猪繁殖与呼吸综合征病毒核衣壳蛋白在病毒复制过程中的作用研究
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摘要
猪繁殖与呼吸综合征病毒(Porcine Reproductive and Respiratory Syndrome Virus, PRRSV)是有囊膜的单股正链RNA病毒,包括两个基因型:I型(欧洲型)和II型(北美型)。PRRSV包含至少9个开放阅读框(open reading frame,ORF),ORF1编码的非结构蛋白负责病毒基因组复制和mRNA合成的病毒复制转录复合体;ORF2-ORF7编码病毒的结构蛋白,其中,ORF7编码的核衣壳蛋白(Nucleocapsid Protein,N蛋白)是一个分子量约为15kD的碱性多功能蛋白。同源性较低的I型和II型PRRSV代表株的N蛋白长度分别为128和123个氨基酸,而且I型N蛋白长度呈现多态性。mRNA7起始密码子前包含一段长达319碱基的前导序列(其中含有两个AUG,但并不起始翻译),所以ORF7的翻译调控机制还不清楚。另外,N蛋白已被证明在病毒粒子的装配中起着重要作用,但是关于N蛋白能够发挥作用的最小功能域还是未知的。
在PRRSV感染性克隆平台的基础上,本研究第一部分是关于N蛋白翻译起始位点的研究,以将ORF6和ORF7之间的重复序列拉开并插入酶切位点的感染性克隆pORF673为模板,分别构建了3个ATG的系列突变体,通过病毒拯救表明N蛋白的翻译优先使用前导序列之后的第一个AUG;在迫使病毒使用导致ORF7移码的人工插入AUG时,PRRSV可以通过碱基插入矫正读码框,使得N蛋白的前11个氨基酸可以完全改变但不影响病毒感染性;N蛋白的N端可以插入至少12个氨基酸也不影响病毒感染性。
第二部分是关于N蛋白末端及中间序列在病毒复制过程中作用的研究。本文首先选取N蛋白的N端和C端进行系列缺失,通过转染MARC-145细胞及病毒拯救,表明N端的5-13位氨基酸和C端的最后4个氨基酸对于病毒感染性是非必需的,意外的是,这些拯救病毒除了保持所操作缺失的稳定存在外,N基因中都出现了额外突变,包括氨基酸的替换、缺失及插入等,重复转染试验证明了额外突变存在的必然性。对于没有感染性的突变体通过免疫荧光及RT-PCR来检测基因组复制及sgmRNA转录特性,表明14-20位之间的保守区域可能包含着病毒复制所必需的元件。
为了研究这些额外突变(替换、缺失、插入)本身对病毒的影响,我们又重新构建了单独包含额外突变的两个突变体pN?39-42和pN?48-52,及同时包含原来所做缺失和额外突变的两个突变体pC?3/Y61C和pC?3/N?48-52。这些突变体转染细胞后都能拯救出病毒,同时拯救病毒vN?48-52和vC?3/N?48-52的N基因中仍有额外突变出现,包括点突变及氨基酸的插入。表明N蛋白的中间区域本身也可以承受氨基酸的缺失。
综上所述,本研究证明PRRSV的N蛋白具有以下特性:(1)其翻译倾向于使用前导序列之后的第一个AUG;(2)N端第1-11位氨基酸序列特异性对病毒复制是非必需的;(3) N端第5-13位氨基酸可以缺失而不影响病毒感染性;(4)N端第14-20位氨基酸序列缺失或所致核苷酸及蛋白结构变化导致PRRSV感染性、基因组复制和mRNA转录的功能丧失;(5)C端最后4个氨基酸的缺失不影响病毒的感染性,进一步缺失虽不影响RNA合成及翻译,但病毒装配或出芽过程受损而导致了病毒感染性的丧失;(6)N蛋白的中间区域(aa39-52)也可以承受氨基酸的缺失。所有的拯救病毒在N基因中都出现了额外突变。这些研究成果对进一步剖析PRRSV N蛋白结构与功能的关系,及研发基因标识疫苗奠定了基础。
Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is enveloped single strand positive sense RNA virus. Two genotypes were identified, type I (European type) and type II (North American type). The genome of PRRSV includes at least 9 open reading frames (ORFs), ORF1 encoded non-structural proteins (nsps) in charge of viral genome replication and suggenomic mRNA (sgmRNA) transcription. Structural proteins were expressed by ORF2 to ORF7. Nucleocapsid protein (N protein) expressed by ORF7 is a basic multifunctional protein with molecular weight of about 15kD. Generally, type I and type II PRRSV N proteins consist of 128 and 123 amino acids (aa), but the length of type I N protein exhibited size polymorphism. It is intrested to dissect the translational control mechanism of ORF7 because of the 319nt leader sequence before the start codon of ORF7. N was documented to play crucial roles in virion assembly, but the minimal requirements of N for virus functionality have not been charactered.
