Sindbis virus nucleocapsid core assembly: Characterization of the initiation complex that promotes genome packaging
In Sindbis virus, initiation of nucleocapsid core assembly begins with recognition of the encapsidation signal of the viral RNA genome by capsid protein. This nucleation event drives the recruitment of additional capsid protein to fully encapsidate the genome, generating an icosahedral nucleocapsid core. The encapsidation signal of the Sindbis virus genome has previously been localized to a 132 nt region of the genome, and the RNA-binding activity of the capsid was previously mapped to a central region of the capsid protein. It is unknown how capsid protein binding to the encapsidation signal leads to ordered oligomerization and nucleocapsid core assembly. To address this question, a mobility shift assay was developed to study this interaction. This assay facilitated the identification of a 32 amino acid peptide capable of recognizing the Sindbis virus encapsidation signal RNA. Binding of this peptide to the encapsidation signal induced a conformational change, observed as a complex of faster mobility. Binding is tight (K dapp = 12 nM), and results in dimerization of the capsid peptide. Mutational analysis reveals that while almost every predicted secondary structure within the encapsidation signal is required for efficient protein binding, the identities of most of the bases within the helices and hairpin turns of the RNA do not need to be maintained. In contrast, two purine-rich loops connecting the helices are essential for binding. Alanine scanning of the peptide identified two residues important for specific binding to the encapsidation signal RNA. These amino acids provide important target sites for in vivo analysis of genome specific packaging. Preliminary structure analysis with NMR spectroscopy demonstrated the ability to measure resonance shifts for 15N labeled peptide upon binding to encapsidation signal RNA. This sets the stage for high-resolution structure analysis of this system. From these data we have developed a model in which the encapsidation signal RNA adopts a highly folded structure that directs early events in nucleocapsid core assembly.
Golden, Purdue University.
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