title: Structural requirements for nucleocapsid protein-mediated dimerization of avian leukosis virus RNA.
abstract: The avian leukosis virus (ALV) belongs to the alpha group of retroviruses that are widespread in nature. The 5'-untranslated region of ALV genome contains the L3 element that is important for virus infectivity and the formation of an unstable RNA dimer in vitro. The L3 sequence is predicted to fold into a long stem-loop structure with two internal loops and an apical one. Phylogenetic analysis predicts that the L3 stem-loop is conserved in alpharetroviruses. Furthermore, a significant selection mechanism maintains a palindrome in the apical loop. The nucleocapsid protein of the alpharetroviruses (NCp12) is required for RNA dimer formation and replication in vivo. It is not known whether L3 can be an NCp12-mediated RNA dimerization site able to bind NCp12 with high affinity. Here, we report that NCp12 chaperones formation of a stable ALV RNA dimer through L3. To investigate the NCp12-mediated L3 dimerization reaction, we performed site-directed mutagenesis, gel retardation and heterodimerization assays and analysis of thermostability of dimeric RNAs. We show that the affinity of NCp12 for L3 is lower than its affinity for the microPsi RNA packaging signal. Results show that conservation of a long stem-loop structure and a loop-loop interaction are not required for NCp12-mediated L3 dimerization. We show that the L3 apical stem-loop is sufficient to form an extended duplex and the whole stem-loop L3 cannot be converted by NCp12 into a duplex extending throughout L3. Three-dimensional modelling of the stable L3 dimer supports the notion that the extended duplex may represent the minimal dimer linkage structure found in the genomic RNA.
authors: CNRS UMR8113, LBPA, Ecole Normale Supérieure de Cachan, 94235 Cachan cedex, France.
authors: Moez Ben
authors: MB
authors: Ali
authors: CNRS UMR8113, LBPA, Ecole Normale Supérieure de Cachan, 94235 Cachan cedex, France.
authors: Françoise
authors: F
authors: Chaminade
authors: CNRS UMR8113, LBPA, Ecole Normale Supérieure de Cachan, 94235 Cachan cedex, France.
authors: Igor
authors: I
authors: Kanevsky
authors: CNRS UPR9002, Institut de Biologie Moléculaire et Cellulaire, 67084 Strasbourg cedex, France.
authors: Eric
authors: E
authors: Ennifar
authors: CNRS UMR8113, LBPA, Ecole Normale Supérieure de Cachan, 94235 Cachan cedex, France.
authors: Laurence
authors: L
authors: Josset
authors: CNRS UMR5086, Institut de Biologie et Chimie des Protéines, 69367 Lyon cedex 07, France.
authors: Damien
authors: D
authors: Ficheux
authors: Laboretro Unité de Virologie Humaine INSERM #758, Ecole Normale Supérieure de Lyon, 69364 Lyon cedex 07, France.
authors: Jean-Luc
authors: JL
authors: Darlix
authors: CNRS UMR8113, LBPA, Ecole Normale Supérieure de Cachan, 94235 Cachan cedex, France. Electronic address: fosse@lbpa.ens-cachan.fr.
authors: Philippe
authors: P
authors: Fossé
mesh_terms: D000818:Animals; D001483:Base Sequence; D019281:Dimerization; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D019590:Nucleocapsid Proteins / Q000378:metabolism*; D012367:RNA, Viral / Q000737:chemistry* / Q000378:metabolism
chemical_list: D019590:Nucleocapsid Proteins; D012367:RNA, Viral