Molecular Mechanism Of Host Factor PCNA In Influenza Virus Replication

Influenza virus is one of the pathogens of zoonosis,and the continuous outbreak of influenza has caused immeasurable loss to human health and breeding industry.The replication process of influenza virus in the host is a process of mutual game.The replication of the virus requires the assistance of the host proteins,and the host has developed a variety of mechanisms to combat the invasion of the virus.Viral ribonucleoprotein complex(vRNP)is a basic functional unit of transcription and replication.And many host proteins are involved in these processes.Therefore,studying the interaction between host proteins and influenza viruses can further elucidate influenza pathogenic mechanisms and provide a theoretical basis for the discovery of host derived anti-influenza drug targets.This study confirmed the interaction of the host proteins proliferating cell nuclear antigen(PCNA)with the polymerase subunit PB2 of the influenza virus PR8 by Co-IP experiments.Further studies showed that the interaction between the host protein PCNA and PB2 affects the nuclear import process of PB2,which leads to inhibition of the vRNP assembly process of the viral polymerase complex and ultimately inhibits the replication process of the virus.The main research contents are as follows:1 Host protein PCNA inhibits viral replication of influenza viruses by interacting with PB2In this study,Co-IP experiments were performed to detect the relationships between exogenous PCNA and PB2 in 293 T cells,and it was also further confirmed in the case of infection.After overexpressing or silencing PCNA in A549 cells,cells were infected with PR8(MOI=0.01),then cells supernatants and protein samples were harvested at 12 hpi,24hpi and 36 hpi.The results showed that the titer of PR8 virus was decreased by 1to 1.5 virus titers in PCNA overexpressed cells compared to control.In contrast,the replication of PR8 virus was significantly increased when PCNA koncked down in 293 T cells.These data indicated that PCNA is one of the limiting factors of influenza virus.2 Effect of PCNA on the assembly of PR8 virus polymerase complexResults of mini genome assay demonstrated that the polymerase activity of PR8 virus was decreased significantly along with the increasing amounts of exogenous PCNA and increased when PCNA silenced in 293 T cells.The polymerase activity of influenza virus mainly depends on the correct assembly of vRNP.PCNA interacts with the subunit PB2 of the vRNP complex which affects the assembly of vRNPs.This data hints that PCNA may affect the assembly of polymerase complexes by binding to PB2 thereby delaying viral replication.In this study,we used a vRNP reconstitution system to construct vRNPs whose NP was HA tagged in 293 T cells.The immunoprecipitate,PA as a indication reflected the level of viral RNP assembly.When overexpressing the polymerase system and host protein PCNA simultaneously in 293 T cells,the amount of PA was reduced in a dose-dependent manner,indicating that PCNA can inhibit the assembly process of viral polymerase complex.It is known that PA and PB1 form dimers then entry the nucleus,and NP and PB2 are separately by transporter family proteins into nucleus then formed complex.The virus-associated RNA is assembled into vRNP and c RNP complexes to complete a new round of transcription and replication.Among them,PCNA interacts with PB2 to inhibit the assembly of polymerase,which may interfere with the nuclear import process of PB2.Then we found PCNA competitively disturbed the interaction between transporter family proteins(importin α1/5/7)and PB2.Indirect immunofluorescence experiments showed that PCNA can increase the cytoplasmic retention of PB2 when coexpressied PCNA and PB2 in He La cells.This result also supported the competitive disturbing of PCNA for impotin α family protein and PB2.3 The area where PB2 interact with PCNAThe nuclear import process of PB2 is mediated by the importin αfamily proteins which recognize and bind to the nuclear localization signal of the cargo protein PB2.Then PB2,importin α family and importin β forms a trimer and then the trimer enters the nucleus through the nuclear pore complex.The nuclear localization signal mutant and deletion of PB2 were separately constructed,and the nuclear distribution of PB2 mutant and deletion was verified.Through Co-IP experiments,we found that the interaction between PCNA and those PB2 nuclear localization signal mutants were significantly attenuated,and the interaction between 738 Q and PCNA almost completely disappeared.4 Viral infection down-regulates PCNA expression but does not affect PCNA nuclear distributionA549 cells were inoculated with different MOI of PR8 influenza virus.Protein samples were collected at hours post infection and it was found that PR8 influenza virus down-regulate the expression of endogenous PCNA in a dose-dependent manner.Otherwise,the expression of endogenous PCNA was detected at 24 h after transfecting the Flag-tagged PB2 plasmids in 293 T cells.The expression of PCNA of was also decreased in accordance with the result under virus infecting.As the amounts of plasmid increased,the decrease of PCNA is more pronounced.These suggested that the influenza virus PR8 down-regulates the expression level of the host protein PCNA may mediated by the viral protein PB2.We also found that PR8 virus does not affect the nuclear distribution of PNCA after infection with A549 cells.Whether infected or not infected with PR8 virus,PCNA is mainly present in the nucleus,and some of it exists in the cytoplasm.This study preliminarily elucidated the molecular mechanisms that host protein PCNA inhibits influenza virus replication,and we found that the interaction between PCNA and PB2 affects importin α family proteins recognizing PB2,thus inhibits the entry process of PB2.Thereby the polymerase complex of influenza virus cannot regularly formed.Eventually the replication of the influenza virus was inhibited.