猪圆环病毒2型ORF5蛋白功能分析和ORF4蛋白拮抗细胞凋亡机制研究
发布时间：2018-07-20 18:20
【摘要】：猪圆环病毒2型(Porcine circovirus type 2,PCV2)是一种已知的能够感染哺乳类动物的最小病毒,是引起猪圆环病毒病(Porcine circovirus associated diseases,PCVAD)的病原体。PCV2是一种无囊膜病毒,属于圆环病毒科(Circoviridae)的圆环病毒属(Circovirus),其基因组是一种单链闭合环状的双义的不分节的DNA。该病毒曾经被预测含有11个开放阅读框,其中4个已经被鉴定能够编码该病毒的蛋白。它们分别是ORF1编码病毒的复制酶相关蛋白Rep和Rep',ORF2编码病毒的衣壳蛋白Cap,ORF3编码病毒的促凋亡蛋白ORF3蛋白以及ORF4编码病毒的抗凋亡蛋白ORF4蛋白(尽管该蛋白的抗凋亡机制尚不清楚)。序列比对分析发现Hamel等曾预测的ORF5基因(nt1016-1177)并不存在,这是因为该假定的蛋白在一些PCV2中国分离株中翻译至第7个氨基酸就提前终止了。然而,许多学者在对不同PCV2毒株序列比较时发现该病毒的基因组中有一段基因(nt553-732)高度保守,推测其很可能编码病毒的某个蛋白,甚至还各自为这个未知蛋白起了名字(如基因序列号O92288、Q77GS3、Q77S04等)。为了探究该蛋白(本文将其命名为ORF5蛋白)存在与否,其所编码产物的性质和功能,以及ORF4蛋白拮抗细胞凋亡的分子机制,开展了研究工作,获得了以下结果:(1)基因与蛋白水平验证PCV2 ORF5蛋白的存在。PCV2杨凌株感染猪肺泡巨噬细胞3D4/2(Porcine alveolar macrophages,PAMs)后,通过RT-PCR和Northern blot的检测发现,ORF5基因能够在PCV2复制过程中转录,其转录本约为180 bp,完全镶嵌在ORF1的内部且与ORF1转录方向一致。此外,借助制备的鼠抗ORF5蛋白多克隆抗体(包括利用GST-ORF5融合蛋白、化学合成法得到的ORF5抗原表位多肽以及真核表达质粒pcDNA-ORF5为抗原免疫Balb/c小鼠制备的多克隆抗体),可在ORF5基因转染的PAM中检测到Western blot信号,也可在PCV2感染的PAM中检测到IFA信号,说明PCV2感染过程中存在ORF5基因编码的病毒蛋白,即ORF5蛋白。尽管如此,ORF5蛋白在翻译水平的表达还需要进一步验证,毕竟Western blot未能检测到特异性ORF5蛋白条带。研究结果分别从基因与蛋白水平初步验证了PCV2 ORF5基因的转录和翻译,为后续该蛋白性质和功能的研究奠定了基础。(2)PCV2 ORF5蛋白功能的研究。为了分析PCV2 ORF5蛋白的功能,构建了缺失ORF5蛋白的PCV2感染性克隆(PCV2Δ),经过对其复制动力学分析发现ORF5蛋白并不是pcv2复制所必需的病毒蛋白。利用拯救出的orf5蛋白缺失体病毒pcv2Δ,重组型病毒rpcv2以及野生型病毒wpcv2分别感染pam后用实时荧光定量pcr方法(rt-qpcr)分析orf1、orf2、orf3、orf4和orf5mrna的表达曲线,结果发现orf5基因在病毒感染细胞后约48h表达量最高,而且起始密码子的突变并不影响orf5基因的转录,提示orf5基因转录的起始位点可能位于更上游的位置。此外,pcv2Δ感染后的orf1和orf2mrna表达量较wpcv2和rpcv2都有显著下降趋势,表明orf5蛋白的缺失可能会影响病毒orf1和orf2基因的转录进程。为了进一步分析orf5蛋白在pcv2感染过程中的作用,构建了orf5蛋白的真核表达载体pegfp-orf5,转染pam细胞后检测该蛋白对pam细胞生理功能的影响,结果发现gfp标记的orf5蛋白能够通过蛋白酶体途径被降解,能够抑制pam细胞的增殖,延长pam细胞周期的s期,定位在内质网并引起细胞内质网应激,激活nf-κb并上调其下游基因的表达。但是,由于gfp-orf5蛋白的功能可能并不能完全代表orf5蛋白自身的功能,因此有关orf5蛋白功能的描述还需要进一步的证实。(3)pcv2orf4和orf5蛋白胞内互作蛋白的筛选。为了筛选pcv2orf4和orf5这两个新型病毒蛋白在宿主细胞内的互作蛋白,构建了猪肺泡巨噬细胞的cdna文库,分别利用pcv2orf4和orf5蛋白为诱饵蛋白通过酵母双杂交的方法筛选出了与pcv2orf4蛋白互作的4个蛋白(fhc,snrpn,cox8a和laminc)和与pcv2orf5蛋白互作的5个蛋白(gpnmb,cyp1a1,ywhab,znf511和srsf3)。(4)pcv2orf4蛋白拮抗细胞凋亡机制的研究。pcv2orf4蛋白曾被推测为抗凋亡蛋白,但迄今为止关于该蛋白抗凋亡的具体机制尚不明确。为了澄清这一问题,选择上述酵母双杂交结果中的重链铁蛋白(fhc)为出发点,先通过gstpull-down和co-ip分别在细胞外和细胞内验证orf4蛋白与fhc能够相互结合,后又用激光共聚焦技术在细胞水平证明orf4蛋白与fhc蛋白在hek293t细胞中存在共定位现象,表明pcv2orf4蛋白与宿主fhc蛋白存在相互作用。考虑到fhc具有抗凋亡的作用且该作用具有剂量依赖性,首先用过表达(lv-fhc)和基因干扰(sh-fhc)的方法证实fhc参与了pcv2诱导的凋亡,然后用过表达(gfp-orf4)和基因缺失(感染性克隆pcv2Δ)的方法证实orf4蛋白虽然不能影响宿主细胞fhc基因的转录,但却能够通过某种方式的蛋白修饰降低细胞内具有生物活性的fhc蛋白的浓度,提示pcv2orf4蛋白很可能通过稳定宿主细胞内fhc蛋白水平的恒定而发挥抗凋亡的作用。综上所述,本研究初步证实了pcv2orf5蛋白是病毒复制的非必需蛋白,但是gfp标记的orf5蛋白却能够通过延长细胞周期的s期而抑制细胞的增殖,能够定位在内质网并且可诱导细胞内质网应激,能够激活nf-κb并上调其下游基因;orf5蛋白可与细胞内的gpnmb、cyp1a1、ywhab、znf511和srsf3互作。orf4蛋白能够与细胞内的fhc、snrpn、cox8a和laminc互作,其中orf4蛋白能够通过与宿主fhc互作而维持细胞内FHC蛋白水平的恒定,从而发挥拮抗细胞凋亡的作用,为病毒的早期复制提供帮助。
[Abstract]:The porcine circovirus type 2 (Porcine circovirus type 2, PCV2) is the smallest virus that is known to be able to infect mammals, and is the pathogen that causes the swine Circovirus Disease (Porcine circovirus associated diseases, PCVAD).PCV2 is a non capsula virus, belonging to the cycovirus family (Circoviridae) of the genus circovirus (Circovirus), its base. The DNA. virus has been predicted to contain 11 open reading frames, 4 of which have been identified as the proteins that can encode the virus. They are replicin related proteins Rep and Rep'of the ORF1 encoding virus, the capsid protein Cap of the ORF2 encoding virus, and the apoptosis promoting of the ORF3 encoded virus. Protein ORF3 protein and ORF4 encoding the anti apoptotic protein ORF4 protein of the virus (although the anti apoptotic mechanism of the protein is not clear). Sequence