Condensation of RNase L promotes its rapid activation in response to viral infection in mammalian cells.

Oligoadenylate synthetase 3 (OAS3) and ribonuclease L (RNase L) are components of a pathway that combats viral infection in mammals. Upon detection of viral double-stranded RNA (dsRNA), OAS3 synthesizes 2'-5'-oligo(A), which activates the RNase domain of RNase L by promoting the homodimerization and oligomerization of RNase L monomers. Activated RNase L rapidly degrades all cellular mRNAs, shutting off several cellular processes. We sought to understand the molecular mechanisms underlying the rapid activation of RNase L in response to viral infection. Through superresolution microscopy and live-cell imaging, we showed that OAS3 and RNase L concentrated into higher-order cytoplasmic complexes known as dsRNA-induced foci (dRIF) in response to dsRNA or infection with dengue virus, Zika virus, or West Nile virus. The concentration of OAS3 and RNase L at dRIF corresponded with the activation of RNase L-mediated RNA decay. We showed that dimerized/oligomerized RNase L concentrated in a liquid-like shell surrounding a core OAS3-dRIF structure and dynamically exchanged with the cytosol. These data establish that the condensation of dsRNA, OAS3, and RNase L into dRIF is a molecular switch that promotes the rapid activation of RNase L upon detection of dsRNA in mammalian cells.

Regulation of microglia-mediated inflammation by host lncRNA Gm20559 upon flaviviral infection.

BACKGROUND: Japanese Encephalitis Virus (JEV) and West Nile Viruses (WNV) are neurotropic flaviviruses which cause neuronal death and exaggerated glial activation in the central nervous system. Role of host long non coding RNAs in shaping microglial inflammation upon flavivirus infections has been unexplored. This study attempted to decipher the role of lncRNA Gm20559 in regulating microglial inflammatory response in context of flaviviruses. METHODS: Antisense oligonucleotide LNA Gapmers designed against lncRNA Gm20559 and non-specific site (negative control) were used for Gm20559 knockdown in JEV and WNV-infected N9 microglial cells. Upon establishing successful Gm20559 knockdown, expression of various proinflammatory cytokines, chemokines, interferon-stimulated genes (ISGs) and RIG-I were checked by qRT-PCR and cytometric bead array. Western Blotting was done to analyse the phosphorylation level of various inflammatory markers and viral non-structural protein expression. Plaque Assays were employed to quantify viral titres in microglial supernatant upon knocking down Gm20559. Effect of microglial supernatant on HT22 neuronal cells was assessed by checking expression of apoptotic protein and viral non-structural protein by Western Blotting. RESULTS: Upregulation in Gm20559 expression was observed in BALB/c pup brains, primary microglia as well as N9 microglia cell line upon both JEV and WNV infection. Knockdown of Gm20559 in JEV and WNV-infected N9 cell led to the reduction of major proinflammatory cytokines - IL-1ß, IL-6, IP-10 and IFN-ß. Inhibition of Gm20559 upon JEV infection in N9 microglia also led to downregulation of RIG-I and OAS-2, which was not the case in WNV-infected N9 microglia. Phosphorylation level of P38 MAPK was reduced in case of JEV-infected N9 microglia and not WNV-infected N9 microglia. Whereas phosphorylation of NF-κB pathway was unchanged upon Gm20559 knockdown in both JEV and WNV-infected N9 microglia. However, treating HT22 cells with JEV and WNV-infected microglial supernatant with and without Gm20559 could not trigger cell death or influence viral replication. CONCLUSION: Knockdown studies on lncRNA Gm20559 suggests its pivotal role in maintaining the inflammatory milieu of microglia in flaviviral infection by modulating the expression of various pro-inflammatory cytokines. However, Gm20559-induced increased microglial proinflammatory response upon flavivirus infection fails to trigger neuronal death.

Type I IFN stimulates lymph node stromal cells from adult and old mice during a West Nile virus infection.

