Abstract The w Mel strain of the insect endosymbiont Wolbachia reduces the potential for Aedes aegypti to transmit mosquito-borne viruses such as dengue (DENV). Field trials that have introgressed w Mel into Ae. aegypti populations have shown this approach significantly reduces dengue incidence. In a laboratory setting some w Mel- Ae. aegypti develop infectious saliva following a viremic blood meal. Additionally, studies have demonstrated that exposing w Mel- Ae. aegypti to heat treatment, particularly during the larval stage, reduces w Mel density in key tissues such as the ovaries, midgut and salivary glands. Here we build on these studies, using viremic blood collected from 13 dengue inpatients at the Hospital for Tropical Diseases in Ho Chi Minh City (Viet Nam), to assess how temperature affects the protection afforded to Ae. aegypti by w Mel. We found that, compared to w Mel- Ae. aegypti reared at 28 ± 4°C, those reared at 31 ± 4°C developed infectious saliva more frequently, but the risk of this occurring was still reduced compared to WT mosquitoes reared at the same temperature. Heat treatment reduced the density of w Mel in all tissues tested, decreased the magnitude of w Mel’s protection against DENV replication in the head/thorax, and significantly increased the amount of DENV replication in w Mel- Ae. aegypti . When comparing cohorts of w Mel- Ae. aegypti that did or did not develop infectious saliva, DENV levels in the head/thorax were associated with increased odds of mosquitoes developing infectious saliva, but w Mel density was not. Overall, these findings show that elevated rearing temperatures increase the risk of patient-derived DENV breakthrough infections in w Mel- Ae. aegypti , potentially due to increased DENV replication in these mosquitoes. This limitation suggests it would be prudent to increase surveillance in regions using w Mel for dengue control when daily mean temperatures remain above 30°C for multiday periods. Author Summary The mosquito species Ae. aegypti can be infected with the bacterium Wolbachia ( w Mel strain), reducing its capacity to transmit viruses like dengue (DENV). Wolbachia is now being used as a biocontrol tool to reduce the burden of dengue in communities. However, some mosquitoes with Wolbachia can still transmit DENV. Here we utilised a natural infection model using dengue patient-derived blood to examine how temperature may increase the risk of DENV transmission occurrence in mosquitoes with w Mel. Mosquitoes with w Mel were more likely to transmit virus when reared at an average temperature of 31°C compared to those reared at an average temperature of 28°C but these mosquitoes still had a lower risk of developing infectious saliva compared to their w Mel-free counterparts. Higher temperatures reduced the amount of w Mel in mosquito tissues and increased the amount of DENV replicating in the head/thorax. Increasing levels of DENV RNA in these tissues were found to be associated with increased risk of mosquitoes with w Mel developing infectious saliva. This finding indicates surveillance is warranted in high temperature settings or during heat waves, to monitor for changes in w Mel frequency and DENV infection in Ae. aegypti .

The tissue culture infectious dose 50 (TCID50) end-point dilution assay is the gold-standard assay to titer viruses with negligible or ambiguous cytopathic effects. The assay's specificity is improved when followed by an Enzyme-Linked Immunosorbent Assay (ELISA) to detect viral antigens. Cells infected with mosquito-borne orthoflavi- and alphaviruses are fixed after TCID50, prior to ELISA, using paraformaldehyde (PFA) or acetone. While 4 % PFA has been shown to effectively inactivate these viruses for safe handling in low biocontainment conditions, equivalent studies have not been reported for standard acetone fixation methods (20 % acetone for 24 hours at 4°C). This study evaluated the inactivation efficacy of acetone on orthoflavi- and alphaviruses using dengue virus (DENV) and Ross River virus (RRV), as exemplar viruses from each genus, respectively. We show that 50 % acetone and 4 % PFA fully inactivate DENV and RRV, but 20 % acetone does not reduce the infectivity of these viruses. Importantly, ELISA-based detection of DENV- and RRV-infected cells fixed with 50 % acetone was effective, with calculated titres comparable to cells treated with 20 % acetone. Together, our results inform a fixation method for titrating orthoflavi- and alphavirus samples by TCID50/ELISA, ensuring the safe handling and processing of these viruses under low biocontainment conditions.

