Three Dimensional Reconstruction Of CarMV, IFV Capsids And Cylindrical Inclusion Of SrMV Determined By Electron Microscopy

Viruses not only do harm to human health, but also infect crops and breeding animals,causing severe economic losing. Structure studies of viruses are important subjects instructural biology and cell pathology. Using traditional experimental methods of electronmicroscopy (EM) combined with structural biological methods to study three dimensionalstructure of virus protein polymers can help us known how these protein polymers exertbiological function, and how viruses complete their life cycle. We report the structuralstudies of virus protein polymers by using cryo-electron microscopy and three dimensionalreconstruction to study the capsids structure of Carnation mottle virus (CarMV) andInfectious flacherie virus (IFV); traditional ultra thin section and electron tomography wereused to study the cylindrical inclusion (CI) structure of Sorghum mosaic virus (SrMV) andthe relationship between CI and plasmodesma (PD).Using multiple methods such as I-ELISA?, nucleic acid sequencing, SDS-PAGE andimmunolabelling in EM, we identified CarMV Yunnan isolation. CarMV particles werefound to be districbuted in cytoplasm and vacuole in EM. CarMV have no cell inclusion incytoplasm and no obvious cytopathological change can be found. The chloroplast andmitochondria of host cell badly swell after CarMV infection. The capsid proteins of CarMVdistributing in chloroplast. By comparing the purification effect of three subtle purificationmethods such as PEG deposition, CsCl density gradient centrifugation and centrifugal filter,we find that CsCl density gradient centrifugation can purified virions suitable for cryoelectron microscopy study. Using cryo-electron microscopy combined with single particlereconstruction method, we obtained the resolution of 18(?) for the capsid structure of CarMVin pH7.0 solution.. After eotest calculation and checking the convergence of FSC curve, weconfirm that the reconstruction is reliable. The diameter of CarMV capsid is 354(?). Thereare shell perforative chanels near 3-fold axis. The arrangement of subunits is incompact. Bycomparing this structural result with the reported crystal structure, we find differences on Tnumber, number of spikes in the surface of the shell and numbers of capsid shell, The cryoEM result of CarMV in solution show T=1, there are 60 spikes in the surface and there aretwo layers of shell. The structure of crystalloid CarMV show T=3, there are 90 spikes in thesurface and there is only one layer of shell. The crystal structures of Tombusviridae andSobemovirus were cpmpared using program 'PROSITE' combined with structural analysis,we find that the 'plant virus icosahedral capsid protein S region signature' motif hascommon chracteristics: the array of conservative sequences is not successive but distant; thedistances between key sites are strict conservative; the amino acids within key sites are notstrict conservative.We purified IFV particles. Using cryo-electron microscopy combined with single particlereconstruction method, the capsid structure of IFV capsid was studied and the resolution of17(?) was obtained. After eotest calculation and checking the convergence of FSC curve, weconfirm that the reconstruction is reliable. The diameter of IFV capsid is 302.4(?), thethickness of shell is 15(?). Three dimensional density map show the subunits of IFV followthe P=3 icosahedral symmetry folding pattern. The capsid shell surface of IFV is smoothwithout spikes or protrudes. By comparing the IFV structures with insect picorna (-like) virus CrPV and picornavirus HRV14, Mengovirus, poliovirus, FMDV, we find that the IFVresembles to CrPV in lacking of 'Rossmann canyon', indicating that the mechanism of IFVescaping from host immunity system tracing is different from the mechanism discovered inHRV14 reported as 'Rossmann canyon'. Using program 'phyre', we predict the polypeptidefolding pattern of IFV-VP2 and IFV-VP3 subunit. The polypeptide topology of these twoIFV subunits and the their location at the capsid surface resemble to CrPV's.The CI structure of SrMV and the relationship between CI and plasmodesma (PD) inSrMV infected maize cell were studied by using traditional ultra thin section and electrontomography. The following results were obtained: many CIs distribute in cytoplasm, thebase part of CI conjunct with ER or cell membrane; linear virus particles disposedlengthwise on the plates of CI; the CI in early infecting stage connect with cell wallvertically, the base of CI connect with PD; there are multiple passageways of virus betweenCI and PD, not only through the central channel of CI; the diameter of PD is enlarged; theCI bodies and the connection of CI and PD are also exist in vascular bundle cells. Threedimensional model of CI obtained by ET reconstruction method also confirmed therelationship between CI and PD. After segmentation and rending to the model, thebiological function of CI were predicted: (a) The cylindrical inclusion of potyviruses playsan important role in virus transportation between cells. CI can help virus lengthwise locatingin its plate and then pass through PD. (b) The central channel of CI is not composed ofindependent configurable components. One single CI can connect with multiple PD tocompose multiple channels to transport viruses. Once the virus transportation finished, CIdisengage cell wall and dissociate in cytoplasm. (c) The virus transportation in companioncell and sieve tube cell is depend on CI. Long distance transportation of virus is by thepattern of intact virus particles.