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Title
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Mutant viral polymerase in the transition of virus to error catastrophe identifies a critical site for RNA binding.
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Authors
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A.Arias,
R.Agudo,
C.Ferrer-Orta,
R.Pérez-Luque,
A.Airaksinen,
E.Brocchi,
E.Domingo,
N.Verdaguer,
C.Escarmís.
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Ref.
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J Mol Biol, 2005,
353,
1021-1032.
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PubMed id
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Abstract
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A foot-and-mouth disease virus (FMDV) polymerase (3D) with amino acid
replacements G118D, V239M and G373D (triple DMD mutant) was obtained from a
molecular clone derived from a virus population treated with ribavirin, in the
transition to error catastrophe (virus extinction through lethal mutagenesis).
DMD 3D was expressed in Escherichia coli, purified, and its activity compared
with that of wild-type enzyme and mutant enzymes with either replacement G118D,
G118A or D338A (the latter affecting the catalytic motif YGDD), generated by
site-directed mutagenesis. No differences among the enzymes were noted in their
interaction with monoclonal antibodies specific for the FMDV polymerase. Mutant
enzymes with G118D or G118A showed a 100-fold decrease in polymerization
activity relative to wild-type 3D, using poly(A)/oligo(dT)15 and poly(A)/VPg as
template-primers, under several reaction conditions. As expected, the activity
of 3D with D338A was undetectable (<0.01 times the value for wild-type 3D).
DMD and the G118 mutants showed impaired binding to template-primer RNA whereas
the D338A mutant showed a binding similar to wild-type 3D. Transfection of cells
with FMDV RNA encoding DMD 3D resulted in selection of revertant viruses that
maintained only substitutions V239M and G373D. Consistently, when infectious
transcripts encoded 3D with either G118D, G118A or D338A, viruses with
reversions to the wild-type sequence were isolated. The implication of G118 in
template-primer binding is supported by the location of this residue in the
template-binding groove of the FMDV polymerase. In addition to identifying an
amino acid residue that is critical for the binding of polymerase to RNA, the
results document the presence of defective genomes in the transition of virus to
error catastrophe.
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