Function Of BmCPV Small ORF Encoded By S5,S10 DsRNA

Small open reading frame(sORF)as a gene which encoded less than 100 consecutive codons,plays an important role in many biological processes that can regulate cell proliferation,differentiation,signal transmission,et al.sORF has a variety of biological effects.Bmbyx mori cypovirus type-1(BmCPV-1,referred to as BmCPV)as a member of the reovirus family,is a typical type of polyhedrosis virus,can specifically infect silkworm intestinal tissue.The genome of BmCPV consists of S1-S10 dsRNA fragments.It is generally believed that each dsRNA fragment is a single cistron,encoding a protein.There have been reported that some viruses can encode functional sORFs,but the research of BmCPV has not been reported.1.The function of BmCPV S5-sORFBmCPV S5 dsRNA encoded the nonstructural protein NS1 and the sORF Finder software predicted that the antisense strand of the S 5 dsRNA has the potential to encode a sORF.The sORF is named S5-sORF-1089,referred to as S5-sORF.The sequence of S5-sORF was amplified by PCR and the amplified product was cloned into prokaryotic expression vector and expressed in Escherichia coli.S5-sORF polyclonal antibody was prepared by immunizing mice with purified recombinant protein.Cell immunofluorescence assay showed that S5-sORF peptides could locate in the nucleus of BmN cells infected by BmCPV,indicating that BmCPV did encode S5-sORF.In order to investigate the function of S5-sORF,we constructed the transformed cells with overexpression of S5-sORF.The result of qPCR showed that the multiplication of BmCPV was inhibited in S5-sORF overexpression cells.Moreover,overexpression of S5-sORF had no significant effect on apoptosis and immune response-related genes.Based on reverse genetic technology,the recombinant BmCPV-S5-sORFmut whose initiation codon of S5-sORF was mutant was constructed.The result showed that the relative expression level of S1 gene of BmCPV-S5-sORFmut was higher than the recombinant BmCPV virus.It was speculated that the small peptide encoded by S5-sORF had an inhibitory effect on the proliferation of the virus.The BmN cells were treated with the synthetic S5-sORF peptide with the penetrating peptide and the result showed that the proliferation of BmCPV was inhibited in a dose-dependent,further confirmed that S5-sORF encoded small peptide can inhibit the proliferation of the virus.With Co-IP to screen the interaction proteins of S5-sORF,there were TFAP4,Akirin protein and small molecule ubiquitination protein and so on identified by mass spectrometry.The result suggested that S5-sORF can interact with these proteins to regulate the proliferation of the virus.In addition,we also found that BmCPV was inhibited by transfecting with the RNA which was in vitro transcribed RNA of S5-sORF with the initial codon deletion.It was speculated that the RNA of S5-sORF might also act as antisense RNA of NS1 gene.2.The function of BmCPV S10-sORFBmCPV S10 dsRNA encoded the polyhedron and predicted that the sense strand of S10 dsRNA had the potential to encode a sORF using the sORF Finder software.The sORF is named S10-sORF-393,referred to as S10-sORF.The sequence of S10-sORF was cloned into prokaryotic expression vector and the recombinant protein was used to prepare S10-sORF polyclonal antibody.The results of immunofluorescence assay showed that the small peptide of S10-sORF was mainly located in cytoplasm.The result of qPCR showed that the mRNA level of S10-sORF was similar to that of polyhedrin gene,suggesting that S10-sORF and polyhedrin gene shared the same mRNA template to translate protein.In order to investigate the effect of S10-sORF on BmCPV itself and host cell,the S10-sORF and S10-sORFmut which was S10 fragment with mutation of S10-sORF the sequence after initiation code transfected BmN cells.The results showed that the relative expression of BmCOCS6(JAK/STAT Pathway),BmPGRP-LB(Imd pathway),Bmago2(RNAi pathway)and Bmdrc2(RNAi)were significantly inhibited and the relative expression of caspase gene were improved in S10-sORF overexpression cells.The recombinant BmCPV-S10-sORFmut virus whose S10-sORF sequence was mutant was constructed by reverse genetic system.The result showed that the proliferation of rBmCPV-S10-sORFmut virus was lower than that of recombinant BmCPV virus.The BmN cells were transfected with RNA which was in vitro transcribed RNA of S10-sORF with the initial codon deletion and BmCPV proliferation had been a certain inhibited.The interaction proteins of S10-SORF were screened by Co-IP method and fourteen candidate host proteins were identified by mass spectrometry,which were mainly associated with transcriptional regulation,protein translation and so on.3.The entry pathway of BmCPV into BmN cellsTo date,the entry mechanism of BmCPV into cells is unclear.Here we used electron microscopy to study the route of entry of BmCPV into cells,and the results demonstrated that the entry of BmCPV into BmN cells was mediated by endocytosis.Blocking the entry pathway with four endocytic inhibitors,including dansylcadaverine,chlorpromazine,genistein,and PP2,significantly decreased the infectivity of BmCPV.The best inhibitoty effect of all drugs is chlorpromazine and this indicates that BmCPV enters BmN cells via clathrin-mediated endocytosis.To further investigate the inhibition of genistein and PP2,by co-localization of BmCPV particles and lysosomes,it was found that the drug-treated cells were able to induce an ineffective infection of BmCPV.The BmCPV particles were transported to lysosomes and degraded by hydrolases to inhibit the infectivity of BmCPV.