BTN3A3 evasion promotes the zoonotic potential of influenza A viruses.

Spillover events of avian influenza A viruses (IAVs) to humans could represent the first step in a future pandemic1. Several factors that limit the transmission and replication of avian IAVs in mammals have been identified. There are several gaps in our understanding to predict which virus lineages are more likely to cross the species barrier and cause disease in humans1. Here, we identified human BTN3A3 (butyrophilin subfamily 3 member A3)2 as a potent inhibitor of avian IAVs but not human IAVs. We determined that BTN3A3 is expressed in human airways and its antiviral activity evolved in primates. We show that BTN3A3 restriction acts primarily at the early stages of the virus life cycle by inhibiting avian IAV RNA replication. We identified residue 313 in the viral nucleoprotein (NP) as the genetic determinant of BTN3A3 sensitivity (313F or, rarely, 313L in avian viruses) or evasion (313Y or 313V in human viruses). However, avian IAV serotypes, such as H7 and H9, that spilled over into humans also evade BTN3A3 restriction. In these cases, BTN3A3 evasion is due to substitutions (N, H or Q) in NP residue 52 that is adjacent to residue 313 in the NP structure3. Thus, sensitivity or resistance to BTN3A3 is another factor to consider in the risk assessment of the zoonotic potential of avian influenza viruses.

Characterisation of the antiviral RNA interference response to Toscana virus in sand fly cells.

Toscana virus (TOSV) (Bunyavirales, Phenuiviridae, Phlebovirus, Toscana phlebovirus) and other related human pathogenic arboviruses are transmitted by phlebotomine sand flies. TOSV has been reported in nations bordering the Mediterranean Sea among other regions. Infection can result in febrile illness as well as meningitis and encephalitis. Understanding vector-arbovirus interactions is crucial to improving our knowledge of how arboviruses spread, and in this context, immune responses that control viral replication play a significant role. Extensive research has been conducted on mosquito vector immunity against arboviruses, with RNA interference (RNAi) and specifically the exogenous siRNA (exo-siRNA) pathway playing a critical role. However, the antiviral immunity of phlebotomine sand flies is less well understood. Here we were able to show that the exo-siRNA pathway is active in a Phlebotomus papatasi-derived cell line. Following TOSV infection, distinctive 21 nucleotide virus-derived small interfering RNAs (vsiRNAs) were detected. We also identified the exo-siRNA effector Ago2 in this cell line, and silencing its expression rendered the exo-siRNA pathway largely inactive. Thus, our data show that this pathway is active as an antiviral response against a sand fly transmitted bunyavirus, TOSV.

Mosquito saliva enhances virus infection through sialokinin-dependent vascular leakage.

Viruses transmitted by Aedes mosquitoes are an increasingly important global cause of disease. Defining common determinants of host susceptibility to this large group of heterogenous pathogens is key for informing the rational design of panviral medicines. Infection of the vertebrate host with these viruses is enhanced by mosquito saliva, a complex mixture of salivary-gland-derived factors and microbiota. We show that the enhancement of infection by saliva was dependent on vascular function and was independent of most antisaliva immune responses, including salivary microbiota. Instead, the Aedes gene product sialokinin mediated the enhancement of virus infection through a rapid reduction in endothelial barrier integrity. Sialokinin is unique within the insect world as having a vertebrate-like tachykinin sequence and is absent from Anopheles mosquitoes, which are incompetent for most arthropod-borne viruses, whose saliva was not proviral and did not induce similar vascular permeability. Therapeutic strategies targeting sialokinin have the potential to limit disease severity following infection with Aedes-mosquito-borne viruses.

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.

The antiviral piRNA response in mosquitoes?

There are several RNA interference (RNAi) pathways in insects. The small interfering RNA pathway is considered to be the main antiviral mechanism of the innate immune system; however, virus-specific P-element-induced Wimpy testis gene (PIWI)-interacting RNAs (vpiRNAs) have also been described, especially in mosquitoes. Understanding the antiviral potential of the RNAi pathways is important, given that many human and animal pathogens are transmitted by mosquitoes, such as Zika virus, dengue virus and chikungunya virus. In recent years, significant progress has been made to characterize the piRNA pathway in mosquitoes (including the possible antiviral activity) and to determine the differences between mosquitoes and the model organism Drosophila melanogaster. The new findings, especially regarding vpiRNA in mosquitoes, as well as important questions that need to be tackled in the future, are discussed in this review.

Culex quinquefasciatus mosquitoes do not support replication of Zika virus.

