Mutational analysis of Aedes aegypti Dicer 2 provides insights into the biogenesis of antiviral exogenous small interfering RNAs

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.

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The Aedes aegypti Domino Ortholog p400 Regulates Antiviral Exogenous Small Interfering RNA Pathway Activity and ago-2 Expression

mSphere ◽

10.1128/msphere.00081-20 ◽

2020 ◽

Vol 5 (2) ◽

Cited By ~ 2

Author(s):

Melanie McFarlane ◽

Floriane Almire ◽

Joy Kean ◽

Claire L. Donald ◽

Alma McDonald ◽

...

Keyword(s):

Drosophila Melanogaster ◽

Antiviral Activity ◽

Aedes Aegypti ◽

Small Interfering Rna ◽

Antiviral Response ◽

Pathway Activity ◽

Content Type ◽

Argonaute 2 ◽

Interfering Rna ◽

Sirna Pathway

ABSTRACT Arboviruses are pathogens of humans and animals. A better understanding of the interactions between these pathogens and the arthropod vectors, such as mosquitoes, that transmit them is necessary to develop novel control measures. A major antiviral pathway in the mosquito vector is the exogenous small interfering RNA (exo-siRNA) pathway, which is induced by arbovirus-derived double-stranded RNA in infected cells. Although recent work has shown the key role played by Argonaute-2 (Ago-2) and Dicer-2 (Dcr-2) in this pathway, the regulatory mechanisms that govern these pathways have not been studied in mosquitoes. Here, we show that the Domino ortholog p400 has antiviral activity against the alphavirus Semliki Forest virus (Togaviridae) both in Aedes aegypti-derived cells and in vivo. Antiviral activity of p400 was also demonstrated against chikungunya virus (Togaviridae) and Bunyamwera virus (Peribunyaviridae) but not Zika virus (Flaviviridae). p400 was found to be expressed across mosquito tissues and regulated ago-2 but not dcr-2 transcript levels in A. aegypti mosquitoes. These findings provide novel insights into the regulation of an important aedine exo-siRNA pathway effector protein, Ago-2, by the Domino ortholog p400. They add functional insights to previous observations of this protein’s antiviral and RNA interference regulatory activities in Drosophila melanogaster. IMPORTANCE Female Aedes aegypti mosquitoes are vectors of human-infecting arthropod-borne viruses (arboviruses). In recent decades, the incidence of arthropod-borne viral infections has grown dramatically. Vector competence is influenced by many factors, including the mosquito’s antiviral defenses. The exogenous small interfering RNA (siRNA) pathway is a major antiviral response restricting arboviruses in mosquitoes. While the roles of the effectors of this pathway, Argonaute-2 and Dicer-2 are well characterized, nothing is known about its regulation in mosquitoes. In this study, we demonstrate that A. aegypti p400, whose ortholog Domino in Drosophila melanogaster is a chromatin-remodeling ATPase member of the Tip60 complex, regulates siRNA pathway activity and controls ago-2 expression levels. In addition, we found p400 to have antiviral activity against different arboviruses. Therefore, our study provides new insights into the regulation of the antiviral response in A. aegypti mosquitoes.

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Unknown Areas of Activity of Human Ribonuclease Dicer: A Putative Deoxyribonuclease Activity

Molecules ◽

10.3390/molecules25061414 ◽

2020 ◽

Vol 25 (6) ◽

pp. 1414 ◽

Cited By ~ 1

Author(s):

Marta Wojnicka ◽

Agnieszka Szczepanska ◽

Anna Kurzynska-Kokorniak

Keyword(s):

