The Role Of F Protein And SeNPC1 In The Adsorption Stage Of Spodoptera Exigua Multiple Nucleopolyhedrovirus Infection And In Baculovirus Superinfection Exclusion

Spodoptera exigua damages a variety of crops such as Cabbage,Cauliflower and Brassica.It is widely distributed and has intermittent explosiveness,causing serious losses to the agricultural economy.Baculovirus specifically infects arthropods,is highly pathogenic and harmless to humans and animals.It is widely used as biological insecticide to control forest and crop pests.Spodoptera exigua multiple nucleopolyhedrovirus(SeMNPV),as an environmentally friendly insecticide,can effectively control the population of Spodoptera exigua attracted much attention.Although virus infection of the host usually causes the death of the host,some viruses do not cause the death of the host and form a latent infection in the host.The latently infected cell will resist the related virus infection,that is,superinfection exclusion.The mechanism study of superinfection exclusion is of great significance to the development of anti-virus strategies.Virus adsorption is the first step for the virus to infect host cells.The interaction between the virus adsorption protein and the host cell surface adsorption receptor,as the rate-limiting step of virus infection,plays an important role in the process of virus infection.In the early stage,our laboratory established SeMNPV latent infection cell line P8-Se301-C1 through undiluted passage of SeMNPV.P8-Se301-C1 has a superinfection exclusion effect on homologous SeMNPV,but does not affect the infection of heterologous Autographa californica multiple nucleopolyhedrovirus(Ac MNPV).It preliminarily shows that the superinfection exclusion of SeMNPV has occurred in the adsorption stage.At present,the mechanism at adsorption stage of SeMNPV infection and the role of virus adsorption in the mechanism of superinfection exclusion is still unclear.This research starts with the virus adsorption protein and host cell surface adsorption receptor to elucidate the role of SeMNPV F protein and SeNPC1 in the host adsorption stage of Spodoptera exigua nucleopolyhedrovirus infection and superinfection exclusion of baculovirus at the virus adsorption stage.To explore the molecular mechanism at adsorption stage of SeMNPV infected Spodoptera exigua cells and preliminarily analyse the superinfection exclusion mechanism of latent infected cells P8-Se301-C1 to baculovirus in the adsorption stage.The main findings are as follows:1.The amount of SeMNPV adsorption on the surface of Se301 cells decreased significantly after the F protein site of SeMNPV blocked by antibody.In addition,recombinant virus v Ac WT-GP64KO-F was constructed which replacing Ac MNPV GP64 with SeMNPV F protein.The replacement of GP64 by F protein enhanced the adsorption of Ac MNPV to Se301 cells and the adsorption capacity of recombinant virus to Se301 was significantly higher than that of wild-type Ac MNPV.It shows that SeMNPV envelope protein F protein not only participates in the adsorption stage of SeMNPV infection of Se301 cells,but also plays an important role in the adsorption stage of ACMNPV infected Se301 cells.2.The adsorption amount of Ac MNPV on the surface of Se301 and P8-Se301-C1 cells is equivalent.The adsorption amount of recombinant virus(v Ac WT-GP64KO-F)on the surface of P8-Se301-C1 cells is significantly lower than that of Se301 cells after replacing Ac MNPV GP64 with SeMNPV F protein.The results indicated that the replacement of Ac MNPV GP64 by SeMNPV F protein affects the adsorption capacity of recombinant Ac MNPV on the surface of P8-Se301-C1 cells,resulting in the rejection of P8-Se301-C1 cells to the heterologous virus Ac MNPV in the adsorption stage.The results show that SeMNPV adsorption protein F protein plays a key role in the superinfection exclusion of baculovirus in P8-Se301-C1 cells.3.Immunofluorescence detection did not observe F protein signal in P8-Se301-C1 cells,indicating that Se8 transcripts may not translated as F protein in P8-Se301-C1 cells.The superinfection exclusion of SeMNPV in P8-Se301-C1 cells is not due to the F protein occupies the cell surface adsorption sites and affects the secondary invasion virus adsorption cells.4.After treating Se301 cells with Proteinase K and Tunicamycin,the adsorption capacity of SeMNPV decreased significantly,indicating that the adsorption receptor of SeMNPV on the surface of Se301 cells is essentially glycosylated protein.Using glycosylated protein as the screening condition,the receptor-related protein NPC1(Niemann-Pick type C1)was mined and screened from the transcriptome data of Se301 cells.The Senpc1 gene of Se301 cells was cloned and analysied by bioinformatics tools.The results showed that the SeNPC1 protein encoded by Senpc1 was a glycosylated protein with multiple transmembrane domains.5.Treatment of Se301 cells with NPC1 inhibitor affects SeMNPV adsorption and progeny virus production.Among them,the amount of SeMNPV adsorbed on the cell surface decreased by 40.12%,the BV production after SeMNPV infection decreased by about 10 times,and the production of polyhedron decreased by 94.28%.After knocking down the npc1 transcript in Se301 cells by RNA interference,the amount of SeMNPV adsorption on the cell surface decreased by 44.76%,but it did not affect the BV and polyhedrin production after SeMNPV infection.The above results indicate that SeNPC1 protein is involved in the adsorption stage of SeMNPV infected Se301 cells.6.The tertiary structure modeling and molecular docking analysis of SeMNPV F protein and SeNPC1 protein Domain C showed that the F protein interacted with SeNPC1 protein Domain C.It suggests that F protein and SeNPC1 act as virus adsorption proteins and host adsorption receptors respectively.Fluorescence quantitative PCR detection showed that the expression of Senpc1 in P8-Se301-C1 cells down regulated by 5times compared with Se301 cells.The results indicated that the down-regulation of SeNPC1 expression reduces the adsorption of SeMNPV to cells,which leads to the superinfection exclusion of SeMNPV in P8-Se301-C1 at the adsorption stage.