alignment analysis found that the ORF5 gene (nt1016-1177), which was predicted by Hamel, did not exist, because the postulate protein was translated to seventh amino acids in some PCV2 Chinese isolates and terminated in advance. However, many scholars have found that one gene (nt553-732) in the genome of the virus is highly conserved when comparing the sequence of different PCV2 strains. It is presumed that it is likely to encode a certain protein of the virus, or even the name of the unknown protein (such as the gene sequence number O92288, Q77GS3, Q77S04, etc.). The properties and functions of the encoded products, as well as the molecular mechanism of ORF4 protein antagonism to cell apoptosis, were named as ORF5 protein. The following results were obtained: (1) gene and protein level verified the presence of PCV2 ORF5 protein in.PCV2 Yangling strain of 3D4/2 (Porcine alveolar macrophages, PAMs) in porcine alveolar macrophages. After the detection of RT-PCR and Northern blot, it was found that the ORF5 gene could be transcribed during the PCV2 replication process, and its transcript was about 180 BP, completely inlaid in the interior of ORF1 and in the same direction as ORF1. In addition, the mouse anti ORF5 protein polyclonal antibody (including the ORF5 antigen table by the use of GST-ORF5 fusion protein and chemical synthesis) The polyclonal and eukaryotic expression plasmid pcDNA-ORF5 is the polyclonal antibody prepared by the antigen immunized Balb/c mice. The Western blot signal can be detected in the PAM transfected ORF5 gene, and the IFA signal can be detected in the PAM infected by PCV2, indicating that the ORF5 gene coding virus protein, that is, the ORF5 protein, is found in the PCV2 infection. The expression of white at the level of translation needs further verification. After all, Western blot failed to detect the specific ORF5 protein bands. The results of the study have preliminarily verified the transcription and translation of the PCV2 ORF5 gene from the gene and protein levels, and laid the foundation for the study of the properties and functions of the protein. (2) the study of the function of the PCV2 ORF5 protein. The function of the PCV2 ORF5 protein was analyzed, and the PCV2 infected clone (PCV2 delta) missing ORF5 protein was constructed. After the analysis of its replication kinetics, it was found that ORF5 protein was not the necessary viral protein for PCV2 replication. Using the saved ORF5 protein deletion somatic virus PCV2 Delta, the recombinant virus rpcv2 and the wild type virus wpcv2 infected PAM. The expression curves of ORF1, ORF2, ORF3, ORF4 and orf5mrna were analyzed by real time fluorescence quantitative PCR method (RT-qPCR). The results showed that the ORF5 gene expressed the highest 48h expression after the virus infected cells, and the mutation of the starting codon did not affect the transcription of the ORF5 gene, suggesting that the starting site of the ORF5 gene transcription may be located in a more upstream position. Besides, PCV. The expression of ORF1 and orf2mrna after 2 delta infection was significantly lower than that of wpcv2 and rpcv2, indicating that the deletion of ORF5 protein may affect the transcriptional process of the ORF1 and ORF2 genes. In order to further analyze the role of ORF5 protein in the PCV2 infection process, the eukaryotic expression carrier of the ORF5 protein was constructed, and then the PAM cells were detected. The effect of the protein on the physiological function of PAM cells shows that the GFP labeled ORF5 protein can be