Transcriptional Response Of Immune-related Genes Induced By CPV Capsid Shell Protein In Lepidopteran Cell Lines

Cytoplasmic Polyhedrosis Virus(CPV)is a typical double-stranded RNA reovirus.Its genome consists of 10 segments of dsRNA.CPV is non-enveloped and only a single layer of capsid formed by capsid shell protein(CSP).CPV has a wide host range,and it has certain selective specificity to host cells that reproduction only in midgut epithelial cells.According to this characteristic,CPV virus can be proliferated in host epithelial cells.The host specificity of CPV capsid is strong,and it has good safety that it dose not infect mammals and humans.The baculovirus expression vector system(BEVS)is currently an important and versatile biotechnological platform for protein expression,particularly for large protein complexes,such as viral-like particles(VLPs).RNA viruses that can be considered are nonenveloped viruses with positive-sence ssRNA genome and with segmented dsRNA genome can be used as carriers of RNA silencing.So,baculoviruses seem to show limited potential to be developed as vectors for virus-induced gene silencing.Engineering of the RNA-dependnet RNA polymerase and the viral suppressor of RNAi proteins may accomplish the generation of viral vectors that differ in their persistence and pathogenicity levels for multiple applications in RNA silencing(from specific gene function analysis to insect pest control).VLPs based on the capsid shell and associated proteins of viruses with segmented dsRNA genome also could provide a safer alternative for dsRNA delivery.In such case,the BEVS and Multi Bac technology may provide an ideal system for production.In insects,RNAi is considered the major antiviral immune defense pathway.Ds RNAs produced during viral infection are processed by Dicer enzymes to small RNAs that function as specificity determinants to silence viral genes.By contrast,in mammals,recognition of molecules associated with viral infection,such as dsRNA,by patttern recognition receptors(PRRs)initiates a signaling cascade that culminates in the production and release of signaling proteins with antiviral function such as interferons.While RNAi is considered as the major immune defense mechanism in insects,evidence exists that viral replication also induces a transcriptional response that includes immune-related genes.However,the hypothesis that components of virions or viral replication can be recognized as pathogen-activated molecular patterns(PAMPs)to activate the innate immune response in insects has not been investigated systematically.In this study,the potential of VP1,that constitutes the major capsid protein of cytoplasmic polyhedrosis virus(CPV;Reoviridae),to activate a collection of immune-related genes was examined in silkworm-derived Bm5 cells.Two different methods of VP1 administration were tested,either through endogenous expression in transformed cell lines,or through addition of purified VP1-based viral-like particles to the extracellular medium.In addition,exposure to CPV virions isolated from purified polyhedra was also performed.In general,our results do not show a robust transcriptional response of immune-related genes to VP1 or CPV virions but two exceptions were noted.First,expression of the antimicrobial peptide gene Att was strongly induced after 24 h of exposure to VP1-based VLPs(CSP-VLPs).This indicates that Att has a special resistance to CSP-VLPs.Second,expression levels of Dcr2,an essential gene in the RNAi pathway,were greatly increased in VP1-expressing transformed Sf21 cells but not transformed Bm5 cells,indicating the existence of species-specific effects.However,the increased expression of Dcr2 did not result in increased silencing efficiency when tested in an RNAi reporter assay.This suggests that Dcr2 triggers transcription in a particular way,rather than acting as an effector of RNAi.The conclusions of this experiment include: the capsid protein VP1 of CPV has the potential to act as a PAMP and to induce a transcriptional response in insect cells that relate both to RNAi and protein effcetors such as antimicrobial peptide gene.A few major immune signaling pathway genes(Ago2 and Ago3)showed corresponding transcriptional responses,and capsid protein VP1 or CPV virions were active as PAMPs.Attacin has special resistance to VLP formed by Bm CPV capsid protein VP1,which may reflect the activation of an immune pathway.The identity of the PRRs and the signaling cascade that are potentially triggered by VP1 remain to be elucidated in future experiments.While this study was performed on a small scale,it can encourage more comprehensive studies with high-throughput approaches(micro-array,deep sequencing)to search more systematically for PAMPs that appear during viral infection in insects.Thus the diversity of defense mechanisms of insect viruses has been revealed.