PRRSV内切核糖核酸酶nsp11结构和功能研究
发布时间：2018-08-29 08:20
【摘要】：猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)属于尼多病毒目动脉炎病毒科成员。PRRSV是引起猪繁殖与呼吸综合征(Porcine reproductive and respiratory syndrome,PRRS)的致病因子,PRRS已经成为威胁世界养猪业重要的感染性疾病。自从1995年,我国分离到第一株PRRSV毒株以来,特别是在2006年爆发(2009-2010年间重新爆发)的高致病性PRRSV(HP-PRRSV)已经给我国养猪业造成重大经济损失。由于PRRSV基因组的不断变异,目前尚缺乏有效的疫苗和药物用于PRRSV的防控。Nsp11作为PRRSV自身编码的内切核糖核酸酶,它具有尼多病毒目病毒特有的能够特异性切割尿嘧啶的内切核糖核酸酶(NendoU)活性,这一功能对动脉炎病毒的复制非常重要。本研究中,我们成功解析了动脉炎病毒科的第一个内切核糖核酸酶nsp11的结构并阐明了它以二聚体方式行使NemdoU功能的作用机制,为抗病毒药物的研发提供新思路。具体的研究内容如下:1.湖北省种猪场PRRSV流行病学调查及分子变异分析我们对湖北地区的14个规模化种猪场PRRSV流行情况分析表明:在检测的668份样品中,PRRSV阳性率为5.24%(35/668),C-PRRSV和HP-PRRSV的阳性率分别为为1.95%(13/668)、3.59%(24/668)。HP-PRRSV已经成为该地区主要流行的PRRSV毒株。同时,我们通过对PRRSV的nsp2、orf5和orf7的全长基因进行测序,并分析了该地区PRRSV毒株基因变异情况。氨基酸序列分析表明:该地区多数HP-PRRSV毒株和JXA1株都在Nsp2编码区域的480位和532-560位不连续缺失30氨基酸表位,该结果证实了本试验调查的7株PRRSV毒株属于HP-PRRSV毒株。同时,Nsp2编码区域缺失59或68个氨基酸表位的毒株也被发现,表明其他类型的PRRSV缺失毒株也流行于该地区。基于orf5基因的进化树分析表明:2009-2010年间的湖北分离株形成一个独立的分支。它们与JXA1株相比,在凋亡表位存在突变(Val29→Ala29)。再者,GP5蛋白中潜在糖基化位点的数目呈增加趋势。同时,我们发现GP5蛋白中也存在大量的变异,发生在初级中和表位的突变(Phe39/Leu39→IIe39、Leu41→Ser41)和增加的糖基化位点数目可能是导致该地区PRRSV野毒株能够逃避疫苗中和作用的原因之一。最后,我们也发现在N蛋白中多数的突变发生在氨基酸表位:arg11→lys11、asp15→asn15、lys46→arg46、thr91→ala91、his109→gln109和val117→ala117。综上所述,我们调查了prrsv在湖北地区种猪场的流行情况,并分析了2006-2012年间prrsv流行毒株的分子变异情况,为有效控制湖北地区prrsv的流行提供理论基础。2.prrsv内切核糖核酸酶nsp11结构和功能研究成功解析了动脉炎病毒科第一个内切核糖核酸酶nsp11的蛋白质晶体结构。nsp11的晶体结构为全新的结构,它存在两个不对称的nsp11单体分子,这与冠状病毒nsp15的六聚体晶体结构完全不同。但是,prrsvnsp11和冠状病毒nsp15结构比较中,羧基端“catalyticdomain”较为保守,特别是与内切核糖核酸酶功能相关的两个loop结构:“activesiteloop”(his129-his144)和“supportingloop”(val162-thr179)。再者,结构同源性分析都证实了nsp11的关键酶活位点his129、his144、lys173、thr177、asp180、asp204和tyr219与冠状病毒nsp15具有较高的保守性。这表明,动脉炎病毒属和冠状病毒属的nendou可能具有相似的rna底物裂解机制。prrsvnsp11以二聚体形式行使nendou功能,这与冠状病毒nsp15以六聚体发挥功能的方式完全不同。首先,我们通过生化试验证实了纯化后的nsp11蛋白主要以二聚体形式存在于溶液中。其次,我们通过pdbepisa在线软件分析了nsp11二聚体界面相互作用氨基酸表位,并通过生化试验证明了ser74和phe76突变为ala后,破坏了nsp11稳定的二聚体结构并导致nendou活性显著降低,这表明nsp11可能以二聚体形式发挥功能。在nsp11的晶体结构中,我们发现“activesiteloop”和“supportingloop”的稳定是nsp11发挥功能的结构基础,而它们结构的稳定需要相邻的nsp11单体通过氢键和疏水作用力提供支撑作用。通过生化试验分析了prrsvnsp11的nendou活性的作用机制。首先,我们通过fret试验分析了nsp11野生型和突变体(h129a、k173a、t177a和y219a)的nendou活性。与野生型蛋白相比,突变体蛋白的nendou活性显著降低。我们的实验结果表明这些氨基酸表位是重要的nendou催化位点。其次,结构分析表明三个潜在的催化氨基酸表位(his129、his144和lys173)位于catalyticdomain并包围一个带正电荷的沟槽,其中thr177位于沟槽的中间。我们发现h129a突变体蛋白的NendoU活性较低,这可能与His129在裂解RNA底物过程中具有接收质子的功能有关。另外,Thr177和Tyr219可能对于RNA底物的识别和绑定起着重要的作用。与野生型蛋白相比,突变体蛋白(T177A和Y219A)的催化活性显著降低。发现PRRSV nsp11潜在的细胞毒性可能抑制了IFN-β启动子的激活。在HEK293T细胞中过表达野生型nsp11显著抑制SEV对IFN-β和IRF3启动子的激活,但突变体(S74A、F76A、H129A、K173A、T177A和Y219A)部分失去了抑制作用。然而,过表达野生型nsp11导致报告基因的内参值(pRL-TK)的显著降低,这表明野生型nsp11抑制了宿主基因表达。巧合的是,突变体基因过表达后没有明显降低pRL-TK值,也没有显著抑制IFN-β启动子的激活。因此,在体外实验中,我们不能排除野生型nsp11潜在的细胞毒性抑制了IFN-β启动子的激活。在PRRSV感染过程中,nsp11的NendoU活性能否抑制宿主的先天性免疫功能需要进一步的研究。综上所述,这些发现可能有助于阐明nsp11在动脉炎病毒科病毒复制过程中的作用机制,并为抗病毒药物的研发提供潜在的药物靶点。
[Abstract]:* Porcine reproductive and respiratory syndrome virus (PRRSV) is a member of NNV arteriitis virus *.PRRSV, which is the cause of porcine reproductive and respiratory syndrome (Porcine reproductive and), and has become an important infection threatening the pig industry worldwide. * Since the first PRRSV strain was isolated in 1995 in China, the outbreak of highly pathogenic PRRSV (HP-PRRSV) in 2006 (re-outbreak between 2009 and 2010) has caused great economic losses to the pig industry in China. As the endonuclease encoded by PRRSV itself, PRRSV-1 has the specific activity of endonuclease (NendoU) that can cleave uracil. This function is very important for the replication of arteritis virus. In this study, we successfully resolved the first endonuclease of the family Arteritis Viridae, nsp11. The structure of PRRSV and the mechanism of its dimer-based function of NemdoU were elucidated to provide new ideas for the development of antiviral drugs. Specific research contents are as follows: 1. Epidemiological investigation and