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

正链编码的多蛋白的蛋白水解加工 登革病毒（以及其他黄病毒）的RNA病毒基因组是一种 病毒基因表达的先决条件。我们部门最近的证据 表明NS 1 C-末端的最小长度为8个氨基酸 在NS 1-NS 2A接合点之前需要切割。 本研究的目的是分析与人类基因组序列相关的 在该区域的处理期间NS 1-NS 2A结的最佳切割 病毒蛋白质。为此目的，几个氨基酸取代， 在NS 1-NS 2A连接的位置-1（Gly或Ala）、-2（Thr）或-3（瓦尔） 的登革4序列，并将所得突变体NS 1-NS 2A 由重组牛痘病毒表达。突变序列分析 由牛痘病毒重组体表达的一种重组体表明， -1位的Ala或-3位的瓦尔产生未切割的NS 1-NS 2A融合体 蛋白质表明，在位置-1和-3的氨基酸，即 在黄病毒中严格保守，最适合切割这种 交界处。另一方面，在位置-2处的Thr的替换， 在黄病毒中不保守，只有轻微到中度的 对卵裂的负面影响。位置处氨基酸序列的变异 +1发生在黄病毒中。尽管如此，在这个位点的替换 导致切割减少42%至90%。的工作正在进行 在其它位置（-4至-8）引入氨基酸取代， 我们对NS 1-NS 2A连接处的序列基序的分析。还有， 构建含有策略性突变的全长登革cDNA NS 1-NS 2A切割位点正在进行中。突变病毒回收自 将评价转染细胞在细胞中的病毒生长情况。 培养的细胞和在受感染的实验动物宿主中的毒力。 最终，这种分析可能导致分离减毒登革病毒 这些突变体具有用于人类活病毒疫苗的潜力。

Proteolytic processing of the polyprotein encoded by the positive strand RNA viral genome of dengue virus (as well as other flaviviruses) is a prerequisite for viral gene expression. Recent evidence from our Section indicates that a minimum length of 8 amino acids at the NS1 C-terminus preceding the NS1-NS2A junction is required for cleavage to take place. This study was initiated to analyze the amino acid sequence motif that is optimal cleavage of the NS1-NS2A junction during processing of this region of the viral protein. For this purpose, several amino acid substitutions, at position -1 (Gly or Ala), -2 (Thr), or -3 (Val) of the NS1-NS2A junction of the dengue 4 sequence were introduced and the resulting mutant NS1-NS2A was expressed by a recombinant vaccinia virus. Analysis of mutant sequences expressed by a vaccinia virus recombinant indicated that substitution of Ala at position -1, or Val at -3, yielded an uncleaved NS1-NS2A fusion protein suggesting that the amino acids at positions -1 and -3, that are strictly conserved among flaviviruses, are optimal for cleavage of this junction. On the other hand, replacement of Thr at position -2, a position that is not conserved among flaviviruses, had only a slight to moderate negative effect on cleavage. Variation in amino acid sequence at position +1 occurs among flaviviruses. Nonetheless, substitutions at this site resulted in 42% to 90% reduction in cleavage. Work is in progress to introduce amino acid substitutions at other positions (-4 to -8) to extend our analysis of the sequence motif at the NS1-NS2A junction. Also, construction of full length dengue cDNA containing mutations at strategic NS1-NS2A cleavage sites is underway. Mutant viruses recovered from transfected cells will be evaluated with respect to viral growth in cultured cells and virulence in an infected experimental animal host. Ultimately, this analysis may lead to isolation of attenuated dengue virus mutants that have the potential for use in a live virus vaccine for humans.