臺灣博碩士論文加值系統

冠狀病毒 (Coronavirus)為正向單股RNA病毒 (Positive-sense single-stranded RNA virus )，其基因體大小在27-32 kb之間，為已知具有最大基因體 (Genome)的RNA病毒。文獻指出，冠狀病毒次級基因體 (Subgenome)5’端序列在感染過程當中會發生刪除及插入等修飾。此外，冠狀病毒基因體及次級基因體5’端都具有大小約65個核苷酸之領導序列 (Leader sequence)。我們利用head-to-tail ligation及primer extension的方法針測病毒感染過程中，其正股基因體及正股次級基因體5’端的序列與負股基因體及負股次級基因體3’端的序列變化。結果發現 (1) head-to-tail ligation方法顯示正股及負股基因體與次級基因體末端序列會有刪除及插入之修飾。(2) head-to-tail ligation方法也顯示負股基因體與負股次級基因體的3’端缺少63個核苷酸。(3) primer extension方法顯示正股基因體與正股次級基因體的5’端缺少54個核苷酸，且此不具領導序列之正股基因體以及正股次級基因體的數目約佔具領導序列族群的25%。為了探討領導序列之修飾對於病毒生物功能上的影響。我們構築了不具領導序列的牛冠狀病毒的天然缺陷株 (Bovine defective interfering RNA, BCoV DI RNA)並將其在轉譯、複製以及轉錄上的效率與具領導序列的DI RNA比較，結果發現 (1)不具領導序列之基因體與具領導序列之基因體相比，不具領導序列的基因體負股生成能力較佳。 (2)具領導序列之基因體於早期感染時，其轉譯能力明顯高於不具領導序列之基因體。 (3)不具領導序列之基因體其轉錄效率 (次級基因體的生成)較具領導序列基因體差。因此得知領導序列之功能在於促進病毒的轉譯以及轉錄。因為冠狀病毒為正股RNA 病毒，它在感染細胞後必須擔任轉譯、負股生成及轉錄之角色。而從本研究中可知不具領導序列之基因體功能為促進負股生成，且可當作模板而進行轉錄以合成次級基因體。因此本研究結果推測不具領導序列基因體存在之意義及目的在於減輕具領導序列基因體之負擔，進而增加病毒的致病能力。

Coronavirus is a positive-sense single-stranded RNA virus with the genome size of 27 kb-32 kb, establishing as the largest known RNA genome. Yet, terminal sequence variations at times are found, but not well documented. Here, with the bovine coronavirus (BCoV) which has a leader sequence of 65 nt on both genome and subgenome, we use two approaches, head-to-tail ligation and primer extension, to examine the 5’-terminal sequences of (+)-strand genomic and N subgenomic mRNA, and 3’-terminal sequences of (-)-strand genomic and N subgenomic mRNA during infections. The major findings are the following: (i) By head-to-tail ligation, the modification of deletion or/and insertion on terminal sequence was found in genome and subgenome of both positive- and negative- strands. (ii) By head-to-tail ligation of the (-)-strands of genomic and N subgenomic mRNA, the (-) strands of both species were found to have a 3’-terminal deletions of 63 nt. (iii) By primer extension on the (+)-strands of genomic and N subgenomic mRNAs, 5’-terminal deletions of 54 nt were found on both RNA species；the ratio of leader-containing genome and leaderless genome was 1/4, and so was leader-containing subgenome and leaderless subgenome. The results identified the previously unfound species of leaderless genome and subgenome in coronaviruses during infecton. To explore the biological functions and then to explain the significance of leaderless genome during infection, leaderless defective interfering RNA (DI RNA) was constructed and tested. We found that (i) the efficiency of negative-strand synthesis was increased in DI RNA without leader sequence in comparison with DI RNA with leader sequence, (ii) compared with DI RNA with no leader sequence, DI RNA with leader sequence resulted in a significant increase in translation efficiency during early infection, and (iii) the transcription (interpreted as subgenome synthesis) efficiency of DI RNA without leader is less than that of DI RNA with leader sequence. Instead of negative-strand RNA synthesis, therefore, it was concluded that leader sequence may function in the enhancement of viral translation and transcription. Accordingly, we speculate that the significance of the identified leaderless genomic RNA is to relieve the loading of leader-containing genomic RNA, which has to play multiple roles of translation, negative-strand synthesis, and transcription during infection, and subsequently to enhance the disease development along with leader-containing genomic RNA.