Advanced age is a significant risk factor during viral infection due to an age-associated decline in the immune response. Older individuals are especially susceptible to severe neuroinvasive disease after West Nile virus (WNV) infection. Previous studies have characterized age-associated defects in hematopoietic immune cells during WNV infection that culminate in diminished antiviral immunity. Situated amongst immune cells in the draining lymph node (DLN) are structural networks of nonhematopoietic lymph node stromal cells (LNSCs). LNSCs are comprised of numerous, diverse subsets, with critical roles in the coordination of robust immune responses. The contributions of LNSCs to WNV immunity and immune senescence are unclear. Here, we examine LNSC responses to WNV within adult and old DLNs. Acute WNV infection triggered cellular infiltration and LNSC expansion in adults. Comparatively, aged DLNs exhibited diminished leukocyte accumulation, delayed LNSC expansion, and altered fibroblast and endothelial cell subset composition, signified by fewer LECs. We established an ex vivo culture system to probe LNSC function. Adult and old LNSCs both recognized an ongoing viral infection primarily through type I IFN signaling. Gene expression signatures were similar between adult and old LNSCs. Aged LNSCs were found to constitutively upregulate immediate early response genes. Collectively, these data suggest LNSCs uniquely respond to WNV infection. We are the first to report age-associated differences in LNSCs on the population and gene expression level during WNV infection. These changes may compromise antiviral immunity, leading to increased WNV disease in older individuals.

Novel tools to study West Nile virus NS3 protease activity.

West Nile Virus (WNV) belongs to a group of pathogenic viruses called flaviviruses. West Nile virus infection can be mild, causing so-called West Nile Fever (WNF) or severe neuroinvasive form of the disease (WNND), and ultimately even death. There are currently no known medications to prevent West Nile virus infection. Only symptomatic treatment is used. To date, there are no unequivocal tests enabling a quick and unambiguous assessment of WN virus infection. The aim of the research was to obtain specific and selective tools for determining the activity of the West Nile virus serine proteinase. Using the methods of combinatorial chemistry with iterative deconvolution, the substrate specificity of the enzyme in non-primed and primed positions was determined. The FRET ABZ-Ala-Lys-Gln-Arg-Gly-Gly-Thr-Tyr(3-NO2)-NH2 substrate was obtained, characterized by kinetic parameters (KM = 4.20 ± 0.32 × 10-5 M) as for the majority of proteolytic enzymes. The obtained sequence was used to develop and synthesize highly sensitive functionalized quantum dot-based protease probes (QD). A QD WNV NS3 protease probe was obtained to detect an increase in fluorescence of 0.05 nmol enzyme in the assay system. This value was at least 20 times lower than that observed with the optimized substrate. The obtained result may be the basis for further research on the potential use of the WNV NS3 protease in the diagnosis of West Nile virus infection.

West Nile virus-associated HLA-DRB1 alleles in the Greek population: A structural perspective.

The HLA system plays a significant role via the regulation of the immune system and contributes to the progression and protection of many diseases. In our previous study, several HLA-DRB1 alleles were found to have a susceptible or protective role toward infection and neuroinvasion of West Nile Virus (WNV) in the Greek population. As expected, the majority of polymorphic positions are located in the peptide-binding region of the molecule. In the present work, the structure of these alleles was studied in silico, to examine the effect of polymorphism on the conformation of DRB1 proteins, with the aspect of WNV association. More specifically, molecular dynamics simulations were used for structural prediction of 23 available alleles. These modeled alleles were evaluated using root-mean-square deviation (RMSD) and root-mean-square fluctuation analysis. Low RMSD values indicate that different alleles have similar structures. Furthermore, low fluctuation was observed in the peptide-binding region between alleles with the higher and the lowest RMSD values. These findings indicate that probably variable residues do not affect the behavior of DRB1 alleles in WNV disease, by causing structural differences between them.

Levels of Circulating NS1 Impact West Nile Virus Spread to the Brain.

