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Chikungunya virus capsid protein contains nuclear import and export signals
Virology Journal
Chikungunya virus capsid protein contains nuclear import and export signals
Saijo Thomas 0
Jagdish Rai
Lijo John 0
Stephan Schaefer 0
Brigitte M Ptzer 0
Ottmar Herchenrder 0
0 Institute of Experimental Gene Therapy and Cancer Research, Rostock University Medical Center , Schillingallee 69, 18057, Rostock , Germany
Background: 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. 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. Methods: EGFP-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. Results: Here we show that CHIKV CP is a nuclear-cytoplasmic shuttling protein with an active NLS that binds to karyopherin (Kar) for its nuclear translocation. We also found that the Kar4 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. Conclusions: We 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.
Chikungunya virus; Capsid protein; Nuclear transport; Nuclear localization signal; Nuclear export signal; Karyopherin; CRM1
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Background
The arthropode-borne Chikungunya virus (CHIKV) of the
genus alphavirus in the Togaviridae family is running
rampant along the Western and Northern Rim of the Indian
Ocean. Outbreaks occurred on its islands including the
Malay Archipelago as well as in several tropical African
countries [1-3]. Alphaviruses express a capsid protein (CP)
as part of a polyprotein which, after autoproteolytic
cleavage by its protease activity, forms viral particles and
packages the viral genomic RNA [3]. Although being small
enough (approx. 30 kDa) for passive transport through
nuclear pores, some alphaviral capsid proteins are reported
to harbor a nuclear localization signal (NLS) with the
proteins transport being energy dependent [4,5].
In general, bidirectional transport of molecules
between the nucleus and the cytoplasm occurs through the
nuclear pore complex (NPC), a supra-molecular
structure of the nuclear envelope [6]. NPCs allow passive
diffusion of ions and small proteins up to a molecular
weight of 40 kDa or less than 9 nm in diameter, but
restrict passage of larger molecules to those containing an
appropriate targeting signal [7]. Protein traffic into the
nucleus is mediated by the interaction of transport
cargoes with karyopherins. Karyopherins are adaptor
proteins that recognize cargo to be conveyed via a NLS
which also interacts with the transport receptor importin
. Together, these proteins form a ternary bundle that
conjointly translocates into the nucleus via the nuclear
pore complex [8]. NLS motifs used by the classical
nuclear import pathway consist of a short stretch of the
positively charged amino acids (aa) arginine and lysine
but lack strict consensus sequences [9]. A monopartite
NLS like that of SV40 large T-antigen is composed of a
cluster of five to seven basic aa, while a typical bipartite
NLS contains two clusters of basic amino acids
separated by a linker of 1011 aa [10-12]. 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 specifically 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, although
other hydrophobic residues such as isoleucine,
methionine, phenylalanine (...truncated)