The rapid spread of Zika virus (ZIKV) in the Americas raised many questions about the role of Culex quinquefasciatus mosquitoes in transmission, in addition to the key role played by the vector Aedes aegypti. Here we analysed the competence of Cx. quinquefasciatus (with or without Wolbachia endosymbionts) for a ZIKV isolate. We also examined the induction of RNA interference pathways after viral challenge and the production of small virus-derived RNAs. We did not observe any infection nor such small virus-derived RNAs, regardless of the presence or absence of Wolbachia. Thus, Cx. quinquefasciatus does not support ZIKV replication and Wolbachia is not involved in producing this phenotype. In short, these mosquitoes are very unlikely to play a role in transmission of ZIKV.

Wolbachia Blocks Viral Genome Replication Early in Infection without a Transcriptional Response by the Endosymbiont or Host Small RNA Pathways.

The intracellular endosymbiotic bacterium Wolbachia can protect insects against viral infection, and is being introduced into mosquito populations in the wild to block the transmission of arboviruses that infect humans and are a major public health concern. To investigate the mechanisms underlying this antiviral protection, we have developed a new model system combining Wolbachia-infected Drosophila melanogaster cell culture with the model mosquito-borne Semliki Forest virus (SFV; Togaviridae, Alphavirus). Wolbachia provides strong antiviral protection rapidly after infection, suggesting that an early stage post-infection is being blocked. Wolbachia does appear to have major effects on events distinct from entry, assembly or exit as it inhibits the replication of an SFV replicon transfected into the cells. Furthermore, it causes a far greater reduction in the expression of proteins from the 3' open reading frame than the 5' non-structural protein open reading frame, indicating that it is blocking the replication of viral RNA. Further to this separation of the replicase proteins and viral RNA in transreplication assays shows that uncoupling of viral RNA and replicase proteins does not overcome Wolbachia's antiviral activity. This further suggests that replicative processes are disrupted, such as translation or replication, by Wolbachia infection. This may occur by Wolbachia mounting an active antiviral response, but the virus did not cause any transcriptional response by the bacterium, suggesting that this is not the case. Host microRNAs (miRNAs) have been implicated in protection, but again we found that host cell miRNA expression was unaffected by the bacterium and neither do our findings suggest any involvement of the antiviral siRNA pathway. We conclude that Wolbachia may directly interfere with early events in virus replication such as translation of incoming viral RNA or RNA transcription, and this likely involves an intrinsic (as opposed to an induced) mechanism.

Mutations conferring a noncytotoxic phenotype on chikungunya virus replicons compromise enzymatic properties of nonstructural protein 2.

UNLABELLED: Chikungunya virus (CHIKV) (genus Alphavirus) has a positive-sense RNA genome. CHIKV nonstructural protein 2 (nsP2) proteolytically processes the viral nonstructural polyprotein, possesses nucleoside triphosphatase (NTPase), RNA triphosphatase, and RNA helicase activities, and induces cytopathic effects in vertebrate cells. Although alphaviral nsP2 mutations can result in a noncytotoxic phenotype, the effects of such mutations on nsP2 enzymatic activities are not well understood. In this study, we introduced a P718G (PG) mutation and selected for additional mutations in CHIKV nsP2 that resulted in a CHIKV replicon with a noncytotoxic phenotype in BHK-21 cells. Combinations of PG and either an E117K (EK) substitution or a GEEGS sequence insertion after residue T647 (5A) markedly reduced RNA synthesis; however, neither PG nor 5A prevented nsP2 nuclear translocation. Introducing PG into recombinant nsP2 inhibited proteolytic cleavage of nsP1/nsP2 and nsP3/nsP4 sites, reduced GTPase and RNA helicase activities, and abolished RNA stimulation of GTPase activity. 5A and EK modulated the effects of PG. However, only the RNA helicase activity of nsP2 was reduced by both of these mutations, suggesting that defects in this activity may be linked to a noncytotoxic phenotype. These results increase our understanding of the molecular basis for the cytotoxicity that accompanies alphaviral replication. Furthermore, adaptation of the CHIKV replicon containing both 5A and PG allowed the selection of a CHIKV replicon with adaptive mutations in nsP1 and nsP3 that enable persistence in human cell line. Such cell lines represent valuable experimental systems for discovering host factors and for screening inhibitors of CHIKV replication at lower biosafety levels. IMPORTANCE: CHIKV is a medically important pathogen that causes febrile illness and can cause chronic arthritis. No approved vaccines or antivirals are available for CHIKV. The attenuation of CHIKV is critical to the establishment of experimental systems that can be used to conduct virus replication studies at a lower biosafety level. We applied a functional selection approach to develop, for the first time, a noncytotoxic CHIKV replicon capable of persisting in human cell lines. We anticipate that this safe and efficient research tool will be valuable for screening CHIKV replication inhibitors and for identifying and analyzing host factors involved in viral replication. We also analyzed, from virological and protein biochemistry perspectives, the functional defects caused by mutations conferring noncytotoxic phenotypes; we found that all known enzymatic activities of CHIKV nsP2, as well as its RNA-binding capability, were compromised by these mutations, which led to a reduced capacity for replication.

