An Improved Focus-Forming Assay for Determination of the Dengue Virus Titer.

Dengue virus (DENV), a common and prevalent mosquito-borne endemic disease, is caused by four serotypes (DENV-1-4) and has spread rapidly on a global scale over the past decade. A crucial step in the development of antiviral therapeutics requires the utilization of in vitro cell-based techniques, such as plaque assays and focus-forming assays (FFA) for virus quantification. Vero cells have been widely used for FFA and plaque assay; however, there are instances when their efficacy and efficiency in the detection of certain clinical DENV isolates are low.

Serotype-Specific Regulation of Dengue Virus NS5 Protein Subcellular Localization.

Dengue virus (DENV) nonstructural protein 5 (NS5), consisting of methyltransferase and RNA-dependent RNA polymerase (RdRp) domains, is critical for viral RNA synthesis within endoplasmic reticulum-derived replication complexes in the cytoplasm. However, a significant proportion of NS5 is localized to the nucleus of infected cells for DENV2, 3, and 4, whereas DENV1 NS5 is localized diffusely in the cytoplasm. We still have an incomplete understanding of how the DENV NS5 subcellular localization is regulated.

Secreted dengue virus NS1 from infection is predominantly dimeric and in complex with high-density lipoprotein.

Severe dengue infections are characterized by endothelial dysfunction shown to be associated with the secreted nonstructural protein 1 (sNS1), making it an attractive vaccine antigen and biotherapeutic target. To uncover the biologically relevant structure of sNS1, we obtained infection-derived sNS1 (isNS1) from dengue virus (DENV)-infected Vero cells through immunoaffinity purification instead of recombinant sNS1 (rsNS1) overexpressed in insect or mammalian cell lines. We found that isNS1 appeared as an approximately 250 kDa complex of NS1 and ApoA1 and further determined the cryoEM structures of isNS1 and its complex with a monoclonal antibody/Fab.

Carbazole to indolazepinone scaffold morphing leads to potent cell-active dengue antivirals.

According to WHO, dengue virus is classed among major threats for future pandemics and remains at large an unmet medical need as there are currently no relevant antiviral drugs whereas vaccine developments have met with safety concerns, mostly due to secondary infections caused by antibody-dependant-enhancement in cross infections among the four dengue serotypes. This adds extra complexity in dengue antiviral research and has impeded the progress in this field. Following through our previous effort which born the allosteric, dual-mode inhibitor SP-471P (a carbazole derivative, EC 1.

Tissue-specific expansion of Zika virus isogenic variants drive disease pathogenesis.

Background: The Asian lineage Zika virus (ZIKV) emerged as a public health emergency in 2016 causing severe neurological pathologies with no apparent historical correlate to the mild, disease-causing innocuous member of the mosquito-borne flavivirus genus that was discovered in Africa in 1947. Replication error rate of RNA viruses combined with viral protein/RNA structural plasticity can lead to evolution of virus-induced pathogenicity that is critical to identify and validate.

Methods: Infection studies in cells and A129 interferon alpha/beta receptor deficient mice with ZIKV French Polynesian H/PF/2013 clinical isolate, plaque-purified isogenic clone derivatives as well as infectious cDNA clone derived wild-type and site-specific mutant viruses, were employed together with Next-Generation Sequencing (NGS) to pin-point the contributions of specific viral variants in neurovirulence recapitulated in our ZIKV mouse model.

A cellular screening platform, stably expressing DENV2 NS5, defines a novel anti-DENV mechanism of action of Apigenin based on STAT2 activation.

Type I interferon (IFN-I) evasion by Dengue virus (DENV) is key in DENV pathogenesis. The non-structural protein 5 (NS5) antagonizes IFN-I response through the degradation of the signal transducer and activator of transcription 2 (STAT2). We developed a K562 cell-based platform, for high throughput screening of compounds potentially counteracting the NS5-mediated antagonism of IFN-I signaling.

Functionalized sulfonyl anthranilic acid derivatives inhibit replication of all the four dengue serotypes.

Dengue virus (DENV), a mosquito-borne flavivirus, continues to be a major public health threat in many countries and no approved antiviral therapeutics are available yet. In this work, we designed and synthesized a series of sulfonyl anthranilic acid (SAA) derivatives using a ligand-based scaffold morphing approach of the 2,1-benzothiazine 2,2-dioxide core, previously used by us to develop DENV polymerase inhibitors resulting devoid of any cell-based antiviral activity. Several derivatives based on the new SAA chemotype exhibited potent inhibition against DENV infection in the cell-based assay but did not inhibit DENV NS5 polymerase activity in the in vitro de novo initiation and elongation assays.

Therapeutics for flaviviral infections.

Flaviviruses are vector-borne pathogens capable of causing devastating human diseases. The re-emergence of Zika in 2016 notoriously led to a widescale epidemic in the Americas. New daunting evidence suggests that a single mutation in Zika virus genome may increase transmission and pathogenesis, further highlighting the need to be prepared for flavivirus outbreaks.