molecular variation analysis of PRRSV in Hubei pig breeding farms. The epidemiological analysis of PRRSV in 14 large-scale pig breeding farms in Hubei showed that PRRSV was detected. The positive rate of PRRSV was 5.24% (35/668), C-PRRSV and HP-PRRSV were 1.95% (13/668) and 3.59% (24/668), respectively. HP-PRRSV has become the main prevalent PRRSV strain in this region. At the same time, we sequenced the full-length genes of nsp2, ORF5 and ORF7 of PRRSV and analyzed the genetic variation of PRRSV strains in this region. Amino acid sequence analysis showed that most HP-PRRSV strains and JXA1 strains in the region were found to have discontinuous deletion of 30 amino acid epitopes at 480 and 532-560 sites in the Nsp2 coding region. The results confirmed that the 7 PRRSV strains investigated in this study belonged to HP-PRRSV strains. Evolutionary tree analysis based on ORF5 gene showed that Hubei isolates formed an independent branch from 2009 to 2010. Compared with JXA1 strain, there were mutations in the apoptotic epitope (Val29-Ala29). Furthermore, the number of potential glycosylation sites in GP5 protein increased. Mutations in the primary neutral epitope (Phe39/Leu39_IIe39, Leu41_Ser41) and the increased number of glycosylation sites may be one of the reasons why PRRSV wild strains in this region can escape vaccine neutralization. Finally, we found that most of the mutations in the N protein occurred in ammonia. In summary, we investigated the epidemic situation of PRRSV in pig farms in Hubei province, and analyzed the molecular variation of PRRSV epidemic strains from 2006 to 2012. Basic. 2. The structure and function of PRRSV endonuclease nsp11 have been successfully elucidated. The crystal structure of nsp11, the first endonuclease in the family Arteritis Viridae, is completely new. It has two asymmetric nsp11 monomer molecules, which are completely different from the hexamer structure of the coronavirus nsp15. Yes, the carboxyl-terminal "catalytic domain" of prrsvnsp11 is more conservative than that of the coronavirus nsp15, especially the two loop structures related to the function of endonuclease: active site loop (his 129-his 144) and supporting loop (val162-thr179). Moreover, structural homology analysis has confirmed the key enzyme activity site h of nsp11. Is129, his144, lys173, thr177, asp180, asp204 and tyr219 are highly conserved with coronavirus nsp15. This suggests that nendou of arteritis virus and coronavirus may have similar RNA substrate cleavage mechanism. prrsvnsp11 performs nendou function in the form of dimer, which is accomplished in a hexamer manner with coronavirus nsp15. Firstly, we confirmed that the purified nsp11 protein existed mainly as a dimer in the solution by biochemical experiments. Secondly, we analyzed the amino acid epitopes of the interface interaction between the two molecules by pdbepisa software. Biochemical experiments showed that ser74 and phe76 mutated into ala and destroyed the stable dimer of nsp11. In the crystal structure of NSP 11, we found that the stability of "active iteloop" and "supporting loop" is the structural basis for the functionality of NSP 11, and the stability of their structures requires the adjacent NSP 11 monomers to act through hydrogen bonding and hydrophobic interaction. The mechanism of the nendou activity of prrsvnsp11 was analyzed by biochemical experiments. Firstly, we analyzed the nendou activity of wild-type and mutant nsp11 (h129a, k173a, t177a and y219a) by FRET test. Compared with wild-type proteins, the nendou activity of the mutant protein was significantly decreased. Secondly, structural analysis showed that three potential amino acid epitopes (his 129, his 144 and Lys 173) were located in the catalytic domain and surrounded a positively charged groove, in which thr 177 was located in the middle of the