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The outbreak of West Nile virus (WNV) in 1999 in the USA, and its continued spread throughout the Americas, parts of Europe, the Middle East and Africa, underscored the need for WNV antiviral development. Here, we review the current status of WNV drug discovery. A number of approaches have been used to search for inhibitors of WNV, including viral infection-based screening, enzyme-based screening, structure-based virtual screening, structure-based rationale design, and antibody-based therapy. These efforts have yielded inhibitors of viral or cellular factors that are critical for viral replication. For small molecule inhibitors, no promising preclinical candidate has been developed; most of the inhibitors could not even be advanced to the stage of hit-to-lead optimization due to their poor drug-like properties. However, several inhibitors developed for related members of the family Flaviviridae, such as dengue virus and hepatitis C virus, exhibited cross-inhibition of WNV, suggesting the possibility to re-purpose these antivirals for WNV treatment. Most promisingly, therapeutic antibodies have shown excellent efficacy in mouse model; one of such antibodies has been advanced into clinical trial. The knowledge accumulated during the past fifteen years has provided better rationale for the ongoing WNV and other flavivirus antiviral development. [ABSTRACT FROM AUTHOR]

The article discusses a study by researchers published in the journal "Science" on the lysosomal enzyme trafficking factor LYSET, conducted by a team of researchers from various institutions, published in the journal "Science, ". Topics include the role of LYSET in enabling nutritional usage of extracellular proteins, and the potential implications for cancer therapy and nutrient recycling.

Zika (ZIKV) and dengue (DENV) virus infections elicit a robust but cross-reactive antibody response against the viral envelope protein, while antibody responses against non-structural proteins (NS) are more virus specific. Building on this premise, we have previously developed a flavivirus multiplex microsphere immunoassay (MIA) for the serologic diagnosis of ZIKV and DENV infections. This assay significantly improved diagnostic accuracy; however, MIA could not differentiate more recent from past infections, which still represents a major diagnostic challenge. Therefore, an immunoglobulin G (IgG) based avidity assay was developed and its diagnostic performance evaluated. Specimens from New York State residents were submitted to the Wadsworth Center New York State Department of Health (NYSDOH) for routine clinical testing by Zika IgM ELISA and plaque reduction neutralization test (PRNT). Using our previously developed flavivirus MIA as a platform, we developed an IgG avidity assay to discriminate recent ZIKV from past DENV infections. Zika IgM positive specimens had an average Zika IgG avidity index of 14.8% (95% CI: 11.0-18.4%), while Zika IgM negative but flavivirus MIA and PRNT positive samples had an average Zika IgG avidity index of 34.9% (95% CI: 31.1-38.7%). Specimens positive for dengue antibodies by flavivirus MIA and PRNT had an average dengue IgG avidity index of 68.7% (95% CI: 62.7-75.0%). The IgG avidity assay accurately distinguished recent ZIKV from past DENV infections in patients who traveled to dengue endemic regions. This assay could be very useful in patients with high risk of Zika complications such as pregnant women and monitoring immune responses in vaccine trials. [ABSTRACT FROM AUTHOR]

Dengue is a mosquito-borne viral hemorrhagic disease that is a major threat to human health in tropical and subtropical regions. Here we report crystal structures of a peptide covalently bound to dengue virus serotype 3 (DENV-3) protease as well as the serine-protease inhibitor aprotinin bound to the same enzyme. These structures reveal, for the first time, a catalytically active, closed conformation of the DENV protease. In the presence of the peptide, the DENV-3 protease forms the closed conformation in which the hydrophilic β-hairpin region of NS2B wraps around the NS3 protease core, in a manner analogous to the structure of West Nile virus (WNV) protease. Our results confirm that flavivirus proteases form the closed conformation during proteolysis, as previously proposed for WNV. The current DENV-3 protease structures reveal the detailed interactions at the P4' to P3 sites of the substrate. The new structural information explains the sequence preference, particularly for long basic residues in the nonprime side, as well as the difference in substrate specificity between the WNV and DENV proteases at the prime side. Structural analysis of the DENV-3 protease-peptide complex revealed a pocket that is formed by residues from NS2B and NS3; this pocket also exists in the WNV NS2B/NS3 protease structure and could be targeted for potential antivirus development. The structural information presented in the current study is invaluable for the design of specific inhibitors of DENV protease. [ABSTRACT FROM AUTHOR]

Interferons (IFNs) are key mediators of the host innate antiviral immune response. To identify IFNstimulated genes (ISGs) that instigate an antiviral state against two medically important flaviviruses, West Nile virus (WNV) and dengue virus (DENV), we tested 36 ISGs that are commonly induced by IFN-α for antiviral activity against the two viruses. We discovered that five ISGs efficiently suppressed WNV and/or DENV infection when they were individually expressed in HEK293 cells. Mechanistic analyses revealed that two structurally related cell plasma membrane proteins, IFITM2 and IFITM3, disrupted early steps (entry and/or uncoating) of the viral infection. In contrast, three IFN-induced cellular enzymes, viperin, ISG20, and double-stranded-RNA-activated protein kinase, inhibited steps in viral proteins and/or RNA biosynthesis. Our results thus imply that the antiviral activity of IFN-α is collectively mediated by a panel of ISGs that disrupt multiple steps of the DENV and WNV life cycles. [ABSTRACT FROM AUTHOR]

