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

Lindsay Lundberg,Kylie M Wagstaff,Kylene Kehn-Hall,Chelsea Pinkham,Cynthia De La Fuente,Sharon Tamir,David A Jans,Ashwini Brahms,Nazly Shafagati,Ann M Powers

doi:10.1371/journal.pntd.0005122

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.

Highlights

Endemic to North, Central, and South America, the New World alphaviruses cause a febrile illness that can progress to encephalitis with accompanying high morbidity and mortality rates in humans and equines [1]
Our data demonstrate that novel selective inhibitor of nuclear export (SINE) compounds reduced viral replication of three related New World alphaviruses, Venezuelan equine encephalitis virus (VEEV), EEEV, and WEEV, indicating that CRM1 is instrumental to their life cycle
Inhibitors have a large selective index and represent a potential pan-antiviral therapeutic that targets the host’s transport proteins, which are hijacked by the New World alphaviruses

Summary

Introduction

Endemic to North, Central, and South America, the New World alphaviruses cause a febrile illness that can progress to encephalitis with accompanying high morbidity and mortality rates in humans and equines [1]. Alphaviruses are divided into New World and Old World groups based on geography, disease progression, and protein function. The nonstructural region encodes a polyprotein from the genomic RNA that is cleaved into the nonstructural proteins 1–4 (nsP1-4). The functional role of the viral proteins diverges between Old and New World alphaviruses. Type I interferons (IFN), IFN-α and IFN-β, attenuate alphaviruses, and Old and New World viruses have evolved to counter the host immune response. The VEEV nonstructural proteins inhibit STAT1 activation, preventing its nuclear localization and STAT1-dependent transcription [6]. The nsP2 protein of the Old World alphavirus Chikungunya (CHIKV) inhibits STAT1 activation by blocking its phosphorylation [7]. VEEV, as a model for the New World viruses, uses its capsid protein to halt host transcription [10]. Capsid most likely achieves this by blocking nuclear import and export [11]

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Results

Discussion

Conclusion