PRRSV內(nèi)切核糖核酸酶nsp11結構和功能研究
發(fā)布時間：2018-08-29 08:20
【摘要】：豬繁殖與呼吸綜合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)屬于尼多病毒目動脈炎病毒科成員。PRRSV是引起豬繁殖與呼吸綜合征(Porcine reproductive and respiratory syndrome,PRRS)的致病因子,PRRS已經(jīng)成為威脅世界養(yǎng)豬業(yè)重要的感染性疾病。自從1995年,我國分離到第一株PRRSV毒株以來,特別是在2006年爆發(fā)(2009-2010年間重新爆發(fā))的高致病性PRRSV(HP-PRRSV)已經(jīng)給我國養(yǎng)豬業(yè)造成重大經(jīng)濟損失。由于PRRSV基因組的不斷變異,目前尚缺乏有效的疫苗和藥物用于PRRSV的防控。Nsp11作為PRRSV自身編碼的內(nèi)切核糖核酸酶,它具有尼多病毒目病毒特有的能夠特異性切割尿嘧啶的內(nèi)切核糖核酸酶(NendoU)活性,這一功能對動脈炎病毒的復制非常重要。本研究中,我們成功解析了動脈炎病毒科的第一個內(nèi)切核糖核酸酶nsp11的結構并闡明了它以二聚體方式行使NemdoU功能的作用機制,為抗病毒藥物的研發(fā)提供新思路。具體的研究內(nèi)容如下:1.湖北省種豬場PRRSV流行病學調(diào)查及分子變異分析我們對湖北地區(qū)的14個規(guī)�；N豬場PRRSV流行情況分析表明:在檢測的668份樣品中,PRRSV陽性率為5.24%(35/668),C-PRRSV和HP-PRRSV的陽性率分別為為1.95%(13/668)、3.59%(24/668)。HP-PRRSV已經(jīng)成為該地區(qū)主要流行的PRRSV毒株。同時,我們通過對PRRSV的nsp2、orf5和orf7的全長基因進行測序,并分析了該地區(qū)PRRSV毒株基因變異情況。氨基酸序列分析表明:該地區(qū)多數(shù)HP-PRRSV毒株和JXA1株都在Nsp2編碼區(qū)域的480位和532-560位不連續(xù)缺失30氨基酸表位,該結果證實了本試驗調(diào)查的7株PRRSV毒株屬于HP-PRRSV毒株。同時,Nsp2編碼區(qū)域缺失59或68個氨基酸表位的毒株也被發(fā)現(xiàn),表明其他類型的PRRSV缺失毒株也流行于該地區(qū)�；趏rf5基因的進化樹分析表明:2009-2010年間的湖北分離株形成一個獨立的分支。它們與JXA1株相比,在凋亡表位存在突變(Val29→Ala29)。再者,GP5蛋白中潛在糖基化位點的數(shù)目呈增加趨勢。同時,我們發(fā)現(xiàn)GP5蛋白中也存在大量的變異,發(fā)生在初級中和表位的突變(Phe39/Leu39→IIe39、Leu41→Ser41)和增加的糖基化位點數(shù)目可能是導致該地區(qū)PRRSV野毒株能夠逃避疫苗中和作用的原因之一。最后,我們也發(fā)現(xiàn)在N蛋白中多數(shù)的突變發(fā)生在氨基酸表位:arg11→lys11、asp15→asn15、lys46→arg46、thr91→ala91、his109→gln109和val117→ala117。綜上所述,我們調(diào)查了prrsv在湖北地區(qū)種豬場的流行情況,并分析了2006-2012年間prrsv流行毒株的分子變異情況,為有效控制湖北地區(qū)prrsv的流行提供理論基礎。2.prrsv內(nèi)切核糖核酸酶nsp11結構和功能研究成功解析了動脈炎病毒科第一個內(nèi)切核糖核酸酶nsp11的蛋白質(zhì)晶體結構。nsp11的晶體結構為全新的結構,它存在兩個不對稱的nsp11單體分子,這與冠狀病毒nsp15的六聚體晶體結構完全不同。但是,prrsvnsp11和冠狀病毒nsp15結構比較中,羧基端“catalyticdomain”較為保守,特別是與內(nèi)切核糖核酸酶功能相關的兩個loop結構:“activesiteloop”(his129-his144)和“supportingloop”(val162-thr179)。