Dengue virus (DENV) and West Nile virus (WNV) are arthropod-transmitted flaviviruses that cause systemic vascular leakage and encephalitis syndromes, respectively, in humans. However, the viral factors contributing to these specific clinical disorders are not completely understood. Flavivirus nonstructural protein 1 (NS1) is required for replication, expressed on the cell surface, and secreted as a soluble glycoprotein, reaching high levels in the blood of infected individuals. Extracellular DENV NS1 and WNV NS1 interact with host proteins and cells, have immune evasion functions, and promote endothelial dysfunction in a tissue-specific manner. To characterize how differences in DENV NS1 and WNV NS1 might function in pathogenesis, we generated WNV NS1 variants with substitutions corresponding to residues found in DENV NS1. We discovered that the substitution NS1-P101K led to reduced WNV infectivity in the brain and attenuated lethality in infected mice, although the virus replicated efficiently in cell culture and peripheral organs and bound at wild-type levels to brain endothelial cells and complement components. The P101K substitution resulted in reduced NS1 antigenemia in mice, and this was associated with reduced WNV spread to the brain. Because exogenous administration of NS1 protein rescued WNV brain infectivity in mice, we conclude that circulating WNV NS1 facilitates viral dissemination into the central nervous system and impacts disease outcomes. IMPORTANCE Flavivirus NS1 serves as an essential scaffolding molecule during virus replication but also is expressed on the cell surface and is secreted as a soluble glycoprotein that circulates in the blood of infected individuals. Although extracellular forms of NS1 are implicated in immune modulation and in promoting endothelial dysfunction at blood-tissue barriers, it has been challenging to study specific effects of NS1 on pathogenesis without disrupting its key role in virus replication. Here, we assessed WNV NS1 variants that do not affect virus replication and evaluated their effects on pathogenesis in mice. Our characterization of WNV NS1-P101K suggests that the levels of NS1 in the circulation facilitate WNV dissemination to the brain and affect disease outcomes. Our findings facilitate understanding of the role of NS1 during flavivirus infection and support antiviral strategies for targeting circulating forms of NS1.

Nuclear localisation of West Nile virus NS5 protein modulates host gene expression.

The involvement of the nucleus during flavivirus infection has been observed in only a small number of cases and can be limited to primarily two viral proteins; the structural protein C and the RNA polymerase NS5. Previously we observed that by blocking nuclear transport, WNV strain Kunjin (WNVKUN) replication is severely affected and through mutation of the identified NLS in WNVKUN NS5 protein. In this study, we interrogated the potential nuclear functions of WNVKUN NS5 has on the host transcriptome, by means of RNA sequencing (RNAseq). In a direct comparison between wild type and mutant NS5, it can also be determined that the nuclear translocation of NS5 results in a significant down-regulation of host genes involved in the innate immune response. When compared to published RNAseq data from WNV infection, many of these genes were overlapping indicting the role of NS5 induced transcription during infection.

Using Accelerated Molecular Dynamics Simulation to elucidate the effects of the T198F mutation on the molecular flexibility of the West Nile virus envelope protein.

The envelope (E) protein is an important target for antibodies in flavivirus. Literature reports that the mutation T198F, located at the domain I-II hinge of the E protein, regulates viral breathing and increases the accessibility of a distal cryptic epitope located on the fusion loop, having a direct impact in the neutralization of West Nile virus (WNV). Our study aimed to describe, using accelerated molecular dynamics simulations, the effects of the T198F mutation in the flexibility of the E protein of WNV and to elucidate the mechanism that regulates epitope accessibility. The simulation results revealed that the mutation favors the formation of alternative hydrogen bonds, hampering the bending movement between domains I and II. We hypothesized that this is the mechanism by which the T198F mutation, located at the middle of the protein, locks the distal cryptc epitope near a single preferred conformation, rendering it more prone to recognition by antibodies.

West Nile Virus fidelity modulates the capacity for host cycling and adaptation.

The fidelity of flaviviruses is thought to be tightly regulated for optimal fitness within and between hosts. West Nile virus (WNV) high-fidelity (HiFi) mutations V793I and G806R within the RNA-dependent RNA polymerase, and low-fidelity (LoFi) mutation T248I within the methyltransferase, were previously shown to attenuate infectivity and replicative fitness in Culex mosquitoes and Culex tarsalis (CXT) cells but not in mammalian cells. We hypothesized that fidelity alterations would modify adaptation and maintenance in a host-specific manner. To test this hypothesis, wild-type (WT), HiFi (V793I/G806R) and LoFi (T248I) variants were sequentially passaged eight times in avian (PDE) or mosquito cells, or alternately between the two. Initial characterization confirmed that fidelity mutants are attenuated in mosquito, but not avian, cells. Deep sequencing revealed mutations unique to both cell lines and fidelity mutants, including ENV G1378A, a mutation associated with avian cell adaptation. To characterize maintenance and adaptation, viral outputs were monitored throughout passaging and viral fitness was assessed. The results indicate that fidelity mutants can at times recover fitness during mosquito cell passage, but remain attenuated relative to WT. Despite similar initial fitness, LoFi mutants were impaired during sequential passage in avian cells. Conversely, HiFi mutants passaged in avian cells showed increased adaptation, suggesting that increased fidelity may be advantageous in avian hosts. Although some adaptation occurred with individual mutants, the output titres of fidelity mutants were on average lower and were often lost during host switching. These data confirm that arbovirus fidelity is likely fine-tuned to maximize survival in disparate hosts.

