新城疫病毒L蛋白VI结构域影响甲基化转移酶功能的分子机制

The Large polymerase protein (L protein) is one of biggest proteins in nonsegmented negative-strand RNA viruses (NNSRVs). It possesses viral mRNA capping methyltransferase (Mtase) function for viral mRNA stability besides the polymerase activity. The L protein was bioinformatically divided six conserved sequence regions (also called domains).Among them, the VI domain has been postulated or evidenced to possess viral mRNA capping MTase activity in some NNSRVs and is related to gene expression, mRNA stability ,viral virulence and so on. Newcastle disease virus (NDV) is a member of NNSRVs and its genome encodes an L protein with a similar literal structure with other L proteins in NNSRVs. However, the Mtase function of NDV L protein is not understood thus far. In this study, we respectively constructed 12 point mutants into the VI domain of NDV L gene by alanine-scanning mutagenesis. Subsequently, 12 L mutants will be cloned into the genotpye VII NDV genome, respectively. The resulting mutant viruses will be rescued using a reverse genetics approach. Currently,the preliminary data from our laboratory showed that we have successfully rescued 4 virues and three of four strains have obivous attenuation phenotypes on the viral virulence tested by mean death time in SPF embryos, intracebreal pathogenicity index in 1-day-old SPF chicks and intravenous pathogenicity index in 6-week-old SPF chickens. Next, the biological properties，MTase activities，host range phenotypes and temperature sensitivity of the rescued virus strains will be systemically evaluated by animal studies, co-immunoprecipitation, and in vitro transcription assays compared to the wild-type virus. Eventually, the aims of our study are to find out the key amino acid residues in NDV L protein which determine cap-dependent MTase activity and viral virulence and investigate how the Mtase impacts the viral replication and gene expression. The data from this project will pave a way for further dissecting the function of NDV L protein and provide profoundly important data for the attenuated viral vaccine development in the future.

包括新城疫病毒在内的单股负链不分节段的RNA病毒L蛋白具有6个保守的结构域（结构域Ⅰ-Ⅵ），每个结构域发挥不同生物学功能，Ⅵ区可能与甲基化转移酶活性及病毒毒力相关，且在新城疫病毒的研究中未见报道。因此，本研究以基因Ⅶ型新城疫病毒L基因为研究对象，将L蛋白结构域Ⅵ区中可能与甲基化转移酶及病毒毒力相关的氨基酸位点丙氨酸扫描技术进行点突变，构建12个L基因突变的全长cDNA，运用反向遗传操作技术进行病毒拯救。通过免疫共沉淀、病毒体外转录、动物实验等技术手段，对获救的突变型和野生型病毒的甲基化转移酶功能和病毒毒力进行比较分析，并分别从细胞和宿主水平上评估L蛋白突变对病毒甲基化转移酶活性及毒力的影响，并找出影响帽依赖的甲基化转移酶活性及病毒毒力的关键位点，进一步探索甲基化转移酶与病毒毒力之间的关系。本研究结果将为深入研究新城疫病毒 L蛋白甲基化转移酶功能奠定基础，为设计和开发减毒活疫苗提供理论依据。

新城疫是由新城疫病毒引起禽类动物的一种急性、高度传染性疾病，给养殖业造成巨大的经济损失。本项目以基因VII型新城疫病毒作为研究对象，对L基因VI结构域的不同位点进行突变，构建12个L突变的全长cDNA，利用反向遗传技术拯救病毒。成功获救了10株L蛋白重组突变型新城疫病毒，分别从病毒毒力、病毒复制动力学分析、遗传稳定性和免疫原性等方面开展试验。结果表明，L蛋白K-D-K-E基序的突变显著降低了病毒的毒力和在DF-1细胞上的增殖能力。同时，对这4株病毒.（rNDV-K1756A, rNDV-D1881A, rNDV-K1917A and rNDV-E1954Q）进行连续传代至15代，测序证明突变位点依然存在，可以稳定遗传。突变弱毒株.（rNDV-K1756A, rNDV-D1881A, rNDV-K1917A and rNDV-E1954Q）免疫SPF鸡21 d后攻毒，均可完全保护；其中rNDV-E1954Q组攻毒后，未检测到病毒排毒。在此毒株的研究基础上，构建F蛋白突变的重组病毒rNDV-F+E1954Q。rNDV-F+E1954Q的病毒毒力显著低于野生型NDV；免疫SPF鸡21 d后攻毒，亦可完全保护；攻毒后检测排毒情况和脏器中病毒含量，发现病毒载量均低于LaSota免疫组。本项目的研究，为研发和创制新城疫减毒疫苗和新型疫苗提供了理论依据。