The Australasian Virology Society (AVS) holds premier biennial virology meetings that foster multidisciplinary research and collaboration and promote equity and inclusion of early-career researchers. The 12th AVS meeting (AVS12), convened by M. Tate, J. Fraser, and G. Moseley, was held from 2 to 5 December 2024 on Dja Dja Wurrung country at the RACV Goldfields Resort in Creswick, Victoria, Australia. In this report, we give a brief overview of the history of AVS and outline the current and developing priorities for the society. We provide a summary of the insightful panel discussions held to address career development and Indigenous virology, highlight the presentations given by international plenary speakers Joe Grove and Chantal Abergel, and celebrate the recipients of the numerous awards.

Abstract Background Introgression of the bacterial endosymbiont Wolbachia into Aedes aegypti populations is a biocontrol approach being used to reduce arbovirus transmission. This requires mass release of Wolbachia-infected mosquitoes. While releases have been conducted using a variety of techniques, egg releases, using water-soluble capsules containing mosquito eggs and larval food, offer an attractive method due to its potential to reduce onsite resource requirements. However, optimisation of this approach is required to ensure there is no detrimental impact on mosquito fitness and to promote successful Wolbachia introgression. Methods We determined the impact of storage time and temperature on wild-type (WT) and Wolbachia-infected (wMel or wAlbB strains) Ae. aegypti eggs. Eggs were stored inside capsules over 8 weeks at 18 °C or 22 °C and hatch rate, emergence rate and Wolbachia density were determined. We next examined egg quality and Wolbachia density after exposing eggs to 4–40 °C to determine how eggs may be impacted if exposed to extreme temperatures during shipment. Results Encapsulating eggs for 8 weeks did not negatively impact egg viability or resulting adult emergence and Wolbachia density compared to controls. When eggs were exposed to temperatures within 4–36 °C for 48 h, their viability and resulting adult Wolbachia density were maintained; however, both were significantly reduced when exposed to 40 °C. Conclusions We describe the time and temperature limits for maintaining viability of Wolbachia-infected Ae. aegypti eggs when encapsulated or exposed to extreme temperatures. These findings could improve the efficiency of mass releases by providing transport and storage constraints to ensure only high-quality material is utilised during field releases. Graphical Abstract

AbstractThe insect endosymbiotic bacteriumWolbachia pipientisis being utilised as a biocontrol tool to reduce the incidence ofAedes aegypti-transmitted viral diseases like dengue. However, the precise mechanisms underpinningWolbachia’s antiviral activity are not well defined. Here we generated a panel ofAe. aegypti-derived cell lines infected with antiviral strainswMel andwAlbB or the non-antiviral strainwPip to understand host cell morphological changes specifically induced by antiviral strains. Antiviral strains were frequently found to be entirely wrapped by the host endoplasmic reticulum (ER) membrane, whilewPip bacteria clustered separately in the host cell cytoplasm. ER-derived lipid droplets (LDs) increased in volume inwMel-andwAlbB-infected cell lines and mosquito tissues compared to cells infected withwPip orWolbachia-free controls. Inhibition of fatty acid synthase (required for triacylglycerol biosynthesis) reduced LD formation and significantly restored ER-associated dengue virus replication in cells occupied bywMel. Together, this suggests that antiviralWolbachiastrains may specifically alter the lipid composition of the ER to preclude the establishment of DENV replication complexes. DefiningWolbachia’s antiviral mechanisms will support the application and longevity of this effective biocontrol tool that is already being used at scale.ImportanceAedes aegyptitransmits a range of important human pathogenic viruses like dengue. However, infection ofAe. aegyptiwith the insect endosymbiotic bacterium,Wolbachia, reduces the risk of mosquito to human viral transmission.Wolbachiais being utilized at field sites across more than 13 countries to reduce the incidence of viruses like dengue, but it is not well understood howWolbachiainduces its antiviral effects. To examine this at the subcellular level, we compared how different strains ofWolbachiawith varying antiviral strengths, associate with and modify host cell structures. Strongly antiviral strains were found to specifically associate with the host endoplasmic reticulum and induce striking impacts on host cell lipid distribution. InhibitingWolbachia-induced lipid redistribution partially restored dengue virus replication demonstrating this is a contributing role forWolbachia’s antiviral activity. These findings provide new insights into how antiviralWolbachiastrains associate with and modifyAe. aegyptihost cells.