West Nile virus activity in United States blood donors and optimizing detection strategies: 2014-2018.

BACKGROUND: Rare transfusion-transmitted West Nile virus (WNV) cases usually occur due to gaps in testing involving converting to more sensitive nucleic acid testing (NAT) formats (referred to as triggering). Using data from 2014 to 2018, we investigated a strategy used to increase detection early in the triggering period and reviewed its yield as the individual donation (ID)-NAT geographic area was decreased. METHODS: Mini-pool-NAT transitioned to ID-NAT following triggering based on one WNV NAT-reactive donation (having an elevated signal, repeat reactive, or in an area with WNV ongoing activity). ID-NAT-triggered geographic areas included an entire state (2014-2017) or collections within a 50-mile radius of the triggering donor's residential zip code (2018). During the MP- to ID-NAT transition, donation samples were retrieved and tested by ID-NAT for those with results not yet released (referred to as in-process testing). Reactive sample confirmation was performed by repeat NAT of an independent sample or antibody testing. RESULTS: ID-NAT included 3.2 million donations of more than 25 million tested year-round, resulting in 684 confirmed positives; all confirmed-positive donations occurred from June to December (0.64/10,000). Overall, 52% (358/684) required ID-NAT for detection, including 68 (19%) antibody negatives. Ten of 19 (53%) identified in-process were ID-NAT-only detectable, including four antibody negatives, or approximately 1 per year (2.8% of ID-NAT-only detectable). With reduced triggering geography, 12 of 19 (63%) were not identified (including 6/10 ID-NAT-only detectable, and 2/4 ID-NAT-only detectable/antibody negative). CONCLUSION: WNV NAT's utility is between June-December; however, abandoning testing outside of this time may increase risk. While in-process testing identified approximately one ID-NAT-only detectable (antibody-negative) donation per year, reducing the geographic triggered area decreased its effectiveness.

Chasing Intracellular Zika Virus Using Proteomics.

Flaviviruses are the most medically relevant group of arboviruses causing a wide range of diseases in humans and are associated with high mortality and morbidity, as such posing a major health concern. Viruses belonging to this family can be endemic (e.g., dengue virus), but can also cause fulminant outbreaks (e.g., West Nile virus, Japanese encephalitis virus and Zika virus). Intense research efforts in the past decades uncovered shared fundamental strategies used by flaviviruses to successfully replicate in their respective hosts. However, the distinct features contributing to the specific host and tissue tropism as well as the pathological outcomes unique to each individual flavivirus are still largely elusive. The profound footprint of individual viruses on their respective hosts can be investigated using novel technologies in the field of proteomics that have rapidly developed over the last decade. An unprecedented sensitivity and throughput of mass spectrometers, combined with the development of new sample preparation and bioinformatics analysis methods, have made the systematic investigation of virus-host interactions possible. Furthermore, the ability to assess dynamic alterations in protein abundances, protein turnover rates and post-translational modifications occurring in infected cells now offer the unique possibility to unravel complex viral perturbations induced in the infected host. In this review, we discuss the most recent contributions of mass spectrometry-based proteomic approaches in flavivirus biology with a special focus on Zika virus, and their basic and translational potential and implications in understanding and characterizing host responses to arboviral infections.

Complex dynamics under tension in a high-efficiency frameshift stimulatory structure.

