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.

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.

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.