degraded through the proteasome pathway, which can inhibit the proliferation of PAM cells, prolong the S phase of the PAM cell cycle, locate the endoplasmic reticulum and induce the endoplasmic reticulum stress, activate the nf- kappa B and regulate the expression of the downstream genes. However, due to the gfp-orf5 protein. The function may not fully represent the function of ORF5 protein itself, so the description of the function of ORF5 protein needs further confirmation. (3) screening of intercellular proteins of pcv2orf4 and ORF5 proteins. In order to screen the interacting proteins of the two new viral proteins of pcv2orf4 and ORF5 in the host cells, the porcine alveolar macrophages were constructed. The cDNA library, using pcv2orf4 and ORF5 protein as bait protein, screened 4 proteins interacting with pcv2orf4 protein (FHC, SNRPN, cox8a and laminc) and the 5 proteins interacting with pcv2orf5 protein (gpnmb, CYP1A1, ywhab, laminc and pcv2orf5). (4) study on the mechanism of apoptosis in antagonistic cells. 4 protein has been presumed to be an anti apoptotic protein, but so far the specific mechanism about the anti apoptosis of the protein is not clear. In order to clarify this problem, we choose the heavy chain ferritin (FHC) in the results of the yeast two hybrid results as the starting point. First, gstpull-down and co-Ip are used to verify that the ORF4 protein can interact with FHC in the fine cell and in the cells respectively. In addition, the co localization of ORF4 protein and FHC protein in HEK293T cells was demonstrated by laser confocal technique at the cell level, indicating the interaction between the pcv2orf4 protein and the host FHC protein. Considering the anti apoptosis effect of FHC and the dose dependent effect, the first use of expression (lv-fhc) and gene interference (sh-fhc) The method confirmed that FHC was involved in PCV2 induced apoptosis, and then using the method of overexpression (gfp-orf4) and gene deletion (infectious clone PCV2 delta), it was proved that ORF4 protein could not affect the FHC gene transcription of the host cell, but it could reduce the concentration of the bioactive FHC protein in the cell by some kind of protein modification, suggesting pcv2orf4. Proteins are likely to play an anti apoptotic role by stabilizing the FHC protein level in the host cells. To sum up, this study has preliminarily confirmed that the pcv2orf5 protein is a non essential protein for viral replication, but the GFP labeled ORF5 protein can inhibit cell proliferation by prolonging the S phase of the cell cycle and can be located in the endoplasmic reticulum and can be located in the endoplasmic reticulum. And it can induce endoplasmic reticulum stress to activate nf- kappa B and up-regulate its downstream genes; ORF5 protein can interact with gpnmb, CYP1A1, ywhab, znf511 and srsf3 in cells to interact with FHC, SNRPN, cox8a, and srsf3 in cells. It can play a role in antagonizing cell apoptosis and provide help for early replication of the virus.
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S852.65
，
本文编号：2134359
