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: 3803254
• 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/3803254
• 关键词: Flaviviridae; chimeric proteins; complementary DNA; dengue virus; molecular biology; molecular pathology; mutant; phenotype; plaque assay; point mutation; protein sequence; proteolysis; tissue /cell culture; transfection; virus RNA; virus genetics; virus protein; virus replication

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.

登革病毒多聚蛋白NS1-NS2A区的缺失分析 表明NS1处的最小长度为8个氨基酸， 非结构蛋白NS1之间的切割需要C-末端 NS2A的出现。 这八个氨基酸序列的比较， 具有其他黄病毒类似序列的登革4型病毒 鉴定了其中Ala（-1），瓦尔（-3），Ser（-5），瓦尔（-7）， 和Met/Leu（-8）是保守的。 其他位置的氨基酸各不相同。 我们 有兴趣确定观察到的序列基序是否是 最适合于在NS1-NS2A连接处切割多蛋白。 构建了NS1-NS2的突变体，其含有一个氨基酸 在切割位点上游的不同位置或在 直接下游位置。 这些氨基酸的作用 然后分析NS1-NS2A切割时的取代。 结果 结果表明，大多数Ala（-1）或瓦尔（-3）的取代产生了 主要是未切割的NS1-NS2A融合蛋白。 另一方面，在一项研究中， Thr（-2）的替换对卵裂几乎没有影响。 这些 结果与由以下推导的建议的共有序列一致： 黄病毒序列的比较。 的几个替换 Gly（+1）产生减少的切割，表明该位置的Gly 也在卵裂中起作用。 含有单一氨基酸的突变体 其余五个职位中的一个已被替换， 构建，工作正在进行中，以确定其NS1-NS2切割 表型 一组氨基酸置换突变， 选择对NS1-NS2A切割效率的不同影响， 掺入全长cDNA和体外衍生的RNA 转录物用于转染细胞并回收活的登革病毒 变种人 这些活的突变体表现出生长限制， 通过斑块分析。 其他病毒生长特性的评价 这些变种人是无辜的