非结构蛋白nsp2调控PRRSV亚基因组转录复制的分子基础

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) has remained a major global threat to pork production. Rapid evolution of this positive-stranded RNA virus has led to frequent emergence of many highly virulent strains in the field in the past 25 years, and these include the most recent Asian HP-PRRSV, which is devastating to the Chinese swine industry. As development of an effective vaccine has been difficult, these somber facts clearly strike an urgent need for a better molecular understanding of PRRSV so that new ways can be found to better fight this pathogen. The focus of this application is nonstructural protein 2 (nsp2), a multidomain replicase protein that represents the fastest evolving region in PRRV genome but with much of its function remaining poorly understood. We have made substantial progress in understanding this molecule, but there are three remarkable observations that provide the foundation for this proposal. First, we discovered that the nsp2 central region was able to tolerate various deletions, ranging from 100 to 400 residues in size. Strikingly, a mutant carrying a 400-aa deletion in nsp2 central region was found to be nearly dead in pigs although it replicates fairly well in cell culture. Second, we found that deletions of certain nsp2 middle regions differentially affected subgenomic (sg) mRNA5 accumulation. This is the first example of another PRRSV replicase protein (nsp), in addition to nsp1, selectively affecting certain viral sg mRNA synthesis. Third, we demonstrated that the N-terminal PL2 domain encodes an active enzyme that possesses both cis-and trans-cleavage activities. This enzyme is responsible for cleaving the site(s) at the nsp2|3 junction during viral replicase maturation, and both activities are absolutely required for PRRSV replication. To pursue further clues of how nsp2 works in the PRRSV replication cycle, we have designed three specific aims. In the first aim, we will investigate how nsp2 and other viral nsps come together to assemble viral replication and transcription complex (RTC). The second aim will elucidate the mechanism of how nsp2 hypervariable region modulates and regulates PRRSV sg mRNA synthesis. We propose an unbiased genetic approach in which we will select for second-site repressors that restore the sg mRNA5 to mutants that have been cripple because of the absence of the nsp2 region. This approach will be seeking a "gain of function" to look for viral factors that coordinate with nsp2 in viral RNA synthesis. The third aim is to determine the molecular mechanism of PRRSV nsp2 PL2 cysteine protease activity and substrate recognition. These three aims will substantially advance our understanding of the molecular mechanisms of nsp2 in PRRSV replication and pathogenesis, which may eventually lead to discovery of novel targets for anti-viral drugs that are needed for treatment of PRRSV infection, and also new means for PRRSV attenuation.

猪繁殖与呼吸综合征病毒在选择压力下持续的变异和演化导致新的高致病性毒株在世界范围内频繁诞生, 其典型的代表为在我国新近分离的高致病性PRRSV, 其毒力增强的分子基础目前仍不清楚。本申请项目以PRRSV 基因组中变异最快的编码区, 非结构蛋白nsp2, 为切入点, 通过发挥我们已有的研究工作基础, 综合应用分子病毒学, 分子生物学、遗传学、细胞生物学、生物化学以及生物学信息学等多学科交叉手段, 系统地研究nsp2如何和其他病毒蛋白相互作用组装病毒复制和转录复合体, 研究nsp2高变区如何选择性调控某些亚基因组RNA的合成, 以研究nsp2氨基端结构域PL2蛋白酶酶活性及底物识别的分子基础。这些研究将进一步揭示PRRSV转录、复制的分子机制, 而且能为诊断和防治PRRS提供新的药物靶点以及为开发新型PRRSV疫苗提供重要的理论基础。

猪繁殖与呼吸综合征病毒（PRRSV）严重危害我国及全球养猪生产，其在选在压力下发生持续的变异和演化，并导致高致病性毒株在世界范围内频繁诞生。本项目以PRRSV基因组变异最快的区域-病毒复制酶非结构蛋白nsp2-为切入点，研究其变异的生物学意义及在病毒复制中的作用机制，主要科学发现有一下5点：① 全面解析了PRRSV非结构蛋白（nsp）相互作用网络，发现其相互作用主要集中在非结构膜蛋白与核心复制酶与之间，且膜蛋白对聚合酶nsp9和解旋酶nsp10的招募受构象调控；②发现nsp2高变区缺失可差异影响PRRSV 特定亚基因组合成；③ 发现高变区nsp2 aa.323-521缺失影响PRRSV巨噬细胞嗜性；④ 发现PRRSV nsp2 氨基端PLP2结构域去泛素化酶活性受氧化还原反应调控，其对底物的识别和切割与顺式活性享有共同点，鉴定出影响去泛素化酶活性的关键氨基酸位点，并揭示PLP2顺式活性为病毒复制非必需，而去泛素化酶活性在病毒复制中起重要作用；⑤ 发现nsp2是PRRSV感染中重要的炎症诱导因子，且此特性不依赖于其去泛素化酶活性。上述的研究结果揭示了PRRSV非结构蛋白之间相互作用的复杂性，预示病毒 RTC的组装是一个高度有序的过程；研究还揭示病毒亚基因组的合成是一个复杂的过程，单个亚基因组的转录合成在调控机制上存在差异，而nsp2高变区变异可影响相应亚基因组的丰度；研究还揭示了nsp2的新功能，即与病毒组织嗜性有关，由于最近有研究表明nsp2为病毒的结构蛋白，这一发现很好的解释了在选择压力下的nsp2高变特性的驱动力，对理解病毒的致病机制具有重要的意义；研究还进一步揭示了PRRSV去泛素华酶的生物学特性及其在病毒复制的地位，为进一步深入研究其具体作用机制奠定了良好的基础； 此外，研究还为nsp2去泛素化活性非依赖性调控炎症因子及关键氨基酸位点的鉴定为理解nsp2参与调控天然免疫和设计新型弱毒疫苗提供了重要的启示。