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.

Antiviral RNAi response against the insect-specific Agua Salud alphavirus

Arboviruses transmitted by mosquitoes are responsible for the death of millions of people each year. In addition to arboviruses, many insect-specific viruses (ISVs) have been discovered in mosquitoes in the last decade. ISVs, in contrast to arboviruses transmitted by mosquitoes to vertebrates, cannot replicate in vertebrate cells even when they are evolutionarily closely related to arboviruses. The alphavirus genus includes many arboviruses, although only a few ISVs have been discovered from this genus so far. Here, we investigate the interactions of a recently isolated insect-specific alphavirus, Agua-Salud alphavirus (ASALV), with its mosquito host. RNAi is one of the essential antiviral responses against arboviruses, although there is little knowledge on the interactions of RNAi with ISVs. Through knock-down of transcripts of the different key RNAi pathway (siRNA, miRNA and piRNA) proteins, we show the antiviral role of Ago2 (siRNA), Ago1 (miRNA), and Piwi4 proteins against ASALV in Aedes aegypti derived cells. ASALV replication increased in Dicer2 and Ago2 knock-out cells, confirming the antiviral role of the siRNA pathway. In infected cells, mainly ASALV-specific siRNAs are produced while piRNAs, with the characteristic nucleotide bias resulting from ping-pong amplification, are only produced in Dicer2 knock-out cells. Taken together, ASALV interactions with the mosquito RNAi response differs from arthropod-borne alphaviruses in some aspects, although they also share some commonalities. Further research is needed to understand whether the identified differences can be generalised to other insect-specific alphaviruses.

When Down Is Up: Heterochromatin, Nuclear Organization and X Upregulation

Organisms with highly differentiated sex chromosomes face an imbalance in X-linked gene dosage. Male Drosophila solve this problem by increasing expression from virtually every gene on their single X chromosome, a process known as dosage compensation. This involves a ribonucleoprotein complex that is recruited to active, X-linked genes to remodel chromatin and increase expression. Interestingly, the male X chromosome is also enriched for several proteins associated with heterochromatin. Furthermore, the polytenized male X is selectively disrupted by the loss of factors involved in repression, silencing, heterochromatin formation or chromatin remodeling. Mutations in many of these factors preferentially reduce male survival or enhance the lethality of mutations that prevent normal recognition of the X chromosome. The involvement of primarily repressive factors in a process that elevates expression has long been puzzling. Interestingly, recent work suggests that the siRNA pathway, often associated with heterochromatin formation and repression, also helps the dosage compensation machinery identify the X chromosome. In light of this finding, we revisit the evidence that links nuclear organization and heterochromatin to regulation of the male X chromosome.

Molecular Characterizations and Functional Analyses of LmR2D2 in the Locusta migratoria siRNA Pathway

Small interfering RNAs (siRNAs) are non-coding RNAs with a length of 21~23 nucleotides (nt) and present in almost all eukaryotes. The formation of siRNA is a highly conserved post-transcriptional gene-silencing mechanism mediated by key proteins, including Dicer2, Argonaute2 (Ago2) and R2D2. R2D2 has been identified as a double-stranded RNA (dsRNA)-binding protein and reported as an integral component of the siRNA pathway in Drosophila. However, the involvement of R2D2 in the siRNA pathway of Locusta migratoria is still unknown. In the present study, we identified an LmR2D2 gene from the transcriptome of L. migratoria. It consists of a 954-bp open reading frame that encodes a protein of 318 amino acid residues. Further sequence analysis revealed that LmR2D2 possesses two tandem dsRNA-binding domains (dsRBD) at the N-terminus. Analysis of the developmental expression profile of LmR2D2 indicated that its transcript level was stable in third-instar nymphs of L. migratoria, whereas the tissue-dependent expression profile exhibited high levels of expression of LmR2D2 in the testis and ovary. When LmR2D2 was silenced by RNAi, the RNAi efficiency against Lmβ-tubulin as a marker gene was significantly diminished, as indicated by the 37.7% increased Lmβ-tubulin transcript level. Additionally, the prokaryotic expression system was used to obtain the LmR2D2 supernatant protein. By incubating the LmR2D2 protein with biotin-dsRNA, we found that LmR2D2 can bind to dsRNA in vitro, which supports our conclusion that LmR2D2 plays an essential role in the siRNA pathway of L. migratoria.

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

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.

Structural insights into the role of Dicer-related helicase 3 in RNAi in Caenorhabditis elegans.