groove. In addition, Thr177 and Tyr219 may play an important role in the recognition and binding of RNA substrates. Compared with wild-type proteins, the catalytic activity of mutant proteins (T177A and Y219A) is significantly reduced. It is found that the potential cytotoxicity of PRRSV nsp11 may inhibit the IFN-beta promoter. Activation. Overexpression of wild-type nsp11 in HEK293T cells significantly inhibited SEV activation of IFN-beta and IRF3 promoters, but the mutants (S74A, F76A, H129A, K173A, T177A and Y219A) partially lost their inhibitory effect. However, overexpression of wild-type nsp11 resulted in a significant decrease in the internal parameter (pRL-TK) of the reporter gene, suggesting that wild-type nsp11 inhibited host activation. Coincidentally, the mutant gene overexpression did not significantly reduce the pRL-TK value, nor significantly inhibit the activation of IFN-beta promoter. Therefore, in vitro experiments, we can not rule out the potential cytotoxicity of wild-type nsp11 inhibits the activation of IFN-beta promoter. In conclusion, these findings may help to elucidate the mechanism of nsp11 in the replication of viral arteritis viruses and provide potential drug targets for the development of antiviral drugs.
【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S852.65
本文编号：2210724
[Abstract]:* Porcine reproductive and respiratory syndrome virus (PRRSV) is a member of NNV arteriitis virus *.PRRSV, which is the cause of porcine reproductive and respiratory syndrome (Porcine reproductive and), and has become an important infection threatening the pig industry worldwide. * Since the first PRRSV strain was isolated in 1995 in China, the outbreak of highly pathogenic PRRSV (HP-PRRSV) in 2006 (re-outbreak between 2009 and 2010) has caused great economic losses to the pig industry in China. As the endonuclease encoded by PRRSV itself, PRRSV-1 has the specific activity of endonuclease (NendoU) that can cleave uracil. This function is very important for the replication of arteritis virus. In this study, we successfully resolved the first endonuclease of the family Arteritis Viridae, nsp11. The structure of PRRSV and the mechanism of its dimer-based function of NemdoU were elucidated to provide new ideas for the development of antiviral drugs. Specific research contents are as follows: 1. Epidemiological investigation and molecular variation analysis of PRRSV in Hubei pig breeding farms. The epidemiological analysis of PRRSV in 14 large-scale pig breeding farms in Hubei showed that PRRSV was detected. The positive rate of PRRSV was 5.24% (35/668), C-PRRSV and HP-PRRSV were 1.95% (13/668) and 3.59% (24/668), respectively. HP-PRRSV has become the main prevalent PRRSV strain in this region. At the same time, we sequenced the full-length genes of nsp2, ORF5 and ORF7 of PRRSV and analyzed the genetic variation of PRRSV strains in this region. Amino acid sequence analysis showed that most HP-PRRSV strains and JXA1 strains in the region were found to have discontinuous deletion of 30 amino acid epitopes at 480 and 532-560 sites in the Nsp2 coding region. The results confirmed that the 7 PRRSV strains investigated in this study belonged to HP-PRRSV strains. Evolutionary tree analysis based on ORF5 gene showed that Hubei isolates formed an independent branch from 2009 to 2010. Compared with JXA1 strain, there were mutations in the apoptotic epitope (Val29-Ala29). Furthermore, the number of potential glycosylation sites in GP5 protein increased. Mutations in the primary neutral epitope (Phe39/Leu39_IIe39, Leu41_Ser41) and the increased number of glycosylation sites may be one of the reasons why PRRSV wild strains in this region can escape vaccine neutralization. Finally, we found that most of the mutations in the N protein occurred in ammonia. In summary, we investigated the epidemic situation of PRRSV in pig farms in Hubei province, and analyzed the molecular variation of PRRSV epidemic strains from 2006 to 2012. Basic. 