Investigating Porcine Circovirus Type 2 Uncoating,Genome Nuclear Entry And Replication

Porcine Circovirus type 2(PCV2),is one of the smallest,nonenveloped,single-stranded DNA(ss DNA)viruses.The viral genome was enclosed in a 17 nm icosahedral capsid(T=1).PCV2 has been recognized as the critical agent responsible for Postweaning Multisystemic Wasting Syndrome(PMWS),which resulted in significantly economic losses to the pig industry.So far,the prevention of PCV2 associated diseases is mainly through vaccination,but epidemiological investigation showed that PCV2 are circulating among most of economic farms with highly positive rates and the virus still keep evolution with a high mutation rate.The study on the molecular mechanisms of the viral infection and reproduction will greatly contribute to appreciation of PCV2-associated pathogenesis and prevention of this virus on farms.In this study,we investigated the processes and mechanisms of PCV2 genome release,entry into nuclear and replication in PK-15.1.Investigating the functions of PCV2 NLS on release of viral genome from capsid.It has reported that NLS-A derived from NLS of PCV2 was a CPP and could enhance the membrane permeability.In this study,we firstly found NLS-A contributing to PCV2 infection by inducing the membrane permeability.Second,PCV2 NLS can exposed at the surface of PCV2 VLPs,which depend on the p H in the solutions,although in a stable virion the NLS is located inside the capsid and involved in viral DNA packaging.Therefore,the results revealed that the virus particles may be switched into a metastable state which the NLS can stretch out from the inner of virus particle.According these cues,it was inferred that the NLS may favor viral genome release from the capsid by stretching out from the inner of virus particle,and simultaneously,the externalized NLS disrupts plasma membranes or intracellular membranes to release the viral genome to cytoplasm.2.By detecting the virus genome in PK-15,the influences of endosome/lysosome system on viral genome release was identified.First,we established a smiFISH assay to detect viral gnome in host cells,and the results suggested that inhibition of endosome/lysosome acidification may enhance PCV2 genome release from the capsid.Therefore it indicates weak acid environment favors the release of the viral genome.3.Dual-labeled the PCV2 genomic DNA and its complementary strand to study the processes of PCV2 genome entry into nuclei and replication.First,inhibit the disassembly of nuclear membrane can completely block the PCV2 genome into nuclei,indicating that PCV2 genome enter nuclei by utilizing disassembly of nuclear membrane.And then,we showed three stages of viral genome replication.At early stage,the viral genome formed small punctate replication sitesthen formed ring replication sites.Finally the viral genome filled the whole nucleus.In addition,co-localization of PCV2 replication sites and capsid protein(Cap)has been verified.4.Study the dynamic properties of PCV2 Rep/Rep’ protein in live cells.PCV2 Rep/Rep’ protein can form clusters in PK-15 and He La cells by fusion.Subsequently,the mobility of the clusters was investigated by FRAP and the results demonstrated the clusters were dynamic.Previous study suggested that both residues of Leu35 and lle37 are important for PCV2 replication.Our results suggested that double mutations of Leu35 and lle37 of Rep/Rep’ proteins reduced the clusters formation,and subsequently led to a detrimental effect on the functions of Rep/Rep’ proteins complexes for PCV2 genomic DNA replication.In conclusion,based on biochemistry experiment,confocal imaging,TEM and FRAP,we investigated the processes including PCV2 uncoating,genome entry into nucleus and replication,and found critical clues responsible for PCV2 infection and reproduction,which will greatly contribute to the design of new drugs and vaccines for prevention of PCV2 infections.