Mutational analysis of Aedes aegypti Dicer 2 provides insights into the biogenesis of antiviral exogenous small interfering RNAs.
Mutational analysis of Aedes aegypti Dicer 2 provides insights into the biogenesis of antiviral exogenous small interfering RNAs.
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埃及伊蚊 Dicer 2 的突变分析为了解抗病毒外源小干扰 RNA 的生物发生提供了见解。
DOI：
10.1371/journal.ppat.1010202
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发表时间：
2022-01
期刊：
影响因子：
6.7
通讯作者：
Kohl A
中科院分区：
文献类型：
--
作者：
Gestuveo RJ;Parry R;Dickson LB;Lequime S;Sreenu VB;Arnold MJ;Khromykh AA;Schnettler E;Lambrechts L;Varjak M;Kohl A
外源性小干扰RNA （exo-siRNA）途径是广泛传播人类致病性虫媒病毒的埃及伊蚊（Aedes aegypti mosquito）的关键抗病毒机制。该途径是由病毒衍生的双链rna （dsRNA）诱导的，该双链rna被核糖核酸酶Dicer 2 （Dcr2）切割成主要由21个核苷酸（nt）病毒衍生的小干扰rna （vsirna）。这些vsirna被RNA诱导沉默复合体中的效应蛋白Argonaute 2用来切割靶病毒RNA。Dcr2包含几个对其活性至关重要的结构域，包括解旋酶和RNase III结构域。在果蝇Dcr2中，解旋酶结构域与具有钝端末端的dsRNA结合和一种进程性siRNA产生机制有关，而平台- paz结构域与具有3 '悬垂的dsRNA结合并随后产生分布式siRNA。本文分析了解旋酶和RNase III结构域在Ae中的作用。埃及伊蚊Dcr2抗病毒活性和外显sirna途径。我们鉴定了解旋酶和RNase III结构域的保守氨基酸，以研究Ae的Dcr2抗病毒活性。通过报告者检测和感染蚊媒塞姆利基森林病毒（Togaviridae, Alphavirus），获得了埃及伊蚊衍生的Dcr2敲除细胞系。功能相关氨基酸在单倍型Dcr2序列中被发现是保守的。埃及伊蚊横跨不同的大陆。解旋酶和RNase III结构域对沉默活性和21nt vsiRNA的产生至关重要，单独的RNase III结构域活性被认为不足以产生抗病毒活性。对21 nt vsiRNA序列（由功能性Dcr2产生）进行分析，以评估其沿病毒基因组的分布和相位，结果显示出多种多样但高度一致的vsiRNA库，主要是短序列或长序列重叠，包括19 nt重叠（后者最有可能代表真正的Dcr2切割产物）。结合Dcr2解旋酶结构域的重要性，这表明大多数21 nt vsirna起源于过程切割。这项研究为伊蚊的研究提供了新的线索。埃及伊蚊Dcr2在这一重要虫媒病毒中的功能和特性。传播人类致病性病毒的埃及伊蚊依靠外源性小干扰RNA （exo-siRNA）途径作为抗病毒反应的一部分。该途径由病毒复制过程中产生的病毒衍生双链RNA （dsRNA）触发，然后被Dicer 2 （Dcr2）切割成病毒衍生的小干扰RNA （vsirna）。这些vsirna靶向病毒RNA，导致病毒复制的抑制。Dcr2在这一途径中的重要性已经在果蝇模型中得到了深入的研究，但在蚊子中很大程度上缺乏研究。在这里，我们已经确定了Ae的解旋酶和RNase III结构域的保守和功能相关的氨基酸。埃及伊蚊Dcr2在其沉默活性和对塞姆利基森林病毒（SFV）的抗病毒应答中起重要作用。对携带功能性或突变Dcr2的sfv感染的蚊子细胞进行小RNA测序，为了解vsirna的性质和起源提供了新的见解。这项研究的发现，加上我们之前开发的用于研究蚊子细胞外sirna途径的不同分子工具，已经开始揭示Dcr2的重要特性，这些特性可能对理解蚊子-虫媒病毒相互作用有价值，并可能在开发或协助媒介控制策略方面有价值。
The exogenous small interfering RNA (exo-siRNA) pathway is a key antiviral mechanism in the Aedes aegypti mosquito, a widely distributed vector of human-pathogenic arboviruses. This pathway is induced by virus-derived double-stranded RNAs (dsRNA) that are cleaved by the ribonuclease Dicer 2 (Dcr2) into predominantly 21 nucleotide (nt) virus-derived small interfering RNAs (vsiRNAs). These vsiRNAs are used by the effector protein Argonaute 2 within the RNA-induced silencing complex to cleave target viral RNA. Dcr2 contains several domains crucial for its activities, including helicase and RNase III domains. In Drosophila melanogaster Dcr2, the helicase domain has been associated with binding to dsRNA with blunt-ended termini and a processive siRNA production mechanism, while the platform-PAZ domains bind dsRNA with 3’ overhangs and subsequent distributive siRNA production. Here we analyzed the contributions of the helicase and RNase III domains in Ae. aegypti Dcr2 to antiviral activity and to the exo-siRNA pathway. Conserved amino acids in the helicase and RNase III domains were identified to investigate Dcr2 antiviral activity in an Ae. aegypti-derived