Experimental evidence for a high rate of maternal-fetal transmission of dengue virus in the presence of antibodies in immunocompromised mice.

Background: Congenital disorders associated with prenatal vertical transmission of Zika virus (ZIKV) is well established since the 2016 outbreak in the Americas. However, despite clinical reports of similar mode of transmission for other flaviviruses such as dengue virus (DENV), the phenomenon has not been experimentally explored.

Methods: Pregnant AG129 mice were infected with DENV1 in the presence or absence of enhancing antibodies at different gestational time points.

A conserved arginine in NS5 binds genomic 3' stem-loop RNA for primer-independent initiation of flavivirus RNA replication.

The commitment to replicate the RNA genome of flaviviruses without a primer involves RNA-protein interactions that have been shown to include the recognition of the stem-loop A (SLA) in the 5' untranslated region (UTR) by the nonstructural protein NS5. We show that DENV2 NS5 arginine 888, located within the carboxy-terminal 18 residues, is completely conserved in all flaviviruses and interacts specifically with the top-loop of 3'SL in the 3'UTR which contains the pentanucleotide 5'-CACAG-3' previously shown to be critical for flavivirus RNA replication. We present virological and biochemical data showing the importance of this Arg 888 in virus viability and de novo initiation of RNA polymerase activity in vitro.

Optimal flexibility of the linker region of Zika virus NS5 methyltransferase-polymerase is critical for virus replication.

The flavivirus NS5 protein contains an N-terminal methyl-transferase (MTase) connected through a flexible linker with a C-terminal RNA-dependent RNA-polymerase (RdRp) domain, that work cooperatively to replicate and methylate the viral genome. In this study we probed the importance of an evolutionary-conserved hydrophobic residue (Val266) located at the start of the ten-residue interdomain linker of Zika virus (ZIKV) NS5. In flavivirus NS5 crystal structures, the start of the linker forms a 3 helix when NS5 adopts a compact conformation, but becomes disordered or extended in open conformations.

Amidoxime prodrugs convert to potent cell-active multimodal inhibitors of the dengue virus protease.

The flavivirus genus of the Flaviviridae family comprises Dengue, Zika and West-Nile viruses which constitute unmet medical needs as neither appropriate antivirals nor safe vaccines are available. The dengue NS2BNS3 protease is one of the most promising validated targets for developing a dengue treatment however reported protease inhibitors suffer from toxicity and cellular inefficacy. Here we report SAR on our previously reported Zika-active carbazole scaffold, culminating prodrug compound SP-471P (EC 1.

F-fluorodeoxyglucose positron emission tomography as a window into human dengue pathophysiology.

In mouse models of dengue virus (DENV) infection, F-FDG PET is able to sensitively detect tissue-specific sites of inflammation and disease activity, as well as track therapeutic response to anti- DENV agents. However, the use of F-FDG PET to study the pathogenesis of inflammation and disease activity in DENV infection in humans, has not been clinically validated. Here we report the F-FDG PET imaging results of two patients during the febrile phase of acute DENV infection, paired with serial serum viral load, NS1 and proinflammatory cytokine measurements.

Sustainable, three-component, one-pot procedure to obtain active anti-flavivirus agents.

The mosquito-borne viruses belonging to the genus Flavivirus such as Dengue virus (DENV) and Zika virus (ZIKV) cause human infections ranging from mild flu-like symptoms to hemorrhagic fevers, hepatitis, and neuropathies. To date, there are vaccines only for few flaviviruses while no effective treatments are available. Pyridobenzothiazole (PBTZ) derivatives are a class of compounds endowed with a promising broad-spectrum anti-flavivirus activity and most of them have been reported as potent inhibitors of the flaviviral NS5 polymerase.

Mutations in the cytoplasmic domain of dengue virus NS4A affect virus fitness and interactions with other non-structural proteins.

The dengue virus (DENV) replication complex is made up of its non-structural (NS) proteins and yet-to-be identified host proteins, but the molecular interactions between these proteins are not fully elucidated. In this work, we sought to uncover the interactions between DENV NS1 and its fellow NS proteins using a yeast two-hybrid (Y2H) approach, and found that domain II of NS1 binds to an N-terminal cytoplasmic fragment of NS4A. Mutations in amino acid residues 41 and 43 in this cytoplasmic region of NS4A disrupted the interaction between NS1 and the NS4A-2K-4B precursor protein.

Pyridobenzothiazolones Exert Potent Anti-Dengue Activity by Hampering Multiple Functions of NS5 Polymerase.

Treatment of dengue virus (DENV) and other flavivirus infections is an unmet medical need. The highly conserved flaviviral NS5 RNA-dependent RNA polymerase (RdRp) is an attractive antiviral target that interacts with NS3 and viral RNA within the replication complex assembly. Biochemical and cell-based evidence indicate that targeting cavity B may lead to dual RdRp and NS5-NS3 interaction inhibitors.

