The N terminus of Rift Valley fever virus nucleoprotein is essential for dimerization.
Rift Valley fever virus (RVFV) is a Phlebovirus in the Bunyaviridae family. The nucleoprotein N is the most abundant component of the virion; numerous copies of N associate with the viral RNA genome and form pseudohelicoidal ribonucleoproteins (RNPs) circularized by a panhandle structure formed by the base-paired RNA sequences at the 3' and 5' termini. These structures play a central role in transcription and replication. We investigated the intermolecular interactions of the RVFV N protein and found that after chemical cross-linking treatment, the nucleoprotein from purified RNPs migrates mainly as dimers. The N-N interaction was studied using the yeast two-hybrid system, the GST pull-down method, and mutational analysis. We demonstrated that the N terminus from residue 1 to 71, and particularly Tyr 4 and Phe 11, which are conserved among phlebovirus N sequences, are involved in the interaction. The C-terminal region did not seem to be essential for the N-N interaction. Moreover, we showed that N(TOS), the N protein of the related Toscana phlebovirus, interacts with itself and forms heterodimers with N(RVF), suggesting that the dimeric form of N may be a conserved feature in phlebovirus RNPs.
Le May
Nicolas
N
Unité de Génétique Moléculaire des Bunyaviridés, Institut Pasteur, 25 rue du Docteur Roux 75015, Paris, France.
Gauliard
Nicolas
N
Billecocq
Agnès
A
Bouloy
Michèle
M
D000595:Amino Acid Sequence; D000818:Animals; D002522:Chlorocebus aethiops; D017124:Conserved Sequence; D003432:Cross-Linking Reagents; D019281:Dimerization; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019590:Nucleocapsid Proteins / Q000737:chemistry* / Q000235:genetics; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D012296:Rift Valley fever virus / Q000737:chemistry* / Q000235:genetics; D029301:Sandfly fever Naples virus / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D020798:Two-Hybrid System Techniques; D014709:Vero Cells
D003432:Cross-Linking Reagents; D019590:Nucleocapsid Proteins; D011993:Recombinant Fusion Proteins