Functional Study Of The Carboxyl Terminus Of Porcine Circovirus Type 2 Capsid Protein (Cap)

PCV2 causes post-weaning multisystemic wasting syndrome and other PCV-associated diseases（PCVADs）,and co-infections with other pathogens.The virus has brought great economic losses to global swine industry,vaccination is a major defense against PCV2 dissemination and its associated diseases.To understand the assembly,cellular internalization,and the replication of PCV2,the main results of this study are as below:1.62 PCV2 isolates were identified from seven farms in southern China from2013 to 2015.The results show that PCV2b was the main genotype in circulation throughout these farms（95%）.Moreover,an emerging mutant（PCV2b-1C）,isolated from PCV2-vaccinated farms,was the predominant strain prevalent on these farms.Furthermore,we discussed evidences that antigenicity and structural variation on surface of the capsid resulted from mutation of the carboxyl terminus loop（Loop CT）of the PCV2b-1C Cap in silico,by means of display and simulation of three-dimensional（3D）structures of the PCV2 capsid.In addition,the PCV2 cap genes of the putative recombinant isolate and its reference strains were further analyzed to determine nucleotide identity,and this new cluster that may represent evolution of the virus through recombination of PCV2b-1A/1B and PCV2b-1C.Finally,the PCV2 strains and the cap gene were obtained for research of Loop CT in this study.2.Unique surface patterns of the icosahedral 5-fold axes of the PCV2 capsid were identified and characterized.A conserved tyrosine phosphorylation motif(²⁰⁶IYD²⁰⁸)in Loop HI that might be recognized by non-receptor tyrosine kinase（s）in vivo was present only in PCV2 isolates.In particular,the concurrent presence of 60pairs of the conserved tyrosine in Loop HI and a canonical P××P motif at the end of the C-terminus on the PCV2 capsid surface could be a potential mechanism for P××P motif binding to and activation of an SH3 domain-containing tyrosine kinase（i.e.Src and Abl）in host cells.And this study will greatly lay a theoretical foundation for reasearch of Loop CT.3.Protein expression vectors were constructed,wild type and mutant Cap proteins were highly expressed in E.coli BL21,and grown at 37℃by inducing 1 mM IPTG for 6 h,and it can be one-step purified from soluble supernatants by Ni²⁺affinity chromatography.4.Removal of Loop CT completely abolished the capacity of the Cap assembly into virus-like particles（VLPs）.Further,replacement of Loop CT with its counterpart from PCV type 1（PCV1）did not affect the capacities of assembly and cell entry of the VLPs.These studies suggested ²²⁵NLKDP²2929 within Loop CT should be necessary for VLPs assembly and cell entry,however two conserved amino acids residues(²²⁷KD²²⁸)in the loop are critical for VLPs assembly.Simultaneous mutations of both of the sites（"AA"or"RE"）led to the failure of PCV2 Cap assembly into VLPs.Furthermore,the lysine(²²⁷K)in the loop plays a crucial role in PCV2 VLPs entry into PK15 cells,and its subsequent mutation to alanine abolishes the capacity of PCV2 VLPs internalization,albeit this mutant is able to self-assemble into VLPs in vitro.Virus rescue experiments also suggest viral titer of this mutant was dramatically decreased in PK15 cell culture,compared to the wild type of PCV2.In conclusion,all the loops on the surface of PCV2 have been further analyzed and predicted by simulation,determining the conserved and divergent amino acid residues within the loops.The mechanisms of PCV2 assembly,cell entry,virus function and pathogenesis will be further investigated through mutation analysis using protein expression of PCV2 Cap,in vitro assembly,electron microscopy,virus rescue and amplification.This work indicated that the Loop CT may participate in the assembly,cellular internalization,and the replication of PCV2.Furthermore,The results will constitute a basis for the research and development of PCV2 vaccine and drug delivery.