The insect endosymbiotic bacterium Wolbachia pipientis is being utilised as a biocontrol tool to reduce the incidence of Aedes aegypti -transmitted viral diseases like dengue. However, the precise mechanisms underpinning Wolbachia ’s antiviral activity are not well defined. Here we generated a panel of Ae. aegypti -derived cell lines infected with antiviral strains w Mel and w AlbB or the non-antiviral strain w Pip to understand host cell morphological changes specifically induced by antiviral strains. Antiviral strains were frequently found to be entirely wrapped by the host endoplasmic reticulum (ER) membrane, while w Pip bacteria clustered separately in the host cell cytoplasm. ER-derived lipid droplets (LDs) increased in volume in w Mel-and w AlbB-infected cell lines and mosquito tissues compared to cells infected with w Pip or Wolbachia -free controls. Inhibition of fatty acid synthase (required for triacylglycerol biosynthesis) reduced LD formation and significantly restored ER-associated dengue virus replication in cells occupied by w Mel. Together, this suggests that antiviral Wolbachia strains may specifically alter the lipid composition of the ER to preclude the establishment of DENV replication complexes. Defining Wolbachia ’s antiviral mechanisms will support the application and longevity of this effective biocontrol tool that is already being used at scale.Importance Aedes aegypti transmits a range of important human pathogenic viruses like dengue. However, infection of Ae. aegypti with the insect endosymbiotic bacterium, Wolbachia , reduces the risk of mosquito to human viral transmission. Wolbachia is being utilized at field sites across more than 13 countries to reduce the incidence of viruses like dengue, but it is not well understood how Wolbachia induces its antiviral effects. To examine this at the subcellular level, we compared how different strains of Wolbachia with varying antiviral strengths, associate with and modify host cell structures. Strongly antiviral strains were found to specifically associate with the host endoplasmic reticulum and induce striking impacts on host cell lipid distribution. Inhibiting Wolbachia -induced lipid redistribution partially restored dengue virus replication demonstrating this is a contributing role for Wolbachia ’s antiviral activity. These findings provide new insights into how antiviral Wolbachia strains associate with and modify Ae. aegypti host cells.### Competing Interest StatementThe authors have declared no competing interest.

Wolbachia are a genus of insect endosymbiotic bacteria which includes strains wMel and wAlbB that are being utilized as a biocontrol tool to reduce the incidence of Aedes aegypti-transmitted viral diseases like dengue. However, the precise mechanisms underpinning the antiviral activity of these Wolbachia strains are not well defined. Here, we generated a panel of Ae. aegypti-derived cell lines infected with antiviral strains wMel and wAlbB or the non-antiviral Wolbachia strain wPip to understand host cell morphological changes specifically induced by antiviral strains. Antiviral strains were frequently found to be entirely wrapped by the host endoplasmic reticulum (ER) membrane, while wPip bacteria clustered separately in the host cell cytoplasm. ER-derived lipid droplets (LDs) increased in volume in wMel- and wAlbB-infected cell lines and mosquito tissues compared to cells infected with wPip or Wolbachia-free controls. Inhibition of fatty acid synthase (required for triacylglycerol biosynthesis) reduced LD formation and significantly restored ER-associated dengue virus replication in cells occupied by wMel. Together, this suggests that antiviral Wolbachia strains may specifically alter the lipid composition of the ER to preclude the establishment of dengue virus (DENV) replication complexes. Defining Wolbachia's antiviral mechanisms will support the application and longevity of this effective biocontrol tool that is already being used at scale. IMPORTANCE Aedes aegypti transmits a range of important human pathogenic viruses like dengue. However, infection of Ae. aegypti with the insect endosymbiotic bacterium, Wolbachia, reduces the risk of mosquito to human viral transmission. Wolbachia is being utilized at field sites across more than 13 countries to reduce the incidence of viruses like dengue, but it is not well understood how Wolbachia induces its antiviral effects. To examine this at the subcellular level, we compared how different strains of Wolbachia with varying antiviral strengths associate with and modify host cell structures. Strongly antiviral strains were found to specifically associate with the host endoplasmic reticulum and induce striking impacts on host cell lipid droplets. Inhibiting Wolbachia-induced lipid redistribution partially restored dengue virus replication demonstrating this is a contributing role for Wolbachia's antiviral activity. These findings provide new insights into how antiviral Wolbachia strains associate with and modify Ae. aegypti host cells.

