D’autres pénètrent dans le noyau pour profiter du système transcriptionnel de la machinerie cellulaire. C'est pourquoi le virus de la grippe de type A fait l'objet de recherches plus intensives. Pandémies passées et futures du virus de la grippe (adapté de Horimoto et Kawaoka, 2005, revue).
D'autres segments génomiques du virus de la grippe codent pour deux protéines virales différentes. La NA est la cible de l'oseltamivir (Tamiflu®, Roche) et du zanamivir (Relenza®, GlaxoSmithKline), analogues de l'acide sialique couramment utilisés contre le virus de la grippe. Fonctions associées aux sous-unités et sous-unités du complexe ARN polymérase du virus de la grippe (adapté de Ishihama, 1996).
Nous disposons d'une reconstruction TEM 23 Å du complexe polymérase associé à un vRNP recombinant (Area et al., 2003) (Figure 2.11). Dès que les vRNP atteignent le noyau, la transcription des ARNm viraux commence (De la Luna et al., 1993). Une particularité du virus de la grippe est la capture d’ARNm cellulaires coiffés et polyadénylés.
Le virus de la grippe se lie à la cellule hôte grâce à l’interaction de l’HA avec l’acide sialique. Le virus de la grippe emprunte également CRM1 lors de l’exportation de son génome dans la phase finale de l’infection nucléaire. Influenza NP est une protéine hautement conservée entre différentes souches de virus grippal (Shu et al., 1993).
Reconstruction tridimensionnelle d'un virus grippal recombinant vRNP par TEM (Martin-Benito et al., 2001). Ou encore le peptide NLS monopartite de l'antigène T SV40 et bipartite de la nucléoplasmine (155KRPAATKKAGQAKKKK170) avec importine α de souris (Fontes et al., 2000). De gauche à droite : NP pour la grippe (virus de la grippe), la rage (virus de la rage) et la rougeole (virus de la rougeole).
Une concentration élevée en sel est nécessaire pour séparer les vRNP de la chromatine (Bui et al., 2000). En bref, M1 semble avoir une fonction régulatrice dans la localisation intracellulaire des vRNP ( Martin et Helenius, 1991 ; Whittaker et al., 1996 ). Expériences de microinjection de protéines du virus de la grippe dans le noyau de l'ovocyte de Xenopus laevis.
MUTAGENESE DIRIGEE ; EXPRESSION ET PURIFICATION DES PROTEINES
2003) Crystal structure of the M1 protein-binding domain of the influenza A virus nuclear export protein (NEP/NS2). 1995) Identification of an RNA-binding region within the N-terminal third of the influenza A virus nucleoprotein. Several protein regions contribute to determining the nuclear and cytoplasmic localization of the influenza A virus nucleoprotein.
Nuclear export of influenza virus ribonucleoproteins: identification of an export intermediate at the nuclear periphery. Studies on the primary structure of influenza virus hemagglutinin. 2002) Analysis of running transport systems. During influenza virus infection, transcription and replication of viral RNA occurs in the nucleus of the cell.
Crystal structure of the M1 protein-binding domain of the influenza A virus nuclear export protein (NEP/NS2).
NEP/NS2)
Four of these (Ile97, Met100, Leu103 and Leu107) form a continuous groove on the surface; the fifth is a prominently exposed tryptophan (Trp78) located in the center of the glutamate cluster. Therefore, we conclude that proteolytic degradation of the N-terminal domain leads to spontaneous dimerization of the C-terminal domain. A likely explanation is that the N-terminal domain packs against the hydrophobic surface of the C-terminal domain in the structure of full-length NEP, sterically hindering access to the dimerization interface; thus, proteolysis would remove this steric hindrance.
Burial of the hairpin's hydrophobic face by the N-terminal domain would also explain why full-length NEP is highly soluble in the absence of detergents. We tested the ability of NEP to bind to M1mutNLS, an M1 mutant in which the four basic residues of the NLS are replaced by alanines (101AALAA105). Failure to bind NEP cannot be attributed to misfolding of the mutant protein, as its N-terminal domain (residues 1±164) has essentially the same crystal structure as that of wild-type M1 (unpublished results).
Whether NEP recognizes the NLS motif of M1 through similar interactions must await the determination of the structure of the M1/NEP complex. In particular, in the C-terminal domain, the amphipathic nature of the helical hairpin and the highly charged character of helix C1 are retained. Interestingly, the Trp78 residue involved in M1 binding is replaced by a leucine in inuenza B, suggesting M1 recognition by NEP. 2. Structure of the NEP C-terminal domain.
Typically, residues of helix C1 interact with those of helix C2 of the same or an adjacent layer. Masking of the NLS motif of M1 by NEP could prevent the complex from being directly reimported to the nucleus when exported to the cytoplasm. We note that of the viral components tested, only NEP binds Crm1 together with RanGTP (Figure 5B).
Each of the helices in the bundle contributes one or more side chains per layer, with symmetry-related residues in the same layer. The two-protein complex purified by Superdex 75 chromatography (Pharmacia) was concentrated to 20 mg/ml. SOLVE (Terwilliger and Berentzen, 1999) located two heavy atom sites in the platinum derivatives, yielding an interpretable map after phase refinement with RESOLVE (Terwilliger, 2000), which automatically tracked most of the main chain.
