Tetraspanin CD81 serves as a functional entry factor for porcine circovirus type 2 infection.

Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus-associated disease, clinically resulting in immunosuppression and co-infections with other pathogens in infected pigs. The mechanism of PCV2 infection remains unclear. In this study, we firstly found that the tetraspanin CD81 in PK-15 cells interacts with PCV2 Cap protein by using virus overlay protein-binding assay combined with mass spectrometry. Knockdown of the CD81 significantly reduces the levels of the viral Cap mRNA and protein, and viral internalization in PK-15 cells. The critical interaction regions locate in the large extracellular loop (LEL) domain of CD81 and the CD loop region (82-91aa) of the Cap protein, and a polyclonal antibody against the CD81 LEL domain significantly inhibits PCV2 infection. The transmembrane proteoglycan Syndecan-1 interacts with both CD81 and PCV2 Cap, and co-operates with CD81 to promote PCV2 infection in PK-15 cells. Furthermore, CD81 facilitates RhoA activation and enhances the viral internalization and replication in PK-15 cells. It was concluded that the tetraspanin CD81 is a key host factor for PCV2 invasion into PK-15 cells, thus providing new insights into PCV2 life cycle and identifying a potential target for antiviral drug development.IMPORTANCEPorcine circovirus type 2 (PCV2), a significant economic pathogen in the swine industry, presents persistent challenges in its prevention and treatment. Despite extensive research, the mechanism of PCV2 invading host cells remains unclear. In this study, we found and identified a novel interaction between the tetraspanin CD81 and the viral Cap protein during the PCV2 invading PK-15 cells. The transmembrane proteoglycan Syndecan-1 and RhoA are involved in the infection process through the CD81. Moreover, this is the first time that the role of Syndecan-1 in the PCV2 infection process has been demonstrated. Also, a polyclonal antibody against the CD81 extracellular domain significantly inhibits PCV2 infection in PK-15 cells. It not only enriches our understanding of PCV2 life cycle but also offers new perspectives for the development of antiviral therapeutics against circovirus.