In the last few decades, dengue virus, an arbovirus, has spread to over 120 countries. Although a vaccine has been approved in some countries, limitations on its effectiveness and a lack of effective antiviral treatments reinforce the need for additional research. The functions of several viral nonstructural proteins are essentially unknown. To better understand the functions of these proteins and thus dengue virus pathogenesis, we embarked on a genomewide transposon mutagenesis screen with next-generation sequencing to determine sites in the viral genome that tolerate 15-nucleotide insertions. Using this approach, we generated support for several published predicted transmembrane and enzymatic domains. Next, we created 7 mutants containing the 15-nucleotide insertion from the original selection and found 6 of them were capable of replication in both mammalian and mosquito tissue culture cells. Interestingly, one mutation had a significant impairment of viral assembly, and this mutation may lead to a better understanding of viral assembly and release. In addition, we created a fully infectious virus expressing a functionally tagged NS4B protein, which will provide a much-needed tool to elucidate the role of NS4B in viral pathogenesis.

IMPORTANCE Dengue virus is a mosquito-borne virus distributed in tropical and subtropical regions globally that can result in hospitalization and even death in some cases. Although a vaccine exists, its limitations and a lack of approved antiviral treatments highlight our limited understanding of dengue virus pathogenesis and host immunity. The functions of many viral proteins are poorly understood. We used a previously published approach using transposon mutagenesis to develop tools to study these proteins' functions by adding insertions randomly throughout the viral genomes. These genomes were transferred into cells, and infectious progeny were recovered to determine sites that tolerated insertions, as only the genomes that tolerated insertions would be able to propagate. Using these results, we created viruses with epitope tags, one in the viral structural protein Capsid and one in the viral nonstructural protein NS4B. Further investigation of these mutants may elucidate the roles of Capsid and NS4B during dengue virus infections.

在过去的几十年里，登革热病毒（一种虫媒病毒）已传播到 120 多个国家。尽管疫苗已在一些国家获得批准，但其有效性的限制和有效抗病毒治疗的缺乏增强了进行额外研究的必要性。几种病毒非结构蛋白的功能基本上是未知的。为了更好地了解这些蛋白质的功能以及登革热病毒的发病机制，我们开始使用新一代测序进行全基因组转座子诱变筛选，以确定病毒基因组中耐受 15 个核苷酸插入的位点。使用这种方法，我们为几个已发表的预测跨膜和酶域提供了支持。接下来，我们创建了 7 个含有来自原始选择的 15 个核苷酸插入的突变体，并发现其中 6 个能够在哺乳动物和蚊子组织培养细胞中复制。有趣的是，一种突变对病毒组装有显着损害，这种突变可能会导致人们更好地了解病毒组装和释放。此外，我们创建了一种表达功能标记 NS4B 蛋白的完全感染性病毒，这将为阐明 NS4B 在病毒发病机制中的作用提供急需的工具。

重要性登革热病毒是一种分布在全球热带和亚热带地区的蚊媒病毒，在某些情况下可能导致住院甚至死亡。尽管存在疫苗，但其局限性和缺乏经批准的抗病毒治疗凸显了我们对登革热病毒发病机制和宿主免疫的了解有限。人们对许多病毒蛋白的功能知之甚少。我们使用之前发表的转座子诱变方法来开发工具，通过在病毒基因组中随机添加插入来研究这些蛋白质的功能。这些基因组被转移到细胞中，并恢复感染性后代以确定耐受插入的位点，因为只有耐受插入的基因组才能繁殖。利用这些结果，我们创建了带有表位标签的病毒，一种位于病毒结构蛋白衣壳中，一种位于病毒非结构蛋白 NS4B 中。对这些突变体的进一步研究可能会阐明衣壳和 NS4B 在登革热病毒感染过程中的作用。