Molecular Mechanism Of Anas Heterogeneous Nuclear Ribonucleoprotein K Interacts With Duck Hepatitis A Virus To Regulate Viral Replication

Duck hepatitis A virus type 1（DHAV-1）is the main pathogen causing duck viral hepatitis,mainly causing hemorrhagic liver lesions and extremely high mortality in young ducklings.DHAV-1 belongs to the Avihepatovirus genus of the Picornaviridae family.Picornaviruses need many host proteins to regulate viral proliferation.This study sheds light on a molecular mechanism that the cellular heterogeneous nuclear ribonucleoprotein K（hnRNPK）protein regulates DHAV-1 replication.The main results are as follows.1.Effect of DHAV-1 infection on hnRNPKWB experiment detected the effect of DHAV-1 on the expression of hnRNPK protein,and the results showed that the expression level of hnRNPK protein in duck embryo fibroblast（DEF）cells significantly increased after infection with DHAV-1.The data of indirect immunofluorescence assay（IFA）and nuclear and cytoplasmic fractionation assay demonstrated that during DHAV-1 infection,hnRNPK translocated from the nucleus to the cytoplasm,where it co-localized with DHAV-1 RNA.2.Effect of hnRNPK on DHAV-1 replicationThe life cycle of picornaviruses is completed in the cytoplasm,and it is speculated that hnRNPK nucleates and participates in the life cycle of DHAV-1.Western Blotting（WB）and RT-qPCR detected the expression of viral protein VP3 and copy numbers after hnRNPK was overexpressed or knockdowned in DEF cells,respectively,then found that overexpression hnRNPK protein significantly promoted the expression of DHAV-1 VP3 protein and viral RNA synthesis（P<0.001）.Knockdown hnRNPK inhibited the expression of DHAV-1 VP3 protein and replication of viral genomic RNA（P<0.0001）.These results indicate that hnRNPK is a positive regulator of DHAV-1.3.hnRNPK interacts with DHAV-1 RNADHAV-1 infected DEF cells overexpressed hnRNPK,and 24 h later samples were collected for RNA immunoprecipitation（RIP）detection.The results showed that the labeled antibody of hnRNPK-His could pulldown DHAV-1 genomic RNA,indicating that hnRNPK interacts with DHAV-1genomic RNA.The DHAV-1 IRES RNA or 3’UTR RNA were co-incubated with hnRNPK protein,and then EMSA experiment showed that hnRNPK protein could combine with DHAV-1 IRES RNA or 3’UTR RNA to form RNA protein complex.The RNA pull-down further detected the binding of DHAV-1 IRES RNA or 3’UTR RNA with hnRNPK protein,and the results showed that both DHAV-1 IRES RNA and 3’UTR RNA could pulldown hnRNPK protein.These results demonstrate that hnRNPK directly interacts with untranslated regions of DHAV-1.4.Mechanism of DHAV-1 non-structural proteins 3CD and 3D affecting hnRNPKScreening viral proteins that promote the expression of hnRNPK protein by WB experiment.To transfect the eukaryotic expression plasmid of DHAV-1 structural and non-structural proteins into DEF cells,and found that structural protein VP0 and non-structural proteins 2BC,2B,3CD and 3D were responsible for upregulating hnRNPK.Considering the important role of 3CD and 3D proteins in virus replication,this study selected3CD and 3D proteins for subsequent experiments.The eukaryotic expression plasmid pCAGGS-3D-HA,pCAGGS-3CD-HA and pCAGGS-hnRNPK-His were co-transfected into HEK293T cells respectively.After 24 hours,samples were collected for co-immunoprecipitation（Co-IP）and IFA detection.The results showed that the labeled antibody of 3CD-HA or 3D-HA could pulldown the hnRNPK protein,indicating that hnRNPK interacts with 3CD and 3D proteins.Meanwhile,hnRNPK co-localized with 3CD and 3D in the cytoplasm after co-transfection with 3CD or 3D,suggesting that 3CD and 3D proteins may play a key role in regulating hnRNPK enter to the cytoplasm.hnRNPK truncated or mutant co-transfected with 3CD or 3D,and the interaction between them was detected by Co-IP.The results showed that KH1 and KI domain of hnRNPK mediated the interaction with DHAV-1 3CD and 3D.DHAV-1（MOI=0.01 or MOI=0.5）infected DEF cells which transfected KH1 and KI domain deletion mutants pCAGGS-hnRNPK_△1-110-His and pCAGGS-hnRNPK_△191-328-His respectively,WB and RT-qPCR experiments compared the copy number and VP3 expression of DHAV-1 in expressed hnRNPK and its mutant hnRNPK_△1-110/hnRNPK_△191-328cells.The results showed that the VP3 expression level and copy numbers of DHAV-1 in hnRNPK_△1-110 group were the same as those in hnRNPK group,while the VP3 expression level and copy numbers of DHAV-1 in the hnRNPK_△191-328 group were significantly lower（P<0.01）than those in the hnRNPK group after virus infection 24 h and 36 h,indicating that the mutant hnRNPK_△1-110retains its promoting effect on DHAV-1 replication,but the mutant hnRNPK_△191-328 attenuates the stimulation of DHAV-1 replication.In conclusion,this study demonstrates that the expression of hnRNPK can be up-regulated by DHAV-1 and viral proteins VP0,2BC,2B,3CD and 3D,and the interaction between hnRNPK and DHAV-1 untranslated regions promotes viral replication.hnRNPK interacts with DHAV-1 3CD and3D via its KH1 and KI domains,this interaction contributes to hnRNPK’s promotion of DHAV-1replication,and the KI domain of hnRNPK plays an important role in the regulation of DHAV-1replication.