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PDBsum entry 3ee7
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Viral protein
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PDB id
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3ee7
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References listed in PDB file
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Key reference
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Title
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Severe acute respiratory syndrome coronavirus nsp9 dimerization is essential for efficient viral growth.
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Authors
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Z.J.Miknis,
E.F.Donaldson,
T.C.Umland,
R.A.Rimmer,
R.S.Baric,
L.W.Schultz.
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Ref.
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J Virol, 2009,
83,
3007-3018.
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PubMed id
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Abstract
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The severe acute respiratory syndrome coronavirus (SARS-CoV) devotes a
significant portion of its genome to producing nonstructural proteins required
for viral replication. SARS-CoV nonstructural protein 9 (nsp9) was identified as
an essential protein with RNA/DNA-binding activity, and yet its biological
function within the replication complex remains unknown. Nsp9 forms a dimer
through the interaction of parallel alpha-helices containing the protein-protein
interaction motif GXXXG. In order to study the role of the nsp9 dimer in viral
reproduction, residues G100 and G104 at the helix interface were targeted for
mutation. Multi-angle light scattering measurements indicated that G100E, G104E,
and G104V mutants are monomeric in solution, thereby disrupting the dimer.
However, electrophoretic mobility assays revealed that the mutants bound RNA
with similar affinity. Further experiments using fluorescence anisotropy showed
a 10-fold reduction in RNA binding in the G100E and G104E mutants, whereas the
G104V mutant had only a 4-fold reduction. The structure of G104E nsp9 was
determined to 2.6-A resolution, revealing significant changes at the dimer
interface. The nsp9 mutations were introduced into SARS-CoV using a reverse
genetics approach, and the G100E and G104E mutations were found to be lethal to
the virus. The G104V mutant produced highly debilitated virus and eventually
reverted back to the wild-type protein sequence through a codon transversion.
Together, these data indicate that dimerization of SARS-CoV nsp9 at the GXXXG
motif is not critical for RNA binding but is necessary for viral replication.
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