Browsing by Author "Zheng, Wenjie"

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    Structural and Functional Studies on the Infectious Salmon Anemia Virus Nucleoprotein
    (2013-10-25) Zheng, Wenjie; Tao, Yizhi Jane; Beckingham, Kathleen M.; Nikonowicz, Edward P.; Suh, Junghae; Wang, Qinghua
    Genome packaging for viruses with segmented genomes is often a complex problem. This is particularly true for influenza viruses and other orthomyxoviruses which are able to cause infectious disease, and even worldwide pandemics. The genome of Orthomyxovirus consists of 6-8 negative-sense RNAs encapsidated as ribonucleoprotein (RNP) complexes which perform multiple essential functions throughout the virus life cycle. To better understand the structural features of orthomyxovirus RNPs that allow them to be specifically packaged, we performed structural/functional studies of the nucleoprotein (NP), the major protein component of the RNPs, from the infectious salmon anemia virus (ISAV). The crystal structure of the ISAV-NP was determined to 2.7Å resolution. The ISAV-NP possesses a 112-aa N-terminal domain and a bi-lobular core structure that strongly resembles the structure of the influenza virus NP. Because the ISAV-NP forms homogenous dimers that are stable in solution, I was able to study the NP:RNA binding affinity as well as stoichiometry with fluorescence polarization, using recombinant proteins and synthetic oligos. Surprisingly, the RNA binding analysis revealed that each NP binds ~12 nts of RNA, shorter than the 24-28 nts originally estimated for the influenza A virus NP. The 12-nt stoichiometry was further confirmed by results from electron microscopy and dynamic light scattering. These results suggest that free RNA exists in the orthomyxovirus RNPs, and selective RNP packaging is likely accomplished through direct RNA-RNA interactions.
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    The Crystal Structure and RNA-Binding of an Orthomyxovirus Nucleoprotein
    (Public Library of Science, 2013) Zheng, Wenjie; Olson, John S.; Vakharia, Vikram; Tao, Yizhi Jane
    Genome packaging for viruses with segmented genomes is often a complex problem. This is particularly true for influenza viruses and other orthomyxoviruses, whose genome consists of multiple negative-sense RNAs encapsidated as ribonucleoprotein (RNP) complexes. To better understand the structural features of orthomyxovirus RNPs that allow them to be packaged, we determined the crystal structure of the nucleoprotein (NP) of a fish orthomyxovirus, the infectious salmon anemia virus (ISAV) (genus Isavirus). As the major protein component of the RNPs, ISAV-NP possesses a bi-lobular structure similar to the influenza virus NP. Because both RNA-free and RNA-bound ISAV NP forms stable dimers in solution, we were able to measure the NP RNA binding affinity as well as the stoichiometry using recombinant proteins and synthetic oligos. Our RNA binding analysis revealed that each ISAV-NP binds ,12 nts of RNA, shorter than the 24ﾖ28 nts originally estimated for the influenza A virus NP based on population average. The 12-nt stoichiometry was further confirmed by results from electron microscopy and dynamic light scattering. Considering that RNPs of ISAV and the influenza viruses have similar morphologies and dimensions, our findings suggest that NP-free RNA may exist on orthomyxovirus RNPs, and selective RNP packaging may be accomplished through direct RNA-RNA interactions.