Spli-221 Cells Research Articles

Functional analysis of UDP-glycosyltransferase genes conferring indoxacarb resistance in Spodoptera litura

UDP-glycosyltransferase (UGT) is the major detoxification enzymes of phase II involved in xenobiotics metabolism, which potentially mediates the formation of insect resistance. Previous transcriptome sequencing studies have found that several UGT genes were upregulated in indoxacarb resistant strains of Spodoptera litura, but whether these UGT genes were involved in indoxacarb resistance and their functions in resistance were unclear. In this study, the UGTs inhibitor, 5-nitrouracil, enhanced the toxicity of indoxacarb against S. litura, preliminarily suggesting that UGTs were participated in indoxacarb resistance. Two UGT genes, UGT33J17 and UGT41D10 were upregulated in the resistant strains and could be induced by indoxacarb. Alignment of UGT protein sequences revealed two conserved donor-binding regions with several key residues that interact with catalytic sites and sugar donors. Further molecular modeling and docking analysis indicated that two UGT proteins were able to stably bind indoxacarb and N-decarbomethoxylated metabolite (DCJW). Furthermore, knockdown of UGT33J17 and UGT41D10 decreased viability of Spli-221 cells and enhanced susceptibility of larvae to indoxacarb. Transgenic overexpression of these genes reduced the toxicity of indoxacarb in Drosophila melanogaster. This work revealed that upregulation of UGT genes significantly contributes to indoxacarb resistance in S. litura, and is of great significance for the development of integrated and sustainable management strategies for resistant pests in the field.

Microplitis bicoloratus bracovirus regulates cyclophilin A-apoptosis-inducing factor interaction to induce cell apoptosis in the insect immunosuppressive process.

Microplitis bicoloratus bracovirus (MbBV) induces apoptosis in hemocytes of the host (Spodoptera litura) via the cyclophilin A (CypA)‐mediated signaling pathway. However, the mechanisms underlying CypA‐mediated signaling during apoptosis remain largely unknown. Therefore, in this study, we investigated how CypA and apoptosis‐inducing factor (AIF) interact during MbBV‐mediated apoptosis. Our findings showed that MbBV induces apoptosis through the CypA‐AIF axis of insect immune suppression. In MbBV‐infected Spli221 cells, both the expression of the cypa gene and the release of AIF from the mitochondria increased the number of apoptotic cells. CypA and AIF underwent concurrent cytoplasm‐nuclear translocation. Conversely, blocking of AIF release from mitochondria not only inhibited the CypA‐AIF interaction but also inhibited the cytoplasmic‐nuclear translocation of AIF and CypA. Importantly, the survival of the apoptotic phenotype was significantly rescued in MbBV‐infected Spli221 cells. In addition, we found that the cyclosporine A‐mediated inhibition of CypA did not prevent the formation of the CypA and AIF complex; rather, this only suppressed genomic DNA fragmentation. In vitro experiments revealed direct molecular interactions between recombinant CypA and AIF. Taken together, our results demonstrate that the CypA‐AIF interaction plays an important role in MbBV‐induced innate immune suppression. This study will help to clarify aspects of insect immunological mechanisms and will be relevant to biological pest control.

Identification of β-chain of Fo F1 -ATPase in apoptotic cell population induced by Microplitis bicoloratus bracovirus and its role in the development of Spodoptera litura.

Two physiological changes of Spodoptera litura parasitized by Microplitis bicoloratus are hemocyte-apoptosis and retarded immature development. β-Chain of Fo F1 -ATPase was found from a S. litura transcriptome. It belongs to a conserved P-loop NTPase superfamily, descending from a common ancestor of Lepidopteran clade. However, the characterization of β-chain of ATPase in apoptotic cells and its involvement in development remain unknown. Here, the ectopic expression and endogenous Fo F1 -ATPase β-chain occurred on S. litura cell membrane: in vivo, at the late stage of apoptotic hemocyte, endogenous Fo F1 -ATPase β-chain was stably expressed during M. bicoloratus larva development from 4 to 7 days post-parasitization; in vitro, at an early stage of pre-apoptotic Spli221 cells by infecting with M. bicoloratus bracovirus particles, the proteins were speedily recover expression. Furthermore, endogenous Fo F1 -ATPase β-chain was localized on the apoptotic cell membrane. RNA interference (RNAi) of Fo F1 -ATPase β-chain led to significantly decreased head capsule width. This suggested that Fo F1 -ATPase β-chain positively regulated the development of S. litura. The RNAi effect on the head capsule width was enhanced with parasitism. Our research found that Fo F1 -ATPase β-chain was expressed and localized on the cell membrane in the apoptotic cells, and involved in the development of S. litura.

