A Study Of The Role Of Cathepsin L In The Process Of PHEV Infection

Viral replication involves virus adsorption,entry,genome transcription and replication,assembly and release,processes that are critical for virus survival and pathogenicity,and most viral replication is dependent on the involvement of host-associated proteins.It was found that during replication of members of variousβ-coronavirus genera,including SARS-Co V-2,lysosomal histone protease L(CTSL)facilitates virus adsorption and entry by cleaving the viral receptor binding protein(S),and later facilitates release of the genome by cleaving the viral particles in the endosome,which then initiates the virus replication process.In addition,CTSL may be involved in virus hijacking of the lysosome to facilitate In addition,CTSL may degrade virus through its hydrolytic activity during the release of daughter viruses,suggesting that CTSL plays a complex regulatory role on β-coronavirus replication.Porcine hemagglutinating encephalomyelitis virus(PHEV)is the first virus in the genus β-coronavirus that has been found to invade the central nervous system of pigs.The risk of epidemic mutation of this virus cannot be ignored in the pig industry.Previous studies have found that PHEV infection leads to abnormal CTSL expression,however,the correlation between CTSL and PHEV replication is not clear.Firstly,to clarify the effect of PHEV infection on CTSL expression and its distribution on lysosomes,this study used PHEV-infected mouse neuroblastoma mother cells(N2a cells)as a model,and detected by Western Blot and fluorescence quantitative PCR(RT-q PCR)that immature and mature CTSL did not show significant changes in protein and m RNA levels at 0-24 hours(hpi)of virus infection.However,at 48 hpi,both immature and mature CTSL expressed abnormally at the protein and m RNA levels;applying antibodies to lysosomal-associated membrane protein(LAMP1)and CTSL,the co-localization of CTSL and LAMP1 was significantly enhanced by indirect immunofluorescence detection after PHEV infection,indicating that PHEV infection led to CTSL expression and its distribution on lysosomes were increased by indirect immunofluorescence detection.Secondly,to reveal the role of CTSL in the replication process of PHEV,si RNAs targeting CTSL were designed and synthesized in this study;furthermore,the CTSL gene sequence from mice was successfully cloned into the p CMV-C-EGFP vector to construct a recombinant plasmid expressing CTSL,p CMV-C-EGFP-CTSL,which was transfected with N2 a cells,inoculated with virus,and The m RNA samples were collected at different time intervals and detected by RT-q PCR.It was found that knockdown of CTSL could inhibit virus adsorption and entry,but did not affect virus proliferation;overexpression of CTSL could promote virus adsorption and entry and inhibit virus proliferation,indicating that CTSL plays a complex regulatory role in the replication process such as PHEV adsorption and entry.In addition,in our previous work,we found that Trehalose had a significant inhibitory effect on PHEV replication,but the exact mechanism was not completely clear.In order to reveal the role of CTSL in the inhibition of PHEV replication by Trehalose,we examined the changes of virus adsorption and entry before and after Trehalose treatment by RT-q PCR,and found that Trehalose significantly inhibited the adsorption and entry of PHEV.In addition,TRE treatment down-regulated the expression of CTSL in N2 a cells as detected by Western Blot and RT-q PCR.Using PHEV-infected N2 a cells with knockdown or overexpression of CTSL as a model,we found that knockdown of CTSL did not affect the inhibition of PHEV adsorption and entry by Trehalose before and after Trehalose treatment,while overexpression of CTSL attenuated the inhibitory effect of Trehalose on virus adsorption and entry,suggesting that Trehalose inhibited PHEV adsorption and entry by downregulating CTSL.Further,previous studies have shown that the lysosomal protein PGRN plays an important role in the inhibition of PHEV replication by Trehalose.To preliminarily investigate the role of CTSL in the PGRN-mediated inhibition of PHEV replication by Trehalose,we used PGRN knockout cell lines that had been constructed in our laboratory,and also inoculated with virus and added Trehalose,and detected by RT-q PCR that PGRN deletion did not alter the effect of Trehalose on CTSL expression during PHEV replication inhibition,implying that PGRN and CTSL each play a regulatory role in PHEV replication inhibition by Trehalose.In conclusion,this study found that PHEV infection resulted in abnormal CTSL expression and its distribution on lysosomes,and that CTSL affected replication processes such as PHEV adsorption and entry.In addition,the correlation study between abnormal CTSL expression and the anti-PHEV effect of Trehalose confirmed that CTSL plays an important role in processes such as PHEV adsorption and entry inhibition by Trehalose,indicating that CTSL is closely related to PHEV replication.These results will help to reveal the mechanism of PHEV replication,and also provide a reference for the development of potential anti-PHEV drugs using CTSL as a target.