Browsing by Author "Guu, Tom S.Y."

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    Biochemical and Structural Evidence in Support of a Coherent Model for the Formation of the Double-Helical Influenza A Virus Ribonucleoprotein
    (American Society for Microbiology, 2012) Ye, Qiaozhen; Guu, Tom S.Y.; Mata, Dougslas A.; Kuo, Rei-Lin; Smith, Bartram; Krug, Robert M.; Tao, Yizhi Jane
    Influenza A virions contain eight ribonucleoproteins (RNPs), each comprised of a negative-strand viral RNA, the viral polymerase, and multiple nucleoproteins (NPs) that coat the viral RNA. NP oligomerization along the viral RNA is mediated largely by a 28-amino-acid tail loop. Influenza viral RNPs, which serve as the templates for viral RNA synthesis in the nuclei of infected cells, are not linear but rather are organized in hairpin-like double-helical structures. Here we present results that strongly support a coherent model for the assembly of the double-helical influenza virus RNP structure. First, we show that NP self-associates much more weakly in the absence of RNA than in its presence, indicating that oligomerization is very limited in the cytoplasm. We also show that once NP has oligomerized, it can dissociate in the absence of bound RNA, but only at a very slow rate, indicating that the NP scaffold remains intact when viral RNA dissociates from NPs to interact with the polymerase during viral RNA synthesis. In addition, we identify a previously unknown NP-NP interface that is likely responsible for organizing the double-helical viral RNP structure. This identification stemmed from our observation that NP lacking the oligomerization tail loop forms monomers and dimers. We determined the crystal structure of this NP dimer, which reveals this new NP-NP interface. Mutation of residues that disrupt this dimer interface does not affect oligomerization of NPs containing the tail loop but does inactivate the ability of NPs containing the tail loop to support viral RNA synthesis in minigenome assays.
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    Structure determination of a human virus by the combination of cryo-EM and X-ray crystallography
    (Springer, 2016) Liu, Zheng; Guu, Tom S.Y.; Cao, Jianhao; Li, Yinyin; Cheng, Lingpeng; Tao, Yizhi Jane; Zhang, Jingqiang
    Virus 3D atomic structures provide insight into our understanding of viral life cycles and the development of antiviral drugs. X-ray crystallography and cryo-EM have been used to determine the atomic structure of viruses. However, limited availability of biological samples, biosafety issues due to virus infection, and sometimes inherent characteristics of viruses, pose difficulties on combining both methods in determining viral structures. These have made solving the high resolution structure of some medically important viruses very challenging. Here, we describe our recently employed protocols for determining the high-resolution structure of the virus-like particle of hepatitis E virus (HEV), a pathogen of viral hepatitis in human. These protocols include utilizing recombinant baculovirus system to generate sufficient amount of virus particles, single-particle cryo-EM to get an intermediate resolution structure as a phasing model, and X-ray crystallography for final atomic structure determination. Our protocols have solved the hepatitis E virus structure to the resolution of 3.5 Å. The combined methodology is generally applicable to other human infectious viruses.