Dengue virus (DENV) threatens almost 70% of the world’s population, with no effective therapeutic currently available and controversy surrounding the one approved vaccine. A key factor in dengue viral replication is the interaction between DENV nonstructural proteins (NS) 5 and 3 (NS3) in the infected cell. Here, we perform a proof-of-principle high-throughput screen to identify compounds targeting the NS5-NS3 binding interface. We use a range of approaches to show for the first time that two small molecules–repurposed drugs I-OMe tyrphostin AG538 (I-OMe-AG238) and suramin hexasodium (SHS)–inhibit NS5-NS3 binding at low μM concentration through direct binding to NS5 that impacts thermostability. Importantly, both have strong antiviral activity at low μM concentrations against not only DENV-2, but also Zika virus (ZIKV) and West Nile virus (WNV). This work highlights the NS5-NS3 binding interface as a viable target for the development of anti-flaviviral therapeutics.

Wolbachia is an endosymbiotic bacterium that can restrict the transmission of human pathogenic viruses by Aedes aegypti mosquitoes. Recent field trials have shown that dengue incidence is significantly reduced when Wolbachia is introgressed into the local Ae. aegypti population. Female Ae. aegypti are anautogenous and feed on human blood to produce viable eggs. Herein, we tested whether people who reside on Tri Nguyen Island (TNI), Vietnam developed antibodies to Wolbachia Surface Protein (WSP) following release of Wolbachia-infected Ae. aegypti, as a measure of exposure to Wolbachia. Paired blood samples were collected from 105 participants before and after mosquito releases and anti-WSP titres were measured by ELISA. We determined no change in anti-WSP titres after ~30 weeks of high levels of Wolbachia-Ae. aegypti on TNI. These data suggest that humans are not exposed to the major Wolbachia surface antigen, WSP, following introgression of Wolbachia-infected Ae. aegypti mosquitoes.

Volume 95, no. 13, e02203-20, 2021, https://doi.org/10.1128/JVI.02203-20. The article was originally published on 10 June 2021 with a standard copyright line ("© 2021 American Society for Microbiology. All Rights Reserved."). We elected to pay for open access for the article after publication, necessitating replacement of the original copyright line with "© 2021 Edenborough et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license." This change was made to the online version of the article on 2 July 2021.

The artificial introduction of the endosymbiotic bacterium, Wolbachia pipientis, into Aedes (Ae.) aegypti mosquitoes reduces the ability of mosquitoes to transmit human pathogenic viruses and is now being developed as a biocontrol tool. Successful introgression of Wolbachia-carrying Ae. aegypti into native mosquito populations at field sites in Australia, Indonesia and Malaysia has been associated with reduced disease prevalence in the treated community. In separate field programs, Wolbachia is also being used as a mosquito population suppression tool, where the release of male only Wolbachia-infected Ae. aegypti prevents the native mosquito population from producing viable eggs, subsequently suppressing the wild population. While these technologies show great promise, they require mass rearing of mosquitoes for implementation on a scale that has not previously been done. In addition, Wolbachia induces some negative fitness effects on Ae. aegypti. While these fitness effects differ depending on the Wolbachia strain present, one of the most consistent and significant impacts is the shortened longevity and viability of eggs. This review examines the body of evidence behind Wolbachia's negative effect on eggs, assesses nutritional parasitism as a key cause and considers how these impacts could be overcome to achieve efficient large-scale rearing of these mosquitoes.