A polydnaviral genome of Microplitis bicoloratus bracovirus and molecular interactions between the host and virus involved in NF-κB signaling.

Polydnaviruses (PDVs) play a critical role in altering host gene expression to induce immunosuppression. However, it remains largely unclear how PDV genes affect host genes. Here, the complete genome sequence of Microplitis bicoloratus bracovirus (MbBV), which is known to be an apoptosis inducer, was determined. The MbBV genome consisted of 17 putative double-stranded DNA circles and 179 fragments with a total size of 336,336 bp and contained 116 open reading frames (ORFs). Based on conserved domains, nine gene families were identified, of which the IκB-like viral ankyrin (vank) family included 28 members and was one of the largest families. Among the 116 ORFs, 13 MbBV genes were expressed in hemocytes undergoing MbBV-induced apoptosis and further analyzed. Three vank genes (vank86, vank92, vank101) were expressed in hemocytes collected from Spodoptera litura larvae parasitized by M. bicoloratus, in which host NF-κB/IκBs, including relish, dorsal, and cactus, were also persistently expressed. When Spli221 cells were infected with MbBV viral particles, mRNA levels of host and viral NF-κB/IκB genes were persistent and also varied in Spli221 cells undergoing virus-induced pre-apoptosis cell from 1 to 5 hours postinfection. Both were then expressed in a time-dependent expression in virus-induced apoptotic cells. These data show that viral IκB-like transcription does not inhibit host NF-κB/IκB expression, suggesting that transcription of these genes might be regulated by different mechanisms.

Transcription factor CAAT/enhancer-binding protein is involved in regulation of expression of sterol carrier protein x in Spodoptera litura.

The Spodoptera litura sterol carrier protein x (SlSCPx) gene is expressed in various tissues throughout the life cycle and plays important role in sterol absorption and transport. In this study, the effects of insect hormones (20-hydroexcdysone and juvenile hormone) and lipids (arachidonic acid, cholesterol) on the expression of SlSCPx was analysed by reverse-transcriptase PCR. The results showed that none of these substances significantly induced the expression of SlSCPx in Spodoptera litura-221 (Spli-221) cells. To identify the transcription factors responsible for regulation of SlSCPx expression, a 3311-bp promoter sequence of the gene was cloned. Transcriptional activity of the promoter was studied using an in vivo promoter/reporter system and a 29-bp sequence between -1000 and -1029 nucleotides (nt) upstream of this gene was found to be responsible for the up-regulation of the gene. Over-expression of CAAT/enhancer-binding protein (C/EBP) in Spli-221 cells increased the promoter activity 5.57-fold. An electrophoretic mobility shift assay showed that two nuclear proteins bound to this sequence. Recombinant C/EBP specifically bound with a putative cis-regulatory element (CRE). Mutation of the C/EBP CRE abolished the binding of the C/EBP with the CRE. These results suggest that the transcription factor C/EBP may regulate the expression of SlSCPx by binding to the CRE in the promoter of this gene.

