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

登革热病毒多蛋白NS3-NS4A-NS4B-NS5结构域的加工

• 批准号: 3790819
• 负责人: C J LAI
• 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/3790819
• 关键词: Flaviviridae; active sites; dengue virus; endopeptidases; exopeptidase; gene expression; genetic manipulation; immunoprecipitation; infection; protein sequence; protein signal sequence; protein structure function; tissue /cell culture; vaccinia virus; virus genetics; virus protein

The cleavage mechanism utilized for processing of the NS3-NS4A-NS4B-NS5 domain of the dengue virus polyprotein was studied by using the vaccinia virus expression system. Recombinant vaccinia viruses vNS2B-NS3-NS4A-NS5, vNS3-NS4A-NS4B-NS5, vNS4A-NS4B-NS5, and vNS4B-NS5 were constructed. These recombinants were used to infect cells and the labelled lysates were analyzed by immunoprecipitation. Recombinant vNS2B-NS3-NS4A-NS4B-NS5 expressed the authentic NS3 and NS5 proteins, but the other recombinants produced uncleaved polyproteins. These findings indicate the NS2B is required for processing of the downstream nonstructural proteins, including the NS3/NS4A and NS4B/NS5 junctions, both of which contain a dibasic amino acid sequence preceding the cleavage site. The flavivirus NS4A/NS4B cleavage site follows a long hydrophobic sequence. The polyprotein NS4A- NS4B-NS5 was cleaved at the NS4A/NS4B junction in the absence of other dengue virus functions. One interpretation for this finding is that NS4A/NS4B cleavage is mediated by a host protease, presumably a signal peptidase. Although vNS3-NS4A-NS4B-NS5 expressed only the polyprotein, earlier results demonstrated that cleavage at the NS4A/NS4B junction occurred when an analogous recombinant, vNS3-NS4A-84%NS4B, was expressed. Thus, it appears that uncleaved NS3 plus NS5 inhibit NS4A/NS4B cleavage presumably because the putative signal sequence is not accessible for recognition by the responsible protease. Finally, recombinants that expressed an uncleaved NS4B-NS5 polyprotein, such as vNS4A-NS4B-NS5 or vNS4B-NS5, produced NS5 when complemented with vNS2B-30%NS3 or with vNS2B plus v30%NS3. These results indicate that cleavage at the NS4B/NS5 junction can be mediated by NS2B and NS3 in trans.

用于加工NS 3-NS 4A-NS 4 B-NS 5的切割机制 用痘苗病毒研究了登革病毒多聚蛋白的结构域， 病毒表达系统 重组牛痘病毒vNS 2B-NS 3-NS 4A-NS 5， 构建vNS 3-NS 4A-NS 4 B-NS 5、vNS 4A-NS 4 B-NS 5和vNS 4 B-NS 5。 这些 重组体用于感染细胞，并将标记的裂解物 通过免疫沉淀分析。 重组vNS 2B-NS 3-NS 4A-NS 4 B-NS 5 表达了真实的NS 3和NS 5蛋白，但其他重组子表达了真实的NS 3和NS 5蛋白， 产生未裂解的多聚蛋白。 这些发现表明NS 2B是 下游非结构蛋白的加工所需的，包括 NS 3/NS 4A和NS 4 B/NS 5连接，两者都含有一个二元氨基 切割位点之前的酸序列。 黄病毒NS 4A/NS 4 B 切割位点遵循长的疏水序列。 多聚蛋白NS 4A- NS 4 B-NS 5在不存在其他酶的情况下在NS 4A/NS 4 B连接处被切割。 登革热病毒的功能。 对这一发现的一种解释是， NS 4A/NS 4 B切割由宿主蛋白酶介导，推测是一种信号 肽酶 虽然vNS 3-NS 4A-NS 4 B-NS 5仅表达多聚蛋白， 早期的结果表明，在NS 4A/NS 4 B连接处的切割 当表达类似的重组体vNS 3-NS 4A-84%NS4B时，发生了突变。 因此，似乎未切割的NS 3加NS 5抑制NS 4A/NS 4 B切割 可能是因为推定的信号序列对于 由负责的蛋白酶识别。 最后， 表达未切割的NS 4 B-NS 5多蛋白，例如vNS 4A-NS 4 B-NS 5或 vNS 4 B-NS 5，当与vNS 2B-30%NS3或与vNS 2B互补时产生NS 5 加上v30%NS3。 这些结果表明，NS 4 B/NS 5处的切割 连接可以由NS 2B和NS 3反式介导。