Dengue virus (DENV) and Zika virus (ZIKV), members of the Flavivirus genus, rearrange endoplasmic reticulum membranes to induce invaginations known as vesicle packets (VPs), which are the assumed sites for viral RNA replication. Mechanistic information on VP biogenesis has so far been difficult to attain due to the necessity of studying their formation under conditions of viral replication, where perturbations reducing replication will inevitably impact VP formation. Here, we report a replication-independent expression system, designated pIRO (plasmid-induced replication organelle formation) that induces bona fide DENV and ZIKV VPs that are morphologically indistinguishable from those in infected cells. Using this system, we demonstrate that sequences in the 3′ terminal RNA region of the DENV, but not the ZIKV genome, contribute to VP formation in a non-replicative manner. These results validate the pIRO system that opens avenues for mechanistically dissecting virus replication from membrane reorganization.

登革热病毒 (DENV) 和寨卡病毒 (ZIKV) 属于黄病毒属，它们会重新排列内质网膜以诱导称为囊泡包 (VP) 的内陷，这是病毒 RNA 复制的假定位点。由于需要在病毒复制条件下研究 VP 的形成，因此迄今为止很难获得 VP 生物发生的机制信息，其中减少复制的扰动将不可避免地影响 VP 的形成。在这里，我们报告了一种复制独立的表达系统，称为 pIRO（质粒诱导的复制细胞器形成），它诱导真正的 DENV 和 ZIKV VP，这些 VP 在形态上与受感染细胞中的 VP 没有区别。使用该系统，我们证明 DENV 3' 末端 RNA 区域中的序列（而非 ZIKV 基因组）以非复制方式促进 VP 形成。这些结果验证了 pIRO 系统为从膜重组中机械剖析病毒复制开辟了途径。