AMINO ACID SUBSTITUTION AT THE NSI-NS2A CLEAVAGE JUNCTION OF DENGUE PROTEIN

登革热蛋白 NSI-NS2A 切割连接处的氨基酸取代

• 批准号: 3790818
• 负责人: M PETHEL
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3790818
• 关键词: Flaviviridae; chemical cleavage; chemical substitution; dengue virus; gene deletion mutation; mutant; point mutation; protein sequence; site directed mutagenesis; tissue /cell culture; virus genetics; virus protein

We have previously shown that proper processing of dengue type 4 virus (DEN 4) NS1 from the NS1-NS2A region of the viral polyprotein requires a hydrophobic N-terminal signal and the downstream NS2A. Results from deletion analysis indicate that a minimum length of eight amino acids at the C-terminus of NS1 is required for cleavage at the NS1-NS2A junction. Comparison of this eight amino acid sequence with the corresponding sequences of other flaviviruses suggests a consensus cleavage sequence of Met/Leu-Val-Xaa-Ser-Xaa-Val-Xaa-Ala. Site-directed mutagenesis was performed to construct mutants of NS1-NS2A that contained a single amino acid substitution at different positions of the consensus cleavage sequence or at the immediate downstream position. Three to eight different substitutions were made at each position. A total of 50 NS1-NS2A mutants were analyzed for their cleavage efficiency relative to that of the wild type DEN 4 sequence. As predicted, nearly all substitutions at positions P-1, P-3, P-5, P-7, and P-8, occupied by conserved amino acids, yielded low levels of cleavage, with the exception that Pro or Ala substituting for Ser (P-5) was tolerated. Substitutions of an amino acid at the remaining positions occupied by nonconserved amino acids generally yielded high levels of cleavage. However, some substitutions at nonconserved positions were not tolerated. For example, substitution of Gly or Glu for Gln (P-4) and substitution of Val or Glu for Lys (P-6) each yielded a low level of cleavage. Overall, these data support the proposed cleavage sequence motif deduced by comparison of sequences among the flaviviruses. In addition to the eight amino acid sequence, this study also showed that the amino acid immediately following the NS1-NS2A cleavage site plays a role in cleavage.

我们以前已经表明，适当的处理登革4型病毒（DEN 4)来自病毒多聚蛋白NS 1-NS 2A区域的NS 1需要一个 疏水N-末端信号和下游NS 2A。 结果 缺失分析表明，最小长度为8个氨基酸， NS 1的C-末端是在NS 1-NS 2A连接处切割所必需的。 将这8个氨基酸序列与相应的 其他黄病毒的序列表明， Met/Leu-Val-Xaa-Ser-Xaa-Val-Xaa-Ala。 定点诱变 进行构建NS 1-NS 2A的突变体，所述突变体含有单个氨基酸 在共有切割序列的不同位置处的酸取代 或者在紧邻下游的位置。 三到八个不同的 在每个位置进行取代。 共50个NS 1-NS 2A突变体 分析它们相对于野生型的切割效率 类型DEN 4序列。 正如所预测的，几乎所有的位置处的取代， P-1、P-3、P-5、P-7和P-8被保守氨基酸占据，产生低产量。 切割水平，除了Pro或Ala取代Ser (P-5)是被容忍的。 在剩余的氨基酸处的氨基酸取代 由非保守氨基酸占据的位置通常产生高的 分裂的水平。 然而，在非保守位置的一些取代， 是不能容忍的。 例如，Gly或Glu取代Gln（P-4） 和用瓦尔或Glu取代Lys（P-6）各自产生低水平的 乳沟 总之，这些数据支持所提出的切割序列基序 通过比较黄病毒之间的序列推断。 除了 八个氨基酸序列，这项研究还表明， 紧接着NS 1-NS 2A切割位点在切割中起作用。