Structural Analysis Of Two Icosahedral Animal Viruses By Cryo-Electron Microscopy

Background and ObjectiveSingle particle cryo-electron microscopy(cryo-EM)has become the most prominent tool in structure biology to determine the structure of bio-macromolecules due to recent advancement in hardware,especially the advent of direct detective detector,and software algorithms.Structure analysis of icosahedral viruses that have high symmetry and large molecular mass is the prominent subject undergoing study by Cryo-EM.Infectious bursal disease virus(IBDV)is an icosahedral double-stranded RNA virus,which causes huge economic losses to the global poultry industry.The present structural study mainly focuses on IBDV capsid.Until now,it is unclear how the dsRNA segments of IBDV are organized inside its capsid.The purpose of this study is to investigate the structure of IBDV and package information about the dsRNA segments and RNA dependent RNA polymerase inside the capsid.Porcine circovirus type 2(PCV2)is the pathogen of porcine circovirus disease and porcine circovirus-associated diseases(PCVD).In essence,the diameter of PCV2 virion is about 15 nm,and its genome consists of circular single-stranded DNA(ssDNA).Although in previous studies,crystal structures of the virus-like particles(VLP)without viral genome have been successfully determined,these structures don't fully represent the native structure of PCV2.So,that partial structure put many obstacles to investigate the interaction between capsid proteins and genome.To analyze the interaction between capsid protein and genome,we study the intact structure of PCV2 by cryo-EM,which provides the structural information for understanding the pathogenic mechanism of PCV2.Methods(1)DF-1 cells were infected with IBDV to obtain IBDV particles.(2)The integrity of purified IBDV particles was detected by indirect immunofluorescence assay.(3)The three-dimensional structure of IBDV was determined by cryo-EM,and compared with that of other dsRNA viruses.(4)The three-dimensional structure of PCV2 was determined by cryo-EM.Results(1)IBDV particles with high purity and homogeneity were obtained.(2)The results of indirect immunofluorescence assay showed that DF-1 cells infected with purified IBDV reacted with anti-VP5 monoclonal antibody and produced green fluorescence.(3)The structure of IBDV was determined by cryo-EM at 6.6 A resolution.(4)The structure of PCV2 was determined by cryo-EM at 2.97 A resolution.Conclusion(1)IBDV T=13 single shell is composed of 260 VP2 trimers,and VP2 trimers lie in five different local environments.At the same time,we did not observe characteristic density of dsRNA and RNA dependent RNA polymerase inside IBDV capsid.Statistical analysis showed that the internal genome packaging density of IBDV was significantly lower than that of other dsRNA viruses.These facts suggest that IBDV might adapt different mechanism to organize its genome(2)PCV2 is a T=1 nonenveloped icosahedral virus,and its capsid is composed of capsid protein.The intact structure of PCV2 showed that the interactions between capsid proteins and genome affected the local conformations of capsid proteins.Amino acids near the 5-fold axes of the capsid interact with the viral ssDNA,suggesting that the release of viral genome is associated with the pores at the 5-fold axes.