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

含有不同长度登革热DNA的重组痘苗病毒 构建了NS2B-NS3-NS4A-NS4B-NS5结构域的编码基因 为了检查用于表达的切割策略 这一地区的。最初，含有抗体的单特异性抗血清 登革热NS3或NS5是通过针对适当的Trp免疫制备的 E融合蛋白。重组痘苗病毒(NS2B-NS3-NS4A-)感染细胞 NS4B-NS5)产生NS2B、NS3和NS5，而重组痘苗病毒 V(NS3-NS4A-NS4B-NS5)产生一个由 登革热DNA序列中的重组。因此，NS2B对于正确的 下游非结构蛋白的加工。卵裂的缺陷 NS3-NS4A-NS4B-NS5多聚蛋白在共感染过程中互补 用v(NS2B-30%NS3)或v(NS2B)加v(30%NS3)表示 产生可由适当的抗血清识别的NS3和NS5。它 登革热NS3-NS4A和NS4B-NS5连接处的裂解似乎 多聚蛋白需要由反式NS2B和N提供的功能 NS3的30%的终端。无论是NS2B还是NS3都不能单独调解这些问题 乳沟。NS4A-NS4B连接处的切割似乎是由一种 因为一段很长的疏水序列先于 预测的卵裂位置。支持这种切割机制的证据 也是通过分析牛痘病毒的登革热蛋白产物而获得的 重组v(NS4A-NS4B-NS5)感染细胞中NS4B-NS5的裂解 从多聚蛋白中检测到。最后，NS5抗血清检测到一种 重组痘苗病毒中部分切割的NS5(1-198)短型 V(NS4B-NS5)或v(NS4B-NS5[1-198])感染细胞。这一发现表明 在没有NS2B和NS3的情况下，NS4B-NS5连接处的切割可以 发生在低水平，推测是由NS4功能调节的。这些结果 提示黄病毒非结构蛋白的加工可能利用 不止一种切割策略，这些切割步骤可能很复杂 并受到严格监管。

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.