[Abstract]:The porcine circovirus type 2 (Porcine circovirus type 2, PCV2) is the smallest virus that is known to be able to infect mammals, and is the pathogen that causes the swine Circovirus Disease (Porcine circovirus associated diseases, PCVAD).PCV2 is a non capsula virus, belonging to the cycovirus family (Circoviridae) of the genus circovirus (Circovirus), its base. The DNA. virus has been predicted to contain 11 open reading frames, 4 of which have been identified as the proteins that can encode the virus. They are replicin related proteins Rep and Rep'of the ORF1 encoding virus, the capsid protein Cap of the ORF2 encoding virus, and the apoptosis promoting of the ORF3 encoded virus. Protein ORF3 protein and ORF4 encoding the anti apoptotic protein ORF4 protein of the virus (although the anti apoptotic mechanism of the protein is not clear). Sequence alignment analysis found that the ORF5 gene (nt1016-1177), which was predicted by Hamel, did not exist, because the postulate protein was translated to seventh amino acids in some PCV2 Chinese isolates and terminated in advance. However, many scholars have found that one gene (nt553-732) in the genome of the virus is highly conserved when comparing the sequence of different PCV2 strains. It is presumed that it is likely to encode a certain protein of the virus, or even the name of the unknown protein (such as the gene sequence number O92288, Q77GS3, Q77S04, etc.). The properties and functions of the encoded products, as well as the molecular mechanism of ORF4 protein antagonism to cell apoptosis, were named as ORF5 protein. The following results were obtained: (1) gene and protein level verified the presence of PCV2 ORF5 protein in.PCV2 Yangling strain of 3D4/2 (Porcine alveolar macrophages, PAMs) in porcine alveolar macrophages. After the detection of RT-PCR and Northern blot, it was found that the ORF5 gene could be transcribed during the PCV2 replication process, and its transcript was about 180 BP, completely inlaid in the interior of ORF1 and in the same direction as ORF1. In addition, the mouse anti ORF5 protein polyclonal antibody (including the ORF5 antigen table by the use of GST-ORF5 fusion protein and chemical synthesis) The polyclonal and eukaryotic expression plasmid pcDNA-ORF5 is the polyclonal antibody prepared by the antigen immunized Balb/c mice. The Western blot signal can be detected in the PAM transfected ORF5 gene, and the IFA signal can be detected in the PAM infected by PCV2, indicating that the ORF5 gene coding virus protein, that is, the ORF5 protein, is found in the PCV2 infection. The expression of white at the level of translation needs further verification. After all, Western blot failed to detect the specific ORF5 protein bands. The results of the study have preliminarily verified the transcription and translation of the PCV2 ORF5 gene from the gene and protein levels, and laid the foundation for the study of the properties and functions of the protein. (2) the study of the function of the PCV2 ORF5 protein. The function of the PCV2 ORF5 protein was analyzed, and the PCV2 infected clone (PCV2 delta) missing ORF5 protein was constructed. After the analysis of its replication kinetics, it was found that ORF5 protein was not the necessary viral protein for PCV2 replication. Using the saved ORF5 protein deletion somatic virus PCV2 Delta, the recombinant virus rpcv2 and the wild type virus wpcv2 infected PAM. The expression curves of ORF1, ORF2, ORF3, ORF4 and orf5mrna were analyzed by real time fluorescence quantitative PCR method (RT-qPCR). The results showed that the ORF5 gene expressed the highest 48h expression after the virus infected cells, and the mutation of the starting codon did not affect the transcription of the ORF5 gene, suggesting that the starting site of the ORF5 gene transcription may be located in a more upstream position. Besides, PCV. The expression of ORF1 and orf2mrna after 2 delta infection was significantly lower than that of wpcv2 and rpcv2, indicating