Dnase Activity ◽

Helicase Domain ◽

Small Interfering Rnas ◽

Rnase Iii ◽

Domain Specific ◽

Dsrna Binding ◽

Small Regulatory Rnas ◽

Paz Domain ◽

Canonical Products ◽

Truncated Variants

The Dicer ribonuclease plays a crucial role in the biogenesis of small regulatory RNAs (srRNAs) by processing long double-stranded RNAs and single-stranded hairpin RNA precursors into small interfering RNAs (siRNAs) and microRNAs (miRNAs), respectively. Dicer-generated srRNAs can control gene expression by targeting complementary transcripts and repressing their translation or inducing their cleavage. Human Dicer (hDicer) is a multidomain enzyme comprising a putative helicase domain, a DUF283 domain, platform, a PAZ domain, a connector helix, two RNase III domains (RNase IIIa and RNase IIIb) and a dsRNA-binding domain. Specific, ~20-base pair siRNA or miRNA duplexes with 2 nucleotide (nt) 3’-overhangs are generated by Dicer when an RNA substrate is anchored within the platform-PAZ-connector helix (PPC) region. However, increasing number of reports indicate that in the absence of the PAZ domain, binding of RNA substrates can occur by other Dicer domains. Interestingly, truncated variants of Dicer, lacking the PPC region, have been found to display a DNase activity. Inspired by these findings, we investigated how the lack of the PAZ domain, or the entire PPC region, would influence the cleavage activity of hDicer. Using immunopurified 3xFlag-hDicer produced in human cells and its two variants: one lacking the PAZ domain, and the other lacking the entire PPC region, we show that the PAZ domain deletion variants of hDicer are not able to process a pre-miRNA substrate, a dsRNA with 2-nt 3ʹ-overhangs, and a blunt-ended dsRNA. However, the PAZ deletion variants exhibit both RNase and DNase activity on short single-stranded RNA and DNAs, respectively. Collectively, our results indicate that when the PAZ domain is absent, other hDicer domains may contribute to substrate binding and in this case, non-canonical products can be generated.

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An Aedes Aegypti-Derived Ago2 Knockout Cell Line to Investigate Arbovirus Infections

Viruses ◽

10.3390/v13061066 ◽

2021 ◽

Vol 13 (6) ◽

pp. 1066

Author(s):

Christina Scherer ◽

Jack Knowles ◽

Vattipally B. Sreenu ◽

Anthony C. Fredericks ◽

Janina Fuss ◽

...

Keyword(s):

Antiviral Activity ◽

Viral Replication ◽

Cell Line ◽

Small Rna Sequencing ◽

Sequencing Data ◽

Rna Sequences ◽

Argonaute 2 ◽

Si Rna ◽

Sirna Pathway ◽

The Impact

Mosquitoes are known as important vectors of many arthropod-borne (arbo)viruses causing disease in humans. These include dengue (DENV) and Zika (ZIKV) viruses. The exogenous small interfering (si)RNA (exo-siRNA) pathway is believed to be the main antiviral defense in arthropods, including mosquitoes. During infection, double-stranded RNAs that form during viral replication and infection are cleaved by the enzyme Dicer 2 (Dcr2) into virus-specific 21 nt vsiRNAs, which are subsequently loaded into Argonaute 2 (Ago2). Ago2 then targets and subsequently cleaves complementary RNA sequences, resulting in degradation of the target viral RNA. Although various studies using silencing approaches have supported the antiviral activity of the exo-siRNA pathway in mosquitoes, and despite strong similarities between the siRNA pathway in the Drosophila melanogaster model and mosquitoes, important questions remain unanswered. The antiviral activity of Ago2 against different arboviruses has been previously demonstrated. However, silencing of Ago2 had no effect on ZIKV replication, whereas Dcr2 knockout enhanced its replication. These findings raise the question as to the role of Ago2 and Dcr2 in the control of arboviruses from different viral families in mosquitoes. Using a newly established Ago2 knockout cell line, alongside the previously reported Dcr2 knockout cell line, we investigated the impact these proteins have on the modulation of different arboviral infections. Infection of Ago2 knockout cell line with alpha- and bunyaviruses resulted in an increase of viral replication, but not in the case of ZIKV. Analysis of small RNA sequencing data in the Ago2 knockout cells revealed a lack of methylated siRNAs from different sources, such as acute and persistently infecting viruses-, TE- and transcriptome-derived RNAs. The results confirmed the importance of the exo-siRNA pathway in the defense against arboviruses, but highlights variability in its response to different viruses and the impact the siRNA pathway proteins have in controlling viral replication. Moreover, this established Ago2 knockout cell line can be used for functional Ago2 studies, as well as research on the interplay between the RNAi pathways.

