Effect Of Loop HI Key Amino Acid Residues On Virus Assembly And In Vitro Amplification

Porcine circovirus type 2（PCV2）is recognized as the pivotal pathogen responsible for postweaning multisystemic wasting syndrome（PMWS）and multiple clinical symptoms and causes huge economic losses globally in swine industry every year.PCV2 consists of 60 of viral capsid protein（Cap）,which are capable of self-assembling into an icosahedral capsid with a diameter of～17-20 nm.The viral genome is packaged into the capsid.PCV2 genome is a single-stranded and circular DNA containing～1.7 kilobases,which is one of the smallest animal viruses.PCV2 is also non-enveloped virus,and the Cap is the sole structural protein of this virus,which plays crucial roles in virus assembly,cell entry,viral replication etc.Previous results have demonstrated that three critical loops（BC,HI and DE）form the surface of the 5-axes of the PCV2 capsid and are involved in virus-host cell interactions and immunorecognitions.In order to study the functions of Loop HI of the PCV2 Cap on virus assembly,cell entry and proliferation in vitro,we designed two Cap mutants（Cap-HI-M1 and Cap-HI-M2）.In Cap-HI-M1,the residue of tyrosine(²⁰⁷Y)was replaced with phenylalanine（F）;while the four resides(²⁰⁵SIYD²⁰⁸)within Loop HI of the PCV2 Cap is substituted by the counterpart(²⁰⁵AATA²⁰⁸)of the PCV1 Cap.The detailed results are listed below:（1）The effect of the key residue mutations within Loop HI of the PCV2 Cap on virus assembly.Both mutated Cap proteins were expressed and soluble in bacteria.Furthermore,they are capable of self-assembling into virus-like particles（VLPs）.These results suggest that residues of Loop HI may not be involved in the virus assembly.（2）The effect of the key residue mutations within Loop HI of the PCV2 Cap on virus proliferation in vitro.We also prepared two PCV2 infectious DNA clones（p SP72-PCV2-IF-HI-M1 and p SP72-PCV2-IF-HI-M2）containing both mutations above,respectively,for virus rescue in vitro.Real-time PCR results showed that both the mutated viruses have been successfully rescued from PK15 cell culture.After consecutive passages of the cell cultures,the genomic copies numbers of the PCV2-M1 mutant in both cell culture media and PK15 cells are similar to the numbers of the control（wild type of PCV2）.However,the genomic copies numbers of the PCV2-M2 mutant in either cell culture media or PK15cells are significantly different,compared to the wild type or the PCV2-M1 mutant,and the genomic copies numbers dramatically decreased.In addition,immunofluorescence assays（IFAs）also confirmed the results above.Altogether,residues within Loop HI of the PCV2 Cap may have a minimal effect on the virus/VLPs assembly,and the residues of ²⁰⁷Y does not affect PCV2 proliferation in vitro.However,the four resides(²⁰⁵SIYD²⁰⁸)within Loop HI of the PCV2 Cap play a critical role in the virus proliferation in vitro,although PCV1 and PCV2 share more than 80%homology on genomic sequences,which further indicates both Caps employ distinct mechanisms to aid the viruses for infections and replications,while the elaborate mechanism remains to be further investigated.