复制缺陷型痘苗病毒天坛株的细胞生物学特性及复制缺陷机制探究
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摘要
痘苗病毒减毒株作为基因表达载体,广泛应用于多种疾病的预防和治疗。复制缺陷型痘苗病毒天坛株(Non-replicating Tiantan vaccinia virus,NTV)作为一株高度减毒的痘苗病毒株,其生物学特性和复制缺陷机制还有待研究。探究NTV细胞生物学特性和复制缺陷机制。在不同种属和组织来源的细胞中测定NTV复制能力、细胞嗜性和毒力;Western Blot检测HeLa细胞中的NTV的A17/A27蛋白表达水平,Real-time PCR检测晚期蛋白A17/A27转录水平。NTV在BHK-21、CEF细胞中可有效复制和扩散,而在Vero细胞及人源细胞HeLa、2BS、Hep-2和143TK-中均不能有效复制;在HeLa细胞中,晚期蛋白A17和A27蛋白表达受阻,而转录水平基本不变。NTV是一株复制缺陷型痘苗病毒,在多种细胞中复制和扩散受限;NTV晚期蛋白A17和A27的表达受阻且与蛋白表达转录水平无关,初步判定蛋白表达受阻发生在蛋白翻译阶段。
As gene expression vectors, attenuated strain of vaccinia virus was widely used in prevention and treatment of various diseases. Non-replicating Tiantan vaccinia virus(NTV) was a highly attenuated vaccinia virus strain while its cytobiological characteristics and replication-defective mechanism remained to be explored.To explore the cytobiological characteristics and replication-defective mechanism of NT V, we detected the replication capacity, cell tropism and virulence of NTV in cells from different generas and tissues. We measured expression of protein A17/A27 in NTV-infected HeLa cells by Western Blotting. We measured the transcription level of late protein A17/A27 by real-time polymerase chain reaction. NTV was capable to replicate and spread effectively in BHK-21 and CEF cells, while the replication and spread of NTV in HeLa, 2 BS, Hep-2 and143 TK-was blocked; in NTV infected HeLa cells, expression of late protein A17 and A27 was inhibited,whereas the transcription level was equal to that of vaccinia virus(Tiantan strain). The replication and spread capacity of NTV, a highly attenuated vaccinia virus( VV) strain, was limited in several cell lines. Blockade of expression of NTV late protein A17 and A27 did not occur at the transcription stage, and we hypothesize that it occurred at the translation stage.
As gene expression vectors, attenuated strain of vaccinia virus was widely used in prevention and treatment of various diseases. Non-replicating Tiantan vaccinia virus(NTV) was a highly attenuated vaccinia virus strain while its cytobiological characteristics and replication-defective mechanism remained to be explored.To explore the cytobiological characteristics and replication-defective mechanism of NT V, we detected the replication capacity, cell tropism and virulence of NTV in cells from different generas and tissues. We measured expression of protein A17/A27 in NTV-infected HeLa cells by Western Blotting. We measured the transcription level of late protein A17/A27 by real-time polymerase chain reaction. NTV was capable to replicate and spread effectively in BHK-21 and CEF cells, while the replication and spread of NTV in HeLa, 2 BS, Hep-2 and143 TK-was blocked; in NTV infected HeLa cells, expression of late protein A17 and A27 was inhibited,whereas the transcription level was equal to that of vaccinia virus(Tiantan strain). The replication and spread capacity of NTV, a highly attenuated vaccinia virus( VV) strain, was limited in several cell lines. Blockade of expression of NTV late protein A17 and A27 did not occur at the transcription stage, and we hypothesize that it occurred at the translation stage.
引文
[1]阮力,朱既明,娄元梅,陆柔剑.复制缺陷型天坛株痘苗病毒[P]中国专利:CN1831122, 2008-06-11.
[2] M. Carmen Sancho, Sibylle Schleich, Gareth Griffiths,Jacomine Krijnse-Locker. The block in assembly of modified vaccinia virus Ankara in HeLa cells reveals new insights into vaccinia virus morphogenesis[J]. J Virol,2002, 76(16):8318.
[3] Carroll M W, Moss B. Host range and cytopathogenicity of the highly attenuated MV A strain of vaccinia virus:propagation and generation of recombinant viruses in a nonhuman mammalian cell line[J]. Virology, 1997, 238(2):198-211.
[4] James Tartaglia,Marion E Perkus,Jill Taylor, Elizabeth K Norton, Jean Christophe Audonnet, William I Cox, Stephen W Davis, Johanna Van Der Hoeven, Bernard Meignier, Michel Riviere, Bernard Languet, Enzo Paolettl. NYVAC:a highly attenuated strain of vaccinia virus[J]. Virology, 1992, 188(1):217-232.
[5] Smith G L, Vanderplasschen A, Law M. The formation and function of extracellular enveloped vaccinia virus[J].J Gen Virol,2002, 83(Pt 12):2915-2931.
[6] Jose'Luis Na'jera,Carmen Elena Go'mez,Elena Domingo-Gil, Mari'a Magdalena Gherardi, Mariano Esteban. Cellular and biochemical differences between two attenuated poxvirus vaccine candidates(MVA and NYVAC)and role of the C7L gene[J]. J Virol, 2006, 80(12):6033-6047.
[7]黄盼盼,赵莉,任皎,赵颖,阮力,谭文杰,田厚文.复制缺陷型痘苗病毒天坛株复制缺陷机制初步探究[J].中华实验和临床病毒学杂志,2018, 32(2):119-123.