中文摘要.....................I
英文摘要.....................II
目次.........................III
表次.........................VI
圖次.........................VII
第一章 .....................前言 1
第二章 文獻探討 ................2
第一節 冠狀病毒之介紹..............2
1.1 冠狀病毒之歸類................2
1.2 冠狀病毒之背景 ...............2
1.3 冠狀病毒之型態結構與基因體結構...2
1.4 冠狀病毒之生活史...............3
第二節 冠狀病毒正股基因體及次級基因體.....4
2.1 冠狀病毒正股基因體之功能.............4
2.2 冠狀病毒正股基因體及次級基因體共同之特徵....4
2.3 次級基因體領導序列之形成機轉及其功能.....4
2.4 領導序列之介紹及其功能.....5
第三節 冠狀病毒天然缺陷株.....6
3.1 冠狀病毒天然缺陷株 (DI RNA)之介紹.......... 6
3.2 冠狀病毒天然缺陷株 (DI RNA)之生物功能利用性........ 6
3.3 牛冠狀病毒天然缺陷株之介紹....... 6
第四節 病毒基因體末端序列修飾之重要性....... 7
第五節 負股基因體偵測之方法....... 8
5.1 負股基因體之偵測........8
5.2 負股基因體偵測方法之改進........ 8
第三章 材料與方法 ............10
第一節 病毒與細胞.......... 10
1.1 病毒與細胞.... 10
1.2 細胞之繼代.... 10
第二節 牛冠狀病毒的急性及持續性感染試驗........ 10
2.1 牛冠狀病毒感染細胞(急性感染)..... 10
2.2 牛冠狀病毒感染細胞(持續性感染)... 11
2.3 細胞及病毒RNA收集及萃取 .......11
2.4 Head-to-tail ligation偵測病毒基因體及次級基因體5’末端序列..... 11
2.4.1 基因體頭端帽(Cap)之去除........ 11
2.4.2 T4 RNA ligase將基因體末端5’ UTR及3’UTR相連...... 11
2.4.3 反轉錄聚合酶連鎖反應......12
2.5 Primer extension assay偵測病毒正股病毒基因體及次級基因體5’末端序列...... 12
第三節 牛冠狀病毒基因體5’端突變DI RNA之構築.... 13
3.1 利用重疊聚合酶連鎖反應構築DI RNA 5’端突變株....... 13
3.2 PCR產物轉殖及聚合酶連鎖反應篩檢....14
3.3 質體細菌增殖及質體DNA萃取....... 14
3.4 酵素切割及溶膠試驗..... 15
3.5 T4 DNA ligase連接Vector及Insert與產物連接後之沉澱法 15
3.6 轉殖及聚合酶連鎖反應篩檢....... 15
3.7 質體細菌增殖及質體DNA萃取...... 16
3.8 利用MluI酵素將質體DNA 線性化及T7 polymerase進行in vitro transcription....... 16
3.9 RNA純化...... 17
3.10 Denaturing gel製備及RNA 樣本定量......17
3.11 RNA transcripts轉染細胞與細胞RNA及蛋白質收集與萃取.. 17
3.12北方墨點法-病毒RNA之偵測.......18
3.13西方墨漬法-偵測病毒之轉譯.......19
第四節 領導序列及不具領導序列基因體DI RNA之構築...... 19
4.1 DI RNA 質體插入表現蛋白質之Histidine序列... 19
4.2 DI RNA 質體插入螢光表現物質之EGFP序列......20
第五節 統計分析.... 20
第四章 結果.......21
第一節 探討牛冠狀病毒在感染細胞過程中，病毒基因體及次級基因體末端序列之修飾與此修飾在病毒生物功能上所扮演角色....21
1.1牛冠狀病毒於病毒感染過程中，病毒基因體及次級基因體末端序列之修飾...21
1.2 病毒基因體5’末端序列修飾對於病毒轉譯之影響.....21
1.3 病毒基因體5’端序列修飾對於病毒複製(正股的形成)之影響22
第二節 探討牛冠狀病毒在感染過程中是否出現不具領導序列的病毒基因體或次級基因體....22
2.1探討在病毒感染過程中，是否出現不具領導序列之負股基因體或
負股次級基因體..... 22
2.2 探討在病毒感染過程中，是否出現不具領導序列之正股基因體或
正股次級基因體..... 22
第三節 牛冠狀病毒領導序列在病毒生活史之生物功能...23
3.1 牛冠狀病毒領導序列在基因體之影響...23
3.1.1 牛冠狀病毒基因體領導序列在病毒轉譯之影響 23
3.1.2 牛冠狀病毒基因體領導序列在病毒負股生成之影響....24
3.1.3 牛冠狀病毒基因體領導序列在次級基因體生成之影響...24
第五章 討論.....26
參考文獻....54

Banerjee S and Makino S. Studies of murine coronavirus DI RNA replication from negative-strand transcripts. Adv Exp Med Biol 440: 241-246, 1998.
