VP3 G-H环中氨基酸突变对O型口蹄疫病毒生物学特性的影响

Effect of amino acid mutation(s) in the G-H loop of VP3 on biological characteristics of foot-and-mouth disease virus serotype O

摘要:

[目的]为了研究O型口蹄疫病毒VP3 G-H环中氨基酸突变对其生物学特性的影响。[方法]借助口蹄疫病毒反向遗传操作技术平台拯救出2株定点突变体rHN^V3174Y和rHN^{D3173N+V3174E+N3179C}。进行蚀斑形成试验、一步生长曲线的绘制、TCID₅₀和LD₅₀的测定、间接免疫荧光与激光共聚焦显微镜检测。[结果]结果显示，与骨架病毒rHN相比，虽然rHN^V3174Y和rHN^{D3173N+V3174E+N3179C}对BHK-21细胞的感染性及其蚀斑表型和复制动力学无显著性差异；但rHN^V3174Y和rHN^{D3173N+V3174E+N3179C}对乳鼠的致病力明显减弱，且均获得了小窝蛋白介导侵染CHO-K1细胞的能力。[结论]VP3上第3174位特征性氨基酸突变影响O型口蹄疫病毒感染宿主细胞的毒力及其内吞作用路径，这有助于我们认知VP3 G-H环在口蹄疫病毒粒子立体空间构象中潜在的作用。

关键词:口蹄疫病毒;VP3;G-H环;定点突变体;生物学特性;内吞作用

Abstract:

[Objective] To study the effect of amino acid mutation(s) in the G-H loop of VP3 of foot-and-mouth disease virus (FMDV) serotype O on its biological characteristics. [Methods] Two site-directed mutants, rHN^V3174Y and rHN^{D3173N+V3174E+N3179C}, were generated by using reverse genetic technique platform of FMDV. The phenotypic properties of the resulting viruses were characterized by plaque forming assays, one-step growth curves, determination of TCID₅₀ and LD₅₀, indirect immunofluorescence assays and laser confocal microscopies. [Results] There were no significant differences in the infectivity, plaque phenotype, and replication kinetics between rHN, rHN^V3174Y and rHN^{D3173N+V3174E+N3179C} for BHK-21 cells. Compared to the backbone virus rHN, however, the pathogenicity of rHN^V3174Y and rHN^{D3173N+V3174E+N3179C} was obvious decreased in suckling mice, and these two rescued viruses gained the ability to infect CHO-K1 cells via caveola-mediated endocytosis. [Conclusion] Individual amino acid mutations at position 3174 in VP3 have an influence on the virulence and endocytic pathway of FMDV serotype O in the infection of host cells, which may help us understand the potentially important functional roles of the G-H loop of VP3 in the 3D spatial conformation of FMDV virion.

Key words:foot-and-mouth disease virus;VP3;G-H loop;site-directed mutant;biological characteristics;endocytosis