Hori H, Lai C J
Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892.
J Virol. 1990 Sep;64(9):4573-7. doi: 10.1128/JVI.64.9.4573-4577.1990.
The length of amino acid sequence at the NS1-NS2A juncture of dengue virus that is required for specific cleavage effected by the cis-acting function of NS2A was identified by deletion analysis. Recombinant DNA sequences of NS1-NS2A, each containing a deletion in NS1 followed by a sequence of 3 to 20 amino acids at the C terminus of NS1 preceding the cleavage site, were constructed and expressed with vaccinia virus as a vector. The NS1 product of recombinant vaccinia virus-infected cells was immunoprecipitated and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The occurrence of cleavage between NS1 and NS2A was indicated by the appearance of shortened NS1. Failure to cleave this site yielded a large NS1-NS2A fusion protein. This analysis indicated that a minimum length of eight amino acids at the NS1 C terminus preceding the NS1-NS2A juncture is required for cleavage to take place. Comparison of this eight-amino-acid sequence of the NS1 C terminus of dengue type 4 virus with the analogous sequences of 12 other flaviviruses suggests that the consensus cleavage site sequence is as follows: (table; see text)
通过缺失分析确定了登革病毒NS1-NS2A交界处的氨基酸序列长度，该长度是NS2A顺式作用功能实现特异性切割所必需的。构建了NS1-NS2A的重组DNA序列，每个序列在NS1中都有一个缺失，随后在切割位点之前的NS1 C末端有一段3至20个氨基酸的序列，并以痘苗病毒为载体进行表达。对重组痘苗病毒感染细胞的NS1产物进行免疫沉淀，并通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳进行分析。NS1缩短表明在NS1和NS2A之间发生了切割。未能切割该位点则产生了一个大的NS1-NS2A融合蛋白。该分析表明，在NS1-NS2A交界处之前的NS1 C末端，至少需要八个氨基酸的长度才能发生切割。将登革4型病毒NS1 C末端的这八个氨基酸序列与其他12种黄病毒的类似序列进行比较，结果表明共有切割位点序列如下：（表格；见正文）