The Australasian Virology Society (AVS) aims to promote, support and advocate for the discipline of virology in the Australasian region. The society was incorporated in 2011 after 10 years operating as the Australian Virology Group (AVG) founded in 2001, coinciding with the inaugural biennial scientific meeting. AVS conferences aim to provide a forum for the dissemination of all aspects of virology, foster collaboration, and encourage participation by students and post-doctoral researchers. The tenth Australasian Virology Society (AVS10) scientific meeting was held on 2–5 December 2019 in Queenstown, New Zealand. This report highlights the latest research presented at the meeting, which included cutting-edge virology presented by our international plenary speakers Ana Fernandez-Sesma and Benjamin tenOever, and keynote Richard Kuhn. AVS10 honoured female pioneers in Australian virology, Lorena Brown and Barbara Coulson. We report outcomes from the AVS10 career development session on “Successfully transitioning from post-doc to lab head”, winners of best presentation awards, and the AVS gender equity policy, initiated in 2013. Plans for the 2021 meeting are underway which will celebrate the 20th anniversary of AVS where it all began, in Fraser Island, Queensland, Australia.

The bacterial endosymbiont Wolbachia is a biocontrol tool that inhibits the ability of the Aedes aegypti mosquito to transmit positive-sense RNA viruses such as dengue and Zika. Growing evidence indicates that when Wolbachia strains w Mel or w AlbB are introduced into local mosquito populations, human dengue incidence is reduced. Despite the success of this novel intervention, we still do not fully understand how Wolbachia protects mosquitoes from viral infection. Here, we demonstrate that the Wolbachia strain w Pip does not inhibit virus infection in Ae. aegypti . We have leveraged this novel finding, and a panel of Ae. aegypti lines carrying virus-inhibitory ( w Mel and w AlbB) and non-inhibitory ( w Pip) strains in a common genetic background, to rigorously test a number of hypotheses about the mechanism of Wolbachia -mediated virus inhibition. We demonstrate that, contrary to previous suggestions, there is no association between a strain’s ability to inhibit dengue infection in the mosquito and either its typical density in the midgut or salivary glands, or the degree to which it elevates innate immune response pathways in the mosquito. These findings, and the experimental platform provided by this panel of genetically comparable mosquito lines, clear the way for future investigations to define how Wolbachia prevents Ae. aegypti from transmitting viruses. Author summary Dengue virus, transmitted by the Aedes aegypti mosquito, is one of the fastest-growing infectious diseases, causing an estimated 390 million human infections per year worldwide. Vaccines have limited efficacy and there are no approved therapeutics. This has driven the rise of novel vector control programs, in particular those that use the bacterium, Wolbachia , which prevents transmission of dengue and other human pathogenic viruses when stably introduced into Ae. aegypti populations. Although this is proving to be a highly effective method, the details of how this biocontrol tool works are not well understood. Here we characterise a new Wolbachia strain, w Pip, and find that Ae. aegypti carrying w Pip are still able to transmit dengue similar to mosquitoes that do not carry Wolbachia . This finding has allowed us to begin a rigorous program of comparative studies to determine which features of a Wolbachia strain determine whether it is antiviral. Understanding these mechanisms will enable us to predict the risk of viral resistance arising against Wolbachia and facilitate preparation of second-generation field release lines.

Dengue virus (DENV) threatens almost 70% of the world’s population, with no effective vaccine or therapeutic currently available. A key contributor to infection is nuclear localisation in the infected cell of DENV nonstructural protein 5 (NS5) through the action of the host importin (IMP) α/β1 proteins. Here, we used a range of microscopic, virological and biochemical/biophysical approaches to show for the first time that the small molecule GW5074 has anti-DENV action through its novel ability to inhibit NS5–IMPα/β1 interaction in vitro as well as NS5 nuclear localisation in infected cells. Strikingly, GW5074 not only inhibits IMPα binding to IMPβ1, but can dissociate preformed IMPα/β1 heterodimer, through targeting the IMPα armadillo (ARM) repeat domain to impact IMPα thermal stability and α-helicity, as shown using analytical ultracentrifugation, thermostability analysis and circular dichroism measurements. Importantly, GW5074 has strong antiviral activity at low µM concentrations against not only DENV-2, but also zika virus and West Nile virus. This work highlights DENV NS5 nuclear targeting as a viable target for anti-flaviviral therapeutics.