that the deletion of ORF5 protein may affect the transcriptional process of the ORF1 and ORF2 genes. In order to further analyze the role of ORF5 protein in the PCV2 infection process, the eukaryotic expression carrier of the ORF5 protein was constructed, and then the PAM cells were detected. The effect of the protein on the physiological function of PAM cells shows that the GFP labeled ORF5 protein can be degraded through the proteasome pathway, which can inhibit the proliferation of PAM cells, prolong the S phase of the PAM cell cycle, locate the endoplasmic reticulum and induce the endoplasmic reticulum stress, activate the nf- kappa B and regulate the expression of the downstream genes. However, due to the gfp-orf5 protein. The function may not fully represent the function of ORF5 protein itself, so the description of the function of ORF5 protein needs further confirmation. (3) screening of intercellular proteins of pcv2orf4 and ORF5 proteins. In order to screen the interacting proteins of the two new viral proteins of pcv2orf4 and ORF5 in the host cells, the porcine alveolar macrophages were constructed. The cDNA library, using pcv2orf4 and ORF5 protein as bait protein, screened 4 proteins interacting with pcv2orf4 protein (FHC, SNRPN, cox8a and laminc) and the 5 proteins interacting with pcv2orf5 protein (gpnmb, CYP1A1, ywhab, laminc and pcv2orf5). (4) study on the mechanism of apoptosis in antagonistic cells. 4 protein has been presumed to be an anti apoptotic protein, but so far the specific mechanism about the anti apoptosis of the protein is not clear. In order to clarify this problem, we choose the heavy chain ferritin (FHC) in the results of the yeast two hybrid results as the starting point. First, gstpull-down and co-Ip are used to verify that the ORF4 protein can interact with FHC in the fine cell and in the cells respectively. In addition, the co localization of ORF4 protein and FHC protein in HEK293T cells was demonstrated by laser confocal technique at the cell level, indicating the interaction between the pcv2orf4 protein and the host FHC protein. Considering the anti apoptosis effect of FHC and the dose dependent effect, the first use of expression (lv-fhc) and gene interference (sh-fhc) The method confirmed that FHC was involved in PCV2 induced apoptosis, and then using the method of overexpression (gfp-orf4) and gene deletion (infectious clone PCV2 delta), it was proved that ORF4 protein could not affect the FHC gene transcription of the host cell, but it could reduce the concentration of the bioactive FHC protein in the cell by some kind of protein modification, suggesting pcv2orf4. Proteins are likely to play an anti apoptotic role by stabilizing the FHC protein level in the host cells. To sum up, this study has preliminarily confirmed that the pcv2orf5 protein is a non essential protein for viral replication, but the GFP labeled ORF5 protein can inhibit cell proliferation by prolonging the S phase of the cell cycle and can be located in the endoplasmic reticulum and can be located in the endoplasmic reticulum. And it can induce endoplasmic reticulum stress to activate nf- kappa B and up-regulate its downstream genes; ORF5 protein can interact with gpnmb, CYP1A1, ywhab, znf511 and srsf3 in cells to interact with FHC, SNRPN, cox8a, and srsf3 in cells. It can play a role in antagonizing cell apoptosis and provide help for early replication of the virus.
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S852.65
，
本文编号：2134359
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