2. The structure and function of PRRSV endonuclease nsp11 have been successfully elucidated. The crystal structure of nsp11, the first endonuclease in the family Arteritis Viridae, is completely new. It has two asymmetric nsp11 monomer molecules, which are completely different from the hexamer structure of the coronavirus nsp15. Yes, the carboxyl-terminal "catalytic domain" of prrsvnsp11 is more conservative than that of the coronavirus nsp15, especially the two loop structures related to the function of endonuclease: active site loop (his 129-his 144) and supporting loop (val162-thr179). Moreover, structural homology analysis has confirmed the key enzyme activity site h of nsp11. Is129, his144, lys173, thr177, asp180, asp204 and tyr219 are highly conserved with coronavirus nsp15. This suggests that nendou of arteritis virus and coronavirus may have similar RNA substrate cleavage mechanism. prrsvnsp11 performs nendou function in the form of dimer, which is accomplished in a hexamer manner with coronavirus nsp15. Firstly, we confirmed that the purified nsp11 protein existed mainly as a dimer in the solution by biochemical experiments. Secondly, we analyzed the amino acid epitopes of the interface interaction between the two molecules by pdbepisa software. Biochemical experiments showed that ser74 and phe76 mutated into ala and destroyed the stable dimer of nsp11. In the crystal structure of NSP 11, we found that the stability of "active iteloop" and "supporting loop" is the structural basis for the functionality of NSP 11, and the stability of their structures requires the adjacent NSP 11 monomers to act through hydrogen bonding and hydrophobic interaction. The mechanism of the nendou activity of prrsvnsp11 was analyzed by biochemical experiments. Firstly, we analyzed the nendou activity of wild-type and mutant nsp11 (h129a, k173a, t177a and y219a) by FRET test. Compared with wild-type proteins, the nendou activity of the mutant protein was significantly decreased. Secondly, structural analysis showed that three potential amino acid epitopes (his 129, his 144 and Lys 173) were located in the catalytic domain and surrounded a positively charged groove, in which thr 177 was located in the middle of the groove. In addition, Thr177 and Tyr219 may play an important role in the recognition and binding of RNA substrates. Compared with wild-type proteins, the catalytic activity of mutant proteins (T177A and Y219A) is significantly reduced. It is found that the potential cytotoxicity of PRRSV nsp11 may inhibit the IFN-beta promoter. Activation. Overexpression of wild-type nsp11 in HEK293T cells significantly inhibited SEV activation of IFN-beta and IRF3 promoters, but the mutants (S74A, F76A, H129A, K173A, T177A and Y219A) partially lost their inhibitory effect. However, overexpression of wild-type nsp11 resulted in a significant decrease in the internal parameter (pRL-TK) of the reporter gene, suggesting that wild-type nsp11 inhibited host activation. Coincidentally, the mutant gene overexpression did not significantly reduce the pRL-TK value, nor significantly inhibit the activation of IFN-beta promoter. Therefore, in vitro experiments, we can not rule out the potential cytotoxicity of wild-type nsp11 inhibits the activation of IFN-beta promoter. In conclusion, these findings may help to elucidate the mechanism of nsp11 in the replication of viral arteritis viruses and provide potential drug targets for the development of antiviral drugs.
【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S852.65
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1 史月军;PRRSV内切核糖核酸酶nsp11结构和功能研究[D];华中农业大学;2016年
，本文编号：2210724
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