Silkworm hemolymph inhibits baculovirus-induced insect cell apoptosis

The effect of silkworm hemolymph on baculovirus-induced insect cell apoptosis was investigated. The addition of silkworm hemolymph into the culture medium either before or during the baculovirus infection increased the host cell longevity; however, its addition after the infection was less effective. This can be explained by the higher transfer rate of silkworm hemolymph which is caused by endocytosis during the virus internalization step. The delayed cell death due to silkworm hemolymph was not caused by an inhibition of the virus attachment and internalization steps. The apoptosis was analyzed using DNA fragmentation and TUNEL assays, and the resulting data confirm that silkworm hemolymph inhibits baculovirus-induced insect cell apoptosis.     

Silkworm hemolymph as a potent inhibitor of apoptosis in Sf9 cells

We have previously shown that silkworm hemolymph exhibits anti-apoptotic activity against baculovirus-induced Sf9 cell apoptosis. In this study, using various chemical inducers, such as actinomycin D, camptothecin, and staurosporine, we found that silkworm hemolymph inhibits insect cell apoptosis induced not only by baculovirus but also by chemical inducers. This indicates that silkworm hemolymph contains anti-apoptotic components that work directly in insect cell apoptosis without any booster expression of baculoviral genes. With the analysis of Sf-caspase-1 activity, it was found that the inhibitory effect of silkworm hemolymph works in a further upstream step than the Sf-caspase-1 activation step.     

Kinetic effect of silkworm hemolymph on the delayed host cell death in an insect cell-baculovirus system

The kinetic effect of silkworm hemolymph on host cell viability during a baculovirus-induced insect cell death process was investigated. Host cell viability after viral infection is important for replication of the baculovirus DNA containing a recombinant gene and expression of the cloned gene. The baculovirus-induced insect cell death process can be divided into a delay phase and a first-order death phase, which are characterized by a delay time (t(d)) and a specific death rate (k(d)), respectively. For 0-10% silkworm hemolymph in the media, higher concentrations resulted in longer delay times and lower specific death rates. By adding 10% silkworm hemolymph, the delay time increased from 72 to 164 h, and the specific death rate was reduced from 13.8 x 10(-)(3) to 6.0 x 10(-)(3) h(-)(1). In addition, host cell viability correlated with DNA fragmentation, which is the biochemical hallmark of apoptosis. This indicates that the silkworm hemolymph inhibits the baculovirus-induced insect cell apoptosis. However, the silkworm hemolymph did not affect the number of hypothetical targets, which represents host cell susceptibility to the baculovirus. The concentration of fetal bovine serum (FBS) in the medium did not affect the delay time, while lower concentrations of silkworm hemolymph resulted in shorter delay times. This means that the substance which increases the longevity of the host cell is not in the FBS but in the silkworm hemolymph.     

Beneficial effect of silkworm hemolymph on a CHO cell system: Inhibition of apoptosis and increase of EPO production

To produce erythropoietin (EPO), Chinese hamster ovary (CHO) cells were first cultured in a medium containing FBS (growth medium) and then in a serum-free medium containing sodium butyrate (production medium). Sodium butyrate increases recombinant protein production, but also induces apoptosis, which reduces cell viability and productivity. In a previous study, we found that silkworm hemolymph (SH), an insect serum, inhibits the apoptosis of insect and mammalian cells. To overcome sodium butyrate-induced apoptosis, we added SH to growth medium. This pretreatment with SH inhibited the sodium butyrate-induced apoptosis of CHO cells and consequently increased their longevity and their ability to produce EPO. As a result, the volumetric productivity of EPO was increased five-fold. SH was found to inhibit cytochrome c release from mitochondria into the cytosol, and prevented the activation of caspase-3 and other subsequent caspase reactions.     