RNA elements within flavivirus genomes are potential targets for antiviral therapy. A panel of phosphorodiamidate morpholino oligomers (PMOs), whose sequences are complementary to RNA elements located in the 5′- and 3′-termini of the West Nile (WN) virus genome, were designed to anneal to important cis-acting elements and potentially to inhibit WN infection. A novel Arg-rich peptide was conjugated to each PMO for efficient cellular delivery. These PMOs exhibited various degrees of antiviral activity upon incubation with a WN virus luciferase-replicon-containing cell line. Among them, PMOs targeting the 5′-terminal 20 nucleotides (5′End) or targeting the 3′-terminal element involved in a potential genome cyclizing interaction (3′CSI) exhibited the greatest potency. When cells infected with an epidemic strain of WN virus were treated with the 5′End or 3′CSI PMO, virus titers were reduced by approximately 5 to 6 logs at a 5 µM concentration without apparent cytotoxicity. The 3′CSI PMO also inhibited mosquito-borne flaviviruses other than WN virus, and the antiviral potency correlated with the conservation of the targeted 3′CSI sequences of specific viruses. Mode-of-action analyses showed that the 5′End and 3′CSI PMOs suppressed viral infection through two distinct mechanisms. The 5′End PMO inhibited viral translation, whereas the 3′CSI PMO did not significantly affect viral translation but suppressed RNA replication. The results suggest that antisense PMO-mediated blocking of cis-acting elements of flavivirus genomes can potentially be developed into an anti-flavivirus therapy. In addition, we report that although a full-length WN virus containing a luciferase reporter (engineered at the 3′ untranslated region of the genome) is not stable, an early passage of this reporting virus can be used to screen for inhibitors against any step of the virus life cycle. [ABSTRACT FROM AUTHOR]

The interferon (IFN) response is the first line of defense against viral infections, and the majority of viruses have developed different strategies to counteract IFN responses in order to ensure their survival in an infected host. In this study, the abilities to inhibit IFN signaling of two closely related West Nile viruses, the New York 99 strain (NY99) and Kunjin virus (KUN), strain MRM61C, were analyzed using reporter plasmid assays, as well as immunofluorescence and Western blot analyses. We have demonstrated that infections with both NY99 and KUN, as well as transient or stable transfections with their replicon RNAs, inhibited the signaling of both alpha/beta IFN (IFN-α/β) and gamma IFN (IFN-γ) by blocking the phosphorylation of STAT1 and its translocation to the nucleus. In addition, the phosphorylation of STAT2 and its translocation to the nucleus were also blocked by KUN, NY99, and their replicons in response to treatment with IFN-α. IFN-α signaling and STAT2 translocation to the nucleus was inhibited when the KUN nonstructural proteins NS2A, NS2B, NS3, NS4A, and NS4B, but not NS1 and NS5, were expressed individually from the pcDNA3 vector. The results clearly demonstrate that both NY99 and KUN inhibit IFN signaling by preventing STAT1 and STAT2 phosphorylation and identify nonstructural proteins responsible for this inhibition. [ABSTRACT FROM AUTHOR]

Recent outbreaks of West Nile Virus (WNV) have been associated with an increase in morbidity and mortality in humans, birds, and many other species. We have initiated studies to define the molecular mechanisms by which a recent pathogenic isolate of WNV evades the host cell innate antiviral response. Biochemical and microarray analyses demonstrated that WNV induced the expression of beta interferon (IFN-β) and several IFN-stimulated genes late in infection of cultured human cells. The late expression of these antiviral genes was due to the delayed activation of the transcription factor IFN regulatory factor 3 (IRF-3). Despite this host response, WNV was still able to replicate efficiently. The effect of the IRF-3 pathway on WNV replication was assessed by examining virus replication and spread in cultures of wild-type or IRF-3-null mouse embryo fibroblasts. The absence of IRF-3 was marked by a significant increase in plaque size and a sustained production of infectious particles. Although the activation of the IRF-3 pathway was not sufficient to block virus replication, our results suggest that IRF-3 target genes function to constrain WNV infection and limit cell-to-cell virus spread. [ABSTRACT FROM AUTHOR]

Superinfection exclusion is the ability of an established viral infection to interfere with a second viral infection. Using West Nile virus (WNV) as a model, we show that replicating replicons in BHK-21 cells suppress subsequent WNV infection. The WNV replicon also suppresses superinfections of other flaviviruses but not nonflaviviruses. Mode-of-action analysis indicates that the exclusion of WNV superinfection occurs at the step of RNA synthesis. The continuous culturing of WNV in the replicon-containing cells generated variants that could overcome the superinfection exclusion. The sequencing of the selected viruses revealed mutations in structural (prM S90R or envelope E138K) and nonstructural genes (NS4a K124R and peptide 2K V9M). Mutagenesis analysis showed that the mutations in structural genes nonselectively enhance viral infection in both naïve and replicon-containing BHK-21 cells; in contrast, the mutations in nonstructural genes more selectively enhance viral replication in the replicon-containing cells than in the naïve cells. Mechanistic analysis showed that the envelope mutation functions through the enhancement of virion attachment to BHK-21 cells, whereas the 2K mutation (and, to a lesser extent, the NS4a mutation) functions through the enhancement of viral RNA synthesis. Furthermore, we show that WNV superinfection exclusion is reversible by the treatment of the replicon cells with a flavivirus inhibitor. The preestablished replication of the replicon could be suppressed by infecting the cells with the 2K mutant WNV but not with the wild-type virus. These results suggest that WNV superinfection exclusion is a result of competition for intracellular host factors that are required for viral RNA synthesis. [ABSTRACT FROM AUTHOR]