This study was based on the platform of full-length PRRSV infectious cDNA clones. The first part focuses on the translational control of N protein. Three mutants were constructed based on the infectious cDNA clone pORF673 which had separated the repeated sequences between ORF6 and ORF7 and inserted three restriction enzyme sites. It proved that the translation of N protein prones to utilize the first AUG before N coding region, and the first 11 aa of N protein could be fully changed without affecting virus infectivity. Also,N terminal could tolerate 12 aa insertion without blocking virus viability.
The second part focuses on the role of terminal and internal regions of N protein for virus replication. We targeted the terminal N protein and constructed a series of N- and C-terminal truncations. The transfection and virus rescue assay indicated that residues 5-13 at the N terminus and the last four residues at the C terminus were non-essential for virus viability. Unexpectedly, the recovered viruses were accompanied by various patterns of second-site mutations, including substitution, in-frame deletion, in- or out-of-frame insertion within N protein. Immunofluorescence assay and RT-PCR analysis for non-viable mutants indicated that the conserved 14-20 aa region may contained the essential elements for virus replication.
To further study whether the N protein can tolerate internal deletion per se, or if the latter has to be accompanied by the terminal deletion, we introduced two internal deletions found in C?3/121-123 ppvs back into the wild-type pAPRRS, named as pN?39-42 and pN?48-52. Meanwhile, two double mutants containing the originally engineered C-terminal 3-aa deletion, together with second-site mutation Y61C and the residue 48-52 deletion were reconstructed. The resultant plasmids were designated as pC?3/N?48-52 and pC?3/Y61C, respectively. These mutants developed visible CPE after transfection, and the growth property exhibited indistinguishable growth kinetics from the wild type APRRS. It was worth noting that the recovered viruses N?48-52 and C?3/N?48-52 still provoked additional mutations, including substitution and aa insertion. The results indicated that the internal regions of N protein could tolerate deletion.
In conclusion, we found that N protein of PRRSV had several properties: (1) its expression proned to utilize the first AUG followed with leader sequences; (2) the sequence specificity of the first 11 aa was non-essential for virus viability; (3) N-terminal residues 5 to13 were non-essential for type II PRRSV viability; (4) the deletion of 14-20 aa at N-terminal rendered virus nonviable and blocked genome replication and sgmRNA transcription; (5) the last 4 residues at C terminus could be deleted without affecting virus infectivity; (6) the internal region (aa39-52) could tolerate deletion. Almost all of the recovered viruses were accompanied by second-site mutations in N protein. This is belived to be the first report that second-site mutations arose in N protein of arterivirus. This study lays a foundation for further molecular dissection of structural and function relationship of N protein, and development of genetically tagged vaccines against PRRS.
在PRRSV感染性克隆平台的基础上,本研究第一部分是关于N蛋白翻译起始位点的研究,以将ORF6和ORF7之间的重复序列拉开并插入酶切位点的感染性克隆pORF673为模板,分别构建了3个ATG的系列突变体,通过病毒拯救表明N蛋白的翻译优先使用前导序列之后的第一个AUG;在迫使病毒使用导致ORF7移码的人工插入AUG时,PRRSV可以通过碱基插入矫正读码框,使得N蛋白的前11个氨基酸可以完全改变但不影响病毒感染性;N蛋白的N端可以插入至少12个氨基酸也不影响病毒感染性。
第二部分是关于N蛋白末端及中间序列在病毒复制过程中作用的研究。本文首先选取N蛋白的N端和C端进行系列缺失,通过转染MARC-145细胞及病毒拯救,表明N端的5-13位氨基酸和C端的最后4个氨基酸对于病毒感染性是非必需的,意外的是,这些拯救病毒除了保持所操作缺失的稳定存在外,N基因中都出现了额外突变,包括氨基酸的替换、缺失及插入等,重复转染试验证明了额外突变存在的必然性。对于没有感染性的突变体通过免疫荧光及RT-PCR来检测基因组复制及sgmRNA转录特性,表明14-20位之间的保守区域可能包含着病毒复制所必需的元件。
为了研究这些额外突变(替换、缺失、插入)本身对病毒的影响,我们又重新构建了单独包含额外突变的两个突变体pN?39-42和pN?48-52,及同时包含原来所做缺失和额外突变的两个突变体pC?3/Y61C和pC?3/N?48-52。这些突变体转染细胞后都能拯救出病毒,同时拯救病毒vN?48-52和vC?3/N?48-52的N基因中仍有额外突变出现,包括点突变及氨基酸的插入。表明N蛋白的中间区域本身也可以承受氨基酸的缺失。
综上所述,本研究证明PRRSV的N蛋白具有以下特性:(1)其翻译倾向于使用前导序列之后的第一个AUG;(2)N端第1-11位氨基酸序列特异性对病毒复制是非必需的;(3) N端第5-13位氨基酸可以缺失而不影响病毒感染性;(4)N端第14-20位氨基酸序列缺失或所致核苷酸及蛋白结构变化导致PRRSV感染性、基因组复制和mRNA转录的功能丧失;(5)C端最后4个氨基酸的缺失不影响病毒的感染性,进一步缺失虽不影响RNA合成及翻译,但病毒装配或出芽过程受损而导致了病毒感染性的丧失;(6)N蛋白的中间区域(aa39-52)也可以承受氨基酸的缺失。所有的拯救病毒在N基因中都出现了额外突变。这些研究成果对进一步剖析PRRSV N蛋白结构与功能的关系,及研发基因标识疫苗奠定了基础。
Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is enveloped single strand positive sense RNA virus. Two genotypes were identified, type I (European type) and type II (North American type). The genome of PRRSV includes at least 9 open reading frames (ORFs), ORF1 encoded non-structural proteins (nsps) in charge of viral genome replication and suggenomic mRNA (sgmRNA) transcription. Structural proteins were expressed by ORF2 to ORF7. Nucleocapsid protein (N protein) expressed by ORF7 is a basic multifunctional protein with molecular weight of about 15kD. Generally, type I and type II PRRSV N proteins consist of 128 and 123 amino acids (aa), but the length of type I N protein exhibited size polymorphism. It is intrested to dissect the translational control mechanism of ORF7 because of the 319nt leader sequence before the start codon of ORF7. N was documented to play crucial roles in virion assembly, but the minimal requirements of N for virus functionality have not been charactered.
This study was based on the platform of full-length PRRSV infectious cDNA clones. The first part focuses on the translational control of N protein. Three mutants were constructed based on the infectious cDNA clone pORF673 which had separated the repeated sequences between ORF6 and ORF7 and inserted three restriction enzyme sites. It proved that the translation of N protein prones to utilize the first AUG before N coding region, and the first 11 aa of N protein could be fully changed without affecting virus infectivity. Also,N terminal could tolerate 12 aa insertion without blocking virus viability.
The second part focuses on the role of terminal and internal regions of N protein for virus replication. We targeted the terminal N protein and constructed a series of N- and C-terminal truncations. The transfection and virus rescue assay indicated that residues 5-13 at the N terminus and the last four residues at the C terminus were non-essential for virus viability. Unexpectedly, the recovered viruses were accompanied by various patterns of second-site mutations, including substitution, in-frame deletion, in- or out-of-frame insertion within N protein. Immunofluorescence assay and RT-PCR analysis for non-viable mutants indicated that the conserved 14-20 aa region may contained the essential elements for virus replication.
To further study whether the N protein can tolerate internal deletion per se, or if the latter has to be accompanied by the terminal deletion, we introduced two internal deletions found in C?3/121-123 ppvs back into the wild-type pAPRRS, named as pN?39-42 and pN?48-52. Meanwhile, two double mutants containing the originally engineered C-terminal 3-aa deletion, together with second-site mutation Y61C and the residue 48-52 deletion were reconstructed. The resultant plasmids were designated as pC?3/N?48-52 and pC?3/Y61C, respectively. These mutants developed visible CPE after transfection, and the growth property exhibited indistinguishable growth kinetics from the wild type APRRS. It was worth noting that the recovered viruses N?48-52 and C?3/N?48-52 still provoked additional mutations, including substitution and aa insertion. The results indicated that the internal regions of N protein could tolerate deletion.
In conclusion, we found that N protein of PRRSV had several properties: (1) its expression proned to utilize the first AUG followed with leader sequences; (2) the sequence specificity of the first 11 aa was non-essential for virus viability; (3) N-terminal residues 5 to13 were non-essential for type II PRRSV viability; (4) the deletion of 14-20 aa at N-terminal rendered virus nonviable and blocked genome replication and sgmRNA transcription; (5) the last 4 residues at C terminus could be deleted without affecting virus infectivity; (6) the internal region (aa39-52) could tolerate deletion. Almost all of the recovered viruses were accompanied by second-site mutations in N protein. This is belived to be the first report that second-site mutations arose in N protein of arterivirus. This study lays a foundation for further molecular dissection of structural and function relationship of N protein, and development of genetically tagged vaccines against PRRS.
引文
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