Specific structures in mRNA can stimulate programmed ribosomal frameshifting (PRF). PRF efficiency can vary enormously between different stimulatory structures, but the features that lead to efficient PRF stimulation remain uncertain. To address this question, we studied the structural dynamics of the frameshift signal from West Nile virus (WNV), which stimulates -1 PRF at very high levels and has been proposed to form several different structures, including mutually incompatible pseudoknots and a double hairpin. Using optical tweezers to apply tension to single mRNA molecules, mimicking the tension applied by the ribosome during PRF, we found that the WNV frameshift signal formed an unusually large number of different metastable structures, including all of those previously proposed. From force-extension curve measurements, we mapped 2 mutually exclusive pathways for the folding, each encompassing multiple intermediates. We identified the intermediates in each pathway from length changes and the effects of antisense oligomers blocking formation of specific contacts. Intriguingly, the number of transitions between the different conformers of the WNV frameshift signal was maximal in the range of forces applied by the ribosome during -1 PRF. Furthermore, the occupancy of the pseudoknotted conformations was far too low for static pseudoknots to account for the high levels of -1 PRF. These results support the hypothesis that conformational heterogeneity plays a key role in frameshifting and suggest that transitions between different conformers under tension are linked to efficient PRF stimulation.

N-linked glycosylation of the West Nile virus envelope protein is not a requisite for avian virulence or vector competence.

The N-linked glycosylation motif at amino acid position 154-156 of the envelope (E) protein of West Nile virus (WNV) is linked to enhanced murine neuroinvasiveness, avian pathogenicity and vector competence. Naturally occurring isolates with altered E protein glycosylation patterns have been observed in WNV isolates; however, the specific effects of these polymorphisms on avian host pathogenesis and vector competence have not been investigated before. In the present study, amino acid polymorphisms, NYT, NYP, NYF, SYP, SYS, KYS and deletion (A'DEL), were reverse engineered into a parental WNV (NYS) cDNA infectious clone to generate WNV glycosylation mutant viruses. These WNV glycosylation mutant viruses were characterized for in vitro growth, pH-sensitivity, temperature-sensitivity and host competence in American crows (AMCR), house sparrows (HOSP) and Culex quinquefasciatus. The NYS and NYT glycosylated viruses showed higher viral replication, and lower pH and temperature sensitivity than NYP, NYF, SYP, SYS, KYS and A'DEL viruses in vitro. Interestingly, in vivo results demonstrated asymmetric effects in avian and mosquito competence that were independent of the E-protein glycosylation status. In AMCRs and HOSPs, all viruses showed comparable viremias with the exception of NYP and KYS viruses that showed attenuated phenotypes. Only NYP showed reduced vector competence in both Cx. quinquefasciatus and Cx. tarsalis. Glycosylated NYT exhibited similar avian virulence properties as NYS, but resulted in higher mosquito oral infectivity than glycosylated NYS and nonglycosylated, NYP, NYF, SYP and KYS mutants. These data demonstrated that amino acid polymorphisms at E154/156 dictate differential avian host and vector competence phenotypes independent of E-protein glycosylation status.

Role of NS1 and TLR3 in Pathogenesis and Immunity of WNV.

West Nile Virus (WNV) is a mosquito-transmitted flavivirus which causes encephalitis especially in elderly and immunocompromised individuals. Previous studies have suggested the protective role of the Toll-like receptor 3 (TLR3) pathway against WNV entry into the brain, while the WNV non-structural protein 1 (NS1) interferes with the TLR3 signaling pathway, besides being a component of viral genome replication machinery. In this study, we investigated whether immunization with NS1 could protect against WNV neuroinvasion in the context of TLR3 deficiency. We immunized mice with either an intact or deleted TLR3 system (TLR3KO) with WNV envelope glycoprotein (gE) protein, NS1, or a combination of gE and NS1. Immunization with gE or gE/NS1, but not with NS1 alone, induced WNV neutralizing antibodies and protected against WNV brain invasion and inflammation. The presence of intact TLR3 signaling had no apparent effect on WNV brain invasion. However, mock-immunized TLR3KO mice had higher inflammatory cell invasion upon WNV brain infection than NS1-immunized TLR3KO mice and wild type mice. Thus, immunization against NS1 may reduce brain inflammation in a context of TLR3 signaling deficiency.