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aBravo Is a Novel Aedes aegypti Antiviral Protein That Interacts with, but Acts Independently of, the Exogenous siRNA Pathway Effector Dicer 2

Viruses ◽

10.3390/v12070748 ◽

2020 ◽

Vol 12 (7) ◽

pp. 748

Author(s):

Margus Varjak ◽

Rommel J. Gestuveo ◽

Richard Burchmore ◽

Esther Schnettler ◽

Alain Kohl

Keyword(s):

Aedes Aegypti ◽

Protein Complexes ◽

Effector Protein ◽

Antiviral Protein ◽

Argonaute 2 ◽

Mosquito Cells ◽

Antiviral Activities ◽

Positive Strand Rna ◽

Interfering Rna ◽

Sirna Pathway

Mosquitoes, such as Aedes aegypti, can transmit arboviruses to humans. The exogenous short interfering RNA (exo-siRNA) pathway plays a major antiviral role in controlling virus infection in mosquito cells. The Dicer 2 (Dcr2) nuclease is a key effector protein in this pathway, which cleaves viral double-stranded RNA into virus-derived siRNAs that are further loaded onto an effector called Argonaute 2 (Ago2), which as part of the multiprotein RNA-induced silencing complex (RISC) targets and cleaves viral RNA. In order to better understand the effector protein Dcr2, proteomics experiments were conducted to identify interacting cellular partners. We identified several known interacting partners including Ago2, as well as two novel and previously uncharacterized Ae. aegypti proteins. The role of these two proteins was further investigated, and their interactions with Dcr2 verified by co-immunoprecipitation. Interestingly, despite their ability to interact with Ago2 and Piwi4, neither of these proteins was found to affect exo-siRNA silencing in a reporter assay. However, one of these proteins, Q0IFK9, subsequently called aBravo (aedine broadly active antiviral protein), was found to mediate antiviral activity against positive strand RNA arboviruses. Intriguingly the presence of Dcr2 was not necessary for this effect, suggesting that this interacting antiviral effector may act as part of protein complexes with potentially separate antiviral activities.

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Bap31 Is a Novel Target of the Human Papillomavirus E5 Protein

Journal of Virology ◽

10.1128/jvi.01240-08 ◽

2008 ◽

Vol 82 (20) ◽

pp. 10042-10051 ◽

Cited By ~ 52

Author(s):

Jennifer A. Regan ◽

Laimonis A. Laimins

Keyword(s):

Amino Acids ◽

Life Cycle ◽

Mutational Analysis ◽

Human Papillomaviruses ◽

Small Interfering Rnas ◽

Binding Partner ◽

C Terminus ◽

Novel Target ◽

Split Ubiquitin ◽

High Risk Hpv

ABSTRACT The E5 proteins of human papillomaviruses (HPVs) are small hydrophobic proteins that are expressed in the early and late stages of the viral life cycle; however, their role in HPV pathogenesis is not clearly understood. In this study, a split-ubiquitin yeast (Saccharomyces cerevisiae) two-hybrid system was used to identify B-cell-associated protein 31 (Bap31) as a binding partner of HPV E5 proteins. The association of these proteins was confirmed by coimmunoprecipitation of complexes of Bap31 with either HPV type 16 (HPV16) or HPV31 E5. In addition, Bap31 and E5 were found to colocalize in perinuclear patterns consistent with localization to the endoplasmic reticulum. Mutational analysis of E5 identified amino acids in the extreme C terminus as important for stabilizing the interaction with Bap31. Deletion of these C-terminal amino acids of E5 in the context of complete HPV31 genomes resulted in impaired proliferative capacity of HPV-positive keratinocytes following differentiation. When small interfering RNAs were used to reduce the levels of Bap31, the proliferative ability of HPV-positive keratinocytes upon differentiation was also reduced, implicating Bap31 as a regulator of this process. These studies identify a novel binding partner of the high-risk HPV E5 proteins and provide insight into how the E5 proteins may modulate the life cycle in differentiating cells.

