Analysis of hepatitis C virus RNA dimerization and core-RNA interactions.
The core protein of hepatitis C virus (HCV) has been shown previously to act as a potent nucleic acid chaperone in vitro, promoting the dimerization of the 3'-untranslated region (3'-UTR) of the HCV genomic RNA, a process probably mediated by a small, highly conserved palindromic RNA motif, named DLS (dimer linkage sequence) [G. Cristofari, R. Ivanyi-Nagy, C. Gabus, S. Boulant, J. P. Lavergne, F. Penin and J. L. Darlix (2004) Nucleic Acids Res., 32, 2623-2631]. To investigate in depth HCV RNA dimerization, we generated a series of point mutations in the DLS region. We find that both the plus-strand 3'-UTR and the complementary minus-strand RNA can dimerize in the presence of core protein, while mutations in the DLS (among them a single point mutation that abolished RNA replication in a HCV subgenomic replicon system) completely abrogate dimerization. Structural probing of plus- and minus-strand RNAs, in their monomeric and dimeric forms, indicate that the DLS is the major if not the sole determinant of UTR RNA dimerization. Furthermore, the N-terminal basic amino acid clusters of core protein were found to be sufficient to induce dimerization, suggesting that they retain full RNA chaperone activity. These findings may have important consequences for understanding the HCV replicative cycle and the genetic variability of the virus.
Ivanyi-Nagy
Roland
R
LaboRetro, Unité INSERM de Virologie Humaine, Ecole Normale Supérieure de Lyon IFR, 128 Biosciences Lyon-Gerland, 69364 Lyon Cedex 07, France.
Kanevsky
Igor
I
Gabus
Caroline
C
Lavergne
Jean-Pierre
JP
Ficheux
Damien
D
Penin
François
F
Fossé
Philippe
P
Darlix
Jean-Luc
JL
D020413:3' Untranslated Regions / Q000737:chemistry*; D001483:Base Sequence; D019281:Dimerization; D016174:Hepacivirus / Q000235:genetics*; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D017434:Protein Structure, Tertiary; D012367:RNA, Viral / Q000737:chemistry*; D014758:Viral Core Proteins / Q000737:chemistry / Q000378:metabolism*
D020413:3' Untranslated Regions; D018832:Molecular Chaperones; D012367:RNA, Viral; D014758:Viral Core Proteins; C069130:nucleocapsid protein, Hepatitis C virus