The Homologous Regions ofSpodoptera litura(Lepidoptera: Noctuidae) Nucleopolyhedrovirus II Have Both the Function as Origin of DNA Replication and Enhancer

In the genome sequence of the Spodoptera litura nucleopolyhedrovirus II (SpltNPVII), seven homologous regions (hrs), Sphr1-7, were identified. Each of them composed of three to eight 64-bp highly conserved sequences, and each contained a 24-bp imperfect palindrome. A transient expression assay demonstrated that the expression of SpltNPVII-ie1 promoter-driven luciferase gene was enhanced between 3- and 13-fold by infection of SpltNPVII in Spli221 cells. Real-time polymerase chain reaction confirmed each of seven hrs could function as origin (ori) of viral DNA replication. This suggests that these hrs are bifunctional, having both ori and enhancer activities for transcription. In addition, the potential of seven hrs as origins had a significantly positive correlation with the number of their palindromes (r = 0.847, Sig: 0.016 < 0.05), and enhancer efficiency had a significantly positive correlation with the number of characteristic motifs (r = 0.893, Sig: 0.007 < 0.01). The efficiency of replication and enhancement of each hr both increased with increasing total numbers of palindromes, repeat sequences, and characteristic motifs. In addition, a single 64-bp highly conserved consensus sequence cannot very good support to the function as origin and enhancer, and require the assistance of other cis-elements in hrs.

Baculovirus induces host cell aggregation via a Rho/Rok-dependent mechanism.

Several baculoviruses can induce host cell aggregation during infection; however, the molecular basis remains unknown. The Rho family of small GTPases, including Rho1, Racs and Cdc42, plays important roles in cell migration and cell-cell contact. Activated GTPases target actin polymerization to discrete sites on the plasma membrane, thereby inducing membrane protrusions. In this study, we demonstrated that Spodoptera litura nucleopolyhedrovirus (SpltNPV) infection induced the amoeboid movement and aggregation of SpLi-221 cells in vitro. The amount of Rho1-GTP increased in the infected cells, which suggested that Rho1 was activated upon infection. RNA interference and superinfection of dominant-negative recombinants revealed that the SpltNPV-induced SpLi-221 cell aggregation was dependent on the Rho1, but not Racs or Cdc42, signalling pathway. Inhibition of Rho-associated protein kinase (Rok) activity by the inhibitor Y-27632 significantly reduced SpLi-221 cell aggregation. Silencing Rho1 expression with RNA interference decreased SpltNPV propagation by approximately 40 % in vitro, when SpLi-221 cells were infected at a low, but not high, m.o.i., suggesting that the SpltNPV-induced cell aggregation may benefit SpltNPV spread.

A role for Innexin2 and Innexin3 proteins from Spodoptera litura in apoptosis.

Gap junctions formed by two hemichannels from two neighboring cells are cell-to-cell communication channels; hemichannels are communication channels between intracellular and extracellular environments. Hemichannels are hexameric proteins formed by connexins, pannexins, innexins and vinnexins. Innexin-hemichannels (innexons) exist in the lepidopteran cell surface, but their component innexins and functions have not been reported. Recent studies by others have demonstrated that hemichannels, connexons and pannexons from vertebrates serve as regulators of apoptosis via inactivating the PI3K/Akt signaling pathway. Here, the apoptogenic properties of innexons are demonstrated using two innexin cDNAs, Spli-inx2 and Spli-inx3, which were isolated from hemocytes of lepidopteran Spodoptera litura. Alignment analysis revealed that these two genes belong to a conserved innexin family, as they contain the insect signature YYQWV motif at the beginning of the second transmembrane domain. Immunofluorescence showed that two fusion proteins, Inx2-V5 and Inx3-V5, were localized predominantly in the cell membrane, cytoplasm and also nuclei. Ectopic expression in Sf9 cells and over-expression of Inx2 and Inx3 in Spli221 cells promoted apoptosis. In the Spli221 cells, apoptotic cells presented remarkable membrane blebbing. This study also showed that Sf9 and Spli221 cells undergo low level apoptosis under normal culture conditions, but not Hi5 cells. In Hi5 stable cell lines, biotinylation was used to isolate surface proteins and confirm Inx2 and Inx3 localization in the cell membrane and also further data showed that Hi5 cells may activate the PI3K signaling pathway via phosphorylating molecular Akt downstream. This result suggests that innexon-promoted apoptosis may be involving the PI3K/Akt signaling pathway. These findings will facilitate further examinations of the apoptotic regulation by the PI3K/Akt signaling pathway and comparative studies of innexons, connexons, pannexons, and vinnexons.