再者,結構同源性分析都證實了nsp11的關鍵酶活位點his129、his144、lys173、thr177、asp180、asp204和tyr219與冠狀病毒nsp15具有較高的保守性。這表明,動脈炎病毒屬和冠狀病毒屬的nendou可能具有相似的rna底物裂解機制。prrsvnsp11以二聚體形式行使nendou功能,這與冠狀病毒nsp15以六聚體發(fā)揮功能的方式完全不同。首先,我們通過生化試驗證實了純化后的nsp11蛋白主要以二聚體形式存在于溶液中。其次,我們通過pdbepisa在線軟件分析了nsp11二聚體界面相互作用氨基酸表位,并通過生化試驗證明了ser74和phe76突變?yōu)閍la后,破壞了nsp11穩(wěn)定的二聚體結構并導致nendou活性顯著降低,這表明nsp11可能以二聚體形式發(fā)揮功能。在nsp11的晶體結構中,我們發(fā)現(xiàn)“activesiteloop”和“supportingloop”的穩(wěn)定是nsp11發(fā)揮功能的結構基礎,而它們結構的穩(wěn)定需要相鄰的nsp11單體通過氫鍵和疏水作用力提供支撐作用。通過生化試驗分析了prrsvnsp11的nendou活性的作用機制。首先,我們通過fret試驗分析了nsp11野生型和突變體(h129a、k173a、t177a和y219a)的nendou活性。與野生型蛋白相比,突變體蛋白的nendou活性顯著降低。我們的實驗結果表明這些氨基酸表位是重要的nendou催化位點。其次,結構分析表明三個潛在的催化氨基酸表位(his129、his144和lys173)位于catalyticdomain并包圍一個帶正電荷的溝槽,其中thr177位于溝槽的中間。我們發(fā)現(xiàn)h129a突變體蛋白的NendoU活性較低,這可能與His129在裂解RNA底物過程中具有接收質(zhì)子的功能有關。另外,Thr177和Tyr219可能對于RNA底物的識別和綁定起著重要的作用。與野生型蛋白相比,突變體蛋白(T177A和Y219A)的催化活性顯著降低。發(fā)現(xiàn)PRRSV nsp11潛在的細胞毒性可能抑制了IFN-β啟動子的激活。在HEK293T細胞中過表達野生型nsp11顯著抑制SEV對IFN-β和IRF3啟動子的激活,但突變體(S74A、F76A、H129A、K173A、T177A和Y219A)部分失去了抑制作用。然而,過表達野生型nsp11導致報告基因的內(nèi)參值(pRL-TK)的顯著降低,這表明野生型nsp11抑制了宿主基因表達。巧合的是,突變體基因過表達后沒有明顯降低pRL-TK值,也沒有顯著抑制IFN-β啟動子的激活。因此,在體外實驗中,我們不能排除野生型nsp11潛在的細胞毒性抑制了IFN-β啟動子的激活。在PRRSV感染過程中,nsp11的NendoU活性能否抑制宿主的先天性免疫功能需要進一步的研究。綜上所述,這些發(fā)現(xiàn)可能有助于闡明nsp11在動脈炎病毒科病毒復制過程中的作用機制,并為抗病毒藥物的研發(fā)提供潛在的藥物靶點。
[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.
【學位授予單位】：華中農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S852.65
本文編號：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.
【學位授予單位】：華中農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S852.65
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1 史月軍;PRRSV內(nèi)切核糖核酸酶nsp11結構和功能研究[D];華中農(nóng)業(yè)大學;2016年
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