Download Crystal Structure of the Nucleoprotein in the Infectious Salmon

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Crystal Structure of the Nucleoprotein in the
Infectious Salmon Anemia Virus
Wenjie Zheng1, Yizhi Tao1
1
Rice University
The infectious salmon anemia virus (ISAV) is the causative agent of the infectious salmon
anemia, a high-mortality multi-systemic syndrome that is associated with many salmonid
species. ISAV is a member of the Orthomyxoviridae family, which also include the
influenza A and B viruses that cause widespread flu epidemics in human. Like the
influenza A virus, ISAV has a segmented genome consisting of eight single-stranded,
negative-sense RNAs, each encapsidated as individual, helical-shaped ribonucleoprotein
complexes. The viral nucleoprotein (NP), which is encoded by the RNA segment 3, binds
to the viral ribonucleoprotein complexes in a stoichiometric manner. Besides genome
encapsidation, the ISAV NP also plays important roles in facilitating the transcription,
replication, and genome-packaging of the virus. To better understand how ISAV NP
interacts with RNAs, we employed X-ray crystallography to determine its 3D structure. The
Full length protein was successfully crystallized but only diffracted to 20Å. Crystal
annealing, dehydration, and cross-linking did not improve the diffraction. Inspection of the
primary sequence of ISAV NP indicated that 12 out of the last 16 residues are either
aspartic acid or glutamic acid and are likely to be structurally disordered. Therefore a
truncation mutant NPΔC16 was made by removing the last 16 amino acid residues at the
C-terminus. Crystals of NPΔC16 diffracted to ~8Å, and was further improved to 2.7Å
resolution by buffer exchange to 20% PEG400. The structure was determined by SAD
(single-wavelength anomalous dispersion) using selenomethionine-substituted proteins.
The overall structure of ISAV NP closely resembles that of the influenza A virus NP, except
that the ISAV NP has an extra N-terminal domain. Electrostatic potential distribution of
ISAV NP shows a similar pattern to that of influenza A virus NP, indicating an external
groove between the head domains and body domains is likely to function as the major
RNA binding site. We are now determining the RNA binding mechanism of ISAV NP using
site-directed mutagenesis and fluorescence-based RNA binding assays.