Baric RS and Yount B. Subgenomic negative-strand RNA function during mouse hepatitis virus infection. J Virol 74: 4039-4046, 2000.
Baric RS, Stohlman SA and Lai MM. Characterization of replicative intermediate RNA of mouse hepatitis virus: presence of leader RNA sequences on nascent chains. J Virol 48: 633-640, 1983.
Barr JN and Fearns R. How RNA viruses maintain their genome integrity. J Gen Virol 91: 1373-1387, 2010.
Bender SJ and Weiss SR. Pathogenesis of murine coronavirus in the central nervous system. J Neuroimmune Pharmacol 5: 336-354, 2010.
Brian DA and Baric RS. Coronavirus genome structure and replication. Curr Top Microbiol Immunol 287: 1-30, 2005.
Brian DA and Spaan WJ. Recombination and Coronavirus Defective Interfering RNAs. Seminars in Virology 8(issue): 101-111, 1997.
Chang RY, Hofmann MA, Sethna PB and Brian DA. A cis-acting function for the coronavirus leader in defective interfering RNA replication. J Virol 68: 8223-8231, 1994.
Chapman NM, Kim KS, Drescher KM, Oka K and Tracy S. 5'' terminal deletions in the genome of a coxsackievirus B2 strain occurred naturally in human heart. Virology 375: 480-491, 2008.
Cowley JA, Dimmock CM and Walker PJ. Gill-associated nidovirus of Penaeus monodon prawns transcribes 3''-coterminal subgenomic mRNAs that do not possess 5''-leader sequences. J Gen Virol 83: 927-935, 2002.
Cowley JA, Dimmock CM, Spann KM and Walker PJ. Gill-associated virus of Penaeus monodon prawns: an invertebrate virus with ORF1a and ORF1b genes related to arteri- and coronaviruses. J Gen Virol 81: 1473-1484, 2000.
de Vries AAF, Horzinek MC, Rottier PJM and de Groot RJ. The genome organization of the Nidovirales: similarities and differences between arteri-, toro-, and coronaviruses. Seminars in Virology 8: 33-47, 1997.
Gonzalez JM, Gomez-Puertas P, Cavanagh D, Gorbalenya AE and Enjuanes L. A comparative sequence analysis to revise the current taxonomy of the family. Arch Virol 148: 2207-2235, 2003.
Gorbalenya AE, Snijder EJ and Spaan WJ. Severe acute respiratory syndrome coronavirus phylogeny: toward consensus. J Virol 78: 7863-7866, 2004.
Habjan M, Andersson I, Klingstrom J, Schumann M, Martin A, Zimmermann P, Wagner V, Pichlmair A, Schneider U, Muhlberger E, Mirazimi A and Weber F. Processing of genome 5’termini as a strategy of negative-strand RNA viruses to avoid RIG-I-dependent interferon induction. PLoS ONE 3: 20-32, 2008.
Hofmann MA, Senanayake SD, and Brian DA. A translation-attenuating intraleader open reading frame is selected on coronavirus mRNAs during persistent infection. Proc Natl Acad Sci U S A 90: 11733-11737, 1993.
Hofmann MA, Sethna PB and Brian DA. Bovine coronavirus mRNA replication continues throughout persistent infection in cell culture. J Virol 64: 4108-4114, 1990.