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In Vivo Identification of Intermediate Stages of the DNA Inversion Reaction Catalyzed by the Salmonella Hin Recombinase

Genetics ◽

10.1093/genetics/149.4.1649 ◽

1998 ◽

Vol 149 (4) ◽

pp. 1649-1663

Author(s):

Oliver Z Nanassy ◽

Kelly T Hughes

Keyword(s):

Amino Acids ◽

Biochemical Analysis ◽

Mutational Analysis ◽

Recombination Reaction ◽

Recombination Site ◽

Dna Inversion ◽

Hin Recombinase ◽

One Step ◽

A Site

Abstract The Hin recombinase catalyzes a site-specific recombination reaction that results in the reversible inversion of a 1-kbp segment of the Salmonella chromosome. The DNA inversion reaction catalyzed by the Salmonella Hin recombinase is a dynamic process proceeding through many intermediate stages, requiring multiple DNA sites and the Fis accessory protein. Biochemical analysis of this reaction has identified intermediate steps in the inversion reaction but has not yet revealed the process by which transition from one step to another occurs. Because transition from one reaction step to another proceeds through interactions between specific amino acids, and between amino acids and DNA bases, it is possible to study these transitions through mutational analysis of the proteins involved. We isolated a large number of mutants in the Hin recombinase that failed to carry out the DNA exchange reaction. We generated genetic tools that allowed the assignment of these mutants to specific transition steps in the recombination reaction. This genetic analysis, combined with further biochemical analysis, allowed us to define contributions by specific amino acids to individual steps in the DNA inversion reaction. Evidence is also presented in support of a model that Fis protein enhances the binding of Hin to the hixR recombination site. These studies identified regions within the Hin recombinase involved in specific transition steps of the reaction and provided new insights into the molecular details of the reaction mechanism.

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Phytophthora effector targets a novel component of small RNA pathway in plants to promote infection

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1421475112 ◽

2015 ◽

Vol 112 (18) ◽

pp. 5850-5855 ◽

Cited By ~ 82

Author(s):

Yongli Qiao ◽

Jinxia Shi ◽

Yi Zhai ◽

Yingnan Hou ◽

Wenbo Ma

Keyword(s):

Small Rna ◽

Rna Silencing ◽

Effector Proteins ◽

Helicase Domain ◽

Small Interfering Rnas ◽

Developmental Defects ◽

Interacting Protein ◽

Homologous Genes ◽

Unidentified Component ◽

Virulence Target

A broad range of parasites rely on the functions of effector proteins to subvert host immune response and facilitate disease development. The notorious Phytophthora pathogens evolved effectors with RNA silencing suppression activity to promote infection in plant hosts. Here we report that the Phytophthora Suppressor of RNA Silencing 1 (PSR1) can bind to an evolutionarily conserved nuclear protein containing the aspartate–glutamate–alanine–histidine-box RNA helicase domain in plants. This protein, designated PSR1-Interacting Protein 1 (PINP1), regulates the accumulation of both microRNAs and endogenous small interfering RNAs in Arabidopsis. A null mutation of PINP1 causes embryonic lethality, and silencing of PINP1 leads to developmental defects and hypersusceptibility to Phytophthora infection. These phenotypes are reminiscent of transgenic plants expressing PSR1, supporting PINP1 as a direct virulence target of PSR1. We further demonstrate that the localization of the Dicer-like 1 protein complex is impaired in the nucleus of PINP1-silenced or PSR1-expressing cells, indicating that PINP1 may facilitate small RNA processing by affecting the assembly of dicing complexes. A similar function of PINP1 homologous genes in development and immunity was also observed in Nicotiana benthamiana. These findings highlight PINP1 as a previously unidentified component of RNA silencing that regulates distinct classes of small RNAs in plants. Importantly, Phytophthora has evolved effectors to target PINP1 in order to promote infection.

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Amino acids of the adamantane series. I. Synthesis and antiviral activity of α-amino acids of the adamantane series and their derivatives

Pharmaceutical Chemistry Journal ◽

10.1007/bf00766686 ◽

1985 ◽

Vol 19 (7) ◽

pp. 474-478

Author(s):

P. A. Krasutskii ◽

I. G. Semenova ◽

M. I. Novikova ◽

A. G. Yurchenko ◽

N. A. Leont'eva ◽

...

