Institute of Virology, Medical Faculty, University of Bonn, Bonn, Germany.
Institute of Clinical Chemistry and Clinical Pharmacology, Medical Faculty, University of Bonn, Bonn, Germany.
PLoS Pathog. 2024 Oct 2;20(10):e1012607. doi: 10.1371/journal.ppat.1012607. eCollection 2024 Oct.
The RNA genome of orthoflaviviruses encodes a methyltransferase within the non-structural protein NS5, which is involved in 2'-O-methylation of the 5'-terminal nucleotide of the viral genome resulting in a cap1 structure. While a 2'-O-unmethylated cap0 structure is recognized in vertebrates by the RNA sensor RIG-I, the cap1 structure allows orthoflaviviruses to evade the vertebrate innate immune system. Here, we analyzed whether the cap0 structure is also recognized in mosquitoes. Replication analyses of 2'-O-methyltransferase deficient yellow fever virus mutants (YFV NS5-E218A) of the vaccine 17D and the wild-type Asibi strain in mosquito cells revealed a distinct downregulation of the cap0 viruses. Interestingly, the level of inhibition differed for various mosquito cells. The most striking difference was found in Aedes albopictus-derived C6/36 cells with YFV-17D cap0 replication being completely blocked. Replication of YFV-Asibi cap0 was also suppressed in mosquito cells but to a lower extent. Analyses using chimeras between YFV-17D and YFV-Asibi suggest that a synergistic effect of several mutations across the viral genome accompanied by a faster initial growth rate of YFV-Asibi cap1 correlates with the lower level of YFV-Asibi cap0 attenuation. Viral growth analyses in Dicer-2 knockout cells demonstrated that Dicer-2 is entirely dispensable for attenuating the YFV cap0 viruses. Translation of a replication-incompetent cap0 reporter YFV-17D genome was reduced in mosquito cells, indicating a cap0 sensing translation regulation mechanism. Further, oral infection of Aedes aegypti mosquitoes resulted in lower infection rates for YFV-Asibi cap0. The latter is related to lower viral loads found in the midguts, which largely diminished dissemination to secondary tissues. After intrathoracic infection, YFV-Asibi cap0 replicated slower and to decreased amounts in secondary tissues compared to YFV-Asibi cap1. These results suggest the existence of an ubiquitously expressed innate antiviral protein recognizing 5'-terminal RNA cap-modifications in mosquitoes, both in the midgut as well as in secondary tissues.
黄病毒的 RNA 基因组编码非结构蛋白 NS5 内的甲基转移酶，该酶参与病毒基因组 5'端核苷酸的 2'-O-甲基化，导致帽 1 结构。虽然脊椎动物中的 RNA 传感器 RIG-I 识别未甲基化的帽 0 结构，但帽 1 结构允许黄病毒逃避脊椎动物先天免疫系统。在这里，我们分析了蚊子是否也能识别帽 0 结构。疫苗 17D 的 2'-O-甲基转移酶缺陷型黄热病毒突变体（YFV NS5-E218A）和野生型 Asibi 株在蚊子细胞中的复制分析表明，帽 0 病毒的表达明显下调。有趣的是，各种蚊子细胞的抑制水平不同。最显著的差异是在 Aedes albopictus 衍生的 C6/36 细胞中发现的，YFV-17D 帽 0 复制完全被阻断。YFV-Asibi 帽 0 的复制在蚊子细胞中也受到抑制，但程度较低。使用 YFV-17D 和 YFV-Asibi 之间的嵌合体进行的分析表明，病毒基因组中几个突变的协同作用，伴随着 YFV-Asibi 帽 1 的初始生长速度更快，与 YFV-Asibi 帽 0 衰减程度较低相关。在 Dicer-2 敲除细胞中的病毒生长分析表明，Dicer-2 完全不需要减弱 YFV 帽 0 病毒。在蚊子细胞中，复制失活的帽 0 报告 YFV-17D 基因组的翻译减少，表明存在帽 0 感应翻译调控机制。此外，埃及伊蚊的口服感染导致 YFV-Asibi 帽 0 的感染率降低。这与中肠中发现的病毒载量较低有关，这大大减少了向二级组织的传播。在胸内感染后，YFV-Asibi 帽 0 在二级组织中的复制速度较慢，数量减少，与 YFV-Asibi 帽 1 相比。这些结果表明，在蚊子中存在一种普遍表达的先天抗病毒蛋白，它可以识别 5'端 RNA 帽修饰，包括中肠和二级组织。