Hornung V, Ellegast J., Kim S., Brzozka K., Jung A., Kato H., Poeck H., Akira S., Conzelmann K.-K., Schlee M., Endres S. and Hartmann G. 5’-triphosphate RNA is the ligand for RIG-I. Science 314: 994-997, 2006.
Hunziker I. P., Cornell C. T. and Whitton J. L. Deletions within the 5’UTR of coxsackievirus B3: consequences for virus translation and replication. Virology 360: 120-128, 2007.
Joo M., Banerjee S and Makino S. Replication of murine coronavirus defective interfering RNA from negative-strand transcripts. J Virol 70: 5769-5776, 1996.
Kim KH and Makino S. Two murine coronavirus genes suffice for viral RNA synthesis. J Virol 69: 2313-2321, 1995.
Kim KS, Chapman NM and Tracy S. Replication of coxsackievirus B3 in primary cell cultures generates novel viral genome deletions. J Virol 82: 2033-2037, 2008.
Kim KS, Tracy S, Tapprich W, Bailey J, Lee CK, Kim K, Barry WH and Chapman NM. 5’-Terminal deletions occur in coxsackievirus B3 during replication in murine hearts and cardiac myocyte cultures and correlate with encapsidation of negative-strand viral RNA. J Virol 79: 7024-7041, 2005.
Kim YN, Jeong YS and Makino S. Analysis of cis-acting sequences essential for coronavirus defective interfering RNA replication. Virology 197: 53-63, 1993.
Lai MM, Patton CD, Baric RS and Stohlman SA. Presence of leader sequences in the mRNA of mouse hepatitis virus. J Virol 46: 1027-1033, 1983.
Lai MM. Coronavirus leader-RNA-primed transcription: An alternative mechanism to RNA splicing. BioEssays 5: 257-260, 1986.
Liao CL and Lai MM. Requirement of the 5’-end genomic sequence as an upstream cis-acting element for coronavirus subgenomic mRNA transcription. J Virol 68: 4727-4737, 1994.
Lin YJ and Lai MM. Deletion mapping of a mouse hepatitis virus defective interfering RNA reveals the requirement of an internal and discontiguous sequence for replication. J Virol 67: 6110-6118, 1993.
Lin YJ, Liao CL and Lai MM. Identification of the cis-acting signal for minus-strand RNA synthesis of a murine coronavirus: implications for the role of minus-strand RNA in RNA replication and transcription. J Virol 68: 8131-8140, 1994.
Maeda A, An S and Makino S. Importance of coronavirus negative-strand genomic RNA synthesis prior to subgenomic RNA transcription. Virus Res 57: 35-42, 1998.
Makino S, Fujioka N and Fujiwara K. Structure of the intracellular defective viral RNAs of defective interfering particles of mouse hepatitis virus. J Virol 54: 329-336, 1985.
Makino S, Shieh CK, Keck JG and Lai MM. Defective-interfering particles of murine coronavirus: mechanism of synthesis of defective viral RNAs. Virology 163: 104-111, 1988.
Makino S, Yokomori K and Lai MM. Analysis of efficiently packaged defective interfering RNAs of murine coronavirus: localization of a possible RNA-packaging signal. J Virol 64: 6045-6053, 1990.
Masters PS. The molecular biology of coronaviruses. Adv Virus Res 66: 193-292, 2006.
Mendez A, Smerdou C, Izeta A, Gebauer F and Enjuanes L. Molecular characterization of transmissible gastroenteritis coronavirus defective interfering genomes: packaging and heterogeneity. Virology 217: 495-507, 1996.
Meyer BJ and Schmaljohn C. Accumulation of terminally deleted RNAs may play a role in seoul virus persistence. J Virol 74: 1321-1331, 2000.
Meyer BJ and Southern PJ. A novel type of defective viral genome suggests a unique strategy to establish and maintain persistent lymphocytic choriomeningitis virus infections. J Virol 71: 6757-6764, 1997.
Meyer BJ and Southern PJ. Sequence heterogeneity in the termini of lymphocytic choriomeningitis virus genomic and antigenomic RNAs. J Virol 68: 7659-7664, 1994.