Inhibition of HeLa Cell apoptosis by storage-protein 2

Apoptosis is a barrier to maintaining high viable cell densities in animal cell culture. Silkworm hemolymph and its 30K protein have been reported to exhibit anti-apoptotic activity in various mammalian and insect cell systems. The 30K protein is thermally unstable at temperatures higher than 60 degrees C; however, the silkworm hemolymph heat-treated at 70-80 degrees C still exhibited anti-apoptotic activity. This indicates that silkworm hemolymph contains another anti-apoptotic compound other than 30K protein. In this article, the anti-apoptotic molecule other than 30K protein was found from the silkworm hemolymph and identified. This molecule was storage-protein 2 (SP2), which has no homology with any known anti-apoptotic protein. This molecule was heat-stable up to 80 degrees C, while 30K protein lost its activity at temperatures higher than 60 degrees C. When apoptosis was induced by staurosporine in HeLa cells, SP2 protein suppressed nuclear fragmentation and apoptotic body formation. Moreover, the generation of reactive oxygen species after apoptosis induction was inhibited, which means the inhibition occurred in an early step of the apoptotic process. Inhibition of apoptosis by the SP2 protein would lead to the minimization of cell death during commercial mammalian cell culture.     

Inhibition of agonist-induced activation of phospholipase C following poxvirus infection.

Recent studies indicate that viruses may influence polyphosphoinositide levels. This study has examined the effects of vaccinia virus infection on phospholipase C activity. Infection of BS-C-1 cells, an African Green Monkey kidney cell line, or A431 cells, a human carcinoma cell line, with vaccinia virus inhibits receptor-mediated phospholipase C activation. As a consequence, agonist-mediated Ca2+ mobilization in BS-C-1 cells also was inhibited by vaccinia virus infection. Alleviation of the inhibition of phospholipase C activation was observed in vaccinia virus-infected cells treated with cycloheximide without influencing uninfected cells. Treatment of infected cells with alpha-amanitin, an inhibitor of host mRNA synthesis but not virus mRNA synthesis, failed to alleviate the inhibition of phospholipase C activation. Together these results suggest that a virus-encoded gene product mediates the inhibition of phospholipase C activation without the need of a virus-induced host factor. Analysis of the processes involved in the formation of inositol (1,4,5)-trisphosphate and mobilization of intracellular Ca2+ indicate that the vaccinia virus gene product exerts its inhibitory effects at the level of phospholipase C activity. This may occur by either directly reducing the amount of phospholipase C, reducing the specific activity of phospholipase C, or by inhibiting the association of phospholipase C with its substrate, phosphatidylinositol 4,5-bisphosphate.     

High-level expression of human acidic fibroblast growth factor and basic fibroblast growth factor in silkworm (Bombyx mori L.) using recombinant baculovirus

A hybrid of Autographa californica nuclear polyhedrosis virus and Bombyx mori nuclear polyhedrosis virus, which is infectious to both Spodoptera frugiperda and Bombyx mori, was prepared in our previous study. Two recombinant hybrid baculoviruses, carrying cDNAs of human acidic and basic fibroblast growth factors, respectively, were successfully constructed in this study, for the large-scale production of human aFGF and bFGF using silkworm as host. These recombinant viruses were used to inoculate silkworm larvae. After the infection, the recombinant proteins were not found in the hemolymph. Such nonsecretion from cells has also been observed in the established insect cell lines, Sf21 and Tn-5. Tissue distribution analysis indicated that the expressed products were mainly located in fat body and the production of the recombinant aFGF and bFGF was maximal at around 80 h postinfection. Therefore, silkworm larvae infected with recombinant viruses were dissected and fat bodies were collected for the purification of recombinant aFGF and bFGF. The expression levels in both cases were estimated to be as high as approximately 600-700 microg per larva. Furthermore, the recombinant proteins were characterized and their biological activities were evaluated by in vitro bioassay using cell culture.     

