J Virol. 2006 Aug;80(16):7894-901.
Crystal structure of nonstructural protein 10 from the severe acute respiratory syndrome coronavirus reveals a novel fold with two zinc-binding motifs.
Joseph JS1, Saikatendu KS, Subramanian V, Neuman BW, Brooun A, Griffith M, Moy K, Yadav MK, Velasquez J, Buchmeier MJ, Stevens RC, Kuhn P.
Abstract
The severe acute respiratory syndrome coronavirus
(SARS-CoV) possesses a large 29.7-kb positive-stranded RNA genome. The
first open reading frame encodes replicase polyproteins 1a and 1ab,
which are cleaved to generate 16 "nonstructural" proteins,
nsp1 to nsp16, involved in viral replication and/or RNA processing.
Among these, nsp10 plays a critical role in minus-strand RNA synthesis
in a related coronavirus,
murine hepatitis virus. Here, we report the crystal structure of
SARS-CoV nsp10 at a resolution of 1.8 A as determined by
single-wavelength anomalous dispersion using phases derived from
hexatantalum dodecabromide. nsp10 is a single domain protein consisting
of a pair of antiparallel N-terminal helices stacked against an
irregular beta-sheet, a coil-rich C terminus, and two Zn fingers. nsp10 represents a novel fold and is the first structural representative of this family of Zn finger proteins found so far exclusively in coronaviruses. The first Zn finger coordinates a Zn2+ ion in a unique conformation. The second Zn finger,
with four cysteines, is a distant member of the "gag-knuckle fold
group" of Zn2+-binding domains and appears to maintain the structural
integrity of the C-terminal tail. A distinct clustering of basic
residues on the protein surface suggests a nucleic acid-binding
function. Gel shift assays indicate that in isolation, nsp10 binds
single- and double-stranded RNA and DNA with high-micromolar affinity
and without obvious sequence specificity. It is possible that nsp10
functions within a larger RNA-binding protein complex. However, its
exact role within the replicase complex is still not clear.
- [Indexed for MEDLINE]
https://www.ncbi.nlm.nih.gov/pubmed/26041293
(2) https://www.ncbi.nlm.nih.gov/pubmed/25074927 Abstract
The RNA-synthesizing machinery of the severe acute respiratory syndrome Coronavirus (SARS-CoV) is composed of 16 non-structural proteins (nsp1-16)
encoded by ORF1a/1b. The 148-amino acid nsp10 subunit contains two zinc
fingers and is known to interact with both nsp14 and nsp16, stimulating
their respective 3'-5' exoribonuclease and 2'-O-methyltransferase
activities. Using alanine-scanning mutagenesis, in cellulo
bioluminescence resonance energy transfer experiments, and in vitro
pulldown assays, we have now identified the key residues on the nsp10
surface that interact with nsp14. The functional consequences of
mutations introduced at these positions were first evaluated
biochemically by monitoring nsp14 exoribonuclease activity. Disruption
of the nsp10-nsp14 interaction abrogated the nsp10-driven activation of
the nsp14 exoribonuclease. We further showed that the nsp10 surface
interacting with nsp14 overlaps with the surface involved in the
nsp10-mediated activation of nsp16 2'-O-methyltransferase activity,
suggesting that nsp10 is a major regulator of SARS-CoV
replicase function. In line with this notion, reverse genetics
experiments supported an essential role of the nsp10 surface that
interacts with nsp14 in SARS-CoV
replication, as several mutations that abolished the interaction in
vitro yielded a replication-negative viral phenotype. In contrast,
mutants in which the nsp10-nsp16 interaction was disturbed proved to be
crippled but viable. These experiments imply that the nsp10 surface that
interacts with nsp14 and nsp16 and possibly other subunits of the viral
replication complex may be a target for the development of antiviral
compounds against pathogenic coronaviruses.
© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
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