Study On The Nucleoprotein HnRNPA2/B1 Promotes Virus Replication Through Binding To Porcine Circovirus Type 2 Genome And Proteins

Porcine Circovirus type 2(PCV2)belongs to the genus Circovirus of the circoviridae family in taxonomy.It is a small nonenveloped virus,with a circular,single-stranded genome of approximately diameter of 17 nm.The PCV2 genome is 1.76 to 1.78 kb in length,containing 11 potential open reading frames(ORFs).Currently,6 ORFs have been shown to encode functional proteins.PCV2 is the primary pathogen of porcine circovirus-associated diseases(PCVAD),which including postweaning multisystemic wasting syndrome,reproductive and respiratory failure,enteric disease,and porcine dermatitis and nephropathy syndrome.It is one of the most harmful pathogens in the pig industry.PCV2 infection lead to subclinical symptoms or chronic systemic syndrome,followed by a mixed infection of multiple pathogens,aggravating the mortality rate and bringing huge economic losses to the pig industry worldwide.The synergy between virus and host factors is essential for virus propagation and host antiviral response.Many studies have shown that host nuclear proteins play a key role in the virus replication process.Multiple virus including influenza A virus,herpes simplex virus type 1 and hepatitis E virus have been shown to be affected by heterogeneous nuclear ribonucleoprotein A2/B1(hnRNPA2/B1).HnRNPA2/B1 is one of the most widely expressed nuclear proteins in mammalian cells.It can regulate multiple steps of RNA metabolism,including alternative splicing,RNA transport,miRNA export,and gene expression.In addition,hnRNPA2/B1 has been shown to mediate virus replication through interaction with various viruses.So,what role can it play in PCV2-infected cells?How does it affect PCV2 replication?And the interaction mechanism between hnRNPA2/B1 and PCV2?These are the issues to be addressed by this research.In this study,we analyzed the function of hnRNPA2/B1 in PCV2-infected PK15 cells.The host hnRNPA2/B1 was overexpressed and knocked down,respectively,to study its regulation of PCV2 virus gene replication,gene transcription and translation,and to explore its effect on PCV2 replication.Overexpression experiments proved that hnRNPA2/B1 promoted rep and cap mRNA transcription and protein translation in the early stage of PCV2 infection.Subsequently,we successfully constructed the hnRNPA2/B1 knockdown cell line using the lentiviral packaging method.Knockdown of hnRNPA2/B1 significantly inhibited the transcription and translation of PCV2 rep,cap and genome replication.In order to further explore the mechanism by which hnRNPA2/B1 affects PCV2,we investigated the interaction between hnRNPA2/B1 and PCV2 genomic DNA and proteins.DNA-IP and qPCR results showed that,the number of overexpressed hnRNPA2/B1 combined with PCV2 DNA increased significantly,proving that hnRNPA2/B1 can directly bind to PCV2 genomic DNA during virus infection.The co-IP experiment proved that the hnRNPA2/B1 can interact with the viral proteins Rep and Cap.The results of confocal experiments further showed that hnRNPA2/B1 can co-localize with Rep in the cytoplasm and co-localize with Cap in the nucleus,which provides a basis for the interaction between hnRNPA2/B1 protein and viral proteins Rep and Cap protein.Our study firstly demonstrated that hnRNPA2/B1 positively regulates PCV2 gene transcription,translation,and genome replication through interaction with the PCV2 genome and viral proteins.Our findings explain and enrich the replication mechanism of PCV2 from the perspective of virus-host interaction,and also provides new scientific data for clarifying the pathogenic mechanism of PCV2.