Fundación Instituto Leloir-CONICET, Buenos Aires, Argentina.
Insect-Virus Interactions Group, Department of Genomes and Genetics, Institut Pasteur, Paris, France.
mBio. 2019 Jan 8;10(1):e02506-18. doi: 10.1128/mBio.02506-18.
Flaviviruses include a diverse group of medically important viruses that cycle between mosquitoes and humans. During this natural process of switching hosts, each species imposes different selective forces on the viral population. Using dengue virus (DENV) as model, we found that paralogous RNA structures originating from duplications in the viral 3' untranslated region (UTR) are under different selective pressures in the two hosts. These RNA structures, known as dumbbells (DB1 and DB2), were originally proposed to be enhancers of viral replication. Analysis of viruses obtained from infected mosquitoes showed selection of mutations that mapped in DB2. Recombinant viruses carrying the identified variations confirmed that these mutations greatly increase viral replication in mosquito cells, with low or no impact in human cells. Use of viruses lacking each of the DB structures revealed opposite viral phenotypes. While deletion of DB1 reduced viral replication about 10-fold, viruses lacking DB2 displayed a great increase of fitness in mosquitoes, confirming a functional diversification of these similar RNA elements. Mechanistic analysis indicated that DB1 and DB2 differentially modulate viral genome cyclization and RNA replication. We found that a pseudoknot formed within DB2 competes with long-range RNA-RNA interactions that are necessary for minus-strand RNA synthesis. Our results support a model in which a functional diversification of duplicated RNA elements in the viral 3' UTR is driven by host-specific requirements. This study provides new ideas for understanding molecular aspects of the evolution of RNA viruses that naturally jump between different species. Flaviviruses constitute the most relevant group of arthropod-transmitted viruses, including important human pathogens such as the dengue, Zika, yellow fever, and West Nile viruses. The natural alternation of these viruses between vertebrate and invertebrate hosts shapes the viral genome population, which leads to selection of different viral variants with potential implications for epidemiological fitness and pathogenesis. However, the selective forces and mechanisms acting on the viral RNA during host adaptation are still largely unknown. Here, we found that two almost identical tandem RNA structures present at the viral 3' untranslated region are under different selective pressures in the two hosts. Mechanistic studies indicated that the two RNA elements, known as dumbbells, contain sequences that overlap essential RNA cyclization elements involved in viral RNA synthesis. The data support a model in which the duplicated RNA structures differentially evolved to accommodate distinct functions for viral replication in the two hosts.
黄病毒属包括一大类医学上重要的病毒，这些病毒在蚊子和人类之间循环。在这种自然的宿主转换过程中，每个物种都会对病毒种群施加不同的选择压力。我们以登革热病毒（DENV）为模型，发现来自病毒 3'非翻译区（UTR）重复的同源 RNA 结构在两种宿主中受到不同的选择压力。这些 RNA 结构，称为哑铃（DB1 和 DB2），最初被提议作为病毒复制的增强子。对从感染蚊子中获得的病毒进行分析表明，选择了映射到 DB2 中的突变。携带鉴定出的变异的重组病毒证实，这些突变大大增加了蚊子细胞中的病毒复制，而对人细胞的影响则很低或没有。使用缺乏每个 DB 结构的病毒揭示了相反的病毒表型。虽然删除 DB1 会使病毒复制减少约 10 倍，但缺乏 DB2 的病毒在蚊子中的适应性大大增加，证实了这些相似 RNA 元件的功能多样化。机制分析表明，DB1 和 DB2 以不同的方式调节病毒基因组环化和 RNA 复制。我们发现，DB2 内形成的假结与长距离 RNA-RNA 相互作用竞争，后者对于负链 RNA 合成是必需的。我们的结果支持这样一种模型，即病毒 3'UTR 中重复 RNA 元件的功能多样化是由宿主特异性要求驱动的。这项研究为理解自然跨越不同物种的 RNA 病毒进化的分子方面提供了新的思路。黄病毒属构成了节肢动物传播病毒中最重要的一组，包括登革热、寨卡、黄热病和西尼罗河病毒等重要的人类病原体。这些病毒在脊椎动物和无脊椎动物宿主之间的自然交替塑造了病毒基因组群体，导致选择具有潜在流行病学适应性和发病机制意义的不同病毒变体。然而，在宿主适应过程中作用于病毒 RNA 的选择压力和机制在很大程度上仍然未知。在这里，我们发现存在于病毒 3'UTR 中的两个几乎相同的串联 RNA 结构在两种宿主中受到不同的选择压力。机制研究表明，这两个 RNA 元件，称为哑铃，包含重叠的序列，这些序列与病毒 RNA 合成中涉及的必需 RNA 环化元件重叠。数据支持这样一种模型，即重复的 RNA 结构以不同的方式进化，以适应两种宿主中病毒复制的不同功能。