The Potential Role of the ZIKV NS5 Nuclear Spherical-Shell Structures in Cell Type-Specific Host Immune Modulation during ZIKV Infection.

The Zika virus (ZIKV) non-structural protein 5 (NS5) plays multiple viral and cellular roles during infection, with its primary role in virus RNA replication taking place in the cytoplasm. However, immunofluorescence assay studies have detected the presence of ZIKV NS5 in unique spherical shell-like structures in the nuclei of infected cells, suggesting potentially important cellular roles of ZIKV NS5 in the nucleus. Hence ZIKV NS5's subcellular distribution and localization must be tightly regulated during ZIKV infection.

Cell-active carbazole derivatives as inhibitors of the zika virus protease.

Zika virus (ZIKV) infection recently resulted in an international health emergency the Americas in and despite its high profile there is currently no approved treatment for ZIKV infection with millions of people being at risk. ZIKV is a member of Flaviviridae family which includes prominent members such as dengue virus (DENV) and West Nile virus (WNV). One of the best validated targets for developing anti-flaviviral treatment for DENV and WNV infection is the NS2B/NS3 protease.

A T164S mutation in the dengue virus NS1 protein is associated with greater disease severity in mice.

Dengue viruses cause severe and sudden human epidemics worldwide. The secreted form of the nonstructural protein 1 (sNS1) of dengue virus causes vascular leakage, a hallmark of severe dengue disease. Here, we reverse engineered the T164S mutation of NS1, associated with the severity of dengue epidemics in the Americas, into a dengue virus serotype 2 mildly infectious strain.

Zika virus nonstructural protein 5 residue R681 is critical for dimer formation and enzymatic activity.

Zika virus (ZIKV) relies on its nonstructural protein 5 (NS5) for capping and synthesis of the viral RNA. Recent small-angle X-ray scattering (SAXS) data of recombinant ZIKV NS5 protein showed that it is dimeric in solution. Here, we present insights into the critical residues responsible for its dimer formation.

Assessing the utility of antivirals for preventing maternal-fetal transmission of zika virus in pregnant mice.

Zika virus (ZIKV) infection during pregnancy has been associated with adverse outcomes and birth defects such as microcephaly in newborn children. Congenital malformations associated with ZIKV are believed to occur via direct infection of the fetus. Unfortunately, there are no licensed therapeutic or preventative tools to block maternal-fetal transmission of ZIKV.

Zika Virus NS5 Forms Supramolecular Nuclear Bodies That Sequester Importin-α and Modulate the Host Immune and Pro-Inflammatory Response in Neuronal Cells.

The Zika virus (ZIKV) epidemic in the Americas was alarming because of its link with microcephaly in neonates and Guillain-Barré syndrome in adults. The unusual pathologies induced by ZIKV infection and the knowledge that the flaviviral nonstructural protein 5 (NS5), the most conserved protein in the flavivirus proteome, can modulate the host immune response during ZIKV infection prompted us to investigate the subcellular localization of NS5 during ZIKV infection and explore its functional significance. A monopartite nuclear localization signal (NLS) sequence within ZIKV NS5 was predicted by the cNLS Mapper program, and we observed localization of ZIKV NS5 in the nucleus of infected cells by immunostaining with specific antibodies.

Dengue Virus and Zika Virus Serological Cross-reactivity and Their Impact on Pathogenesis in Mice.

Preexisting immunity to Zika virus (ZIKV) or dengue virus (DENV) may alter the course of their infection, and here we use robust mouse models to examine pathological outcomes following passive immunization, sequential cross-infection, or vaccination with inactivated virus. DENV infection was enhanced (through antibody-dependent enhancement [ADE]) or was suppressed by both DENV and ZIKV immunity. Notably, inactivated ZIKV vaccination enhanced dengue disease severity, although it was highly protective against ZIKV infection.

Luteolin escape mutants of dengue virus map to prM and NS2B and reveal viral plasticity during maturation.

We previously showed that luteolin, a well-known plant-derived component found in the "heat clearing" class of Traditional Chinese Medicine (TCM) herbs, is an uncompetitive inhibitor (Ki 58.6 μM) of the host proprotein convertase furin, an endoprotease that is required for maturation of flaviviruses in the trans-Golgi compartment. Luteolin also weakly inhibited recombinant dengue virus NS2B/NS3 protease (Ki 140.

Dengue Virus Selectively Annexes Endoplasmic Reticulum-Associated Translation Machinery as a Strategy for Co-opting Host Cell Protein Synthesis.

A primary question in dengue virus (DENV) biology is the molecular strategy for recruitment of host cell protein synthesis machinery. Here, we combined cell fractionation, ribosome profiling, and transcriptome sequencing (RNA-seq) to investigate the subcellular organization of viral genome translation and replication as well as host cell translation and its response to DENV infection. We report that throughout the viral life cycle, DENV plus- and minus-strand RNAs were highly partitioned to the endoplasmic reticulum (ER), identifying the ER as the primary site of DENV translation.