PROCESSING OF DENGUE VIRUS POLYPROTEIN NS3-NS4A-NS4B-NS5 DOMAIN

含有不同长度登革DNA的重组牛痘病毒 构建了编码NS 2B-NS 3-NS 4A-NS 4 B-NS 5结构域的多聚蛋白 为了检查用于表达的切割策略， 这一地区。最初，含有抗 通过针对适当的trp免疫制备登革NS 3或NS 5 E融合蛋白。用牛痘重组v（NS 2B-NS 3-NS 4A-NS 4A）感染的细胞 NS 4 B-NS 5）产生NS 2B、NS 3和NS 5，而重组的牛痘病毒产生NS 2B、NS 3和NS 5。 v（NS 3-NS 4A-NS 4 B-NS 5）产生未切割的多聚蛋白，其由 重组体中的登革DNA序列。因此，NS 2B是必要的， 下游非结构蛋白的加工。卵裂缺陷 NS 3-NS 4A-NS 4 B-NS 5多聚蛋白在共感染期间被互补 用v（NS 2B-30%NS3）或v（NS 2B）加v（30%NS3）表示， 产生可通过适当的抗血清鉴定的NS 3和NS 5。它 似乎登革热病毒NS 3-NS 4A和NS 4 B-NS 5连接处的切割 多聚蛋白需要由反式作用NS 2B和N NS 3的末端30%。NS 2B和NS 3都不能单独介导这些。 裂缝NS 4A-NS 4 B连接处的切割似乎是由一种蛋白质介导的。 因为长疏水序列延伸先于 预测的切割位点。支持这种裂解机制的证据 也从分析牛痘的登革蛋白产物中获得 重组v（NS 4A-NS 4 B-NS 5）感染的细胞，其中NS 4 B-NS 5 从多聚蛋白被检测到。最后，NS 5抗血清检测到一种 重组牛痘中部分切割的NS 5（1-198）的缩短形式 v（NS 4 B-NS 5）或v（NS 4 B-NS 5 [1-198]）感染的细胞。这一发现表明 在不存在NS 2B和NS 3的情况下，在NS 4 B-NS 5连接处的切割可以 发生在低水平，推测由NS 4功能介导。这些结果 这表明黄病毒非结构蛋白的加工可以利用 超过一种切割策略，并且这些切割步骤可能是复杂的 并且受到高度监管。

Recombinant vaccinia viruses that contain varying lengths of dengue DNA coding for the polyprotein NS2B-NS3-NS4A-NS4B-NS5 domain were constructed in order to examine the cleavage strategy that is utilized for expression of this region. Initially, mono-specific antiserum containing antibodies to dengue NS3 or NS5 were prepared by immunization against the appropriate trp E fusion protein. Cells infected with vaccinia recombinant v(NS2B-NS3-NS4A- NS4B-NS5) produced NS2B, NS3, and NS5, whereas vaccinia recombinant v(NS3-NS4A-NS4B-NS5) produced an uncleaved polyprotein encoded by the dengue DNA sequence in the recombinant. Thus, NS2B is necessary for proper processing of the downstream nonstructural proteins. The defect in cleavage of the NS3-NS4A-NS4B-NS5 polyprotein was complemented during coinfection with v(NS2B-30%NS3) or v(NS2B) plus v(30%NS3) as indicated by the production of NS3 and NS5 identifiable by the appropriate antiserum. It appears that cleavage at the NS3-NS4A and NS4B-NS5 junctions of the dengue polyprotein requires functions provided by trans acting NS2B and the N terminal 30% of NS3. Neither NS2B nor NS3 alone is able to mediate these cleavages. Cleavage at the NS4A-NS4B junction appears to be mediated by a specific signalase since a long stretch of hydrophobic sequence precedes the predicted cleavage site. Evidence supporting this cleavage mechanism was also obtained from analysis of the dengue protein products of vaccinia recombinant v(NS4A-NS4B-NS5) infected cells in which cleavage of NS4B-NS5 from the polyprotein was detected. Finally, NS5 antiserum detected a partially cleaved shortened form of NS5 (1-198) in vaccinia recombinant v(NS4B-NS5) or v(NS4B-NS5[1-198]) infected cells. This finding indicates that in the absence of NS2B and NS3, cleavage at the NS4B-NS5 junction can occur at a low level, presumably mediated by an NS4 function. These results suggest that processing of flavivirus nonstructural proteins may utilize more than one cleavage strategy and these cleavage steps are likely complex and highly regulated.