Regulation Of The Growth Of Classical Swine Fever Virus By Hemoglobin β-subunit And Its Regulatory Mechanism

One of the initial responses of an organism to infection by pathogenic viruses is the synthesis ofproinflammatory and antiviral cytokines, which cause cell death by the apoptosis mechanism. Someanimals can resist viral infection by antiviral or proinflammatory cytokines and eventually eliminate thevirus by animal body’s specific immune response. Many viruses have evolved strategies for inhibitingthe inflammatory response and blocking apoptosis and can survive to develop disease in the host.Excessive inflammatory reactions may cause pathological changes during antiviral responses.Virus–host cells interaction plays an important role in pathogenic mechanisms during virus infection. Inrecent years, a host cellular receptor for Classical swine fever virus (CSFV), proteins that influence theCSFV virulence and several interactions between CSFV and cellular proteins have been identified.CSFV is the causative agent of classical swine fever (CSF), a highly contagious and economicallyimportant disease of pigs. It is very important to develop effective prevention and treatment strategies ofCSF. To this end, it is necessary to understand the mechanisms of CSFV infection and pathogenesis. Todate, studies on CSFV-host interaction(s) are limited. This study is aimed to identify interactionsbetween the CSFV C protein with host cell proteins and to explore the possible effects of cellularproteins on the CSFV growth and genomic replication and the underlying mechanisms of regulation.In this study, the hemoglobin β-subunit (HB) was identified as a C protein-binding protein usingglutathione S-transferase (GST) pulldown and subsequent mass spectrometry analysis of PK-15cells,which are permissive cells of CSFV. Coimmunoprecipitation and GST pulldown assays confirmed thatHB interacted with the CSFV C protein. And confocal microscopy confirmed that HB colocalized withthe CSFV C protein in the cytoplasm. It was shown that HB was expresssed in different swine tissuesand cells, and was significantly downregulated in the tissues from CSFV-infected pigs. Silencing of HBwith small interfering RNAs promoted the CSFV growth and replication and C expression usingWestern blotting and immunofluorescence, whereas overexpression of HB suppressed CSFV replicationand growth and C expression. Western blotting confirmed that CSFV inhibited cellular HB expressionin infected CSFV PK-15sells. The HB protein can active interferon β (IFN-β) and enhance IFN-βmRNA expression level, as demonstrated by luciferase reporter assay and real-time RT-PCR.Interestingly, HB was found to interact with retinoic acid inducible gene I (RIG-I) and increase itsexpression, resulting in increased production of type I interferon. The expression levels of both HB andRIG-I were reduced with the increased expression of the C protein. Furthermore, knockdown of RIG-Iincreased CSFV growth and replication in PK-15cells and SK6cells that were more efficiently infectedwith CSFV than those pretreated with IFN-β. And IFN-β can enhance HB protein expression and inhibitCSFV C protein expression. Whereas, HB was unable to suppress CSFV growth and genomicreplication when the RIG-I pathway was blocked.Overall, CSFV or C protein alone inhibits the expression of HB, HB interacts with RIG-I andupregulates its expression, RIG-I activates IFN-β, and IFN-β inhibits CSFV growth and genomicreplication and increases HB expression. Our results suggest that the cellular HB antagonizes CSFV growth and replication by triggering the IFN signaling, and might represent a novel antiviral restrictionfactor. This study reports for the first time the novel role of HB in innate immunity.