Posaconazole inhibits multiple steps of the alphavirus replication cycle

Abstract

Repurposing drugs is a promising strategy to identify therapeutic interventions against novel and re-emerging viruses. Posaconazole is an antifungal drug used to treat invasive aspergillosis and candidiasis. Recently, posaconazole and its structural analog, itraconazole were shown to inhibit replication of multiple viruses by modifying intracellular cholesterol homeostasis. Here, we show that posaconazole inhibits replication of the alphaviruses Semliki Forest virus (SFV), Sindbis virus and chikungunya virus with EC 50 values ranging from 1.4 μM to 9.5 μM. Posaconazole treatment led to a significant reduction of virus entry in an assay using a temperature-sensitive SFV mutant, but time-of-addition and RNA transfection assays indicated that posaconazole also inhibits post-entry stages of the viral replication cycle. Virus replication in the presence of posaconazole was partially rescued by the addition of exogenous cholesterol. A transferrin uptake assay revealed that posaconazole considerably slowed down cellular endocytosis. A single point mutation in the SFV E2 glycoprotein, H255R, provided partial resistance to posaconazole as well as to methyl-β-cyclodextrin, corroborating the effect of posaconazole on cholesterol and viral entry. Our results indicate that posaconazole inhibits multiple steps of the alphavirus replication cycle and broaden the spectrum of viruses that can be targeted in vitro by posaconazole, which could be further explored as a therapeutic agent against emerging viruses.

The DEAD-box RNA helicase Dhx15 controls glycolysis and arbovirus replication in Aedes aegypti mosquito cells

“…All genes were individually silenced in Aag2 cells followed by infection with a recombinant Sindbis virus expressing a nano-luciferase reporter gene as a fusion protein with nsP3 (34) (Fig. 1A).…”

“…SINV-nLuc, expressing a Nano-luciferase (nLuc) reporter as fusion protein with the SINV non-structural protein 3 (nsP3), was prepared on BHK-21 cells as previously described (34). The CHIKV expression plasmid encoding the Leiden synthetic (LS3) wildtype strain (35) was kindly provided by Dr. M.J. van Hemert (Leiden University Medical Center) and viral RNA was obtained by in vitro transcription on linearized plasmids using T7 mMessage mMachine (Invitrogen).…”

The Antifungal Itraconazole Is a Potent Inhibitor of Chikungunya Virus Replication

“…Recently, Posaconazole (PCZ), a structural analog of ITZ, was identified as a potent inhibitor of alphaviruses replication, showing comparable levels of Semliki Forest virus (SFV) replication inhibition when added at the time of inoculation or at 3 h post-inoculation (h.p.i), suggesting that this molecule acts on entry or early post-entry steps in the viral life cycle. Moreover, PCZ showed no toxic effects up to 100 µM concentration [ 48 ]. These findings are in agreement with the anti-CHIKV activity identified herein for ITZ but not for PCZ, as replicon-based screenings allow only the discovery of molecules affecting RNA replication, but not viral entry or assemble/release [ 12 ].…”

The DEAD-box RNA helicase Dhx15 controls glycolysis and arbovirus replication in Aedes aegypti mosquito cells

“…All genes were individually silenced in Aag2 cells followed by infection with a SINV infectious clone expressing a nano-luciferase reporter gene as a fusion protein with nsP3 [ 35 ] ( Fig 1A ). In the initial screening round, knockdown of 38 and 49 genes resulted in a ≥ 2-fold increase or decrease of luciferase levels, respectively, compared to a non-targeting control knockdown ( Fig 1B and 1C ).…”

“…The infectious SINV-nLuc clone, expressing a nano-luciferase (nLuc) reporter as fusion protein with non-structural protein 3 (nsP3), was grown in BHK-21 cells as previously described [ 35 ]. The CHIKV expression plasmid encoding the Leiden synthetic (LS3) wildtype strain [ 36 ] was kindly provided by Dr. M.J. van Hemert (Leiden University Medical Center) and viral RNA was obtained by in vitro transcription on linearized plasmids using T7 mMessage mMachine (Invitrogen).…”