Magnetic fractionation and proteomic dissection of cellular organelles occupied by the late replication complexes of Semliki Forest virus.

Alphavirus replicase complexes are initially formed at the plasma membrane and are subsequently internalized by endocytosis. During the late stages of infection, viral replication organelles are represented by large cytopathic vacuoles, where replicase complexes bind to membranes of endolysosomal origin. In addition to viral components, these organelles harbor an unknown number of host proteins. In this study, a fraction of modified lysosomes carrying functionally intact replicase complexes was obtained by feeding Semliki Forest virus (SFV)-infected HeLa cells with dextran-covered magnetic nanoparticles and later magnetically isolating the nanoparticle-containing lysosomes. Stable isotope labeling with amino acids in cell culture combined with quantitative proteomics was used to reveal 78 distinct cellular proteins that were at least 2.5-fold more abundant in replicase complex-carrying vesicles than in vesicles obtained from noninfected cells. These host components included the RNA-binding proteins PCBP1, hnRNP M, hnRNP C, and hnRNP K, which were shown to colocalize with the viral replicase. Silencing of hnRNP M and hnRNP C expression enhanced the replication of SFV, Chikungunya virus (CHIKV), and Sindbis virus (SINV). PCBP1 silencing decreased SFV-mediated protein synthesis, whereas hnRNP K silencing increased this synthesis. Notably, the effect of hnRNP K silencing on CHIKV- and SINV-mediated protein synthesis was opposite to that observed for SFV. This study provides a new approach for analyzing the proteome of the virus replication organelle of positive-strand RNA viruses and helps to elucidate how host RNA-binding proteins exert important but diverse functions during positive-strand RNA viral infection.

Phenoloxidase activity acts as a mosquito innate immune response against infection with Semliki Forest virus.

Several components of the mosquito immune system including the RNA interference (RNAi), JAK/STAT, Toll and IMD pathways have previously been implicated in controlling arbovirus infections. In contrast, the role of the phenoloxidase (PO) cascade in mosquito antiviral immunity is unknown. Here we show that conditioned medium from the Aedes albopictus-derived U4.4 cell line contains a functional PO cascade, which is activated by the bacterium Escherichia coli and the arbovirus Semliki Forest virus (SFV) (Togaviridae; Alphavirus). Production of recombinant SFV expressing the PO cascade inhibitor Egf1.0 blocked PO activity in U4.4 cell- conditioned medium, which resulted in enhanced spread of SFV. Infection of adult female Aedes aegypti by feeding mosquitoes a bloodmeal containing Egf1.0-expressing SFV increased virus replication and mosquito mortality. Collectively, these results suggest the PO cascade of mosquitoes plays an important role in immune defence against arboviruses.

The Timing and Magnitude of the Type I Interferon Response Are Correlated with Disease Tolerance in Arbovirus Infection.

Infected hosts possess two alternative strategies to protect themselves against the negative impact of virus infections: resistance, used to abrogate virus replication, and disease tolerance, used to avoid tissue damage without controlling viral burden. The principles governing pathogen resistance are well understood, while less is known about those involved in disease tolerance. Here, we studied bluetongue virus (BTV), the cause of bluetongue disease of ruminants, as a model system to investigate the mechanisms of virus-host interactions correlating with disease tolerance. BTV induces clinical disease mainly in sheep, while cattle are considered reservoirs of infection, rarely exhibiting clinical symptoms despite sustained viremia. Using primary cells from multiple donors, we show that BTV consistently reaches higher titers in ovine cells than cells from cattle. The variable replication kinetics of BTV in sheep and cow cells were mostly abolished by abrogating the cell type I interferon (IFN) response. We identified restriction factors blocking BTV replication, but both the sheep and cow orthologues of these antiviral genes possess anti-BTV properties. Importantly, we demonstrate that BTV induces a faster host cell protein synthesis shutoff in primary sheep cells than cow cells, which results in an earlier downregulation of antiviral proteins. Moreover, by using RNA sequencing (RNA-seq), we also show a more pronounced expression of interferon-stimulated genes (ISGs) in BTV-infected cow cells than sheep cells. Our data provide a new perspective on how the type I IFN response in reservoir species can have overall positive effects on both virus and host evolution. IMPORTANCE The host immune response usually aims to inhibit virus replication in order to avoid cell damage and disease. In some cases, however, the infected host avoids the deleterious effects of infection despite high levels of viral replication. This strategy is known as disease tolerance, and it is used by animal reservoirs of some zoonotic viruses. Here, using a virus of ruminants (bluetongue virus [BTV]) as an experimental system, we dissected virus-host interactions in cells collected from species that are susceptible (sheep) or tolerant (cow) to disease. We show that (i) virus modulation of the host antiviral type I interferon (IFN) responses, (ii) viral replication kinetics, and (iii) virus-induced cell damage differ in tolerant and susceptible BTV-infected cells. Understanding the complex virus-host interactions in disease tolerance can allow us to disentangle the critical balance between protective and damaging host immune responses.

