Chikungunya virus capsid protein contains nuclear import and export signals

Saijo Thomas,Stephan Schaefer,Ottmar Herchenröder,Jagdish Rai,Brigitte M Pützer,Lijo John

doi:10.1186/1743-422x-10-269

Abstract

BackgroundChikungunya virus (CHIKV) is an alphavirus of the Togaviridae family. After autoproteolytic cleavage, the CHIKV capsid protein (CP) is involved in RNA binding and assembly of the viral particle. The monomeric CP is approximately 30 kDa in size and is small enough for passive transport through nuclear pores. Some alphaviruses are found to harbor nuclear localization signals (NLS) and transport of these proteins between cellular compartments was shown to be energy dependent. The active nuclear import of cytoplasmic proteins is mediated by karyopherins and their export by exportins. As nuclear and cytoplasmic trafficking may play a role in the life cycle of CHIKV, we have sought to identify nuclear localization and nuclear export signals in CHIKV CP in a virus-free system.MethodsEGFP-fusion proteins of CHIKV CP and mutants thereof were created and used to monitor their intracellular localization. Binding of cellular proteins was confirmed in pull-down assays with purified CP using co-immuoprecipitation. Nuclear localization was demonstrated in a virus-free system using fluorescence microscopy.ResultsHere we show that CHIKV CP is a nuclear-cytoplasmic shuttling protein with an active NLS that binds to karyopherin α (Karα) for its nuclear translocation. We also found that the Karα4 C-terminal NLS binding site is sufficient for this interaction. We further demonstrate that CHIKV CP interacts directly with the export receptor CRM1 to transport this viral protein out of the nucleus via a nuclear export signal (NES). The CHIKV CP NES was mapped between amino acids 143 and 155 of CP. Deduced from in silico analyses we found that the NES has a mode of binding similar to the snurportin-1 CRM1 complex.ConclusionsWe were able to show that in a virus-free system that the CHIKV capsid protein contains both, a NLS and a NES, and that it is actively transported between the cytoplasma and the nucleus. We conclude that CHIKV CP has the ability to shuttle via interaction with karyopherins for its nuclear import and, vice versa, by CRM1-dependent nuclear export.

Highlights

Chikungunya virus (CHIKV) is an alphavirus of the Togaviridae family
We show that CHIKV capsid protein (CP) contains a Chromosomal Region Maintenance 1 (CRM1)-mediated nuclear export signal (NES), which is mapped to a leucine rich region between amino acids 143 and 155 and docks similar to snurportin-1 CRM1 complex in silico
To validate the role of the corresponding region in CHIKV CP, the putative nuclear localization signals (NLS) containing sequence was cloned in fusion with EGFP and HEK293 were transfected with pNLS-EGFP

Summary

Introduction

Chikungunya virus (CHIKV) is an alphavirus of the Togaviridae family. After autoproteolytic cleavage, the CHIKV capsid protein (CP) is involved in RNA binding and assembly of the viral particle. Karyopherins are adaptor proteins that recognize cargo to be conveyed via a NLS which interacts with the transport receptor importin β Together, these proteins form a ternary bundle that conjointly translocates into the nucleus via the nuclear pore complex [8]. Nuclear export signals (NES) are recognized by exportins and allow proteins to be actively carried from the nucleus to the cytoplasm through the NPC [13]. NES are bound by exportin known as exportin 1 or Chromosomal Region Maintenance 1 (CRM1) This transport is mediated via binding to the GTP-bound form of the guanine nucleotide-binding protein Ran. The most commonly identified NES are short leucine-rich stretches, other hydrophobic residues such as isoleucine, methionine, phenylalanine, and valine can contribute to this entity [14]

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