The Proliferation Of CSFV Promoted By Beclin1 Protein And The Establishment Of Its Stable Transgenic Cell Line

Classical swine fever (CSF) is a highly contagious and clinically characterized by high fever,bleeding,and immunosuppression caused by the classical swine fever virus (CSFV),which has caused significant economic losses to the pig industry.At present,China’s prevention and control of swine fever still relies on the widespread use of vaccines.The rabbit attenuated swine fever vaccine developed by Chinese scientists has played a crucial role in global swine fever prevention and control.In the production process,the rabbit attenuated swine fever vaccine is produced using a porcine testicular cell line (ST),which has limited proliferation efficiency and high production costs.Research has found that CSFV infection can effectively activate the autophagy response of host cells and utilize autophagy to promote self proliferation.The Beclinl protein positively regulates autophagy in host cells,further participates in regulating viral replication.The CSFV NS5A protein,as a polyphosphate protein,directly participates in the replication of the virus genome and has the function of regulating cellular signaling pathways through interaction with host proteins.However,its interaction with Beclinl protein has not been reported yet.Therefore,this study aims to investigate the effect of Beclinl on CSFV replication and its interaction with NS5A.Based on this,ST cell lines overexpressing Beclinl protein were constructed to investigate its effect on the proliferation of classical swine fever virus.The following results were obtained.(1) Beclinl protein promotes CSFV replication.First,we studied the effect of CSFV infection on endogenous Beclinl expression,and used RT-qPCR and Western blot methods to detect ST cells infected with swine fever virus at different time points,and found that compared with the control group,the expression of Beclinl was significantly increased at both transcriptional and protein levels after CSFV infection.On this basis,we utilized overexpression and siRNA mediated knockdown of Beclin1 protein expression.After infection with CSFV,the results showed that overexpression of Beclin1 significantly promoted CSFV replication,while knockdown of Beclin1 protein expression significantly inhibited CSFV replication.(2) There is an interaction between Beclin1 and CSFV NS5A protein.The NS5A protein,as an important component of the viral replication complex,directly participates in the proliferation process of CSFV.And our research found that Beclinl protein has a promoting effect on virus proliferation.Based on this,we investigated the interaction relationship between Beclin1 and CSFV NS5A.A series of expression vectors of Beclin1 and NS5A were constructed respectively,and the interaction between Beclin1 and CSFV NS5A protein in cells was confirmed by immuno coprecipitation experiment;Using the GSTpull-down experiment,there is still interaction between Beclinl and CSFV NS5A protein in the extracellular environment;Finally,a laser confocal microscope was used to observe the colocalization of Beclinl and CSFV NS5A proteins within the cell.(3) Establishment of ST cell line overexpressing Beclin1 protein and its effect on CSFV proliferation.Construct a GFP-Beclinl eukaryotic expression vector,transfect ST cells,and screen for stable overexpression of Beclin1 protein using G418 antibiotics.At the same time,set ST cells transfected with blank GFP vector as control.Through continuous drug screening and passage,two ST cell lines stably expressing Beclinl-GFP and GFP empty vector were obtained.Both fluorescence observation and Western blot detection can confirm the correct expression of the target protein.The CCK-8 method was used to determine the activity of this cell line,which did not change compared to the original ST cells;Cell line tumorigenicity experiments showed that both the ST Beclin1 cell line and the original ST cells did not possess tumorigenicity.Through RT-qPCR detection,it was found that the replication of the swine fever rabbit attenuated vaccine virus was significantly enhanced on this cell line,and its proliferation efficiency could be increased by about 20%.In summary,this study demonstrates that the host Beclinl protein can significantly promote the replication of CSFV,and there is an interaction between the Beclinl protein and CSFV NS5A;A ST cell line capable of stably overexpressing Beclinl protein has been screened,which can further improve the proliferation efficiency of the rabbit attenuated swine fever vaccine strain and has potential practical application value.