[8] Zhang Y F, Moss B. Vaccinia virus morphogenesis is interrupted when expression of the gene encoding an 11-kilodalton phosphorylated protein is prevented by the Escherichia coli lac repressor[J]. J Virol, 1991, 65(11):6101-6110.
[9] Wickramasekera N T, Traktman P. Structure/Function analysis of the vaccinia virus F18 phosphoprotein, an abundant core component required for virion maturation and infectivity[J]. J Virol, 2010, 84(13):6846-6860.
[10] Rodriguez D, Esteban M., Rodriguez JR. Vaccinia virus A17L gene product is essential for an early step in virion morphogenesis[J]. J Virol,1995,69(8):4640-4648.
[11]管茜茜,赵莉,任皎,黄盼盼,王慧娟,陈迎珠,朱娜,谭文杰,阮力,田厚文.猴痘病毒特异性单克隆抗体制备及鉴定[J].中华实验和临床病毒学杂志,2017, 31(2):153-156.
[12]庞聪,田厚文.非复制型痘苗病毒的生物学性状研究进展[J].生物技术通讯,2014, 25(3):415-420.
[13] Bethany Unger, Jason Mercer, Kathleen A. Boyle, Pau-la Traktman. Biogenesis of the vaccinia virus membrane:genetic and ultrastructural analysis of the contributions of the A14 and A17 proteins[J]. J Virol,2013,87(2):1083.
[14] Jia L, Grant M F. SAMD9 is an innate antiviral host factor with stress response properties that can be antagonized by poxviruses[J]. J Virol, 2015, 89(3):1925-1931.
[15] Gilad Sivan, Shira G Glushakow-Smith, George C Katsafanas, Jeffrey L Americo, Bernard Moss. Human hostrange restriction of the vaccinia virus C7/K1 double deletion mutant is mediated by an atypical mode of translation inhibition[J]. J Virol, 2018, 92(23):1-18.
[2] M. Carmen Sancho, Sibylle Schleich, Gareth Griffiths,Jacomine Krijnse-Locker. The block in assembly of modified vaccinia virus Ankara in HeLa cells reveals new insights into vaccinia virus morphogenesis[J]. J Virol,2002, 76(16):8318.
[3] Carroll M W, Moss B. Host range and cytopathogenicity of the highly attenuated MV A strain of vaccinia virus:propagation and generation of recombinant viruses in a nonhuman mammalian cell line[J]. Virology, 1997, 238(2):198-211.
[4] James Tartaglia,Marion E Perkus,Jill Taylor, Elizabeth K Norton, Jean Christophe Audonnet, William I Cox, Stephen W Davis, Johanna Van Der Hoeven, Bernard Meignier, Michel Riviere, Bernard Languet, Enzo Paolettl. NYVAC:a highly attenuated strain of vaccinia virus[J]. Virology, 1992, 188(1):217-232.
[5] Smith G L, Vanderplasschen A, Law M. The formation and function of extracellular enveloped vaccinia virus[J].J Gen Virol,2002, 83(Pt 12):2915-2931.
[6] Jose'Luis Na'jera,Carmen Elena Go'mez,Elena Domingo-Gil, Mari'a Magdalena Gherardi, Mariano Esteban. Cellular and biochemical differences between two attenuated poxvirus vaccine candidates(MVA and NYVAC)and role of the C7L gene[J]. J Virol, 2006, 80(12):6033-6047.
[7]黄盼盼,赵莉,任皎,赵颖,阮力,谭文杰,田厚文.复制缺陷型痘苗病毒天坛株复制缺陷机制初步探究[J].中华实验和临床病毒学杂志,2018, 32(2):119-123.
[8] Zhang Y F, Moss B. Vaccinia virus morphogenesis is interrupted when expression of the gene encoding an 11-kilodalton phosphorylated protein is prevented by the Escherichia coli lac repressor[J]. J Virol, 1991, 65(11):6101-6110.
[9] Wickramasekera N T, Traktman P. Structure/Function analysis of the vaccinia virus F18 phosphoprotein, an abundant core component required for virion maturation and infectivity[J]. J Virol, 2010, 84(13):6846-6860.
[10] Rodriguez D, Esteban M., Rodriguez JR. Vaccinia virus A17L gene product is essential for an early step in virion morphogenesis[J]. J Virol,1995,69(8):4640-4648.
[11]管茜茜,赵莉,任皎,黄盼盼,王慧娟,陈迎珠,朱娜,谭文杰,阮力,田厚文.猴痘病毒特异性单克隆抗体制备及鉴定[J].中华实验和临床病毒学杂志,2017, 31(2):153-156.
[12]庞聪,田厚文.非复制型痘苗病毒的生物学性状研究进展[J].生物技术通讯,2014, 25(3):415-420.
[13] Bethany Unger, Jason Mercer, Kathleen A. Boyle, Pau-la Traktman. Biogenesis of the vaccinia virus membrane:genetic and ultrastructural analysis of the contributions of the A14 and A17 proteins[J]. J Virol,2013,87(2):1083.
[14] Jia L, Grant M F. SAMD9 is an innate antiviral host factor with stress response properties that can be antagonized by poxviruses[J]. J Virol, 2015, 89(3):1925-1931.
[15] Gilad Sivan, Shira G Glushakow-Smith, George C Katsafanas, Jeffrey L Americo, Bernard Moss. Human hostrange restriction of the vaccinia virus C7/K1 double deletion mutant is mediated by an atypical mode of translation inhibition[J]. J Virol, 2018, 92(23):1-18.