Study On Structures Of Two Icosahedral MCMV, CarMV Capsids And MCMV Replication Site By Three-dimensional Electron Microscopy

Maize chlorotic mottle virus (MCMV) and Carnation mottle virus (CarMV) are representative members of the genera Machlomovirus and Carmovirus respectively in the family Tombusviridaes. In this study, the three-dimensional structures of MCMV and CarMV particles were reconstructed by Cryo-EM single particle analysis, and the three-dimensional structure of MCMV replication site and the cytopathology were studied by combining traditional electron microscopy as wellThe three-dimensional structure of MCMV with 3.6 A was obtained by Cryo-EM single particle analysis. The spatial location of subunit C-terminal and obvious opening at 2-fold axis demonstrated the compactness of the asymmetric unit, which reveals the contribution of the asymmetric unit to the stability of the intact virion. Through comparing the surface features with other viruses, the exposed residues on the surface of MCMV were analyzed and speculated to be related to MCMV transmission by insect vectors, which would provide essential structure evidences for further study on MCMV transmission. Meantime, based on the comparison among the 12 viruses in the Tombusviridae family and Sobemovirus genus, MCMV was found to have close phylogenetic relationship with the Panicum mosaic virus (PMV) in the genus Panicovirus of the Tombusviridae family. The three-dimensional structures of the CarMV under four conditions (Ca-pH7; EDTA-pH7, Ca-pH5 and EDTA-pH5) were obtained by Cryo-EM single particle analysis as well, the differences among which indicated that the role of the calcium ions in the CarMV was not for structure preservation. Based on the electrostatic potential on the inner surface of the CarMV capsid, the potential mechanism of the CarMV genome uncoating was suggested that the center of the asymmetric unit might serve as the potential passage of the genomic RNA. The insight into the CarMV genome uncoating mechanism would be of great importance for the virus control caused by the CarMV.The subcellular structure of the maize leaves infected with MCMV was analyzed by TEM observation, which showed that the virus particles were accumulated largely in the cytoplasm, and peroxisome and mitochondrion were shown to have obvious pathology. The peroxisomes were proliferated and gathered, with small vesicles inside the membrane, which then developed into multi-vesicle bodies during late stage of viral infection. The replication site of the MCMV was located in small vesicles at the periphery of multi-vesicle bodies through the location of viral replication intermediate dsRNA and replicase using immuno-gold labeling method. The three-dimensional reconstruction of the small vesicles at the periphery suggests that these vesicles are formed due to the invagination of the peroxisome membrane. At the same time, the vesicles with bigger dimension inside the multi-vesicular bodies were found with a large number of amorphous materials. The immuno-gold labeling result further demonstrated that viral capsid protein was located in the bigger vesicles not only in the cytoplasm. The electron tomography reconstruction of the linkage between two neighboring multi-vesicle bodies showed that the bigger vesicles inside originated from local invagination of the membrane with small vesicles from multi-vesicle bodies. Due to the finding of crystal-like viral particles in close proximity to the multi-vesicle bodies, it was speculated that these bigger vesicles were potential compartment of MCMV particle assembly. The orientation of viral particles inside the crystal-like structure suggested that the interactions between two neighboring particles happened between the loops around the five-fold axis, which were speculated to play a critical role in viral assembly and particle release into cytoplasm. The major pathology of the mitochondrion was vesiculation, with accumulating filamentous materials aggravated in the inter-membrane space with the progress of viral infection. The structure from high-pressure freezing method suggested that the accumulation of filaments seemed to result in the division of the mitochondrion into small mitochondria, which indicated that MCMV infection might lead to abnormal proliferation of mitochondria. The study on cytopathology and replication mechanism of the MCMV would provide important theoretical basis for viral life cycle and infection mechanism, which would be great significant on the control of the virus disease caused by MCMV...