Studies On The Nuclear Entry Mechanism Of Porcine Circovirus In Host Cells

Porcine circovirus,the smallest virus known to be autonomously replicable,are major etiological agents of post-weaning multisystemic wasting syndrome(PMWS),porcine dermatitis and nephropathy syndrome(PDNS),porcine proliferative and necrotizing pneumonia(PNP).Previous studies demonstrated the process of binding,internalization,genome replication,and transcriptional regulation,but the mechanisms of intracellular transport and nuclear entry of porcine circovirus into host cell nuclei remain unknown.The successful transport and nuclear entry of viral circular DNA to the nucleus are crucial to proceed a full replication for circovirus.Virus-host interaction is a tug of war between pathogenesis and immunity,followed by either activating the host immune defense system to eliminate virus or manipulating host immune control mechanisms to survive and facilitate virus propagation.Comprehensive knowledge of interactions between host and viral proteins might provide hints for developing novel antiviral strategies.In the study,we demonstrated that viral protein Cap-triggered a-tubulin acetylation improves the stability of microtubules and facilitates PCV2 replication,which is beneficial to the nuclear-targeted transport of PCV2 virions.PCV2 Cap inhibits HDAC6 deacetylase activity by interaction with HDAC6,which antagonizes the antiviral function of HDAC6,and finally promotes circovirus replication.We then employed a coimmunoprecipitation combined with liquid chromatography mass spectrometry approach and confirmed that the intranuclear protein NPM1 interacts with PCV2 and PCV3 Cap.When PCV2 virions transport along the microtubule with intact capsid and reach the MTOC at the peripheral region,the viral capsid recruits and binds to NPM1 at the early stage of infection and induces NPM1 to shuttle between the nucleus and cytoplasm and translocates from the nucleolus to the nucleoplasm/cytoplasm,thus facilitating the circovirus replication.In addition,the NPM1 is essential for the nucleolar localization and binding to PCV3 Cap.The charge property of serine-48 residue within NPM1-OligoD domain is essential for its subcellular localization and physical interaction with PCV3 Cap.1.Circovirus capsid protein facilitates replication by inhibiting histone deacetylase 6 activity.The previous results indicated that circovirus transport proceeds via direct interaction of the cytoplasmic dynein IC1 subunit with the viral capsid protein at the early stage of PCV2 infection.In order to analyze whether other host proteins can participate in the regulation of the intracellular transport process of PCV2 virions by affecting the function of microtubules and whether PCV2 can regulate the acetylation modification of tubulin to improve the stability of microtubules,we found that viral Cap protein-triggered ?-tubulin acetylation facilitates PCV2 replication.The selective inhibitor of histone deacetylase 6 Tubacin would increase the acetylation modification level of ?-tubulin can promotePCV2 replication.In addition,we proved that PCV2 Cap colocalized and interacted directly with HDAC6,and the N-terminal region(42-100aa)of Cap protein was involved in the interaction with full-length HDAC6.PCV2 Cap can suppress the deacetylase activity of HDAC6 in vitro.HDAC6 inhibits PCV2 replication by inducing ?-tubulin undergoing deacetylation modification and thus exerts an antiviral effect.In turn,PCV2 Cap can also inhibit the deacetylase activity of HDAC6 to antagonize the antiviral function of HDAC6.In summary,these results showed that PCV2 Cap can inhibit the deacetylase activity of HDAC6 by interaction with HDAC6,sustaining the level of acetylation modification of ?-tubulin and ultimately promote circovirus replication.2.Protein interactions network of porcine circovirus type 2 capsid with host proteins.To gain a more detailed knowledge of the interactions with porcine circovirus type 2 capsid protein,we employed a coimmunoprecipitation combined with liquid chromatography mass spectrometry(LC-MS)approach and 222 putative PCV2 Cap-interacting host proteins were identified in the infected porcine kidney(PK-15)cells.Further,a protein-protein interactions(PPIs)network was plotted,and the PCV2 Cap-interacting host proteins were potentially involved in protein binding,DNA transcription,metabolism and innate immune response based on the gene ontology annotation and Kyoto Encyclopedia of Genes and Genomes database enrichment.Verification in vitro assay demonstrated that four cellular proteins,namely heterogeneous nuclear ribonucleoprotein C,nucleophosmin-1,DEAD-box RNA helicase 21,and intermediate chain 1 interacted directly with PCV2 Cap.The PCV2 Cap-interacting cellular proteins may assemble novel replication-transcription complex and play vital roles in the PCV2 replication and pathogenesis.3.Nucleolar protein NPM1 is essential for circovirus replication by binding to viral capsid.Entry of circovirus into the host cell nucleus is essential for viral replication during the early stage of infection.However,the mechanisms by which nucleolar shuttle proteins are used during viral replication is still not well understood.In the study,we report a previously unidentified nucleolar localization signal in circovirus capsid protein(Cap),and that circovirus hijacks the nucleolar phosphoprotein nucleophosmin-1(NPM1)to facilitate its replication.Colocalization analysis showed that NPM1 translocates from the nucleolus to the nucleoplasm and cytoplasm during viral infection.Coimmunoprecipitation and glutathione S-transferase pull-down assays showed that Cap interacts directly with NPM1.Binding domain mapping showed that the arginine-rich N-terminal motif 1MTYPRRRYRRRRHRPRSHLG20 of Cap,and serine-48 residue of the N-terminal oligomerization domain of NPM1,are essential for the interaction.Virus rescue experiments showed that all arginine to alanine mutation in the N-terminal arginine-rich motif of Cap resulted in diminished viral replication.Knockdown of NPM1 and mutation of serine-48 residue in NPM1 to glutamic acid also decreased viral replication.In addition,binding assays showed that the arginine-rich motif of Cap is a nucleolar localization signal.Taken together,our findings demonstrate that circovirus protein Cap is a nucleolus-located,and regulates viral replication by directly binding to NPM1.4.The charge property of a conserved serine-48 residue in nucleolar protein NPM1 is essential for its subcellular localization and physical interaction with porcine circovirus type 3 capsid protein.The role of nucleolar shuttle proteins during PCV3 capsid protein import is still not well understood.Here,we report a previously unidentified nucleolar localization signal in PCV3 Cap,which hijacks the nucleolar phosphoprotein nucleophosmin-1(NPM1)to facilitate nucleolar localization of PCV3 Cap.Colocalization analysis showed that NPM1 colocalizes with PCV3 Cap into nucleolus during transfection.Coimmunoprecipitation and glutathione S-transferase pull-down assays showed that PCV3 Cap interacts with NPM1.Binding domain mapping showed that the nuclear localization signals of Cap of circoviruses from terrestrial,aquatic and avian species,including pigs,canaries,canines,minks,dragonflies,pigeons,ducks,bats,geese,and parrots,and serine-48 residue of the N-terminal oligomerization domain of NPM1,are essential for the interaction.Knockdown of NPM1 impaired the nucleolar localization PCV3 Cap.In addition,binding assays showed that the NLS of PCV3 Cap is also a nucleolar localization signal and the charge property of serine-48 residue in NPM1 is indispensable for binding to PCV3 Cap.Taken together,our findings demonstrate that PCV3 Cap is located into the nucleolus,and the charge property of serine-48 residue of NPM1 is essential for its subcellular localization and physical interaction with porcine circovirus type 3 capsid protein.The study is aimed at clarifying the transport and nuclear entry strategy and "nuclear shuttle" mechanisms utilized by HDAC6 and NPM1 proteins for PCV2 replication,and to further analyze the molecular mechanism of viral replication and identify potential antiviral drug targets.