Rabies virus chaperone: identification of the phosphoprotein peptide that keeps nucleoprotein soluble and free from non-specific RNA.
The genomic RNA of rabies virus is always complexed with the viral nucleoprotein (N). This N-RNA complex is the template for viral transcription and replication. The viral phosphoprotein (P) has two functions during the infection process: it binds through its carboxy-terminus to N in the N-RNA complex and at the same time with an amino-terminal domain to the polymerase and in this way fixes the polymerase to its template. The second function of P is to bind to newly produced N in the infected cell in order to prevent that N binds non-specifically and irreversibly to cellular RNA. In order to identify the part of the phosphoprotein that binds to N and keeps the latter soluble, we isolated the N-P complex, performed sequential protease digestions, and determined the identity of the remaining N and P peptides in the purified digested complex. Although the digestion steps removed short sequences of N, most of N remained intact and soluble, indicating that the overall structure was not affected. Most of P, including the carboxy-terminal N-RNA-binding domain, was removed during the first digestion step. N-terminal sequencing and mass spectrometry analysis identified a P peptide containing residues 4-40 that remained associated with N. Coexpression and coimmunoprecipitation experiments and yeast two-hybrid experiments showed that this peptide alone could bind to N in vivo.
Mavrakis
Manos
M
EMBL Grenoble Outstation, B.P. 181, 38042 Grenoble cedex 9, France.
Méhouas
Stéphanie
S
Réal
Eléonore
E
Iseni
Frédéric
F
Blondel
Danielle
D
Tordo
Noël
N
Ruigrok
Rob W H
RW
D000595:Amino Acid Sequence; D047468:Immunoprecipitation; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D019590:Nucleocapsid Proteins / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017433:Protein Structure, Secondary; D011820:Rabies virus / Q000235:genetics / Q000502:physiology*; D016415:Sequence Alignment; D014357:Trypsin / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D015678:Viral Structural Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D018832:Molecular Chaperones; D019590:Nucleocapsid Proteins; C103961:P phosphoprotein, Rabies virus; D010446:Peptide Fragments; D010750:Phosphoproteins; D015678:Viral Structural Proteins; C099601:nucleocapsid protein, Rabies virus; D014357:Trypsin