Crystal Structure of the Japanese Encephalitis Virus Capsid Protein.

Japanese encephalitis (JE) is inflammation and swelling of the brain caused by the JE virus (JEV), a mosquito-borne member of the Flavivirus family. There are around 68,000 JE cases worldwide each year, many of which result in permanent brain damage and death. There is no specific treatment for JE. Here we present the crystal structure of the JEV capsid protein, a potential drug target, at 1.98 Å, and compare it to other flavivirus capsid proteins. The JEV capsid has a helical secondary structure (α helixes 1-4) and a similar protein fold to the dengue virus (DENV), the West Nile virus (WNV), and the Zika virus (ZIKV) capsid proteins. It forms a homodimer by antiparallel pairing with another subunit (') through α-helix 1-1', 2-2', and 4-4' interactions. This dimeric form is believed to be the building block of the nucleocapsid. The flexibility of the N-terminal α helix-1 allows the formation of closed and open conformations with possible functional importance. The basic C-terminal pairing of α4-4' forms a coiled-coil-like structure, indicating possible nucleic acid binding functionality. However, a comparison with other nucleic acid interacting domains indicates that homodimerization would preclude binding. This is the first JEV capsid protein to be described and is an addition to the structural biology of the Flavivirus.

Flavivirus NS1 Triggers Tissue-Specific Vascular Endothelial Dysfunction Reflecting Disease Tropism.

Flaviviruses cause systemic or neurotropic-encephalitic pathology in humans. The flavivirus nonstructural protein 1 (NS1) is a secreted glycoprotein involved in viral replication, immune evasion, and vascular leakage during dengue virus infection. However, the contribution of secreted NS1 from related flaviviruses to viral pathogenesis remains unknown. Here, we demonstrate that NS1 from dengue, Zika, West Nile, Japanese encephalitis, and yellow fever viruses selectively binds to and alters permeability of human endothelial cells from lung, dermis, umbilical vein, brain, and liver in vitro and causes tissue-specific vascular leakage in mice, reflecting the pathophysiology of each flavivirus. Mechanistically, each flavivirus NS1 leads to differential disruption of endothelial glycocalyx components, resulting in endothelial hyperpermeability. Our findings reveal the capacity of a secreted viral protein to modulate endothelial barrier function in a tissue-specific manner both in vitro and in vivo, potentially influencing virus dissemination and pathogenesis and providing targets for antiviral therapies and vaccine development.

Structure-based prediction of West Nile virus-human protein-protein interactions.

In recent years, West Nile virus (WNV) has posed a great threat to global human health due to its explosive spread. Studying the protein-protein interactions (PPIs) between WNV and human is beneficial for understanding the pathogenesis of WNV and the immune response mechanism of human against WNV infection at the molecular level. In this study, we identified the human target proteins which interact with WNV based on protein structure similarity, and then the interacting pairs were filtered by the subcellular co-localization information. As a result, a network of 3346 interactions was constructed, involving 6 WNV proteins and 1970 human target proteins. To our knowledge, this is the first predicted interactome for WNV-human. By analyzing the topological properties and evolutionary rates of the human target proteins, it was demonstrated that these proteins tend to be the hub and bottleneck proteins in the human PPI network and are more conserved than the non-target ones. Triplet analysis showed that the target proteins are adjacent to each other in the human PPI network, suggesting that these proteins may have similar biological functions. Further, the functional enrichment analysis indicated that the target proteins are mainly involved in virus process, transcription regulation, cell adhesion, and so on. In addition, the common and specific targets were identified and compared based on the networks between WNV-human and Dengue virus II (DENV2)-human. Finally, by combining topological features and existing drug target information, we identified 30 potential anti-WNV human targets, among which 11 ones were reported to be associated with WNV infection. Communicated by Ramaswamy H. Sarma.

Direct Activation of Adenosine Monophosphate-Activated Protein Kinase (AMPK) by PF-06409577 Inhibits Flavivirus Infection through Modification of Host Cell Lipid Metabolism.