Expression of Bombyx mori 30Kc19 protein in Escherichia coli and its anti-apoptotic effect in Sf9 cell

The silkworm hemolymph has an anti-apoptotic activity in insect, mammalian, and human cell systems. The protein from silkworm hemolymph with the highest apoptosis inhibiting activity was found to be 30Kc19 protein, which was one of the ‘30K proteins’. In this study, 30Kc19 protein encoded by the 30Kc19 gene of the silkworm was expressed in Escherichia coli with (pET-22b(+)) and without (pET-3a) pelB leader sequence. 30Kc19 protein was over-expressed largely as a soluble form by pET-3a and both as soluble and insoluble forms by pET-22b(+). The medium was supplemented with each of the recombinant 30Kc19 proteins, and their presence was found to inhibit nuclear fragmentation and apoptotic body formation in actinomycin D-induced Sf9 cell apoptosis. Moreover, 30Kc19 protein repressed the activation of Sf-caspase-1. The 30Kc19 protein obtained from periplasm showed the most effective anti-apoptotic activity. This protein holds great potential for industrial and pharmaceutical applications since mass production and easy purification of this protein is possible.     

Enhancement of recombinant protein production in Chinese hamster ovary cells through anti-apoptosis engineering using 30Kc6 gene

It was previously reported that silkworm hemolymph (SH) inhibits apoptosis and increases the production of recombinant human erythropoietin (EPO) in Chinese hamster ovary (CHO) cells. The apoptosis-inhibiting component in SH is a member of 30K protein family. In this study, the CHO cell line producing EPO was manipulated genetically to express the 30Kc6 gene encoding a 30K protein in the hemolymph of the silkworm, Bombyx mori. The transient expression of 30Kc6 significantly suppressed the cell death induced by serum deprivation. A stable cell line expressing 30Kc6 with an anti-apoptotic property was established. The stable expression of 30Kc6 inhibited serum-deprivation-induced apoptosis and increased the cell density and EPO titer by 5- and 10-fold, respectively. The positive effects of the 30Kc6 expression on cell viability and productivity were due to the stable maintenance of the mitochondrial activity. The 30Kc6 expression efficiently suppressed the depolarization of the mitochondrial membrane and subsequently balanced the generation/consumption of ATP. The use of the 30Kc6 gene is expected to provide a new method of host cell engineering for improving the productivity of the recombinant protein.     

Inhibition of apoptosis by a Bombyx mori gene

An apoptosis-inhibiting component of silkworm hemolymph, isolated and characterized in our previous study, showed 95% N-terminal amino acid sequence homology with one of the 30K proteins, a group of structurally related proteins. The 30K protein was expressed in mammalian HEK293 cells and CHOK1 cells by transfection with 30Kc6. The expression of 30Kc6 inhibited apoptosis comparably to that of whole silkworm hemolymph, indicating that both intracellular expression and external supplementation inhibited apoptosis. The expression of 30Kc6 resulted in lower intracellular activity for caspase 3. However, the results of in vitro assay of caspase 3 show that the 30Kc6 protein does not inhibit caspase 3 activity. This indicates that the 30Kc6 protein inhibits the apoptosis by working in a further upstream event than caspase 3 activation.     

Mechanism of Inhibition of Vaccinia Virus Replication in Adenovirus-Infected HeLa Cells

The ability of vaccinia virus to replicate in HeLa cells which had been previously infected with adenovirus type 2 (Ad2) was studied in order to gain insight into the mechanism by which adenovirus inhibits the expression of host cell functions. Vaccinia virus was employed in these studies because it replicates in the cytoplasm, whereas Ad2 replicates in the nucleus of the cell. It was found that vaccinia deoxyribonucleic acid (DNA) synthesis is greatly inhibited in adeno-preinfected HeLa cells provided that vaccinia superinfection does not occur before 18 hr after adeno infection. The inhibition of vaccinia DNA synthesis can be traced to an inhibition of vaccinia protein synthesis and viral uncoating. Vaccinia ribonucleic acid (RNA) synthesis is not inhibited in adeno-preinfected cells, but the vaccinia RNA does not become associated with polysomes.     