Wolbachia pipientis from Drosophila melanogaster (wMel) is an endosymbiotic bacterium that restricts transmission of human pathogenic flaviviruses and alphaviruses, including dengue, Zika, and chikungunya viruses, when introduced into the mosquito vector Aedes aegypti. To date, wMel-infected Ae. aegypti have been released in field trials in 5 countries to evaluate the effectiveness of this strategy for disease control. Despite the success in establishing wMel-infected mosquitoes in wild populations, and the well-characterized antiviral capabilities of wMel, transinfecting different or additional Wolbachia strains into Ae. aegypti may improve disease impact, and perhaps more importantly, could provide a strategy to account for the possible evolution of resistant arboviruses. Here, we report the successful transinfection of Ae. aegypti with the Wolbachia strains wMelCS (D. melanogaster), wRi (D. simulans) and wPip (Culex quinquefasciatus) and assess the effects on Ae. aegypti fitness, cytoplasmic incompatibility, tissue tropism and pathogen blocking in a laboratory setting. The results demonstrate that wMelCS provides a similar degree of protection against dengue virus as wMel following an infectious blood meal, and significantly reduces viral RNA levels beyond that of wMel following a direct challenge with infectious virus in mosquitoes, with no additional fitness cost to the host. The protection provided by wRi is markedly weaker than that of wMelCS, consistent with previous characterisations of these lines in Drosophila, while wPip was found to substantially reduce the fitness of Ae. aegypti. Thus, we determine wMelCS as a key candidate for further testing in field-relevant fitness tests and viremic blood feeding challenges in a clinical setting to determine if it may represent an alternative Wolbachia strain with more desirable attributes than wMel for future field testing.

Infections by dengue virus (DENV) are increasing worldwide, with an urgent need for effective anti-DENV agents. We recently identified N-(4-hydroxyphenyl) retinamide (4-HPR), an anti-DENV agent effective against all 4 serotypes of DENV in cell culture, and in a lethal mouse model for DENV infection (Fraser et al., 2014b). Although identified as an inhibitor of DENV non-structural protein 5 (NS5) recognition by host nuclear import proteins, the precise impact and mode of action of 4-HPR in effecting DENV clearance remains to be defined. Significantly, concurrent with decreased viral RNA and infectious DENV in 4-HPR-treated cells, we previously observed specific up-regulation of transcripts representing the Protein Kinase R-like Endoplasmic Reticulum Kinase (PERK) arm of the unfolded protein response (UPR) pathway upon 4-HPR addition. Here we pursue these findings in detail, examining the role of specific PERK pathway components in DENV clearance. We demonstrate that 4-HPR-induced nuclear localization of Activating Transcription Factor 4 (ATF4), a pathway component downstream from PERK, occurs in a PERK-independent manner, implying activation instead occurs through Integrated Stress Response (ISR) kinases. Significantly, ATF4 does not appear to be required for the antiviral activity of 4-HPR, suggesting transcriptional events induced by ATF4 do not drive the 4-HPR-induced antiviral state. Instead, we demonstrate that 4-HPR induces phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2 alpha), a target of ISR kinases which controls translation attenuation, and confirm the importance of phosphorylated-eIF2 alpha. in DENV infection using guanabenz, a specific inhibitor of eIF2 alpha. dephosphorylation. This study provides the first detailed insight into the cellular effects modulated by 4-HPR in DENV-infected cells, critical to progressing 4-HPR towards the clinic. (C) 2016 Elsevier B.V. All rights reserved.

