| Silkworm is a model insect of Lepidoptera and sericulture is a crucial industry of agriculture economy in some developing countries.However,the silkworm densovirus disease caused by BmNPV has done severe damage to sericulture,which is highly contagious and difficult to control.Its infection leads to about 50% annual loss in sericulture in the world.Therefore,it’s very urgent and necessary for researchers to explore some new effective agents against BmNPV.However,thymosin from Bombyx mori(BmTHY)is crucial for the defense against Bombyx mori nucleopolyhedrovirus(BmNPV).Our previous study has shown that BmTHY was an actin-sequestering protein which located mainly in nucleus and rarely in the cytoplasm in BmN cells.BmTHY was also distributed in most tissues of fifth-instar.This suggests that BmTHY play an essential role in many biological events or Bombyx mori.In a recent study,we also found that recombinant BmTHY not only increased the viability of BmNPV-infected BmN cells but also improved the survival rate of Bombyx mori infected with BmNPV.So BmTHY exerted immunomodulatory effects on Bombyx mori,rendering them resistant to viral infection.However,the molecular mechanism of Bm THY antivirus remains poorly understood.Firstly,to test the effects of BmTHY on BmNPV proliferation and replication,we successfully constructed the system of over-expression and knock-down of BmTHY in BmN cells.Over-expression and knock-down of BmTHY BmN cells were infected with wildtype BmNPV and analyzed the viral proliferation by the method of TCID50.For the viral titer detection,at 168 h post-infection of wild BmNPV in BmN cells,over-expression of BmTHY resulted in an over 50% decrease in the viral titer compared with the control.However,At 168 h post-infection of wildtype BmNPV,TCID50 mean value in the knock-down of BmTHY group climbed up,with increase of 2.37-fold to that of the control group.Secondly,when Over-expression and knock-down of BmTHY BmN cells were infected with BmNPV-RFP,the results showed that the red fluorescence intensity in over-expression of BmTHY group remarkably reduced than the control group,but significantly increased in with BmTHY down-regulation in BmN cells.QRT-PCR analysis also showed the copies of gene BmNPV gp41 decreased when BmTHY expression level was up-regulated,but increased with BmTHY down-regulation in BmN cells infected with BmNPV.These results indicated that BmTHY can inhibit Bm NPV replication in BmN cells.Thirdly,we further investigated the molecular mechanism of BmTHY antivirus.In the present study,after the total protein was extracted from BmN cells,co-immunoprecipitation assay was carried out using BmTHY antibody as the bait,with rabbit IgG as a negative control.The result of Co-IP suggested a physical interaction between BmTHY and actin in BmN cells.These results suggest that interaction between BmTHY and actin maybe affect cytoskeleton,which was involved in BmTHY antiviral mechanism against BmNPV.A wealth of evidence has demonstrated that viral manipulation of the actin cytoskeleton both during nucleocapsid transport and after viral gene expression is important for baculovirus infection and replication.Here,BmNPV infected over expression and knock-down of BmTHY BmN cells for 48 h,and then the ratio of F/G-actin was performed to analyze the effect of BmTHY on the polymerization of actin.For BmTHY over-expressing BmN cells infected with BmNPV,the ratio of F/G-actin was increased to 20.4% of the corresponding control group.However,BmTHY konck-down BmN cells infected with BmNPV showed a 39.8% reduction in the ratio of F/G-actin compared to the control group.These results showed that BmTHY shifted the ratio of F/G-actin in BmN cells infected with BmNPV.This indicated that BmTHY affect viral manipulation of actin in BmN cells infected with BmNPV,and then influence the structure and function of actin cytoskeleton leading to reduce BmNPV infection and replication.In conclusion,these results demonstrated that Bm THY might be one of the key host factors against BmNPV by inhibiting viral proliferation and replication via interacting with actin in BmN cells. |
