Voßmann Stephanie, Wieseler Janett, Kerber Romy, Kümmerer Beate Mareike
Department of Virology, Bernhard-Nocht-Institute for Tropical Medicine, Hamburg, Germany.
Institute of Virology, University of Bonn Medical Centre, Bonn, Germany.
J Virol. 2015 May;89(9):4951-65. doi: 10.1128/JVI.03351-14. Epub 2015 Feb 18.
The flavivirus NS2A protein is involved in the assembly of infectious particles. To further understand its role in this process, a charged-to-alanine scanning analysis was performed on NS2A encoded by an infectious cDNA clone of yellow fever virus (YFV). Fifteen mutants containing single, double, or triple charged-to-alanine changes were tested. Five of them did not produce infectious particles, whereas efficient RNA replication was detectable for two of the five NS2A mutants (R22A-K23A-R24A and R99A-E100A-R101A mutants). Prolonged cultivation of transfected cells resulted in the recovery of pseudorevertants. Besides suppressor mutants in NS2A, a compensating second-site mutation in NS3 (D343G) arose for the NS2A R22A-K23A-R24A mutant. We found this NS3 mutation previously to be suppressive for the NS2Aα cleavage site Q189S mutant, also deficient in virion assembly. In this study, the subsequently suggested interaction between NS2A and NS3 was proven by coimmunoprecipitation analyses. Using selectively permeabilized cells, we could demonstrate that the regions encompassing R22A-K23A-R24A and Q189S in NS2A are localized to the cytoplasm, where NS3 is also known to reside. However, the defect in particle production observed for the NS2A R22A-K23A-R24A and Q189S mutants was not due to a defect in physical interaction between NS2A and NS3, as the NS2A mutations did not interrupt NS3 interaction. In fact, a region just upstream of R22-K23-R24 was mapped to be critical for NS2A-NS3 interaction. Taken together, these data support a complex interplay between YFV NS2A and NS3 in virion assembly and identify a basic cluster in the NS2A N terminus to be critical in this process.
Despite an available vaccine, yellow fever remains endemic in tropical areas of South America and Africa. To control the disease, antiviral drugs are required, and an understanding of the determinants of virion assembly is central to their development. In this study, we identified a basic cluster of amino acids in the N terminus of YFV NS2A which inhibited virion assembly upon mutation. The defect was rescued by a spontaneously occurring mutation in NS3. Our study proves an interaction between NS2A and NS3, which, remarkably, was maintained for the NS2A mutant in the presence and absence of the NS3 mutation. This suggests a role for other viral and/or cellular proteins in virion assembly. Residues important for YFV virion production reported here only partially coincided with those reported for other flaviviruses, suggesting that the determinants for particle production are virus specific. Reconstruction of a YFV encoding tagged NS2A paves the way to identify further NS2A interaction partners.
黄病毒NS2A蛋白参与感染性颗粒的组装。为了进一步了解其在此过程中的作用，对黄热病毒（YFV）感染性cDNA克隆编码的NS2A进行了电荷到丙氨酸的扫描分析。测试了15个含有单、双或三个电荷到丙氨酸变化的突变体。其中5个不产生感染性颗粒，而5个NS2A突变体中的2个（R22A-K23A-R24A和R99A-E100A-R101A突变体）可检测到有效的RNA复制。转染细胞的长时间培养导致假回复体的恢复。除了NS2A中的抑制突变体外，NS2A R22A-K23A-R24A突变体在NS3中出现了一个补偿性的第二位点突变（D343G）。我们之前发现这个NS3突变对NS2Aα切割位点Q189S突变体具有抑制作用，该突变体在病毒体组装方面也存在缺陷。在本研究中，随后提出的NS2A与NS3之间的相互作用通过共免疫沉淀分析得到了证实。使用选择性通透细胞，我们可以证明NS2A中包含R22A-K23A-R24A和Q189S的区域定位于细胞质，NS3也已知存在于该区域。然而，NS2A R22A-K23A-R24A和Q189S突变体在颗粒产生方面的缺陷并不是由于NS2A与NS3之间的物理相互作用缺陷，因为NS2A突变并未中断NS3相互作用。事实上，R22-K23-R24上游的一个区域被确定为NS2A-NS3相互作用的关键区域。综上所述，这些数据支持YFV NS2A和NS3在病毒体组装中存在复杂的相互作用，并确定NS2A N端的一个碱性簇在这个过程中至关重要。
尽管有可用的疫苗，但黄热病在南美洲和非洲的热带地区仍然流行。为了控制这种疾病，需要抗病毒药物，而了解病毒体组装的决定因素对于其开发至关重要。在本研究中，我们在YFV NS2A的N端鉴定了一个氨基酸碱性簇，该簇在突变时抑制病毒体组装。NS3中的一个自发突变挽救了该缺陷。我们的研究证明了NS2A与NS3之间的相互作用，值得注意的是，在存在和不存在NS3突变的情况下，NS2A突变体的这种相互作用都得以维持。这表明其他病毒和/或细胞蛋白在病毒体组装中发挥作用。此处报道的对YFV病毒体产生重要的残基仅部分与其他黄病毒报道的残基一致，这表明颗粒产生的决定因素是病毒特异性的。编码带标签NS2A的YFV的重建为鉴定进一步的NS2A相互作用伙伴铺平了道路。
