Wolfgang Fischl

Synthesis and biological evaluation of α-ketoamides as inhibitors of the Dengue virus protease with antiviral activity in cell-culture

The development of small molecule inhibitors of the viral protease is of considerable interest for the treatment of emergent flaviviral diseases such as Dengue or West Nile fever. Until today little progress has been made in finding drug-like compounds that inhibit the protease and provide a starting point for lead optimization. We describe here the initial steps of a drug discovery effort that focused on the styryl pharmacophore, combined with a ketoamide function to serve as electrophilic trap for the catalytic serine. This resulted in a fragment-like lead compound with reasonable target affinity and good ligand efficiency, which was extensively modified to explore structure-activity relationships. Selected compounds were cross-tested against the West Nile virus protease and thrombin, indicating that selectivity for one or more flaviviral proteases can be achieved. Finally, the antiviral activity of several protease inhibitors was confirmed in a cell-culture model of Dengue virus replication. The SAR presented here may serve as starting point for further drug discovery efforts with the aim of targeting flaviviral proteases.

Exploitation of cellular pathways by Dengue virus.

Dengue virus (DENV) is the causative agent of the most prevalent arthropod-borne viral disease, thus representing a significant global health burden. Because of its limited coding capacity, DENV exploits components and pathways of the host cell to assure productive replication. In the past few years, important insights into this intimate interaction between DENV and the host cell have been gained. These include the identification of the ER-associated degradation pathway, autophagy, the unfolded protein response or lipid droplets that all play a crucial role for efficient DENV replication. In addition, strategies used by the virus to combat innate antiviral responses have been unraveled. Improving our understanding of the DENV-host cell relation will facilitate our attempts to develop efficient antiviral strategies.

Dengue Virus Perturbs Mitochondrial Morphodynamics to Dampen Innate Immune Responses.

Summary With no antiviral drugs or widely available vaccines, Dengue virus (DENV) constitutes a public health concern. DENV replicates at ER-derived cytoplasmic structures that include substructures called convoluted membranes (CMs); however, the purpose of these membrane alterations remains unclear. We determine that DENV nonstructural protein (NS)4B, a promising drug target with unknown function, associates with mitochondrial proteins and alters mitochondria morphology to promote infection. During infection, NS4B induces elongation of mitochondria, which physically contact CMs. This restructuring compromises the integrity of mitochondria-associated membranes, sites of ER-mitochondria interface critical for innate immune signaling. The spatio-temporal parameters of CM biogenesis and mitochondria elongation are linked to loss of activation of the fission factor Dynamin-Related Protein-1. Mitochondria elongation promotes DENV replication and alleviates RIG-I-dependent activation of interferon responses. As Zika virus infection induces similar mitochondria elongation, this perturbation may protect DENV and related viruses from innate immunity and create a favorable replicative environment.

Dengue Virus Non-structural Protein 1 Modulates Infectious Particle Production via Interaction with the Structural Proteins

Non-structural protein 1 (NS1) is one of the most enigmatic proteins of the Dengue virus (DENV), playing distinct functions in immune evasion, pathogenesis and viral replication. The recently reported crystal structure of DENV NS1 revealed its peculiar three-dimensional fold; however, detailed information on NS1 function at different steps of the viral replication cycle is still missing. By using the recently reported crystal structure, as well as amino acid sequence conservation, as a guide for a comprehensive site-directed mutagenesis study, we discovered that in addition to being essential for RNA replication, DENV NS1 is also critically required for the production of infectious virus particles. Taking advantage of a trans-complementation approach based on fully functional epitope-tagged NS1 variants, we identified previously unreported interactions between NS1 and the structural proteins Envelope (E) and precursor Membrane (prM). Interestingly, coimmunoprecipitation revealed an additional association with capsid, arguing that NS1 interacts via the structural glycoproteins with DENV particles. Results obtained with mutations residing either in the NS1 Wing domain or in the β-ladder domain suggest that NS1 might have two distinct functions in the assembly of DENV particles. By using a trans-complementation approach with a C-terminally KDEL-tagged ER-resident NS1, we demonstrate that the secretion of NS1 is dispensable for both RNA replication and infectious particle production. In conclusion, our results provide an extensive genetic map of NS1 determinants essential for viral RNA replication and identify a novel role of NS1 in virion production that is mediated via interaction with the structural proteins. These studies extend the list of NS1 functions and argue for a central role in coordinating replication and assembly/release of infectious DENV particles.

