N-Phenylpyridine-3-Carboxamide and 6-Acetyl-1H-Indazole Inhibit the RNA Replication Step of the Dengue Virus Life Cycle.

Dengue virus (DENV) is a Flavivirus that causes the most prevalent arthropod-borne viral disease. Clinical manifestation of DENV infection ranges from asymptomatic to severe symptoms that can lead to death. Unfortunately, no antiviral treatments against DENV are currently available. In order to identify novel DENV inhibitors, we screened a library of 1,604 chemically diversified fragment-based compounds using DENV reporter viruses that allowed quantification of viral replication in infected cells. Following a validation screening, the two best inhibitor candidates were N-phenylpyridine-3-carboxamide (NPP3C) and 6-acetyl-1H-indazole (6A1HI). The half maximal effective concentration of NPP3C and 6A1H1 against DENV were 7.1 µM and 6.5 µM, respectively. 6A1H1 decreased infectious DENV particle production up to 1,000-fold without any cytotoxicity at the used concentrations. While 6A1HI was DENV-specific, NPP3C also inhibited the replication of other flaviviruses such as West Nile virus and Zika virus. Structure-activity relationship (SAR) studies with 151 analogues revealed key structural elements of NPP3C and 6A1HI required for their antiviral activity. Time-of-drug-addition experiments identified a postentry step as a target of these compounds. Consistently, using a DENV subgenomic replicon, we demonstrated that these compounds specifically impede the viral RNA replication step and exhibit a high genetic barrier-to-resistance. In contrast, viral RNA translation and the de novo biogenesis of DENV replication organelles were not affected. Overall, our data unveil NPP3C and 6A1H1 as novel DENV inhibitors. The information revealed by our SAR studies will help chemically optimize NPP3C and 6A1H1 in order to improve their anti-flaviviral potency and to challenge them in in vivo models.

Valosin-containing protein ATPase activity regulates the morphogenesis of Zika virus replication organelles and virus-induced cell death.

With no available therapies, infections with Zika virus (ZIKV) constitute a major public health concern as they can lead to congenital microcephaly. In order to generate an intracellular environment favourable to viral replication, ZIKV induces endomembrane remodelling and the morphogenesis of replication factories via enigmatic mechanisms. In this study, we identified the AAA+ type ATPase valosin-containing protein (VCP) as a cellular interaction partner of ZIKV non-structural protein 4B (NS4B). Importantly, its pharmacological inhibition as well as the expression of a VCP dominant-negative mutant impaired ZIKV replication. In infected cells, VCP is relocalised to large ultrastructures containing both NS4B and NS3, which are reminiscent of dengue virus convoluted membranes. Moreover, short treatment with the VCP inhibitors NMS-873 or CB-5083 drastically decreased the abundance and size of ZIKV-induced convoluted membranes. Furthermore, NMS-873 treatment inhibited ZIKV-induced mitochondria elongation previously reported to be physically and functionally linked to convoluted membranes in case of the closely related dengue virus. Finally, VCP inhibition resulted in enhanced apoptosis of ZIKV-infected cells strongly suggesting that convoluted membranes limit virus-induced cytopathic effects. Altogether, this study identifies VCP as a host factor required for ZIKV life cycle and more precisely, for the maintenance of viral replication factories. Our data further support a model in which convoluted membranes regulate ZIKV life cycle by impacting on mitochondrial functions and ZIKV-induced death signals in order to create a cytoplasmic environment favourable to viral replication.

Amaryllidaceae Alkaloid Cherylline Inhibits the Replication of Dengue and Zika Viruses.

Dengue fever, caused by dengue virus (DENV), is the most prevalent arthropod-borne viral disease and is endemic in many tropical and subtropical parts of the world, with an increasing incidence in temperate regions. The closely related flavivirus Zika virus (ZIKV) can be transmitted vertically in utero and causes congenital Zika syndrome and other birth defects. In adults, ZIKV is associated with Guillain-Barré syndrome. There are no approved antiviral therapies against either virus. Effective antiviral compounds are urgently needed. Amaryllidaceae alkaloids (AAs) are a specific class of nitrogen-containing compounds produced by plants of the Amaryllidaceae family with numerous biological activities. Recently, the AA lycorine was shown to present strong antiflaviviral properties. Previously, we demonstrated that Crinum jagus contained lycorine and several alkaloids of the cherylline, crinine, and galanthamine types with unknown antiviral potential. In this study, we explored their biological activities. We show that C. jagus crude alkaloid extract inhibited DENV infection. Among the purified AAs, cherylline efficiently inhibited both DENV (50% effective concentration [EC50], 8.8 µM) and ZIKV replication (EC50, 20.3 µM) but had no effect on HIV-1 infection. Time-of-drug-addition and -removal experiments identified a postentry step as the one targeted by cherylline. Consistently, using subgenomic replicons and replication-defective genomes, we demonstrate that cherylline specifically hinders the viral RNA synthesis step but not viral translation. In conclusion, AAs are an underestimated source of antiflavivirus compounds, including the effective inhibitor cherylline, which could be optimized for new therapeutic approaches.

Rapid On-Site Detection of Arboviruses by a Direct RT-qPCR Assay.