ABSTRACT The nucleolar subcompartment of the nucleus is increasingly recognized as an important target of RNA viruses. Here we document for the first time the ability of dengue virus (DENV) polymerase, nonstructural protein 5 (NS5), to accumulate within the nucleolus of infected cells and to target green fluorescent protein (GFP) to the nucleolus of live transfected cells. Intriguingly, NS5 exchange between the nucleus and nucleolus is dynamically modulated by extracellular pH, responding rapidly and reversibly to pH change, in contrast to GFP alone or other nucleolar and non-nucleolar targeted protein controls. The minimal pH-sensitive nucleolar targeting region (pHNTR), sufficient to target GFP to the nucleolus in a pH-sensitive fashion, was mapped to NS5 residues 1 to 244, with mutation of key hydrophobic residues, Leu-165, Leu-167, and Val-168, abolishing pHNTR function in NS5-transfected cells, and severely attenuating DENV growth in infected cells. This is the first report of a viral protein whose nucleolar targeting ability is rapidly modulated by extracellular stimuli, suggesting that DENV has the ability to detect and respond dynamically to the extracellular environment. IMPORTANCE Infections by dengue virus (DENV) threaten 40% of the world's population yet there is no approved vaccine or antiviral therapeutic to treat infections. Understanding the molecular details that govern effective viral replication is key for the development of novel antiviral strategies. Here, we describe for the first time dynamic trafficking of DENV nonstructural protein 5 (NS5) to the subnuclear compartment, the nucleolus. We demonstrate that NS5's targeting to the nucleolus occurs in response to acidic pH, identify the key amino acid residues within NS5 that are responsible, and demonstrate that their mutation severely impairs production of infectious DENV. Overall, this study identifies a unique subcellular trafficking event and suggests that DENV is able to detect and respond dynamically to environmental changes.

Dengue virus (DENV) nonstructural protein 5 (NS5) plays a central role in viral replication in the cytoplasm of infected cells. Despite this, NS5 is predominantly located in the nucleus of infected cells where it is thought to play a role in suppression of the host antiviral response. We have investigated the nuclear localization of NS5 using immunofluorescent staining for NS5 in infected cells, showing that NS5 nuclear localization is significantly inhibited by Ivermectin, a general inhibitor of nuclear transport mediated by the cellular nuclear transport proteins importin alpha/beta (IMP alpha/beta). Experiments in living mammalian cells transfected to express green fluorescent protein (GFP)-tagged NS5 protein confirm that NS5 is predominantly nuclear and that this localization is inhibited by Ivermectin, demonstrating that NS5 contains an Ivermectin-sensitive IMP alpha/beta-recognized nuclear localization signal [Pryor et al. Traffic 8:795-807, 2007]. Consistent with this observation, mutation of critical residues within the nuclear localization signal (the A2 mutant; [Pryor et al. Traffic 8: 795-807, 2007]) results in an 80 % reduction in nuclear localization of NS5. Finally we demonstrate direct, high-affinity binding of NS5 to IMP alpha/beta using an AlphaScreen protein-protein binding assay.

Background. Dengue virus (DENV) is estimated to cause 390 million infections each year, but there is no licensed vaccine or therapeutic currently available.Methods. We describe a novel, high-throughput screen to identify compounds inhibiting the interaction between DENV nonstructural protein 5 and host nuclear transport proteins. We document the antiviral properties of a lead compound against all 4 serotypes of DENV, antibody-dependent enhanced (ADE) infection, and ex vivo and in vivo DENV infections. In addition, we use quantitative reverse-transcription polymerase chain reaction to examine cellular effects upon compound addition.Results. We identify N-(4-hydroxyphenyl) retinamide (4-HPR) as effective in protecting against DENV-1-4 and DENV-1 ADE infections, with 50% effective concentrations in the low micromolar range. 4-HPR but not the closely related N-(4-methoxyphenyl) retinamide (4-MPR) could reduce viral RNA levels and titers when applied to an established infection. 4-HPR but not 4-MPR was found to specifically upregulate the protein kinase R-like endoplasmic reticulum kinase arm of the unfolded protein response. Strikingly, 4-HPR but not 4-MPR restricted infection in peripheral blood mononuclear cells and in a lethal ADE-infection mouse model.Conclusions. 4-HPR is a novel antiviral that modulates the unfolded protein response, effective against DENV1-4 at concentrations achievable in the plasma in a clinical setting, and provides protection in a lethal mouse model.