., The functional genomics of noncoding RNA. Science, 2005. 309(5740): p. 1527-8.
44.Batchelor, A.H. and P. O''Hare, Regulation and cell-type-specific activity of a promoter located upstream of the latency-associated transcript of herpes simplex virus type 1. J Virol, 1990. 64(7): p. 3269-79.
45.Jones, C., et al., Analysis of the transcriptional promoter which regulates the latency-related transcript of bovine herpesvirus 1. J Virol, 1990. 64(3): p. 1164-70.
46.Gussow, A.M., et al., Tissue-specific splicing of the herpes simplex virus type 1 latency-associated transcript (LAT) intron in LAT transgenic mice. J Virol, 2006. 80(19): p. 9414-23.
47.Wilcox, C.L., et al., The herpes simplex virus type 1 immediate-early protein ICP0 is necessary for the efficient establishment of latent infection. J Virol, 1997. 71(9): p. 6777-85.
48.Arthur, J.L., et al., Disruption of the 5'' and 3'' splice sites flanking the major latency-associated transcripts of herpes simplex virus type 1: evidence for alternate splicing in lytic and latent infections. J Gen Virol, 1998. 79 ( Pt 1): p. 107-16.
49.Pyle, A.M., O. Fedorova, and C. Waldsich, Folding of group II introns: a model system for large, multidomain RNAs? Trends Biochem Sci, 2007. 32(3): p. 138-45.
50.Copertino, D.W. and R.B. Hallick, Group II twintron: an intron within an intron in a chloroplast cytochrome b-559 gene. Embo J, 1991. 10(2): p. 433-42.
51.Wu, T.T., et al., Atypical splicing of the latency-associated transcripts of herpes simplex type 1. Virology, 1998. 243(1): p. 140-9.
52.Stahley, M.R. and S.A. Strobel, RNA splicing: group I intron crystal structures reveal the basis of splice site selection and metal ion catalysis. Curr Opin Struct Biol, 2006. 16(3): p. 319-26.
53.Kang, W., et al., Characterization of a spliced exon product of herpes simplex type-1 latency-associated transcript in productively infected cells. Virology, 2006. 356(1-2): p. 106-14.
54.Cui, C., et al., Prediction and identification of herpes simplex virus 1-encoded microRNAs. J Virol, 2006. 80(11): p. 5499-508.
55.Krek, A., et al., Combinatorial microRNA target predictions. Nat Genet, 2005. 37(5): p. 495-500.
56.Rehmsmeier, M., et al., Fast and effective prediction of microRNA/target duplexes. Rna, 2004. 10(10): p. 1507-17.
57.Ravichandran, V., et al., Interactions between c-Jun, nuclear factor 1, and JC virus promoter sequences: implications for viral tropism. J Virol, 2006. 80(21): p. 10506-13.
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