Mosquito-borne flaviviruses are a group of RNA viruses that constitute global threats for human and animal health. Replication of these pathogens is strictly dependent on cellular lipid metabolism. We have evaluated the effect of the pharmacological activation of AMP-activated protein kinase (AMPK), a master regulator of lipid metabolism, on the infection of three medically relevant flaviviruses, namely, West Nile virus (WNV), Zika virus (ZIKV), and dengue virus (DENV). WNV is responsible for recurrent outbreaks of meningitis and encephalitis, affecting humans and horses worldwide. ZIKV has caused a recent pandemic associated with birth defects (microcephaly), reproductive disorders, and severe neurological complications (Guillain-Barré syndrome). DENV is the etiological agent of the most prevalent mosquito-borne viral disease, which can induce a potentially lethal complication called severe dengue. Our results showed, for the first time, that activation of AMPK using the specific small molecule activator PF-06409577 reduced WNV, ZIKV, and DENV infection. This antiviral effect was associated with an impairment of viral replication due to the modulation of host cell lipid metabolism exerted by the compound. These results support that the pharmacological activation of AMPK, which currently constitutes an important pharmacological target for human diseases, could also provide a feasible approach for broad-spectrum host-directed antiviral discovery.

Arsenite-induced stress granule formation is inhibited by elevated levels of reduced glutathione in West Nile virus-infected cells.

Oxidative stress activates the cellular kinase HRI, which then phosphorylates eIF2α, resulting in stalled translation initiation and the formation of stress granules (SGs). SG assembly redirects cellular translation to stress response mRNAs and inhibits cap-dependent viral RNA translation. Flavivirus infections were previously reported to induce oxidative stress in infected cells but flavivirus-infected cells paradoxically develop resistance to arsenite (Ars)-induced SG formation with time after infection. This resistance was previously postulated to be due to sequestration of the SG protein Caprin1 by Japanese encephalitis virus capsid protein. However, Caprin1 did not co-localize with West Nile virus (WNV) capsid protein in infected cells. Other stressors induced SGs with equal efficiency in mock- and WNV-infected cells indicating the intrinsic ability of cells to assemble SGs was not disabled. Induction of both reactive oxygen species (ROS) and the antioxidant response was detected at early times after WNV-infection. The transcription factors, Nrf2 and ATF4, which activate antioxidant genes, were upregulated and translocated to the nucleus. Knockdown of Nrf2, ATF4 or apoptosis-inducing factor (AIF), a mitochondrial protein involved in regenerating intracellular reduced glutathione (GSH) levels, with siRNA or treatment of cells with buthionine sulphoximine, which induces oxidative stress by inhibiting GSH synthesis, decreased intracellular GSH levels and increased the number of SG-positive, infected cells. Mitochondria were protected from Ars-induced damage by WNV infection until late times in the infection cycle. The results indicate that the increase in virus-induced ROS levels is counterbalanced by a virus-induced antioxidant response that is sufficient to also overcome the increase in ROS induced by Ars treatment and prevent Ars-induced SG assembly and mitochondrial damage. The virus-induced alterations in the cellular redox status appear to provide benefits for the virus during its lifecycle.

Regional astrocyte IFN signaling restricts pathogenesis during neurotropic viral infection.

Type I IFNs promote cellular responses to viruses, and IFN receptor (IFNAR) signaling regulates the responses of endothelial cells of the blood-brain barrier (BBB) during neurotropic viral infection. However, the role of astrocytes in innate immune responses of the BBB during viral infection of the CNS remains to be fully elucidated. Here, we have demonstrated that type I IFNAR signaling in astrocytes regulates BBB permeability and protects the cerebellum from infection and immunopathology. Mice with astrocyte-specific loss of IFNAR signaling showed decreased survival after West Nile virus infection. Accelerated mortality was not due to expanded viral tropism or increased replication. Rather, viral entry increased specifically in the hindbrain of IFNAR-deficient mice, suggesting that IFNAR signaling critically regulates BBB permeability in this brain region. Pattern recognition receptors and IFN-stimulated genes had higher basal and IFN-induced expression in human and mouse cerebellar astrocytes than did cerebral cortical astrocytes, suggesting that IFNAR signaling has brain region-specific roles in CNS immune responses. Taken together, our data identify cerebellar astrocytes as key responders to viral infection and highlight the existence of distinct innate immune programs in astrocytes from evolutionarily disparate regions of the CNS.