YXXM 基序对J 亚群禽白血病病毒复制的影响

Effect of YXXM motif on viral replication of subgroup J avian leukosis virus

摘要:

摘要:【目的】毒株NX0101 是骨髓瘤病变型J 亚群禽白血病病毒，其早期感染细胞能诱导PI3K /Akt 信号转导通路的激活，本文针对NX0101 毒株是否存在YXXM 基序及其作用进行了探讨。【方法】利用TMpred 软件对NX0101 毒株囊膜蛋白(Env)的氨基酸序列进行生物信息学分析，通过搭桥PCR 方法将YXXM 基序相应的核苷酸序列突变后，构建突变质粒并转染DF-1细胞，拯救出YXXM 突变体毒株NX0101mt(Y/F，M/A)，利用real-time PCR 和ELISA 方法检测并比较YXXM 突变前后毒株在RNA 水平和蛋白水平的复制情况。 【结果】NX0101 毒株Env 胞浆区554－557 位氨基酸存在典型的PI3K 结合基序YXXM。YXXM 基序突变后，病毒RNA 转录水平和病毒蛋白合成水平都显著下降。【结论】YXXM 基序对NX0101 毒株在体外宿主细胞中复制发挥重要的作用。

Abstract:

Abstract:［Objective］ It was reported that subgroup J avian leukosis virus strain NX0101 activates PI3K /Akt pathway during early infection in DF-1 cells． Whether there is YXXM motif in the amino acid sequence of NX0101 and the function of YXXM motif were studied．［Methods］ The presence of internal transmembrane domains in the envelope protein of NX0101 was analyzed by Tmpred． Point mutation was introduced to change the YXXM motif in the NX0101 strain to FXXA． The plasmid containing the full genome of NX0101 with mutation within the YXXM motif in pMD18-T vector was constructed and transfected into DF-1 cells． Viral replication levels of NX0101 strain and the mutation one were tested and compared by real-time PCR and ELISA． ［Results］ The amino acid sequence of NX0101 strain had one YXXM motif (amino acids 554－557) in the cytoplasmic tail of envelope protein． The mutated NX0101 strain (Y554F，M557A) was rescued by reverse genetics technique． Viral replication of the mutated NX0101 strain was significantly lower than that of NX0101 strain in the level of either RNA or protein synthesis．［Conclusion］ The results revealed that theYXXM motif was important for virus NX0101 replication in DF-1 cells．