核糖体移码对乙型脑炎病毒基因组表达调控作用解析

The NS1' protein of flavivirus in the Japanese encephalitis virus (JEV) serogroup derives frome -1 ribosomal frameshift in the coding region of NS2A. Currently, the function of NS1' is not completely clear, and the role of ribosomal frameshift in the life cycles of JEV has not been determined. Here, the role of ribosomal frameshift in genome expression of JEV would be studied. The signal sequences for ribosomal frameshift and the coding region of C terminal of NS1' would be changed based on JEV infectious clone pHJEV, respectively. The rescued mutant viruses would be used to infect vero cells and the morphology of plaques formed on vero cells were characterized. Both extracellular and intracellular progeny virus would be analyzed with real time RT-PCR. The amount of IFNs expressed in Ana-1 cells and 3T3-swiss albino cells would be determined during the infection of mutant and parent virus HEN0701. Three-week old mice would be innoculated with mutant virus or HEN0701 to evaluate the virulence of these virus. Complement studies in vitro would be performed to clarify the function of NS1'. These results may clarify the effect of ribosomal frameshift on JEV replication, morphogenesis and virulence.Therefor, the role of ribosomal frameshift in the life cycles of JEV would be elucidated.

乙型脑炎血清群黄病毒通过核糖体-1移码表达NS1′蛋白，但NS1′蛋白功能尚不明确，核糖体移码调控病毒基因组表达对病毒感染复制循环的影响也不清楚。本申请以乙型脑炎病毒为研究对象，操作病毒基因组中核糖体移码信号和改变移码后编码氨基酸序列，测定不同突变病毒核糖体移码效率、空斑形态、感染细胞内外病毒基因组的变化、感染细胞干扰素合成水平及病毒毒力等指标，并以NS1′蛋白体外互补NS1蛋白，探讨核糖体移码对病毒基因组复制、病毒粒子形成及病毒毒力的影响，并研究移码所产生的NS1′蛋白功能，进而明确核糖体移码对JEV基因组表达调控作用及其对病毒感染复制循环的意义。

乙型脑炎病毒（Japanese encephalitis virus，JEV）及其同血清群黄病毒在基因组表达过程中存在核糖体-1移码表达NS1’蛋白的现象，但核糖体移码对病毒基因组表达的调控作用和NS1’蛋白的功能目前没有清晰的认识。本研究以JEV为研究对象，通过分析JEV基因组中核糖体移码元件序列，在对NS2A基因实施同义突变基础上，设计8对引物，通过定点突变PCR方法在核糖体移码元件序列中引入突变，并在JEV HEN0701株感染性克隆pJEHEN基础上，将突变序列置换如感染性克隆中，成功拯救出8株突变病毒：vJETA、vJECT、vJETC、vJESTC、vJESGA、vJE2R、vJET1和vJET2。将8株突变病毒感染Vero细胞后，Western Blot检测显示，8株突变病毒均表达NS1蛋白，但NS1’蛋白表达出现差异：vJETC株、vJECT株和vJESTC株表达正常的NS1’蛋白；vJETA株和vJESGA株不表达NS1’蛋白；vJE2R株、vJET1株和vJET2株则表达异常的NS1’蛋白，vJE2R株表达编码框缩短即蛋白分子量减小的NS1’蛋白，vJET1株与vJET2株分别表达NS1’蛋白编码框不同程度延伸即蛋白分子量不同程度增加的NS1’蛋白。生长曲线和空斑试验显示，突变对病毒复制能力造成了不同程度的影响。与亲本病毒vTHen相比，vJE2R株与vJESGA株复制能力与vTHEN株基本一致；vJETC株与vJESTC株复制能力有所下降；而vJECT、vJETA、vJET1和vJET2复制能力明显降低。动物实验发现，突变对病毒的毒力也造成了不同程度的影响。神经毒力试验中，vJE2R株和vJESGA株与对亲本株vTHEN相比基本一致，vJESTC株与vJETC株神经毒力有所下降，vJETA株、vJECT株、vJET1株和vJET2株神经毒力明显下降。神经侵袭力试验中，vJESGA株、vJE2R株和vJETC株与亲本株vTHEN相比基本一致，但vJETA株、vJECT株、vJESTC株、vJET1株和vJET2株神经侵袭力明显下降。上述试验结果显示，NS1’是否表达，不影响病毒在体外生长和病毒对小鼠的毒力。本研究表明，JEV基因组表达核糖体移码及其产生的NS1’蛋白，对病毒在体外哺乳细胞上的生长和对小鼠毒力没有影响。