Nucleocytoplasmic shuttling of the rabies virus P protein requires a nuclear localization signal and a CRM1-dependent nuclear export signal.
Rabies virus P protein is a co-factor of the viral RNA polymerase. It has been shown previously that P mRNA directs the synthesis of four N-terminally truncated P products P2, P3, P4, and P5 due to translational initiation by a leaky scanning mechanism at internal Met codons. Whereas P and P2 are located in the cytoplasm, P3, P4, and P5 are found in the nucleus. Here, we have analyzed the molecular basis of the subcellular localization of these proteins. Using deletion mutants fused to GFP protein, we show the presence of a nuclear localization signal (NLS) in the C-terminal part of P (172-297). This domain contains a short lysine-rich stretch ((211)KKYK(214)) located in close proximity with arginine 260 as revealed by the crystal structure of P. We demonstrate the critical role of lysine 214 and arginine 260 in NLS activity. In the presence of Leptomycin B, P is retained in the nucleus indicating that it contains a CRM1-dependent nuclear export signal (NES). The subcellular distribution of P deletion mutants indicates that the domain responsible for export is the amino-terminal part of the protein. The use of fusion proteins that have amino terminal fragments of P fused to beta-galactosidase containing the NLS of SV40 T antigen allows us to identify a NES between residues 49 and 58. The localization of NLS and NES determines the cellular distribution of the P gene products.
Pasdeloup
David
D
Unité Mixte de Virologie Moléculaire et Structurale UMR2472 CNRS, UMR1157 INRA, 91198 Gif sur Yvette Cedex, France.
Poisson
Nicolas
N
Raux
Hélène
H
Gaudin
Yves
Y
Ruigrok
Rob W H
RW
Blondel
Danielle
D
D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D006224:Cricetinae; D028884:Karyopherins / Q000378:metabolism*; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019913:Nuclear Localization Signals / Q000737:chemistry / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011820:Rabies virus / Q000378:metabolism* / Q000472:pathogenicity; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D015678:Viral Structural Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000097606:Exportin 1 Protein
D028884:Karyopherins; D018832:Molecular Chaperones; D019913:Nuclear Localization Signals; D010750:Phosphoproteins; D018160:Receptors, Cytoplasmic and Nuclear; D015678:Viral Structural Proteins; D000097606:Exportin 1 Protein; C103961:P phosphoprotein, Rabies virus