Chatel-Chaix Laurent, Fischl Wolfgang, Scaturro Pietro, Cortese Mirko, Kallis Stephanie, Bartenschlager Marie, Fischer Bernd, Bartenschlager Ralf
Department of Infectious Diseases, Molecular Virology, University of Heidelberg, Heidelberg, Germany.
Computational Genome Biology, German Cancer Research Center, Heidelberg, Germany.
J Virol. 2015 Jul;89(14):7170-86. doi: 10.1128/JVI.00867-15. Epub 2015 Apr 29.
Dengue virus (DENV) infection causes the most prevalent arthropod-borne viral disease worldwide. Approved vaccines are not available, and targets suitable for the development of antiviral drugs are lacking. One possible drug target is nonstructural protein 4B (NS4B), because it is absolutely required for virus replication; however, its exact role in the DENV replication cycle is largely unknown. With the aim of mapping NS4B determinants critical for DENV replication, we performed a reverse genetic screening of 33 NS4B mutants in the context of an infectious DENV genome. While the majority of these mutations were lethal, for several of them, we were able to select for second-site pseudoreversions, most often residing in NS4B and restoring replication competence. To identify all viral NS4B interaction partners, we engineered a fully viable DENV genome encoding an affinity-tagged NS4B. Mass spectrometry-based analysis of the NS4B complex isolated from infected cells identified the NS3 protease/helicase as a major interaction partner of NS4B. By combining the genetic complementation map of NS4B with a replication-independent expression system, we identified the NS4B cytosolic loop-more precisely, amino acid residue Q134-as a critical determinant for NS4B-NS3 interaction. An alanine substitution at this site completely abrogated the interaction and DENV RNA replication, and both were restored by pseudoreversions A69S and A137V. This strict correlation between the degree of NS4B-NS3 interaction and DENV replication provides strong evidence that this viral protein complex plays a pivotal role during the DENV replication cycle, hence representing a promising target for novel antiviral strategies.
With no approved therapy or vaccine against dengue virus infection, the viral nonstructural protein 4B (NS4B) represents a possible drug target, because it is indispensable for virus replication. However, little is known about its precise structure and function. Here, we established the first comprehensive genetic interaction map of NS4B, identifying amino acid residues that are essential for virus replication, as well as second-site mutations compensating for their defects. Additionally, we determined the NS4B viral interactome in infected cells and identified the NS3 protease/helicase as a major interaction partner of NS4B. We mapped residues in the cytosolic loop of NS4B as critical determinants for interaction with NS3, as well as RNA replication. The strong correlation between NS3-NS4B interaction and RNA replication provides strong evidence that this complex plays a pivotal role in the viral replication cycle, hence representing a promising antiviral drug target.
登革病毒（DENV）感染是全球最常见的节肢动物传播的病毒性疾病。目前尚无获批的疫苗，且缺乏适合开发抗病毒药物的靶点。一个可能的药物靶点是非结构蛋白4B（NS4B），因为它是病毒复制绝对必需的；然而，其在DENV复制周期中的确切作用在很大程度上尚不清楚。为了绘制对DENV复制至关重要的NS4B决定簇图谱，我们在感染性DENV基因组背景下对33个NS4B突变体进行了反向遗传筛选。虽然这些突变中的大多数是致死性的，但对于其中一些突变，我们能够选择第二位点假回复突变，这些突变大多位于NS4B中并恢复了复制能力。为了鉴定所有病毒NS4B相互作用伙伴，我们构建了一个编码亲和标签NS4B的完全可行的DENV基因组。对从感染细胞中分离的NS4B复合物进行基于质谱的分析，确定NS3蛋白酶/解旋酶是NS4B的主要相互作用伙伴。通过将NS4B的遗传互补图谱与一个不依赖复制的表达系统相结合，我们确定NS4B胞质环——更确切地说是氨基酸残基Q134——是NS4B与NS3相互作用的关键决定因素。该位点的丙氨酸取代完全消除了相互作用和DENV RNA复制，而A69S和A137V假回复突变恢复了这两者。NS4B与NS3相互作用程度和DENV复制之间的这种严格相关性提供了强有力的证据，表明这种病毒蛋白复合物在DENV复制周期中起关键作用，因此是新型抗病毒策略的一个有前景的靶点。
由于没有针对登革病毒感染的获批治疗方法或疫苗，病毒非结构蛋白4B（NS4B）是一个可能的药物靶点，因为它对病毒复制不可或缺。然而，对其精确结构和功能知之甚少。在这里，我们建立了首个全面的NS4B遗传相互作用图谱，鉴定了对病毒复制至关重要的氨基酸残基，以及补偿其缺陷的第二位点突变。此外，我们确定了感染细胞中的NS4B病毒相互作用组，并确定NS3蛋白酶/解旋酶是NS4B的主要相互作用伙伴。我们将NS4B胞质环中的残基定位为与NS3相互作用以及RNA复制的关键决定因素。NS3与NS4B相互作用和RNA复制之间的强相关性提供了强有力的证据，表明这种复合物在病毒复制周期中起关键作用，因此是一个有前景的抗病毒药物靶点。
