Effect Of Biscoumarin Against Rabies Virus And Antiviral Mechanism Based On SQSTM1/p62

Rabies,caused by rabies virus(RABV),is a zoonotic disease which attacks the brain nervous system of all warm-blooded mammals and causes acute progressive encephalitis.Rabies is widely epidemic in developing countries.Mortality of rabies is in the second place in China after India,around the world.Until now,there is no any effective treatment for rabies.The only effectual method is vaccination before or after exposure.Once the clinical symptom appears,the mortality rate is almost 100%.Therefore,it is of profound significance to find effective drugs for treating rabies.Coumarin(CM)belongs to the benzopyrone family.It is mainly found in the roots,stems and leaves of plants such as Aceae,Umbelliferae and Oleaceae,also found in the metabolites of bacteria and fungi.CMs present a variety of biological activities,offer an extended therapeutic profile such as anti-tumor,anti-inflammatory,anti-oxidation and anti-virus.Simple CM osthole can effectively inhibit the proliferation,metastasis and invasion of breast cancer and gastric cancer cells;Pyridine-type CM-Calanolides A(isolation from Calophyllum lanigerum)has an effective anti human immunodeficiency virus(anti-HIV)activity,which is becoming a hot spot of anti-HIV drug and entering clinical phase II trials in the United States;Biscoumarin(BCM),a derivative of CM,has been reported to act as an antibacterial agent for Staphylococcus aureus.However,whether BCM has anti-RABV effect has not been reported yet.In this study,the BCM(labeled as BCM-1?9)is made from 4-hydroxycoumarin,processed by a series of chemical methods involved in heating,dissolving with anhydrous ethanol,and mixing with diverse aromatic aldehydes containing different substituents.The purpose of this study was to screen for drugs with anti-RABV effects from BCMs and to elucidate the molecular mechanisms.1.Study on the effect of Biscoumarin against rabies virusFirstly,the toxicity of nine BCMs in BHK-21 cells(half of concentration of cytotoxicity,CCso)was tested by SRB viability assay.Among them,BCM-8 and BCM-9 significantly inhibited the proliferation of BHK-21 cells at very low concentrations(2.5 ?M),so they were given up for the further study.Subsequently,Direct immunofluorescence assay(DFA)were used to detect cellular toxicity rate of the other 7 BCMs,and the selectivity index(SI=CC50/IC50)was performed to indicate the anti-RABV effect of BCM.Our result demonstrated that BCM-3 with the highest SI of 3.81,CC50 and IC50 respectively are 12.38±3.56 UM and 3.253±0.512 ?M.In addition,DFA,Western-blot and quantitative real-time fluorescent quantitative PCR methods were adopted in order to investigate the mechanism of BCM-3 for inhibiting viral replication,and the effect of BCM-3 on viral replication at different time after infection.The data showed that BCM-3 could decrease the virus titer about 40 folds at the concentration of 5 ?M(P<0.01),and could significantly inhibit the expression of RABV CVS-P gene up to 60.0%(P<0.01).Besides,BCM-3 could inhibit the expression of CVS-P mRNA with the highest suppressive rate of 60%within 48 h after application of the drug.In addition,between 3?12 h after viral infection,the antiviral replication effect was more significant.The drug was administered at 12 h after infection,which inhibited the protein expression of CVS-P by 60%(P<0.01),suggesting that early administration may be more effective against RABV infection;BCM-3 did not directly inactivate the replication of RABV and did not prevent the adsorption of BHK-21 cells by RABV.Compared with the negative control group and the virus-infected untreated control group,the virus titer and RABV CVS-P protein level of the BCM-3 treatment group had no significant change(P>0.05).Finally,RABV infected mice models were constructed with the 10 LD50 of 106.5 TCID50/mL CVS-11(Challenge virus standard-11).Our results showed that the infected mice were crippled and their weights were declined.Treatment of infected mice with 50 mg/kg BCM-3 increased the survival rate of mice from 0%to 12.5%,suggesting that BCM-3 has a certain therapeutic effect on the mice RABV infection model.2.Study on the mechanism of Biocoumarin exerting anti-RABV by regulating SQSTM1/p62It has been reported that autophagy plays an important role in immune escape and viral replication.RABV can eliminate cell apoptosis by inducing autophagy,thereby maintaining cell homeostasis.But there was evidence showed that incomplete autophagy can be induced after RABV infection,and the expression of endogenous LC3 was up-regulated,but SQSTM1/p62 levels did not change significantly.The role of SQSTM1/p62 expression in anti-RABV by exogenous transfection remains unclear.SQSTM1/p62 is an essential substrate for autophagy.It is worth noting that SQSTM1/p62 is also a multi-functional backbone protein which is widely involved in a variety of signaling pathways.Previous trials have confirmed that BCM-3 can inhibit RABV replication,is this inhibition related to autophagy or SQSTM1/p62?Therefore,we carried out the following research.Firstly,we chose N2a cells as a cell model for subsequent mechanism studies and used Western-blot to detect autophagy and apoptosis-related proteins.We wanted to screen the proteins involved in the inhibition of RABV replication by BCM-3.It was found that BCM-3 could increase the protein expression of SQSTM1/p62 after 24 h of treatment of RABV-infected N2a cells.Further,after 48h treatment with BCM-3,it was further confirmed that the SQSTM1/p62 protein could be increased while the expression level of CVS-P protein was decreased 40%(P<0.01).Therefore,we hypothesized that BCM-3 may exert anti-RABV effects by blocking SQSTM1/p62-dependent cellular autophagy.In order to further confirm the role of SQSTM1/p62 in BCM-3 anti-RABV replication,we successfully constructed SQSTM1/p62 over-expression plasmid and transfected N2a cells with these plasmids as well as specific siRNA.The results showed that over-expressed SQSTM1/p62 down-regulated the viral CVS-P mRNA level by 50%(P<0.01),and down-regulated the viral CVS-P protein expression by only 25%;Using the specific siRNA targeting the SQSTM1/p62 gene,the expression of SQSTM1/p62 was knocked down,and the viral CVS-P mRNA level was up-regulated by 2.1-fold(P<0.01),but the up-regulated viral CVS-P protein level was only 30%.It is suggested that BCM-3 may exert its anti-RABV effect through SQSTM1/p62-dependent pathway,but the correlation between BCM-3 regulated SQSTM1/p62-dependent viral replication and autophagy has yet to be further explored.In summary,we screened and identified a novel coumarin compound BCM-3 which can inhibit the anti-rabies virus at least in vitro,and further confirmed that this compound could exert its anti-RABV effect by up-regulating the expression of SQSTM1/p62.Our study showed BCM-3 had a significant anti-RABV effect,indicating that this compound could be a possible candidate for development of an anti-RABV drug.