Selective Inhibitor of Nuclear Export (SINE) Compounds Alter New World Alphavirus Capsid Localization and Reduce Viral Replication in Mammalian Cells

Abstract

The capsid structural protein of the New World alphavirus, Venezuelan equine encephalitis virus (VEEV), interacts with the host nuclear transport proteins importin α/β1 and CRM1. Novel selective inhibitor of nuclear export (SINE) compounds, KPT-185, KPT-335 (verdinexor), and KPT-350, target the host’s primary nuclear export protein, CRM1, in a manner similar to the archetypical inhibitor Leptomycin B. One major limitation of Leptomycin B is its irreversible binding to CRM1; which SINE compounds alleviate because they are slowly reversible. Chemically inhibiting CRM1 with these compounds enhanced capsid localization to the nucleus compared to the inactive compound KPT-301, as indicated by immunofluorescent confocal microscopy. Differences in extracellular versus intracellular viral RNA, as well as decreased capsid in cell free supernatants, indicated the inhibitors affected viral assembly, which led to a decrease in viral titers. The decrease in viral replication was confirmed using a luciferase-tagged virus and through plaque assays. SINE compounds had no effect on VEEV TC83_Cm, which encodes a mutated form of capsid that is unable to enter the nucleus. Serially passaging VEEV in the presence of KPT-185 resulted in mutations within the nuclear localization and nuclear export signals of capsid. Finally, SINE compound treatment also reduced the viral titers of the related eastern and western equine encephalitis viruses, suggesting that CRM1 maintains a common interaction with capsid proteins across the New World alphavirus genus.

Verdinexor (KPT-335), a Selective Inhibitor of Nuclear Export, Reduces Respiratory Syncytial Virus Replication In Vitro

“…We have previously shown that RSV replication is negatively affected by XPO1-mediated nuclear transport inhibition using LMB at nanomolar concentrations (15). Targeting XPO1 with KPT-335 has also proven effective both in vitro and in vivo against several strains of the influenza virus (26,27) and against the Venezuelan equine encephalitis virus (VEEV) in vitro (28). siRNAs were used to inhibit expression of XPO1 in A549 cells, followed by infection with RSV A2, and this was associated with substantial reduction in RSV replication in human epithelial cells.…”

“…In these studies, KPT-335 inhibited nuclear export of the vRNP, resulting in reduced influenza virus replication and decreased disease pathogenesis (26,27). KPT-335 has also proven effective against Venezuelan equine encephalitis virus (VEEV) in vitro by inhibiting the nuclear export of the capsid protein (28). In a previous study conducted as a randomized, double-blind, placebo-controlled, dose-escalating phase 1 clinical trial in healthy human volunteers, KPT-335 was found to be generally safe and well tolerated, with adverse events occurring in similar numbers and grades as placebo (ClinicalTrials.gov registration number NCT02431364).…”

β- d - N 4 -Hydroxycytidine Is a Potent Anti-alphavirus Compound That Induces a High Level of Mutations in the Viral Genome

“…Compounds that indirectly interfere with VEEV replication by modifying intracellular processes in the host were also investigated as potential therapeutic options to modulate the course of alphavirus infections (17)(18)(19)(20). These included (i) an inhibitor of glycogen synthase kinase-3␤ (GSK-3␤) that governs proinflammatory responses in the host cell (17); (ii) acriflavine, an inhibitor of Ago2 (18); (iii) D-(Ϫ) enantiomer of carbodine, which inhibited cellular CTP synthetase and was capable of decreasing the levels of intracellular CTP (19); (iv) VX-497, a reversible uncompetitive inhibitor of inosine-5=-monophosphate dehydrogenase, that demonstrated a moderately negative effect on VEEV replication in vitro (20); and (v) selective inhibitors of nuclear export, which enhanced accumulation of the capsid protein in cell nuclei and thus decreased the titers of released VEEV (21). Ribavirin, a broad-spectrum antiviral drug, exhibited a very limited effect against VEEV even if applied at concentrations higher than 500 M (20).…”

Magnetic Nanotrap Particles Preserve the Stability of Venezuelan Equine Encephalitis Virus in Blood for Laboratory Detection

“…VEEV-TC83 viral stocks were produced from electroporation of in vitro transcribed viral RNA generated from the pTC83 plasmid [a kind gift from Ilya Frolov, The University of Texas Medical Branch at Galveston] as described (22). All experiments were performed under BSL-2 conditions.…”