High-Throughput Screening Using Dengue Virus Reporter Genomes

Large-scale antiviral drug discovery and systematic identification of host factors promoting or restricting virus replication require robust, scalable, and cost-effective assay systems that allow for high throughput and automation. Here we describe the construction and application of full-length infectious dengue virus reporter genomes for such screening applications.

A Combined Genetic-Proteomic Approach Identifies Residues within Dengue Virus NS4B Critical for Interaction with NS3 and Viral Replication

ABSTRACT Dengue virus (DENV) infection causes the most prevalent arthropod-borne viral disease worldwide. Approved vaccines are not available, and targets suitable for the development of antiviral drugs are lacking. One possible drug target is nonstructural protein 4B (NS4B), because it is absolutely required for virus replication; however, its exact role in the DENV replication cycle is largely unknown. With the aim of mapping NS4B determinants critical for DENV replication, we performed a reverse genetic screening of 33 NS4B mutants in the context of an infectious DENV genome. While the majority of these mutations were lethal, for several of them, we were able to select for second-site pseudoreversions, most often residing in NS4B and restoring replication competence. To identify all viral NS4B interaction partners, we engineered a fully viable DENV genome encoding an affinity-tagged NS4B. Mass spectrometry-based analysis of the NS4B complex isolated from infected cells identified the NS3 protease/helicase as a major interaction partner of NS4B. By combining the genetic complementation map of NS4B with a replication-independent expression system, we identified the NS4B cytosolic loop—more precisely, amino acid residue Q134—as a critical determinant for NS4B-NS3 interaction. An alanine substitution at this site completely abrogated the interaction and DENV RNA replication, and both were restored by pseudoreversions A69S and A137V. This strict correlation between the degree of NS4B-NS3 interaction and DENV replication provides strong evidence that this viral protein complex plays a pivotal role during the DENV replication cycle, hence representing a promising target for novel antiviral strategies. IMPORTANCE With no approved therapy or vaccine against dengue virus infection, the viral nonstructural protein 4B (NS4B) represents a possible drug target, because it is indispensable for virus replication. However, little is known about its precise structure and function. Here, we established the first comprehensive genetic interaction map of NS4B, identifying amino acid residues that are essential for virus replication, as well as second-site mutations compensating for their defects. Additionally, we determined the NS4B viral interactome in infected cells and identified the NS3 protease/helicase as a major interaction partner of NS4B. We mapped residues in the cytosolic loop of NS4B as critical determinants for interaction with NS3, as well as RNA replication. The strong correlation between NS3-NS4B interaction and RNA replication provides strong evidence that this complex plays a pivotal role in the viral replication cycle, hence representing a promising antiviral drug target.

Live Cell Analysis and Mathematical Modeling Identify Determinants of Attenuation of Dengue Virus 2’-O-Methylation Mutant

Dengue virus (DENV) is the most common mosquito-transmitted virus infecting ~390 million people worldwide. In spite of this high medical relevance, neither a vaccine nor antiviral therapy is currently available. DENV elicits a strong interferon (IFN) response in infected cells, but at the same time actively counteracts IFN production and signaling. Although the kinetics of activation of this innate antiviral defense and the timing of viral counteraction critically determine the magnitude of infection and thus disease, quantitative and kinetic analyses are lacking and it remains poorly understood how DENV spreads in IFN-competent cell systems. To dissect the dynamics of replication versus antiviral defense at the single cell level, we generated a fully viable reporter DENV and host cells with authentic reporters for IFN-stimulated antiviral genes. We find that IFN controls DENV infection in a kinetically determined manner that at the single cell level is highly heterogeneous and stochastic. Even at high-dose, IFN does not fully protect all cells in the culture and, therefore, viral spread occurs even in the face of antiviral protection of naïve cells by IFN. By contrast, a vaccine candidate DENV mutant, which lacks 2’-O-methylation of viral RNA is profoundly attenuated in IFN-competent cells. Through mathematical modeling of time-resolved data and validation experiments we show that the primary determinant for attenuation is the accelerated kinetics of IFN production. This rapid induction triggered by mutant DENV precedes establishment of IFN-resistance in infected cells, thus causing a massive reduction of virus production rate. In contrast, accelerated protection of naïve cells by paracrine IFN action has negligible impact. In conclusion, these results show that attenuation of the 2’-O-methylation DENV mutant is primarily determined by kinetics of autocrine IFN action on infected cells.