Arthropod-borne diseases currently constitute a source of major health concerns worldwide. They account for about 50% of global infectious diseases and cause nearly 700,000 deaths every year. Their rapid increase and spread constitute a huge challenge for public health, highlighting the need for early detection during epidemics, to curtail the virus spread, and to enhance outbreak management. Here, we compared a standard quantitative polymerase chain reaction (RT-qPCR) and a direct RT-qPCR assay for the detection of Zika (ZIKV), Chikungunya (CHIKV), and Rift Valley Fever (RVFV) viruses from experimentally infected-mosquitoes. The direct RT-qPCR could be completed within 1.5 h and required 1 µL of viral supernatant from homogenized mosquito body pools. Results showed that the direct RT-qPCR can detect 85.71%, 89%, and 100% of CHIKV, RVFV, and ZIKV samples by direct amplifications compared to the standard method. The use of 1:10 diluted supernatant is suggested for CHIKV and RVFV direct RT-qPCR. Despite a slight drop in sensitivity for direct PCR, our technique is more affordable, less time-consuming, and provides a better option for qualitative field diagnosis during outbreak management. It represents an alternative when extraction and purification steps are not possible because of insufficient sample volume or biosecurity issues.

The Biogenesis of Dengue Virus Replication Organelles Requires the ATPase Activity of Valosin-Containing Protein.

The dengue virus (DENV) causes the most prevalent arthropod-borne viral disease worldwide. While its incidence is increasing in many countries, there is no approved antiviral therapy currently available. In infected cells, the DENV induces extensive morphological alterations of the endoplasmic reticulum (ER) to generate viral replication organelles (vRO), which include convoluted membranes (CM) and vesicle packets (VP) hosting viral RNA replication. The viral non-structural protein NS4B localizes to vROs and is absolutely required for viral replication through poorly defined mechanisms, which might involve cellular protein partners. Previous interactomic studies identified the ATPase valosin-containing protein (VCP) as a DENV NS4B-interacting host factor in infected cells. Using both pharmacological and dominant-negative inhibition approaches, we show, in this study, that VCP ATPase activity is required for efficient DENV replication. VCP associates with NS4B when expressed in the absence of other viral proteins while in infected cells, both proteins colocalize within large DENV-induced cytoplasmic structures previously demonstrated to be CMs. Consistently, VCP inhibition dramatically reduces the abundance of DENV CMs in infected cells. Most importantly, using a recently reported replication-independent plasmid-based vRO induction system, we show that de novo VP biogenesis is dependent on VCP ATPase activity. Overall, our data demonstrate that VCP ATPase activity is required for vRO morphogenesis and/or stability. Considering that VCP was shown to be required for the replication of other flaviviruses, our results argue that VCP is a pan-flaviviral host dependency factor. Given that new generation VCP-targeting drugs are currently evaluated in clinical trials for cancer treatment, VCP may constitute an attractive broad-spectrum antiviral target in drug repurposing approaches.

Fragment-Based Phenotypic Lead Discovery To Identify New Drug Seeds That Target Infectious Diseases.

Fragment-based lead discovery has emerged over the last decades as one of the most powerful techniques for identifying starting chemical matter to target specific proteins or nucleic acids in vitro. However, the use of such low-molecular-weight fragment molecules in cell-based phenotypic assays has been historically avoided because of concerns that bioassays would be insufficiently sensitive to detect the limited potency expected for such small molecules and that the high concentrations required would likely implicate undesirable artifacts. Herein, we applied phenotype cell-based screens using a curated fragment library to identify inhibitors against a range of pathogens including Leishmania, Plasmodium falciparum, Neisseria, Mycobacterium, and flaviviruses. This proof-of-concept shows that fragment-based phenotypic lead discovery (FPLD) can serve as a promising complementary approach for tackling infectious diseases and other drug-discovery programs.

Very-long-chain fatty acid metabolic capacity of 17-beta-hydroxysteroid dehydrogenase type 12 (HSD17B12) promotes replication of hepatitis C virus and related flaviviruses.

Flaviviridae infections represent a major global health burden. By deciphering mechanistic aspects of hepatitis C virus (HCV)-host interactions, one could discover common strategy for inhibiting the replication of related flaviviruses. By elucidating the HCV interactome, we identified the 17-beta-hydroxysteroid dehydrogenase type 12 (HSD17B12) as a human hub of the very-long-chain fatty acid (VLCFA) synthesis pathway and core interactor. Here we show that HSD17B12 knockdown (KD) impairs HCV replication and reduces virion production. Mechanistically, depletion of HSD17B12 induces alterations in VLCFA-containing lipid species and a drastic reduction of lipid droplets (LDs) that play a critical role in virus assembly. Oleic acid supplementation rescues viral RNA replication and production of infectious particles in HSD17B12 depleted cells, supporting a specific role of VLCFA in HCV life cycle. Furthermore, the small-molecule HSD17B12 inhibitor, INH-12, significantly reduces replication and infectious particle production of HCV as well as dengue virus and Zika virus revealing a conserved requirement across Flaviviridae virus family. Overall, the data provide a strong rationale for the advanced evaluation of HSD17B12 inhibition as a promising broad-spectrum antiviral strategy for the treatment of Flaviviridae infections.

The Interplay between Dengue Virus and the Human Innate Immune System: A Game of Hide and Seek.

With 40% of the world population at risk, infections with dengue virus (DENV) constitute a serious threat to public health. While there is no antiviral therapy available against this potentially lethal disease, the efficacy of the only approved vaccine is not optimal and its safety has been recently questioned. In order to develop better vaccines based on attenuated and/or chimeric viruses, one must consider how the human immune system is engaged during DENV infection. The activation of the innate immunity through the detection of viruses by cellular sensors is the first line of defence against those pathogens. This triggers a cascade of events which establishes an antiviral state at the cell level and leads to a global immunological response. However, DENV has evolved to interfere with the innate immune signalling at multiple levels, hence dampening antiviral responses and favouring viral replication and dissemination. This review elaborates on the interplay between DENV and the innate immune system. A special focus is given on the viral countermeasure mechanisms reported over the last decade which should be taken into consideration during vaccine development.