Dengue virus (DENV) is a major global pathogen that causes significant morbidity and mortality in tropical and subtropical areas worldwide. An improved understanding of the regions within the DENV genome and its encoded proteins that are required for the virus replication cycle will expedite the development of urgently required therapeutics and vaccines. We subjected an infectious DENV genome to unbiased insertional mutagenesis and used next-generation sequencing to identify sites that tolerate 15-nucleotide insertions during the virus replication cycle in hepatic cell culture. This revealed that the regions within capsid, NS1, and the 3′ untranslated region were the most tolerant of insertions. In contrast, prM- and NS2A-encoding regions were largely intolerant of insertions. Notably, the multifunctional NS1 protein readily tolerated insertions in regions within the Wing, connector, and β-ladder domains with minimal effects on viral RNA replication and infectious virus production. Using this information, we generated infectious reporter viruses, including a variant encoding the APEX2 electron microscopy tag in NS1 that uniquely enabled high-resolution imaging of its localization to the surface and interior of viral replication vesicles. In addition, we generated a tagged virus bearing an mScarlet fluorescent protein insertion in NS1 that, despite an impact on fitness, enabled live cell imaging of NS1 localization and traffic in infected cells. Overall, this genome-wide profile of DENV genome flexibility may be further dissected and exploited in reporter virus generation and antiviral strategies.

IMPORTANCE Regions of genetic flexibility in viral genomes can be exploited in the generation of reporter virus tools and should arguably be avoided in antiviral drug and vaccine design. Here, we subjected the DENV genome to high-throughput insertional mutagenesis to identify regions of genetic flexibility and enable tagged reporter virus generation. In particular, the viral NS1 protein displayed remarkable tolerance of small insertions. This genetic flexibility enabled generation of several novel NS1-tagged reporter viruses, including an APEX2-tagged virus that we used in high-resolution imaging of NS1 localization in infected cells by electron microscopy. For the first time, this analysis revealed the localization of NS1 within viral replication factories known as “vesicle packets” (VPs), in addition to its acknowledged localization to the luminal surface of these VPs. Together, this genetic profile of DENV may be further refined and exploited in the identification of antiviral targets and the generation of reporter virus tools.

登革热病毒（DENV）是一种主要的全球病原体，在全球热带和亚热带地区引起大量发病和死亡。对病毒复制周期所需的DENV基因组内的区域及其编码的蛋白质的进一步了解将加快迫切需要的治疗剂和疫苗的开发。我们对传染性DENV基因组进行了无偏插入诱变，并使用了下一代测序来鉴定在肝细胞培养中病毒复制周期中能耐受15个核苷酸插入的位点。这揭示了衣壳，NS1和3'非翻译区域内的区域对插入的耐受性最高。相反，prM和NS2A编码区在很大程度上不能插入。尤其，翼，连接子和β-梯形结构域，对病毒RNA复制和感染性病毒产生的影响极小。利用这些信息，我们产生了感染性报告病毒，包括一个编码NS1中APEX2电子显微镜标签的变体，该变体独特地使高分辨率定位于病毒复制囊泡表面和内部的成像成为可能。此外，我们产生了一种标记的病毒，该病毒在NS1中带有mScarlet荧光蛋白插入，尽管对健康有影响，但仍可以对NS1定位和感染细胞中的运输进行活细胞成像。总体而言，DENV基因组灵活性的全基因组概况可在报告病毒的产生和抗病毒策略中进一步剖析和利用。

重要性病毒基因组中遗传灵活性的区域可用于生成报告病毒工具，并且在抗病毒药物和疫苗设计中应避免使用。在这里，我们对DENV基因组进行了高通量插入诱变，以鉴定遗传柔性区域并启用标记的报告病毒生成。尤其是，病毒NS1蛋白表现出对小插入的显着耐受性。这种遗传上的灵活性使得能够产生几种新型的带有NS1标签的报告病毒，包括我们通过电子显微镜对NS1定位在感染细胞中的高分辨率成像中使用的带有APEX2标签的病毒。这项分析首次揭示了NS1在病毒复制工厂（称为“囊泡包”（VP））中的定位，除了公认的定位在这些VP的腔表面。总之，DENV的这种遗传特征可以进一步完善和用于抗病毒靶标的鉴定和报告病毒工具的产生。