Keyword(s):

Amino Acids ◽

Antiviral Activity ◽

Adamantane Series

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Subgenomic flavivirus RNA binds the mosquito DEAD/H-box helicase ME31B and determines Zika virus transmission by Aedes aegypti

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1905617116 ◽

2019 ◽

Vol 116 (38) ◽

pp. 19136-19144 ◽

Cited By ~ 17

Author(s):

Giel P. Göertz ◽

Joyce W. M. van Bree ◽

Anwar Hiralal ◽

Bas M. Fernhout ◽

Carmen Steffens ◽

...

Keyword(s):

Aedes Aegypti ◽

Zika Virus ◽

Affinity Purification ◽

Mechanistic Model ◽

Virus Transmission ◽

Protein Translation ◽

Mass Spectrometry Analysis ◽

Small Interfering Rnas ◽

Spectrometry Analysis ◽

Mosquito Infection

Zika virus (ZIKV) is an arthropod-borne flavivirus predominantly transmitted by Aedes aegypti mosquitoes and poses a global human health threat. All flaviviruses, including those that exclusively replicate in mosquitoes, produce a highly abundant, noncoding subgenomic flavivirus RNA (sfRNA) in infected cells, which implies an important function of sfRNA during mosquito infection. Currently, the role of sfRNA in flavivirus transmission by mosquitoes is not well understood. Here, we demonstrate that an sfRNA-deficient ZIKV (ZIKVΔSF1) replicates similar to wild-type ZIKV in mosquito cell culture but is severely attenuated in transmission by Ae. aegypti after an infectious blood meal, with 5% saliva-positive mosquitoes for ZIKVΔSF1 vs. 31% for ZIKV. Furthermore, viral titers in the mosquito saliva were lower for ZIKVΔSF1 as compared to ZIKV. Comparison of mosquito infection via infectious blood meals and intrathoracic injections showed that sfRNA is important for ZIKV to overcome the mosquito midgut barrier and to promote virus accumulation in the saliva. Next-generation sequencing of infected mosquitoes showed that viral small-interfering RNAs were elevated upon ZIKVΔSF1 as compared to ZIKV infection. RNA-affinity purification followed by mass spectrometry analysis uncovered that sfRNA specifically interacts with a specific set of Ae. aegypti proteins that are normally associated with RNA turnover and protein translation. The DEAD/H-box helicase ME31B showed the highest affinity for sfRNA and displayed antiviral activity against ZIKV in Ae. aegypti cells. Based on these results, we present a mechanistic model in which sfRNA sequesters ME31B to promote flavivirus replication and virion production to facilitate transmission by mosquitoes.

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Isolation and Characterization of Mutations in theEscherichia coli Regulatory Protein XapR

Journal of Bacteriology ◽

10.1128/jb.181.14.4397-4403.1999 ◽

1999 ◽

Vol 181 (14) ◽

pp. 4397-4403 ◽

Cited By ~ 22

Author(s):

Casper Jørgensen ◽

Gert Dandanell

Keyword(s):

Amino Acids ◽

Purine Nucleoside Phosphorylase ◽

Mutational Analysis ◽

Regulatory Protein ◽

Three Dimensional ◽

Dimensional Structure ◽

Wild Type ◽

Isolation And Characterization ◽

In The Wild

ABSTRACT In this work, the LysR-type protein XapR has been subjected to a mutational analysis. XapR regulates the expression of xanthosine phosphorylase (XapA), a purine nucleoside phosphorylase inEscherichia coli. In the wild type, full expression of XapA requires both a functional XapR protein and the inducer xanthosine. Here we show that deoxyinosine can also function as an inducer in the wild type, although not to the same extent as xanthosine. We have isolated and characterized in detail the mutants that can be induced by other nucleosides as well as xanthosine. Sequencing of the mutants has revealed that two regions in XapR are important for correct interactions between the inducer and XapR. One region is defined by amino acids 104 and 132, and the other region, containing most of the isolated mutations, is found between amino acids 203 and 210. These regions, when modelled into the three-dimensional structure of CysB from Klebsiella aerogenes, are placed close together and are most probably directly involved in binding the inducer xanthosine.

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