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

登革病毒多聚蛋白NS1-NS2A区缺失分析 指出NS1上至少有8个氨基酸 非结构蛋白NS1之间的切割需要C末端 和NS2A将会发生。这8个氨基酸序列的比较 登革4型病毒与其他黄病毒的相似序列 鉴定了其中Ala(-1)、Val(-3)、Ser(-5)、Val(-7)、 和Met/Leu(-8)是保守的。其他位置的氨基酸是不同的。我们 感兴趣的是确定观察到的序列基序是否 最适合在NS1-NS2a结合处切割多聚蛋白。 构建了含有一种氨基酸的NS1-NS2突变体 在裂解位点上游的不同位置或在 紧靠下游的位置。这些氨基酸的作用 然后分析了NS1-NS2a裂解上的取代。结果是 结果表明，Ala(-1)或Val(-3)的大多数取代得到 主要是未裂解的NS1-NS2a融合蛋白。另一方面， 替换Thr(-2)对卵裂影响很小或没有影响。这些 结果与建议的共识序列是一致的 黄病毒之间的序列比较。的几个替换 Gly(+1)产生的裂解减少，表明Gly在这个位置 也在乳沟中发挥作用。含有单一氨基酸的突变体 其余五个职位中的一个职位的替补是 已构建，正在进行确定其NS1-NS2裂解的工作 表型。一组氨基酸替代突变具有 选择了不同对NS1-NS2A切割效率的影响 掺入全长c DNA，以及体外衍生的RNA 转录本被用来转染细胞并恢复活的登革热病毒 变种人。这些可存活的突变体显示出生长受限。 通过斑块分析。沙门氏菌其他病毒生长特性的评价 这些突变体正在进行中。

Deletion analysis of the NS1-NS2A region of the dengue virus polyprotein indicated that a minimum length of eight amino acids at the NS1 C-terminus is required for cleavage between non-structural proteins NS1 and NS2A to take place. Comparison of this eight amino acid sequence of dengue type 4 virus with the analogous sequences of other flaviviruses identified a sequence motif in which Ala(-1), Val(-3), Ser(-5), Val(-7), and Met/Leu(-8) are conserved. Amino acids at other positions vary. We were interested in determining whether the observed sequence motif is optimal for cleavage of the polyprotein at the NS1-NS2A junction. Mutants of NS1-NS2 were constructed that contained an amino acid substitution at different positions upstream of the cleavage site or at the immediate downstream position. The effect of these amino acid substitutions on cleavage of NS1-NS2A was then analyzed. The results showed that most substitutions of Ala(-1) or Val(-3) yielded predominantly uncleaved NS1-NS2A fusion protein. On the other hand, replacement of Thr(-2) had little or no effect on cleavage. These results are in agreement with the proposed consensus sequence deduced by comparison of sequences among flaviviruses. Several substitutions of Gly(+1) yielded reduced cleavage indicating that Gly at this position also plays a role in cleavage. Mutants that contain a single amino acid substitution in one of the remaining five positions have been constructed, and work is in progress to define their NS1-NS2 cleavage phenotype. A panel of amino acid substitution mutations that had a varying effect upon efficiency of NS1-NS2A cleavage were selected for incorporation into the full-length cDNA, and in vitro derived RNA transcripts were used to transfect cells and recover viable dengue virus mutants. These viable mutants exhibited growth restriction as revealed by plaque analysis. Evaluation of other viral growth properties of these mutants is in progess.