Changing the Protease Specificity for Activation of a Flavivirus, Tick-Borne Encephalitis Virus

ABSTRACT The infectivity of flavivirus particles depends on a maturation process that is triggered by the proteolytic cleavage of the precursor of the M protein (prM). This activation cleavage is naturally performed by ubiquitous cellular proteases of the furin family, which typically recognize the multibasic sequence motif R-X-R/K-R. Previously, we demonstrated that a tick-borne encephalitis virus (TBEV) mutant with an altered cleavage motif, R-X-R, produced immature, noninfectious particles that could be activated by exogenous trypsin, which cleaves after single basic residues. Here, we report the adaptation of this mutant to chymotrypsin, a protease specific for large, hydrophobic amino acid residues. Using selection pressure in cell culture, two different mutations conferring a chymotrypsin-dependent phenotype were identified. Surprisingly, one of these mutations (Ser85Phe) occurred three positions upstream of the natural cleavage site. The other mutation (Arg89His) arose at the natural cleavage position but involved a His residue, which is not a typical chymotrypsin cleavage site. Efficient cleavage of protein prM and activation by the heterologous protease were confirmed using various recombinant TBEV mutants. Mutants with only the originally selected mutations exhibited unimpaired export kinetics and were genotypically stable during at least six cell culture passages. However, in contrast to the wild-type virus or trypsin-dependent mutants, chymotrypsin-dependent mutants were not neurovirulent in suckling mice. Our results demonstrate that flaviviruses with altered protease specificities can be generated and suggest that this approach can be used for the construction of viral mutants or vectors that can be activated on demand and have restricted tissue tropism and virulence.

Cellular N-myristoyltransferases play a crucial picornavirus genus-specific role in viral assembly, virion maturation, and infectivity

In nearly all picornaviruses the precursor of the smallest capsid protein VP4 undergoes co-translational N-terminal myristoylation by host cell N-myristoyltransferases (NMTs). Curtailing this modification by mutation of the myristoylation signal in poliovirus has been shown to result in severe assembly defects and very little, if any, progeny virus production. Avoiding possible pleiotropic effects of such mutations, we here used pharmacological abrogation of myristoylation with the NMT inhibitor DDD85646, a pyrazole sulfonamide originally developed against trypanosomal NMT. Infection of HeLa cells with coxsackievirus B3 in the presence of this drug decreased VP0 acylation at least 100-fold, resulting in a defect both early and late in virus morphogenesis, which diminishes the yield of viral progeny by about 90%. Virus particles still produced consisted mainly of provirions containing RNA and uncleaved VP0 and, to a substantially lesser extent, of mature virions with cleaved VP0. This indicates an important role of myristoylation in the viral maturation cleavage. By electron microscopy, these RNA-filled particles were indistinguishable from virus produced under control conditions. Nevertheless, their specific infectivity decreased by about five hundred fold. Since host cell-attachment was not markedly impaired, their defect must lie in the inability to transfer their genomic RNA into the cytosol, likely at the level of endosomal pore formation. Strikingly, neither parechoviruses nor kobuviruses are affected by DDD85646, which appears to correlate with their native capsid containing only unprocessed VP0. Individual knockout of the genes encoding the two human NMT isozymes in haploid HAP1 cells further demonstrated the pivotal role for HsNMT1, with little contribution by HsNMT2, in the virus replication cycle. Our results also indicate that inhibition of NMT can possibly be exploited for controlling the infection by a wide spectrum of picornaviruses.