Michel YM, Poncet D, Piron M, Kean KM and Borman AM. Cap-poly(A) synergy in mammalian cell-free extracts. J Biol Chem 275: 32268-32276, 2000.
Penzes Z, Tibbles K, Shaw K, Britton P, Brown TD and Cavanagh D. Characterization of a Replicating and Packaged Defective RNA of Avian Coronavirus Infectious Bronchitis. Virus Virology 203: 286-293, 1994.
Sawicki SG and Sawicki DL. A new model for coronavirus transcription. Adv Exp Med Biol 440: 215-219, 1998.
Sawicki SG and Sawicki DL. Coronavirus transcription: a perspective a coronavirus replication and reverse genetics. Curr Top Microbiol Immunol 287: 31-55, 2005.
Sawicki SG and Sawicki DL. Coronavirus transcription: subgenomic mouse hepatitis virus replicative intermediates function in RNA synthesis. J Virol 64: 1050-1056, 1990.
Schenborn ET and Mierendorf RC Jr. A novel transcription property of SP6 and T7 RNA polymerases: dependence on template structure. Nucleic Acids Res 13: 6223-6236, 1985.
Sethna PB, Hung SL and Brian DA. Coronavirus subgenomic minus-strand RNAs and the potential for mRNA replicons. Proc Natl Acad Sci U S A 86: 5626-5630, 1989.
Snijder EJ and Meulenberg JJ. The molecular biology of arteriviruses. J Gen Virol 79, 1998, 961-979, 1998.
Snijder EJ, Bredenbeek PJ, Dobbe JC, Thiel V, Ziebuhr J, Poon LL, Guan Y, Rozanov M, Spaan WJ and Gorbalenya AE. Unique and conserved features of genome and proteome of SARS-coronavirus, an early split-off from the coronavirus group 2 lineage. J Mol Bio 331: 991-1004, 2003.
Spaan W, Delius H, Skinner M, Armstrong J, Rottier P, Smeekens S, van der Zeijst BA and Siddell SG. Coronavirus mRNA synthesis involves fusion of non-contiguous sequences. EMBO J 2: 1839-1844, 1983.
Spagnolo JF and Hogue BG. Host protein interactions with the 3’end of bovine coronavirus RNA and the requirement of the poly(A) tail for coronavirus defective genome replication. J Virol 74: 5053-5065, 2000.
Szymkowiak C, Kwan WS, Su Q, Toner TJ, Shaw AR and Youil R. Rapid method for the characterization of 3’ and 5’ UTRs of influenza viruses. J Virol Methods 107: 15-20, 2003.
Thiel V, Ivanov KA, Putics A, Hertzig T, Schelle B, Bayer S, Weissbrich B, Snijder EJ, Rabenau H, Doerr HW, Gorbalenya AE and Ziebuhr J. Mechanisms and enzymes involved in SARS coronavirus genome expression. J Gen Virol 84: 2305-2315, 2003.
van der Most RG, Bredenbeek PJ and Spaan WJ. A domain at the 3’end of the polymerase gene is essential for encapsidation of coronavirus defective interfering RNAs. J Virol 65: 3219-3226, 1991.
van der Most RG, de Groot RJ and Spaan WJ. Subgenomic RNA synthesis directed by a synthetic defective interfering RNA of mouse hepatitis virus: a study of coronavirus transcription initiation. J Virol 68: 3656-3666, 1994.
van Vliet AL, Smits SL, Rottier PJ and de Groot RJ. Discontinuous and non-discontinuous subgenomic RNA transcription in a nidovirus. EMBO J 21: 6571-6580, 2002.
Wu HY and Brian DA. 5’-proximal hot spot for an inducible positive-to-negative-strand template switch by coronavirus RNA-dependent RNA polymerase. J Virol 81: 3206-3215, 2007.
Wu HY and Brian DA. Subgenomic messenger RNA amplification in coronaviruses Proc Natl Acad Sci U S A 107: 12257-12262, 2010.
Wu HY, Ozdarendeli A and Brian DA. Bovine coronavirus 5’-proximal genomic acceptor hotspot for discontinuous transcription is 65 nucleotides wide. J Virol 80: 2183-2193, 2006.