Dcr2 knockout cell line by reporter assays and infection with mosquito-borne Semliki Forest virus (Togaviridae, Alphavirus). Functionally relevant amino acids were found to be conserved in haplotype Dcr2 sequences from field-derived Ae. aegypti across different continents. The helicase and RNase III domains were critical for silencing activity and 21 nt vsiRNA production, with RNase III domain activity alone determined to be insufficient for antiviral activity. Analysis of 21 nt vsiRNA sequences (produced by functional Dcr2) to assess the distribution and phasing along the viral genome revealed diverse yet highly consistent vsiRNA pools, with predominantly short or long sequence overlaps including 19 nt overlaps (the latter representing most likely true Dcr2 cleavage products). Combined with the importance of the Dcr2 helicase domain, this suggests that the majority of 21 nt vsiRNAs originate by processive cleavage. This study sheds new light on Ae. aegypti Dcr2 functions and properties in this important arbovirus vector species. Aedes aegypti mosquitoes that transmit human-pathogenic viruses rely on the exogenous small interfering RNA (exo-siRNA) pathway as part of antiviral responses. This pathway is triggered by virus-derived double-stranded RNA (dsRNA) produced during viral replication that is then cleaved by Dicer 2 (Dcr2) into virus-derived small interfering RNAs (vsiRNAs). These vsiRNAs target viral RNA, leading to suppression of viral replication. The importance of Dcr2 in this pathway has been intensely studied in the Drosophila melanogaster model but is largely lacking in mosquitoes. Here, we have identified conserved and functionally relevant amino acids in the helicase and RNase III domains of Ae. aegypti Dcr2 that are important in its silencing activity and antiviral responses against Semliki Forest virus (SFV). Small RNA sequencing of SFV-infected mosquito cells with functional or mutated Dcr2 gave new insights into the nature and origin of vsiRNAs. The findings of this study, together with the different molecular tools we have previously developed to investigate the exo-siRNA pathway of mosquito cells, have started to uncover important properties of Dcr2 that could be valuable in understanding mosquito-arbovirus interactions and potentially in developing or assisting vector control strategies.
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影响因子：
4.2
作者：
Campbell CL;Keene KM;Brackney DE;Olson KE;Blair CD;Wilusz J;Foy BD
通讯作者：
Foy BD
DOI：
10.3390/v7020820
发表时间：
2015-02-17
期刊：
Viruses
影响因子：
--
作者：
Blair CD;Olson KE
通讯作者：
Olson KE
DOI：
10.1073/pnas.0813412106
发表时间：
2009-03-31
影响因子：
11.1
作者：
Flynt, Alex;Liu, Na;Lai, Eric C.
通讯作者：
Lai, Eric C.
DOI：
10.1073/pnas.1412984112
发表时间：
2015-01-13
影响因子：
11.1
作者：
Carissimo, Guillaume;Pondeville, Emilie;Vernick, Kenneth D.
通讯作者：
Vernick, Kenneth D.
DOI：
10.4049/jimmunol.1800597
发表时间：
2018-11-15
期刊：
Journal of immunology (Baltimore, Md. : 1950)
影响因子：
--
作者：
Harsh S;Ozakman Y;Kitchen SM;Paquin-Proulx D;Nixon DF;Eleftherianos I
通讯作者：
Eleftherianos I