Novel functions of the alphavirus nonstructural protein nsP3 C-terminal region.

The functions of the alphavirus-encoded nonstructural protein nsP3 during infection are poorly understood. In contrast, nsP1, nsP2, and nsP4 have known enzymatic activities and functions. A functional analysis of the C-terminal region of nsP3 of Semliki Forest virus revealed the presence of a degradation signal that overlaps with a sequence element located between nsP3 and nsP4 that is required for proteolytic processing. This element was responsible for the short half-life (1 h) of individually expressed nsP3, and it also was functionally transferable to other proteins. Inducible cell lines were used to express native nsP3 or truncated mutants. The removal of 10 C-terminal amino acid (aa) residues from nsP3 increased the half-life of the protein approximately 8-fold. While the deletion of 30 C-terminal aa residues resulted in a similar stabilization, this deletion also changed the cellular localization of nsP3. This truncated mutant no longer exhibited a punctate localization in the cytoplasm, but instead filamentous stretches could be formed around the nuclei of induced cells, suggesting the existence of an additional functional element upstream of the degradation signal. C-terminally truncated uncleavable polyprotein P12(CA)3del30 was localized diffusely, which is in contrast to P12(CA)3, which is known to be associated with vesicle membranes. The induction of nsP3 or its truncated forms reduced the efficiency of virus multiplication in corresponding cells by affecting different steps of the infection cycle. The expression of nsP3 or a mutant lacking the 10 C-terminal aa residues repressed the establishment of infection, while the expression of nsP3 lacking 30 C-terminal aa residues led to the reduced synthesis of subgenomic RNA.

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.

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.

A prenylated dsRNA sensor protects against severe COVID-19.

Inherited genetic factors can influence the severity of COVID-19, but the molecular explanation underpinning a genetic association is often unclear. Intracellular antiviral defenses can inhibit the replication of viruses and reduce disease severity. To better understand the antiviral defenses relevant to COVID-19, we used interferon-stimulated gene (ISG) expression screening to reveal that 2'-5'-oligoadenylate synthetase 1 (OAS1), through ribonuclease L, potently inhibits severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We show that a common splice-acceptor single-nucleotide polymorphism (Rs10774671) governs whether patients express prenylated OAS1 isoforms that are membrane-associated and sense-specific regions of SARS-CoV-2 RNAs or if they only express cytosolic, nonprenylated OAS1 that does not efficiently detect SARS-CoV-2. In hospitalized patients, expression of prenylated OAS1 was associated with protection from severe COVID-19, suggesting that this antiviral defense is a major component of a protective antiviral response.

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.

Generation of a reporter yellow fever virus for high throughput antiviral assays.

Yellow fever virus (YFV), a member of the Flaviviridae family, is an arthropod-borne virus that can cause severe disease in humans with a lethality rate of up to 60%. Since 2017, increases in YFV activity in areas of South America and Africa have been described. Although a vaccine is available, named strain 17D (Theiler and Smith, 1937), it is contraindicated for use in the elderly, expectant mothers, immunocompromised people, among others. To this day there is no antiviral treatment against YFV to reduce the severity of viral infection. Here, we used a circular polymerase extension reaction (CPER)-based reverse genetics approach to generate a full-length reporter virus (YFVhb) by introducing a small HiBit tag in the NS1 protein. The reporter virus replicates at a similar rate to the parental YFV in HuH-7 cells. Using YFVhb, we designed a high throughput antiviral screening luciferase-based assay to identify inhibitors that target any step of the viral replication cycle. We validated our assay by using a range of inhibitors including drugs, immune sera and neutralizing single chain variable fragments (scFv). In light of the recent upsurge in YFV and a potential spread of the virus, this assay is a further tool in the development of antiviral therapy against YFV.

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.