Degenerate RNA Packaging Signals in the Genome of Satellite Tobacco Necrosis Virus: Implications for the Assembly of a T=1 Capsid

TY - JOUR

T1 - Degenerate RNA Packaging Signals in the Genome of Satellite Tobacco Necrosis Virus: Implications for the Assembly of a T=1 Capsid

AU - Bunka, David H. J.

AU - Lane, Stephen W.

AU - Lane, Claire L.

AU - Dykeman, Eric C.

AU - Ford, Robert J.

AU - Barker, Amy M.

AU - Twarock, Reidun

AU - Phillips, Simon E. V.

AU - Stockley, Peter G.

PY - 2011/10/14

Y1 - 2011/10/14

N2 - Using a recombinant, T=1 Satellite Tobacco Necrosis Virus (STNV)-like particle expressed in Escherichia coli, we have established conditions for in vitro disassembly and reassembly of the viral capsid. In vivo assembly is dependent on the presence of the coat protein (CP) N-terminal region, and in vitro assembly requires RNA. Using immobilised CP monomers under reassembly conditions with "free" CP subunits, we have prepared a range of partially assembled CP species for RNA aptamer selection. SELEX directed against the RNA-binding face of the STNV CP resulted in the isolation of several clones, one of which (B3) matches the STNV-1 genome in 16 out of 25 nucleotide positions, including across a statistically significant 10/10 stretch. This 10-base region folds into a stem loop displaying the motif ACAA and has been shown to bind to STNV CP. Analysis of the other aptamer sequences reveals that the majority can be folded into stem loops displaying versions of this motif. Using a sequence and secondary structure search motif to analyse the genomic sequence of STNV-1, we identified 30 stem loops displaying the sequence motif AxxA. The implication is that there are many stem loops in the genome carrying essential recognition features for binding STNV CP. Secondary structure predictions of the genomic RNA using Mfold showed that only 8 out of 30 of these stem loops would be formed in the lowest-energy structure. These results are consistent with an assembly mechanism based on kinetically driven folding of the RNA. (C) 2011 Elsevier Ltd. All rights reserved.

AB - Using a recombinant, T=1 Satellite Tobacco Necrosis Virus (STNV)-like particle expressed in Escherichia coli, we have established conditions for in vitro disassembly and reassembly of the viral capsid. In vivo assembly is dependent on the presence of the coat protein (CP) N-terminal region, and in vitro assembly requires RNA. Using immobilised CP monomers under reassembly conditions with "free" CP subunits, we have prepared a range of partially assembled CP species for RNA aptamer selection. SELEX directed against the RNA-binding face of the STNV CP resulted in the isolation of several clones, one of which (B3) matches the STNV-1 genome in 16 out of 25 nucleotide positions, including across a statistically significant 10/10 stretch. This 10-base region folds into a stem loop displaying the motif ACAA and has been shown to bind to STNV CP. Analysis of the other aptamer sequences reveals that the majority can be folded into stem loops displaying versions of this motif. Using a sequence and secondary structure search motif to analyse the genomic sequence of STNV-1, we identified 30 stem loops displaying the sequence motif AxxA. The implication is that there are many stem loops in the genome carrying essential recognition features for binding STNV CP. Secondary structure predictions of the genomic RNA using Mfold showed that only 8 out of 30 of these stem loops would be formed in the lowest-energy structure. These results are consistent with an assembly mechanism based on kinetically driven folding of the RNA. (C) 2011 Elsevier Ltd. All rights reserved.

UR - http://www.scopus.com/inward/record.url?scp=80053306574&partnerID=8YFLogxK

U2 - 10.1016/j.jmb.2011.07.063

DO - 10.1016/j.jmb.2011.07.063

M3 - Article

SN - 0022-2836

VL - 413

SP - 51

EP - 65

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

IS - 1

ER -