Fowlpox virus encodes a Bcl-2 homologue that protects cells from apoptotic death through interaction with the proapoptotic protein Bak

Poxviruses are renowned for encoding numerous immunomodulatory proteins capable of undermining potent immune defenses. One effective barrier against infection is apoptosis, a process controlled at the mitochondria by pro- and antiapoptotic members of the highly conserved Bcl-2 family of proteins. Although poxviruses are known to encode an array of effective inhibitors of apoptosis, members of the Avipoxvirus genus, which includes fowlpox virus, encode proteins with Bcl-2 homology. Here, we show that FPV039, a fowlpox virus protein with limited Bcl-2 homology, inhibited apoptosis in response to a variety of cytotoxic stimuli, including virus infection itself. Similar to other antiapoptotic Bcl-2 proteins, FPV039 localized predominantly to the mitochondria in both human and chicken cells and protected human cells from tumor necrosis factor alpha-induced loss of the mitochondrial membrane potential. In addition, coimmunoprecipitation revealed that FPV039 interacted constitutively with the proapoptotic Bcl-2 protein, Bak, in both human and chicken cells. Concordantly, FPV039 also inhibited apoptosis induced by the transient overexpression of Bak. To confirm these results in the context of virus infection, we generated a recombinant vaccinia virus lacking F1L, the endogenous apoptotic inhibitor in vaccinia virus, and expressing FPV039. In the context of vaccinia virus infection, FPV039 retained the ability to localize to the mitochondria and interacted with Bak. Moreover, FPV039 prevented the activation of Bak and protected infected cells from apoptosis induced by staurosporine and virus infection. Together, our data indicate that FPV039 is a functional Bcl-2 homologue that inhibits apoptosis by neutralizing the proapoptotic Bcl-2 family member Bak.     

Isolation and characterization of an apoptosis-inhibiting component from the hemolymph of Bombyx mori.

Silkworm (Bombyx mori) hemolymph showed an apoptosis-inhibiting activity in insect cells (Sf 9) infected with baculovirus (AcNPV). The addition of silkworm hemolymph into the culture medium increased the host cell longevity due to its apoptosis-inhibition activity. Components with an apoptosis-inhibiting effect were purified from the silkworm hemolymph by heat treatment, gel-filtration chromatography, and ion-exchange chromatography. The component with highest activity was characterized by periodic acid-Schiff staining, isoelectric focusing, MALDI-TOF-mass spectrometry, and N-terminal sequencing and was found to be a nonglycosylated monomeric protein with a molecular weight of ca. 28,000 Da.     

Comparative proteomics analysis of silkworm hemolymph during the stages of metamorphosis via liquid chromatography and mass spectrometry

The silkworm is a lepidopteran insect that has an open circulatory system with hemolymph consisting of blood and lymph fluid. Hemolymph is not only considered as a depository of nutrients and energy, but it also plays a key role in substance transportation, immunity response, and proteolysis. In this study, we used LC‐MS/MS to analyze the hemolymph proteins of four developmental stages during metamorphosis. A total of 728 proteins were identified from the hemolymph of the second day of wandering stage, first day of pupation, ninth day of pupation, and first day as an adult moth. GO annotations and categories showed that silkworm hemolymph proteins were enriched in carbohydrate metabolism, proteolysis, protein binding, and antibacterial humoral response. The levels of nutrient, immunity‐related, and structural proteins changed significantly during development and metamorphosis. Some, such as cuticle, odorant‐binding, and chemosensory proteins, showed stage‐specific expression in the hemolymph. In addition, the expression of several antimicrobial peptides exhibited their highest level of abundance in the hemolymph of the early pupal stage. These findings provide a comprehensive proteomic insight of the silkworm hemolymph and suggest additional molecular targets for studying insect metamorphosis.     

A new inhibitor of apoptosis from vaccinia virus and eukaryotes

A new apoptosis inhibitor is described from vaccinia virus, camelpox virus, and eukaryotic cells. The inhibitor is a hydrophobic, multiple transmembrane protein that is resident in the Golgi and is named GAAP (Golgi anti-apoptotic protein). Stable expression of both viral GAAP (v-GAAP) and human GAAP (h-GAAP), which is expressed in all human tissues tested, inhibited apoptosis induced by intrinsic and extrinsic apoptotic stimuli. Conversely, knockout of h-GAAP by siRNA induced cell death by apoptosis. v-GAAP and h-GAAP display overlapping functions as shown by the ability of v-GAAP to complement for the loss of h-GAAP. Lastly, deletion of the v-GAAP gene from vaccinia virus did not affect virus replication in cell culture, but affected virus virulence in a murine infection model. This study identifies a new regulator of cell death that is highly conserved in evolution from plants to insects, amphibians, mammals, and poxviruses.     