Functional analysis of Spodoptera litura nucleopolyhedrovirus p49 gene during Autographa californica nucleopolyhedrovirus infection of SpLi-221 cells

The Spodoptera litura nucleopolyhedrovirus (SpltNPV) antiapoptotic gene Splt-p49 is able to rescue replication of a p35-null Autographa californica nucleopolyhedrovirus (AcMNPV) in AcMNPV-permissive Sf9 cells. In this study, an AcMNPV p35 knockout bacmid was generated through ET homologous recombination in Escherichia coli and designated as vAc(P35-KO). The Splt-p49 gene was transposed into the polyhedrin locus of vAc(P35-KO) to investigate if Splt-p49 has any antiapoptotic activity in the context of AcMNPV infection of AcMNPV-nonpermissive SpLi-221 cells. Our results demonstrated that Splt-p49 could not inhibit the apoptosis induced by AcMNPV infection of SpLi-221 cells when it was under the control of its native promoter. Western blot analysis showed that Splt-P49 was poorly expressed. When the expression of Splt-P49 was under the control of Drosophila hsp70 promoter, the expression of Splt-P49 was advanced, and a higher level of Splt-P49 was detected. As a result, the apoptosis of SpLi-221 cells was inhibited; however, budded-virus production did not improve in comparison with that in AcMNPV-infected SpLi-221 cells. These data indicated that there are other barrier(s) to AcMNPV replication in the nonpermissive SpLi-221 cells besides apoptosis.

The sterol carrier protein 2/3-oxoacyl-CoA thiolase (SCPx) is involved in cholesterol uptake in the midgut of Spodoptera litura: gene cloning, expression, localization and functional analyses.

BackgroundSterol carrier protein-2/3-oxoacyl-CoA thiolase (SCPx) gene has been suggested to be involved in absorption and transport of cholesterol. Cholesterol is a membrane component and is a precursor of ecdysteroids, but cannot be synthesized de novo in insects. However, a direct association between SCPx gene expression, cholesterol absorption and development in lepidopteran insects remains to be experimentally demonstrated.ResultsAn SCPx cDNA (SlSCPx) cloned from the common cutworm, Spodoptera litura, was characterized. The SlSCPx cDNA encoded a 535-amino acid protein consisting of a 3-oxoacyl-CoA thiolase (SCPx-t) domain and a SCP-2 (SCPx-2) domain. SlSCPx mRNA was expressed predominately in the midgut, while SlSCPx-2 mRNA was detected in the midgut, fat body and epidermis and no SlSCPx-t mRNA was detected. A 58-kDa full-length SCPx protein and a 44-kDa SCPx-t protein were detected in the midgut of sixth instar larvae when the anti-SlSCPx-t antibody was used in western blotting analysis; a 16-kDa SCP-2 protein was detected when anti-SlSCPx-2 antibody was used. SlSCPx protein was post-translationally cleaved into two smaller proteins, SCPx-t and SCPx-2. The gene appeared to be expressed into two forms of mRNA transcripts, which were translated into the two proteins, respectively. SlSCPx-t and SlSCPx-2 proteins have distinct and different locations in the midgut of sixth instar larvae. SlSCPx and SlSCPx-t proteins were detected predominately in the cytoplasm, whereas SlSCPx-2 protein was detected in the cytoplasm and nuclei in the Spli-221 cells. Over-expression of SlSCPx and SlSCPx-2 proteins enhanced cholesterol uptake into the Spli-221 cells. Knocking-down SlSCPx transcripts by dsRNA interference resulted in a decrease in cholesterol level in the hemolymph and delayed the larval to pupal transition.ConclusionSpatial and temporal expression pattern of this SlSCPx gene during the larval developmental stages of S. litura showed its specific association with the midgut at the feeding stage. Over-expression of this gene increased cholesterol uptake and interference of its transcript decreased cholesterol uptake and delayed the larval to pupal metamorphosis. All of these results taken together suggest that this midgut-specific SlSCPx gene is important for cholesterol uptake and normal development in S. litura.