DRH-3 belongs to the family of duplex RNA-dependent ATPases (DRAs), which include Dicer and RIG-I-like receptors (RLRs). DRH-3 is critically involved in germline development and RNAi-facilitated chromosome segregation via the 22G-siRNA pathway in C. elegans. The molecular understanding of DRH-3 and its function in endogenous RNAi pathways remains elusive. In this study, we solved the crystal structures of the DRH-3 N-terminal domain (NTD) and the C-terminal domains (CTDs) in complex with 5'-triphosphorylated RNAs. The NTD of DRH-3 adopts a distinct fold of tandem Caspase Activation and Recruitment Domains (CARDs) structurally similar to the CARDs of RIG-I and MDA5, suggesting a signaling function in the endogenous RNAi biogenesis. The CTD preferentially recognizes 5'-triphosphorylated double-stranded RNAs bearing the typical features of secondary siRNA transcripts. The full-length DRH-3 displays unique structural dynamics upon binding to RNA duplexes that differ from RIG-I or MDA5. These unique molecular features of DRH-3 help explain its function in RNAi in worms and the evolutionary divergence of the Dicer-like helicases.

The Interplay Between Viruses and RNAi Pathways in Insects

As an overarching immune mechanism, RNA interference (RNAi) displays pathogen specificity and memory via different pathways. The small interfering RNA (siRNA) pathway is the primary antiviral defense mechanism against RNA viruses of insects and plays a lesser role in defense against DNA viruses. Reflecting the pivotal role of the siRNA pathway in virus selection, different virus families have independently evolved unique strategies to counter this host response, including protein-mediated, decoy RNA–based, and microRNA-based strategies. In this review, we outline the interplay between insect viruses and the different pathways of the RNAi antiviral response; describe practical application of these interactions for improved expression systems and for pest and disease management; and highlight research avenues for advancement of the field.

Characterization of a Novel Mitovirus of the Sand Fly Lutzomyia longipalpis Using Genomic and Virus–Host Interaction Signatures

Hematophagous insects act as the major reservoirs of infectious agents due to their intimate contact with a large variety of vertebrate hosts. Lutzomyia longipalpis is the main vector of Leishmania chagasi in the New World, but its role as a host of viruses is poorly understood. In this work, Lu. longipalpis RNA libraries were subjected to progressive assembly using viral profile HMMs as seeds. A sequence phylogenetically related to fungal viruses of the genus Mitovirus was identified and this novel virus was named Lul-MV-1. The 2697-base genome presents a single gene coding for an RNA-directed RNA polymerase with an organellar genetic code. To determine the possible host of Lul-MV-1, we analyzed the molecular characteristics of the viral genome. Dinucleotide composition and codon usage showed profiles similar to mitochondrial DNA of invertebrate hosts. Also, the virus-derived small RNA profile was consistent with the activation of the siRNA pathway, with size distribution and 5′ base enrichment analogous to those observed in viruses of sand flies, reinforcing Lu. longipalpis as a putative host. Finally, RT-PCR of different insect pools and sequences of public Lu. longipalpis RNA libraries confirmed the high prevalence of Lul-MV-1. This is the first report of a mitovirus infecting an insect host.

Transgenerational Regulation of Sexual Attractiveness in C. elegans Nematodes

C. elegans nematodes transmit responses to environmental cues transgenerationally, however it is unknown whether this non-DNA-based inheritance impacts the genome and consequently the process of evolution. Here we show that inherited small RNAs regulate the crucial decision of whether to self-fertilize or outcross, and thus indirectly control genetic variation. Under standard growth conditions, hermaphrodites secrete a male-attracting pheromone only when they are old and their supply of self-produced sperm is depleted. Since heat compromises sperm functions, from worms to humans, we examined young hermaphrodites that were cultivated at a mildly stressful temperature (25°C), and discovered that they become more attractive due to the premature secretion of the pheromone. Moreover, we discovered that the enhanced attractiveness transmits transgenerationally to unstressed progeny via heritable small RNAs and the Argonaute protein Heritable RNAi Deficient-1 (HRDE-1). We identified a specific endogenous small interfering RNA (endo-siRNA) pathway, enriched in endosiRNAs which target sperm genes, that can transgenerationally regulate sexual attraction, prevalence of males, and the rate of successful mating. Mathematical simulations and multigenerational competition experiments revealed that over generations, animals that inherit attractiveness mate more, and their alleles spread in the population. We propose that the sperm serves as a “stress sensor” which, via small RNA inheritance, can enhance outcrossing in challenging environments, when increasing genetic variation is advantageous.