The Effects Of Disulfide Bond On The Interaction Of PCV2 Capsid Protein(Cap),Assembly Of PCV2 VLPs And Viral Replication In Vitro

Porcine circovirus type 2(PCV2)is a single-stranded circular DNA Virus which causes PCV2-associated diseases(PCVADs)and have great threats to pig health,thereby lead to huge economic losses to global pig industry.The capsid protein(Cap)is the only structural protein of PCV2 and 60 PCV2 Caps can self-assemble into virus-like particles(VLPs).The VLPs are capable of inducing a strong immunoprotection against PCV2 infection for animals.Therefore,it has been used as the key antigen for many commercial vaccines against PCV2 infections.Analyzing the interactions between subunits of PCV2 Cap,the assembly mechanism of VLPs and the replication characteristics of PCV2 in vitro will greatly prompt the development of PCV2 vaccine and the understanding of PCV2 molecular pathogenesis.The main goals of this study are as follows:1)To study the effects of a disulfide bond on the interaction between subunits of PCV2 Cap,the assembly of VLPs,and subsequent cellular entry,two recombinant PCV2 Cap(hCap-WT,wild type and hCap-Mt,mutant: C108G)with histidine tag(His-tag)were successfully expressed in E.coli and purified by nickel affinity chromatography,respectively,it was observed that the disulfide bond was formed between subunits of wild type Cap through conserved cysteine,and further formed the dimer of Cap.Both Caps are able to self-assemble into VLPs in oxidative or reductive buffers.Furthermore,molecular sieve validation showed that there was no significant difference of the assembly efficiencies of both the Caps assembling into VLPs and indirect immunofluorescence assays(IFA)also proved that both VLPs assembled by the two Cap have a similar capacity to enter PK-15 cells.Therefore,the disulfide bond involve in the formation of Caps dimer,but it does not have negative effects on assembly and cellular entry of the VLPs.2)We have established a protein co-expression system of E.coli to study interaction between PCV2 Cap subunits.nhCap-WT(without any tag)were co-expressed with hCap-WT in E.coli and importantly,the nhCap-WT are able to co-purified with the hCap-WT by nickel affinity chromatography,indicating there are other intermolecular interactions between the PCV2 Cap subunits.In addition,both VLPs assembled by the two Caps in either oxidative or reductive buffers are also ableto enter PK-15 cells.Altogether,we proved again that the disulfide bond did not affect the assembly of PCV2 VLPs,and further revealed the existence of non-disulfide bond interactions between subunits of Cap,which contributed to the assembly of the VLPs in vitro.The method of co-assembly of PCV2 Cap subunits favor to understand the assembly mechanism of VLPs and to develop a chimeric vaccine based on the PCV2 VLPs.3)To explore the effect of the disulfide bond on PCV2 replication in PK15 cell culture.We constructed a mutated infectious PCV2 DNA clone in which the conserved cysteine residue was replace with a glycine residue(C108G)in the PCV2 Cap.PCV2 was successfully rescued by this infectious DNA clone.Furthermore,the viral DNA copy number is comparable to that of the wild type in PK15 cell culture,which suggested that this conserved cysteine residue in the PCV2 Cap does not play a critical role in the life cycle(i.e.infection and replication)of PCV2 in vitro.