Functional analysis of the putative antiapoptotic genes, p49 and iap4, of Spodoptera litura nucleopolyhedrovirus with RNAi

A homology search of a public database revealed that Spodoptera litura nucleopolyhedrovirus (SpltNPV) possesses two putative, antiapoptotic genes, p49 and inhibitor of apoptosis 4 (iap4), but their function has not been investigated in its native host cells. In the present study, we used RNA interference (RNAi) to silence the expression of Splt-iap4 and Splt-p49, independently or together, to determine their roles during the SpltNPV life cycle. RT-PCR analysis and Western blot analysis showed the target gene expression had been knocked out in the SpltNPV-infected SpLi-221 cells after treatment with Splt-p49 or Splt-iap4 double-stranded RNA (dsRNA), respectively, confirming that the two genes were effectively silenced. In SpltNPV-infected cells treated with Splt-p49 dsRNA, apoptosis was observed beginning at 14 h, and almost all cells had undergone apoptosis by 48 h. In contrast, budded virus production and polyhedra formation progressed normally in infected cells treated with Splt-iap4 dsRNA. Cell viability analysis showed that Splt-IAP4 had no synergistic effect on the inhibition of apoptosis of SpLi-221 cells induced by SpltNPV infection. Interestingly, after Splt-iap4 dsRNA treatment, cells did not congregate like those infected with SpltNPV in the early infection phase, implying an unknown role of baculovirus iap4. Our results determine that Splt-p49 is necessary to prevent apoptosis; however, Splt-iap4 has no antiapoptotic function during SpltNPV infection.

Identification and characterization of odv-e25 of Spodoptera litura multicapsid nucleopolyhedrovirus

Spodoptera litura multicapsid nucleopolyhedrovirus (SpltMNPV) odv-e25 is 684 bp long, potentially encoding 227 amino acids with a predicted molecular weight of 24.9 kDa. Homology analysis indicated that SpltMNPV ODV-E25 has 35-65% amino acid identity with that of other known baculoviruses. RT-PCR results revealed that the odv-e25 is transcribed actively at the late stage of infection and the mRNA start site was mapped within a consensus baculovirus late promoter sequence (TTAAG). Western blot analysis of odv-e25 expression with an antiserum made against 6 x His tagged ODV-E25 expressed in Escherichia coli indicated that it was present as a doublet of approximately 27 kDa from 24 h through 96 h in SpltMNPV-infected Spli-221 cells. Similar results were seen on Western blots of Spodoptera exigua (Se)MNPV-infected Se301 cells. Immunofluorescence analysis showed that ODV-E25 was predominantly present in the cytoplasm of SpltMNPV-infected cells and localized to the envelopes of occlusion-derived virus.

Identification of Spodoptera Litura Multicapsid Nucleopolyhedrovirus ORF97, A Novel Protein Associated with Envelope of Occlusion-derived Virus

Open reading frame (ORF) 97 of Spodoptera litura multicapsid nucleopolyhedrovirus (Splt97) is a member of 11k gene family (InterPro database accession number: IPR009313) in insect viruses, potentially encoding 112 amino acids with a predicted molecular weight of 13.2 kDa. Sequence analysis indicated that Splt97 has 8-50% amino acid identity with the homologues of other known baculoviruses including Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) ORF108. Splt97 transcription in SpltMNPV-infected Spli-221 cells was detected from 6 to 96 h postinfection (p.i.) and the mRNA start site was mapped within a consensus baculovirus early promoter motif (ACATT). Time course of Splt97 expression in SpltMNPV-infected cells showed that Splt97 was expressed from 48 through 96 h p.i. as a 13 kDa protein. Western blot analysis revealed that Splt97 was present in occlusion-derived virus (ODV). Furthermore, when ODV was fractionated into nucleocapsid and envelope components, Splt97 was shown to be present only in the envelope. These results suggested that Splt97 was a baculovirus ODV-associated protein.