The Aedes aegypti RNA interference response against Zika virus in the context of co-infection with dengue and chikungunya viruses.

Since its detection in 2015 in Brazil, Zika virus (ZIKV) has remained in the spotlight of international public health and research as an emerging arboviral pathogen. In addition to single infection, ZIKV may occur in co-infection with dengue (DENV) and chikungunya (CHIKV) viruses, with whom ZIKV shares geographic distribution and the mosquito Aedes aegypti as a vector. The main mosquito immune response against arboviruses is RNA interference (RNAi). It is unknown whether or not the dynamics of the RNAi response differ between single arboviral infections and co-infections. In this study, we investigated the interaction of ZIKV and DENV, as well as ZIKV and CHIKV co-infections with the RNAi response in Ae. aegypti. Using small RNA sequencing, we found that the efficiency of small RNA production against ZIKV -a hallmark of antiviral RNAi-was mostly similar when comparing single and co-infections with either DENV or CHIKV. Silencing of key antiviral RNAi proteins, showed no change in effect on ZIKV replication when the cell is co-infected with ZIKV and DENV or CHIKV. Interestingly, we observed a negative effect on ZIKV replication during CHIKV co-infection in the context of Ago2-knockout cells, though his effect was absent during DENV co-infection. Overall, this study provides evidence that ZIKV single or co-infections with CHIKV or DENV are equally controlled by RNAi responses. Thus, Ae. aegypti mosquitoes and derived cells support co-infections of ZIKV with either CHIKV or DENV to a similar level than single infections, as long as the RNAi response is functional.

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.

Sugar feeding protects against arboviral infection by enhancing gut immunity in the mosquito vector Aedes aegypti.

As mosquito females require a blood meal to reproduce, they can act as vectors of numerous pathogens, such as arboviruses (e.g. Zika, dengue and chikungunya viruses), which constitute a substantial worldwide public health burden. In addition to blood meals, mosquito females can also take sugar meals to get carbohydrates for their energy reserves. It is now recognised that diet is a key regulator of health and disease outcome through interactions with the immune system. However, this has been mostly studied in humans and model organisms. So far, the impact of sugar feeding on mosquito immunity and in turn, how this could affect vector competence for arboviruses has not been explored. Here, we show that sugar feeding increases and maintains antiviral immunity in the digestive tract of the main arbovirus vector Aedes aegypti. Our data demonstrate that the gut microbiota does not mediate the sugar-induced immunity but partly inhibits it. Importantly, sugar intake prior to an arbovirus-infected blood meal further protects females against infection with arboviruses from different families. Sugar feeding blocks arbovirus initial infection and dissemination from the gut and lowers infection prevalence and intensity, thereby decreasing the transmission potential of female mosquitoes. Finally, we show that the antiviral role of sugar is mediated by sugar-induced immunity. Overall, our findings uncover a crucial role of sugar feeding in mosquito antiviral immunity which in turn decreases vector competence for arboviruses. Since Ae. aegypti almost exclusively feed on blood in some natural settings, our findings suggest that this lack of sugar intake could increase the spread of mosquito-borne arboviral diseases.

Analysis of Zika virus capsid-Aedes aegypti mosquito interactome reveals pro-viral host factors critical for establishing infection.

The escalating global prevalence of arboviral diseases emphasizes the need to improve our understanding of their biology. Research in this area has been hindered by the lack of molecular tools for studying virus-mosquito interactions. Here, we develop an Aedes aegypti cell line which stably expresses Zika virus (ZIKV) capsid proteins in order to study virus-vector protein-protein interactions through quantitative label-free proteomics. We identify 157 interactors and show that eight have potentially pro-viral activity during ZIKV infection in mosquito cells. Notably, silencing of transitional endoplasmic reticulum protein TER94 prevents ZIKV capsid degradation and significantly reduces viral replication. Similar results are observed if the TER94 ortholog (VCP) functioning is blocked with inhibitors in human cells. In addition, we show that an E3 ubiquitin-protein ligase, UBR5, mediates the interaction between TER94 and ZIKV capsid. Our study demonstrates a pro-viral function for TER94/VCP during ZIKV infection that is conserved between human and mosquito cells.

A plasmid DNA-launched SARS-CoV-2 reverse genetics system and coronavirus toolkit for COVID-19 research.

The recent emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the underlying cause of Coronavirus Disease 2019 (COVID-19), has led to a worldwide pandemic causing substantial morbidity, mortality, and economic devastation. In response, many laboratories have redirected attention to SARS-CoV-2, meaning there is an urgent need for tools that can be used in laboratories unaccustomed to working with coronaviruses. Here we report a range of tools for SARS-CoV-2 research. First, we describe a facile single plasmid SARS-CoV-2 reverse genetics system that is simple to genetically manipulate and can be used to rescue infectious virus through transient transfection (without in vitro transcription or additional expression plasmids). The rescue system is accompanied by our panel of SARS-CoV-2 antibodies (against nearly every viral protein), SARS-CoV-2 clinical isolates, and SARS-CoV-2 permissive cell lines, which are all openly available to the scientific community. Using these tools, we demonstrate here that the controversial ORF10 protein is expressed in infected cells. Furthermore, we show that the promising repurposed antiviral activity of apilimod is dependent on TMPRSS2 expression. Altogether, our SARS-CoV-2 toolkit, which can be directly accessed via our website at https://mrcppu-covid.bio/, constitutes a resource with considerable potential to advance COVID-19 vaccine design, drug testing, and discovery science.

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

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.

Glucose-Regulated Protein 78 Interacts with Zika Virus Envelope Protein and Contributes to a Productive Infection.

Zika virus (ZIKV; Flaviviridae) is a mosquito-borne flavivirus shown to cause fetal abnormalities collectively known as congenital Zika syndrome and Guillain-Barré syndrome in recent outbreaks. Currently, there is no specific treatment or vaccine available, and more effort is needed to identify cellular factors in the viral life cycle. Here, we investigated interactors of ZIKV envelope (E) protein by combining protein pull-down with mass spectrometry. We found that E interacts with the endoplasmic reticulum (ER) resident chaperone, glucose regulated protein 78 (GRP78). Although other flaviviruses are known to co-opt ER resident proteins, including GRP78, to enhance viral infectivity, the role ER proteins play during the ZIKV life cycle is yet to be elucidated. We showed that GRP78 levels increased during ZIKV infection and localised to sites coincident with ZIKV E staining. Depletion of GRP78 using specific siRNAs significantly reduced reporter-virus luciferase readings, viral protein synthesis, and viral titres. Additionally, GRP78 depletion reduced the ability of ZIKV to disrupt host cell translation and altered the localisation of viral replication factories, though there was no effect on viral RNA synthesis. In summary, we showed GRP78 is a vital host-factor during ZIKV infection, which may be involved in the coordination of viral replication factories.

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

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 melanogasterIMPORTANCE 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.