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

我们之前已经证明，对登革4型病毒(DEN)的正确处理 4)来自病毒多蛋白NS1-NS2a区域的NS1需要 疏水的N-末端信号和下游的NS2a。结果来自 缺失分析表明，至少有8个氨基酸位于 NS1的C末端需要在NS1-NS2A连接处进行切割。 这8个氨基酸序列与相应的 其他黄病毒的序列表明有一个共同的裂解序列 Met/Leu-Val-Xaa-Ser-Xaa-Val-Xaa-Ala。定点突变是 用于构建含有单一氨基的NS1-NS2a突变体 共识裂解序列不同位置的酸取代 或在紧邻下游的位置。三到八个不同的 每个位置都进行了替换。共有50个NS1-NS2a突变体 分析了它们相对于野生动物的卵裂效率 键入DEN 4 SEQUENCE。正如预测的那样，几乎所有的位置上的替换 被保守氨基酸占据的P-1、P-3、P-5、P-7和P-8产量较低 卵裂水平，但Pro或Ala替代Ser (P-5)是耐受的。剩余部分氨基酸的取代 非保守氨基酸占据的位置通常会产生很高的产率 乳沟的水平。然而，非保守位置上的一些替换 是不被容忍的。例如，用Gly或Glu代替Gln(P-4) 和用Val或Glu替代Lys(P-6)时，均产生较低水平的 乳沟。总体而言，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而推断。除了……之外 这八个氨基酸序列，本研究还表明，氨基酸 紧跟在NS1-NS2A之后，裂解位点在裂解中起作用。

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.