Effect Of Host Cell SRSF3 On PCV2 Replication And Identification Of Interaction Proteins

Porcine circovirus type 2(PCV2)is the main pathogen of Porcine circovirus associated disease(PCVAD),which is a viral infectious disease seriously harmful to pig industry.PCV2 mainly impinges on the immune system of pigs and leads to immunosuppression,which increases the susceptibility of infected pigs to other pathogens and induces a variety of diseases.PCV2 is one of the most serious pathogens to the pig industry at present.In previous studies,we found and identified a new gene ORF5 in the PCV2 genome.Its coding product,ORF5 protein,causes the arrest cell cycle and induces endoplasmic reticulum stress and autophagy.ORF5 can interact with multiple proteins of host cells and take effect in virus infection and replication.Yeast two-hybrid test shows that many intracellular proteins were interacting with ORF5 protein in porcine alveolar macrophages(PAMs).Serine/arginine-rich splicing factor 3(SRSF3)is a screened intracellular protein interacting with ORF5 protein,and its role in PCV2 infection has not been researched.Members of the serine/arginine rich splicing factor(SR)family have one or two N-terminal RNA recognition motif(RRM)domains and a C-terminal domain rich in arginine(R)and serine(S)(Amino acid sequences,RS domain).RRM domain recognizes RNA,and the RS domain is involved in protein-protein and protein-RNA interactions.SRSF3 is the smallest protein in the SR family.It is highly expressed in a variety of tumor cells and regulates cell proliferation,cell cycle,apoptosis,autophagy,and cell aging.It is found that SRSF3 can regulate the expression of human papillomavirus(HPV)capsid protein,thus affecting virus replication and proliferation.It can also selectively cut natural immune factors and affect the host’s immune response induced by herpesvirus infection.Based on clarifying the effect of SRSF3 on PCV2 replication,this study screened and identified the intracellular proteins interacting with SRSF3,to provide basic data for the study of PCV2-SRSF3-intracellular protein interaction.The transcription and translation levels of SRSF3 in PCV2 infected and ORF5-transfected cells in vitro were detected,and then cell lines with overexpression and knockdown expression of SRSF3 were constructed,determine the impact of SRSF3 on PCV2 replication.The interaction between ORF5 and SRSF3 was clarified by Co-Immunoprecipitation(Co-IP)and laser confocal,and then the intracellular proteins that may interact with SRSF3 were screened by liquid chromatography-tandem mass spectrometry(LC-MS)technology,and the key intracellular proteins were screened and studied.The following results were obtained:(1)Transcriptional and expression levels of SRSF3 in porcine alveolar macrophages infected with PCV2 and expressing ORF5 protein were significantly increased by RT-qPCR and Western blot;Co-IP and laser confocal detection showed that ORF5 protein was co-located with SRSF3.It is suggested that PCV2 induces the expression of SRSF3 and interacts with SRSF3 through the ORF5 protein.(2)PCV2 Cap transcription and expression increased in porcine alveolar macrophages overexpressing SRSF3(P<0.05).After knocking down SRSF3 expression,the transcription level of the Cap gene decreased(P<0.05).The results showed that SRSF3 could promote the replication of PCV2.(3)In cells overexpressing SRSF3,six intracellular proteins that may interact with SRSF3 were screened by liquid chromatography with LC-MS.(4)PCV2 infection and SRSF3 overexpression up-regulated the expression of Hemoglobin subunit beta(HBB)(P<0.05).Co-localization of SRSF3 and HBB after PCV2 infection.The results shown:SRSF3 is up-regulated in PCV2 infected cells and promotes viral replication,and interacts with PCV2 ORF5 protein.Infection of PCV2 induces co-localization of SRSF3 and HBB.These results provide basic data for further study on the mechanism of SRSF3 infection in PCV2.