Silkworm hemolymph as a substitute for fetal bovine serum in insect cell culture

Summary The effectiveness of silkworm hemolymph was investigated as a substitute for fetal bovine serum (FBS) in insect cell culture. Cells were adapted to grow in reduced FBS medium supplemented with silkworm hemolymph through a gradual adaptation process. FBS concentration in the medium could be reduced to 1% without decrease in cell growth rate and maximum cell concentration by adding 5% silkworm hemolymph.     

Increase of host cell longevity by the expression of 30K protein originating from silkworm hemolymph in an insect cell–baculovirus system

A recombinant baculovirus was constructed by the homologous recombination between wild-type AcMNPV DNA and a baculovirus transfer vector containing a gene coding for the 30K protein originating from silkworm hemolymph. The 30K protein was successfully expressed in Sf9 cells infected with the recombinant baculovirus (AcMNPV/30K). To investigate the effect produced by the expression of the 30K protein, host cell viability after infection was compared with that of Sf9 cells infected with AcMNPV/β-gal. The viability of the cells infected with AcMNPV/β-gal began to decrease exponentially 3 days after infection, whereas that of the cells infected with AcMNPV/30K remained at a high level until 5 days after infection. This indicates that the 30K protein increases cell longevity after viral infection. This increased cell longevity is considered to be due to the inhibition of host cell apoptosis induced by a baculovirus, and the extent of apoptosis was measured by the flow cytometric method. The percentage of the sub-G1 fraction, which represents the extent of apoptosis, was decreased by the expression of the 30K protein. This indicates that the expression of the 30K protein in insect cells increases host cell longevity by inhibiting apoptosis.     

High-level expression of orange fluorescent protein in the silkworm larvae by the Bac-to-Bac system

This novel orange fluorescent protein (OFP) emits brilliant orange fluorescent light. OFP has high fluorescence quantum yield, fast maturation rate, and stability, which imply this protein should be the most favorable biotechnological tools used to investigate the function of target gene by visualizing, monitoring, and quantifying in living cells. B. mori, silkworm has been used as an important bioreactor for the production of recombinant proteins through baculovirus expression system (BES). In this paper, we used infection technique which introduced the baculovirus DNA into silkworms using a cationic lipofectin reagent instead of directly injecting the virus, and demonstrated a high-level expression of the orange fluorescent protein (OFP) gene in the Bombyx mori, silkworm larvae. When recombinant rBacmid/BmNPV/OFP DNA ranging from 50–100 ng/larval was injected, a sufficient OFP expression in hemolymph was harvested. The recombinant viruses could be obtained from the hemolymph of infected larvae and stored as seed which could be used for the large-scale expression. This procedure omitted the costly and labor-consumed insect cell culture. Further investigation of OFP should provide us with more insight in unlocking the mystery of the mechanisms of autocatalytic bioluminescence and its utilization in biotechnology.     

Open reading frame 5 of porcine reproductive and respiratory syndrome virus as a cause of virus-induced apoptosis.

The gene product of open reading frame 5 (p25) of porcine reproductive and respiratory syndrome (PRRS) virus has been expressed by coinfection of culture cells with vaccinia virus expressing the T7 RNA polymerase and a recombinant vaccinia virus encoding the open reading frame 5 gene under the T7 promoter and the encephalomyocarditis virus internal ribosome entry site. In spite of the reported efficiency of the expression system, very poor accumulation of p25 protein was observed and a strong cytotoxicity was produced in the doubly infected cells. This cell toxicity was shown to occur by induction of apoptosis, as indicated by nucleosome ladder formation, chromatin condensation, and rRNA degradation. Apoptosis induction was also observed after infection of cultured cells with an adapted PRRS virus strain and after infection of swine macrophage cells with a PRRS virus field strain. Contrary to the observations made for other cases of virus-induced apoptosis, we could not prevent p25 protein-induced apoptosis by using a cell line permanently expressing Bcl-2 protein.