Secretion of PRRS virrus GP3 glycoprotein controversy: GP3 characterization and role identification in vitro and in vivo

猪繁殖与呼吸综合征（PRRS）是造成全球养猪业经济损失最大的病毒性疾病。PRRS的病原PRRS病毒（PRRSV）分为两种不同的基因型，北美（NA）和欧洲（EU），两者的核苷酸序列同源性为63%。目前，商业疫苗的效力相当低。因此，明确PRRSV病毒蛋白的作用和功能，对提高预防方法具有重要意义。这些改进可能对兽药和养猪业都有益。到目前为止，基于两种基因型GP3之间存在的相互矛盾的数据，PRRSV ORF3基因编码的GP3糖蛋白的功能尚不清楚。1996年，欧盟参考菌株Lelystad （LV）的GP3首次被报道为病毒粒子的结构蛋白，但在1998年，经过我们团队对病毒纯化技术的修改和改进，我们的NA参考菌株IAF-Klop的GP3被报道为PRRSV感染细胞的非结构分泌蛋白。即使两个菌株的GP3之间只有54%的序列同源性，四分之三的蛋白质组学和生物信息学模型（计算机）表明LV GP3是一种细胞外蛋白。由于NA参考菌株的GP3是非结构性的，并且是从感染细胞分泌的，那么它的功能是什么？众所周知，其他病毒分泌的蛋白质（如疱疹、痘）与细胞因子有相似之处，并随后调节受感染宿主的免疫反应。本研究的假设是两种基因型的GP3具有相同的特征，分泌的GP3 （sGP3）在PRRSV感染的宿主中具有特定的活性。为了证明这一假设，将实现不同的目标，如：与IAF-Klop GP3相比，建立LV GP3的细胞内和细胞外定位；评价sp3对猪新分离免疫细胞的影响；评估ORF3突变的PRRSV （GP3分泌被抑制）在感染猪体内的致病性。

The most important viral disease in term of economical lost in swine industry worldwide is the Porcine Reproductive and Respiratory Syndrome (PRRS). The etiological agent of PRRS, PRRS virus (PRRSV) is classified in two distinct genotypes, the North American (NA) and the European (EU), which possess a nucleotide sequence identity of 63% when compared one to another. Presently, the efficacy of commercial vaccines is quite mitigated. Consequently, to improve prophylactic methods, it is important to characterize and establish the roles and functions of the viral proteins of PRRSV. Those improvement could be beneficial for both veterinary pharmaceuticals and porcine industries. Until now, the function of the GP3 glycoprotein, encoded by the ORF3 gene of PRRSV is unknown, based on conflicting data existing between the GP3 of both genotypes. At first in 1996, the GP3 of the EU reference strain Lelystad (LV) was reported to be a structural protein of the virion, but in 1998, following the modification and improvement that was made by our team in the virus purification techniques, the GP3 of our NA reference strain IAF-Klop was reported to be a non structural and secreted protein from PRRSV infected cells. Even if there is only a sequence identity (homology) of 54% between the GP3 of both strains, three out of four proteomic and bioinformatic models (in silico) suggest that LV GP3 is an extracellular protein. Since the GP3 of a NA reference strain is non structural and secreted from infected cells then what is her function? It is well known that other viral secreted proteins (ex: herpes, pox) possess similarities with cytokines and subsequently modulate the immune reponse of infected hosts. The hypothesis of the present proposal is that GP3 of both genotypes share the same characteristics and that secreted GP3 (sGP3) possesses specific activities in PRRSV infected hosts. To prove this hypothesis, different objectives will be realized like: establishing the LV GP3 intra and extracellular localization in comparison with IAF-Klop GP3; evaluating the effect of sGP3 on freshly isolated porcine immune cells; evaluating the pathogenicity of ORF3 mutated PRRSV (which GP3 secretion is suppress) in infected swines.