The Nucleocapsid Protein of Potato Yellow dwarf Virus: Protein Interactions and Nuclear Import Mediated by a Non-Canonical Nuclear Localization Signal

Gavin Anderson,Michael Goodin,Anindya Bandyopadhyay,Renyuan Wang

doi:10.3389/fpls.2012.00014

Abstract

Potato yellow dwarf virus (PYDV) is the type species of the genus Nucleorhabdovirus and, like all members of this genus, replication and morphogenesis occurs inside the nuclei of infected cells. Protein localization prediction algorithms failed to identify a nuclear localization signal (NLS) in PYDV nucleocapsid (N) protein, although PYDV-N has been shown to localize exclusively to the nucleus when expressed as a green fluorescent protein (GFP):N fusion in plant cells. Deletion analysis using fragments of PYDV-N identified a karyophilic region in the carboxy-terminal 122 amino acids. Alanine-scanning mutagenesis was performed across this region in the context of the full-length N protein. Mutants were assayed for their ability to nuclear localize using live-cell imaging and a yeast-based assay. Two amino acid motifs, 419QKR421 and 432KR433 were shown to be essential for nuclear import and interaction with importin-α. Additional bimolecular fluorescence complementation showed that the PYDV-N-NLS mutants cannot be ferried into the nucleus via interaction with PYDV-P or -M. In contrast, interaction with N-NLS mutants appeared to retard the nuclear import of PYDV-P. GFP fused to aa 419–434 established that the PYDV-N-NLS can function outside the context of this protein. Taken together, it was determined that PYDV-N contains the bipartite NLS 419QKRANEEAPPAAQKR433.

Highlights

Rhabdoviruses are structurally complex enveloped viruses, the plant-adapted members of which are separated into the genera Cytorhabdovirus, which replicate in the cytoplasm of infected cells, and Nucleorhabdovirus, the viruses considered here, which replicate in nuclei of infected cells
We report the characterization of the nuclear localization signal (NLS) responsible for nuclear targeting of the nucleocapid protein of Potato yellow dwarf virus (PYDV), which is the type species of the nucleorhabdovirus genus (Hsu and Black, 1973, 1974; Bandyopadhyay et al, 2010)
We have shown previously that despite the fact that protein localization prediction algorithms failed to identify an NLS in PYDV-N, this protein is fully capable of directing Green fluorescent protein (GFP) exclusively to the nucleus in plant cells expressing green fluorescent protein (GFP):PYDV-N fusions (Bandyopadhyay et al, 2010)

Summary

Introduction

Rhabdoviruses are structurally complex enveloped viruses, the plant-adapted members of which are separated into the genera Cytorhabdovirus, which replicate in the cytoplasm of infected cells, and Nucleorhabdovirus, the viruses considered here, which replicate in nuclei of infected cells. The N proteins of viruses belonging to the genus Nucleorhabdovirus contain nuclear localization signals (NLSs) that direct the protein itself to the nucleus and the viral nucleocapsid, which is required to establish sites of replication and viral assembly in nuclei of infected plant cells (Wagner et al, 1996; Wagner and Jackson, 1997; Jackson et al, 2005; Kuzmin et al, 2009) In this regard, the N protein-mediated transport of nucleocapsids parallels the role of VirE2 in the transfer of transfer DNA (T-DNA) from Agrobacterium tumefaciens to nuclei of plant cells to which it is attached. As such the identification and mapping of NLSs in N proteins is critically required to understand the mechanism by which these proteins, and their ribonucleoprotein complexes are targeted to the nuclei (Citovsky et al, 1992, 2004; Deng et al, 2007)

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