Role of NS1 for Dengue virus replication and pathogenicity and ways to counteract it

登革病毒（DENV）是登革热的病原体，属于黄病毒科黄病毒属。DENV每年感染约3.9亿人，其中约500.000人患有严重疾病，约20.000人死亡。在过去20年中，向世卫组织报告的登革热病例数量从2000年的50万例稳步增加到2019年的400多万例。尽管这种惊人的增加和医学相关性，但没有直接的抗病毒疗法可用于治疗DENV感染，并且唯一批准的疫苗具有有限的功效，并且取决于疫苗接受者的基线血清状态。登革病毒感染的一个已知致病因子是多功能病毒蛋白NS 1，其是RNA复制所需的并且与登革疾病的严重程度相关。因此，在这项联合提案中，我们联手解开了NS 1产生和分泌的机制。在我们的实验室独立获得的初步结果确定了几个宿主细胞因子（蛋白质和脂质）可能参与NS 1的生产和分泌。其中一个因素是importin-β 1（IMPβ1），它可以被临床批准的药物伊维菌素靶向，我们发现它在体外和感染患者中影响NS 1的丰度和分泌。基于这些结果，我们建议深入表征IMPβ1和伊维菌素在NS 1产生和分泌中的作用，并确定其他关键参与DENV NS 1合成和从细胞释放的宿主因子和途径。将密切合作，努力实现四个相辅相成、高度相关的目标。这些措施如下：首先，研究NS 1的分泌途径和IMPβ1对NS 1稳定性和分泌的作用机制，其次，研究伊维菌素的作用机制和提高其抗NS 1效价的方法;第三，为了超越IMPβ1和伊维菌素，并寻找参与NS 1产生和分泌的其他宿主细胞因子和途径，我们将利用我们拥有的丰富数据源收集（互补的蛋白质组和脂质组数据）;第四，将我们关于NS 1分泌和所涉及的宿主因子的体外数据与临床数据相关联。两个实验室合作的原因一方面是对登革热病毒的共同兴趣，特别是NS 1在病毒复制周期和发病机制中的作用，另一方面是互补的专业知识和基础设施。这些包括尖端的分子病毒学和成像方法（光学和电子显微镜）和NS 1致病性模型，临床病毒学和患者队列。鉴于NS 1是一个重要的致病因素，获得的知识可能有助于改善感染患者的治疗，以降低严重的登革热疾病表现。

Dengue virus (DENV), the causative agent of dengue fever, belongs to the Flavivirus genus within the Flaviviridae family. DENV infects ~390 million people per year, of which ~500.000 experience severe disease and ~20.000 die. Within the last 20 years the number of dengue cases reported to WHO increased steadily from 500.000 in 2000 to over 4 million in 2019. Despite this alarming increase and the medical relevance, there is no direct antiviral therapy available to treat DENV infections and the only approved vaccine has limited efficacy and depends on baseline serostatus of the vaccine recipient. One known pathogenicity factor of Dengue virus infection is the multifunctional viral protein NS1, which is required for RNA replication and associated with dengue disease severity. Therefore, in this joint proposal we teamed up to unravel the mechanisms underlying NS1 production and secretion. Preliminary results obtained independently in our laboratories identified several host cell factors (proteins and lipids) possibly being involved in NS1 production and secretion. One of these factors is importin-beta 1 (IMPβ1) that can be targeted by the clinically approved drug ivermectin, which we found to affect NS1 abundance and secretion both in vitro and in infected patients. Based on these results, here we propose to characterize in-depth the role of IMPβ1 and ivermectin in NS1 production and secretion and to identify additional host factors and pathways critically involved in DENV NS1 synthesis and release from cells. Four complementary and highly inter-related aims will be pursued in close collaboration. These are as follows: First, to study the secretory pathway of NS1 and the mechanism how IMPβ1 contributes to NS1 stability and secretion; second, to decipher the mode-of-action of ivermectin and ways to increase its anti-NS1 potency; third, to go beyond IMPβ1 and ivermectin and search for additional host cell factors and pathways involved in NS1 production and secretion for which we will take advantage of the rich data source we have collected (complementary proteome and lipidome data); forth, to correlate our in vitro data on NS1 secretion and involved host factor(s) with clinical data. The reasons to join forces by the two laboratories are on the one hand the common interest in Dengue virus, especially the role of NS1 in the viral replication cycle and pathogenesis, on the other hand the complementary expertise and infrastructures. These include cutting-edge molecular virology and imaging approaches (light and electron microscopy) and NS1 pathogenicity models, clinical virology and patient cohorts. Given that NS1 is an important pathogenicity factor, gained knowledge might help to improve treatment of infected patients in order to lower severe dengue disease manifestations.