| Attachment of the virus on the surface of the host cell is the initial step of an effective infection.In order to explain the mechanism by which virus-infected cells form infection,it has been reported that enveloped viruses complete invasion by altering the conformation of viral proteins,and there is no analysis of a mechanism similar to that of non-encapsulated viruses.Heparan sulfate(HS),which is widely distributed on the cell surface,is often used as an attachment receptor for virus adsorption and infection.Porcine circovirus type 2 uses its capsid protein Capsid to directly interact with Heparan sulfate.To achieve the adsorption of target cells,the crystal structure of the PCV2 Capsid protein that has been resolved shows that the HS binding site in the Capsid protein is encapsulated inside the virus particles.The specific mechanism of how to achieve interaction is still unclear.Single molecule energy resonance transfer technology(smFRET)is a new technology for real-time detection of conformational changes of biological macromolecules in recent years.This study uses smFRET technology to present the conformational distribution of PCV2 Capsid protein to explore the mechanism of the host cell surface receptor-Capsid interaction during the PCV2 infection,facilitating further understanding of the circovirus infection process.1.Establishment of dual fluorescent labeling Capsid method for smFRETBased on the crystal structure of protein of PCV2 Capsid and VLP,3XA and 3XC mutants were designed for smFRET experiments.Recombinant Capsid mutant proteins were obtained by prokaryotic expression and purification in vitro.The results of circular dichroism showed that 3XA and 3XC retained protein secondary structures similar with Wild-type,as well as self-assembly activity to form VLPs in vitro,demonstrate that modification of Capsid at the chosen site does not affect the high-level structure of the protein.The E.coli ACPs enzyme was prokaryotic expressed in vitro,and its enzymatic activity was verified to catalyze the formation of a chemical linkage between CoA and specific serine of Al-tag,combined with the covalent formation between Cystein sulfhydryl and maleimide,an in vitro one-step Capsid dual-fluorescent molecular labeling method was established.The labeling rate of donor and acceptor fluorescent reached to?40%and 90%by optimizing the parameters of the labeling reaction system,demonstrating the dual fluorescent labeling method for Capsd is feasible.2.The smFRET imaging and conformational distribution of CapsidUsing high concentration of salt ions to inhibit the oligomerization of PCV2 Capsid in vitro,a method for pretreatment of Capsid 1500 mM NaCl to depolymerize was established to achieve smFRET imaging of single molecule level Capsid protein.The results of smFRET experiments show that the conformation of PCV2 Capsid protein is not static.It can be seen that there are three conformations in the mainstream.The states can be defined as low FRET value,intermediate FRET value and high FRET value state based on the E value of FRET.In the natural state,the Capsid conformation distribution is dominated by the low E value state.3.Regulation of capsid conformational feature by receptorsHS is used as an attachment receptor for PCV2 during infection to host cells,it could competitively bind PCV2 virus or Capsid protein in vitro to inhibit the attachment of cells by viruses or proteins when its concentration reaches 2500 ?g/ml;The N-terminal sulfation modification is critical for its binding to Capsid,and the N-terminal sulfated DeN-sulfated heparin does not competitively inhibit the viral or protein-adhering cells.HS can induce the change of Capsid conformation distribution.Capsid transitions from the low FRET value state to the high value FRET state,and finally shows the dynamic equilibrium distribution with the high value FRET state as the dominant state.While the DeN-sulfated heparin which is not fully sulfated could also promote the conformational change of Capsid.The protein is converted from the low FRET value state to the middle FRET value state,and cannot be further converted to the high FRET value state.Finally,the intermediate FRET value state was mainstream,causing the virus to not attach to the surface of the host cell.4.Regulation of capsid conformational feature by antibodies and drugsNeutralizing antibodies 3F6 and 6H9 inhibited the conformational change of Capsid from a low FRET state to other conformations,thereby constrainting it to a low FRET state distribution,and inhibiting the cell surface attachment of virions.,while non-neutralizing antibodies have no similar regulatory effects.In addition,the small molecule drug EGCG,which has anti-infective activity against PCV2,although it is verified can compete with HS for binding to Capsid to inhibit the virus adsorption process,it does not achieve the antiviral effect by regulating the conformational feature of Capsid.Finally,we propose the following model for the relationship between the change of the conformation distribution of Capsid and the cell surface adsorption of PCV2 virions:the conformation of the Capsid protein of PCV2 is naturally multi-conformed and the dynamic balance of mutual transformation,when it is not in contact with the host cell surface,the receptor binding site is encapsulated inside the virus particle;when the virion contacts the cell surface,the electrostatic interaction between the internal receptor binding site and the cell surface receptor molecule causes the binding site to move outward to be exposed to the surface of the virus particle,it facilitates the interaction with the cell surface receptor heparan sulfate to achieve the adsorption of virions;during this process,the binding of the neutralizing antibody targeting Capsid can inhibit the conformational transition of Capsid,thereby inhibiting the receptor binding site exposure.On the surface of the virus particles,the blocking antigen establishes interaction with the cell surface receptor molecules,and finally inhibits cell surface adhesion of the virions. |
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