Bombyx Mori Nuclear Polyhedrosis Virus Infection Research Articles

Glucose-regulated protein 94 facilitates the proliferation of the Bombyx mori nucleopolyhedrovirus via inhibiting apoptosis

Glucose regulatory protein 94 (GRP94) is an endoplasmic reticulum (ER)-resident member of the heat shock protein 90 (HSP90) family, that plays an important role in secreted protein folding. Bombyx mori nuclear polyhedrosis virus (BmNPV) is one of the main pathogens in sericulture, causing serious economic losses every year. Previous studies showed that HSP90 members promote BmNPV replication in silkworm, but the function of BmGRP94 in BmNPV infection and proliferation is still not understood. In this study, we investigated the interplay between BmGRP94 and BmNPV infection in silkworm. We first identified a single gene of BmGRP94 in the Bombyx mori genome, which encodes a polypeptide with 810 amino acids in length. Spatio-temporal expression profiles showed that BmGRP94 was highly expressed in hemocytes and midgut, and was significantly induced by BmNPV infection. Furthermore, overexpression of BmGRP94 facilitates viral proliferation, while BmGRP94 inhibition evidently decreased BmNPV proliferation in BmN cells and in silkworm midgut. Mechanistically, BmGRP94 inhibition triggers ER stress, as judged by increased expression of PERK/ATF4/ERO1, H2O2 production, and ER calcium efflux, which promotes cell apoptosis to restrict BmNPV replication in silkworm. These results suggest that BmGRP94 plays an important role in facilitating BmNPV proliferation, and provides a potential molecular target for BmNPV prevention.

Bombyx mori nucleopolyhedrovirus downregulates transcription factor BmFoxO to elevate virus infection

Forkhead-box O (FoxO) is the primary transcriptional effector of the insulin-like signaling pathway that enhances gluconeogenesis through transcriptional activation of PEPCK and G6Pase in mammals. We have previously demonstrated the involvement of phosphoenolpyruvate carboxykinase (BmPEPCK-2) in antiviral immunity against the multiplication of Bombyx mori nuclearpolyhedrosisvirus (BmNPV) in silkworm. Therefore, we speculated that BmFoxO might suppress BmNPV by regulating the expression of PEPCK in silkworm. In the present study, we found that the expression of BmFoxO decreased after BmNPV infection in Bombyx mori; this finding was consistent with BmPEPCK-2 expression. In addition, the expression of BmFoxO was altered, and it was found that reduced expression of BmFoxO (dsBmFoxO) downregulated the expression of BmPEPCK-2 and increased the viral fluorescence and content in silkworm embryonic cell line BmE cells, and vice versa. BmFoxO could upregulate the expression of BmPEPCK-2 by binding to the BmPEPCK-2 promoter. Moreover, overexpression of BmFoxO significantly increased the expression of autophagy genes ATG6/7/8 after infection with BmNPV, consistent with BmPEPCK-2. These results indicate that BmNPV downregulates transcription factor BmFoxO to elevate virus infection, and BmFoxO overexpression upregulates BmPEPCK-2 expression and enhances silkworm antiviral resistance.

Increased expression of Suppressor of cytokine signaling 2 (BmSOCS2) is correlated with suppression of Bombyx mori nucleopolyhedrovirus replication in silkworm larval tissues and cells

The resistance of silkworm to infection by Bombyx mori nuclear polyhedrosis virus (BmNPV) is a main focus of sericultural research. Previously, a BmNPV-resistant strain, NB, was identified among a collection of Chinese silkworm strains in our lab. To better understand the molecular mechanism of NB strain resistance, the patterns of host immune response gene transcription in resistant (NB) and susceptible (306) strains were examined. Quantative real-time PCR (qRT-PCR) revealed that multiple insect innate immune signaling pathways (Toll, Imd and JAK/STAT) were strongly activated upon infection with BmNPV. Notably, Suppressor of cytokine signaling 2 (BmSOCS2) mRNA expression was significantly up-regulated in midgut tissues of the resistant NB strain, suggesting that the BmSOCS2 gene product may be involved in host immune defense against BmNPV infection. A significant inhibition of BmNPV replication was also observed in BmN cells transfected with a vector encoding BmSOCS2. The results suggest that BmSOCS2 is a key gene involved in the resistance of the NB silkworm strain to BmNPV infection.

Virus induced global protein synthesis shutdown in Antheraea mylitta infected with Bombyx mori nuclear polyhedrosis virus

An abortive infection system was studied involving Antheraea mylitta and Bombyx mori nuclear polyhedrosis virus (BmNPV). SDS-PAGE of healthy and infected A. mylitta silkworm's hemolymph proteins was analysed and mortality rate was used as index to verify infection. In A. mylitta cells, BmNPV infection resulted in a severe global protein synthesis shutdown during early post infection i.e. within 24 hrs. These results indicate that the restriction of BmNPV multiplication in A. mylitta is caused by a global protein synthesis shutdown resulting in non-productive infection evident from our observation that there was no significant mortality of A. mylitta silkworms administered with BmNPV.

Role of Toll-receptors in the inhibition of BmNPV proliferation and their interplay with antimicrobial seroin proteins in Bombyx mori

The domesticated silkworm, Bombyx mori, is an economically important insect that majorly contributes in financially viable sericulture industry. The baculovirus, Bombyx mori nuclear polyhedrosis virus (BmNPV) is a deadly virus that infects B. mori and inflicts heavy loss to silk production by causing high mortality of silkworms. A number of studies have been done to identify antiviral genes and pathways against BmNPV infection in B. mori . Toll-pathway is a well-known immune pathway responsible for combating bacterial and fungal infections in silkworm. However, the role of Toll-receptors in inhibiting BmNPV has not been explored. In the present study, we have attempted to examine the role of Toll-receptors in inhibiting BmNPV proliferation in B. mori larvae. The effect of Toll-receptors on viral proliferation was determined by quantifying viral DNA and counting viral occlusion bodies upon RNA interference-mediated knockdown of Toll-receptors in B. mori larvae. Knockdown of BmToll, BmToll-6, BmToll-7, BmToll-8 and BmToll-9 led to higher accumulation of BmNPV. Further, interplay between Toll-receptors and the antimicrobial proteins of B. mori, called seroins were studied using RNA interference. As a result, we found a downregulation of Seroin1 transcript level upon knockdown of BmToll , BmToll-7 , BmToll-8 , BmToll-10 , BmToll-11 and BmToL K2, whereas Seroin2 expression was found to decrease only upon BmToll-7 knockdown. Moreover, knockdown of seroins did not result in clearly interpretable expression patterns of Toll-receptors. These results suggest the involvement of B. mori Toll-pathway in defense against BmNPV infection and provides a new insights into the antiviral defense mechanism of B. mori. • BmNPV proliferation increases upon knockdown of B. mori Toll-receptors in larvae. • Knockdown of B. mori Toll-7 significantly enhances viral load. • Seroins are likely to function downstream of Toll-7-mediated signaling pathway in B. mori.

Label-free proteomic analysis of silkworm midgut infected by Bombyx mori nuclear polyhedrosis virus

Bombyx mori nuclear polyhedrosis virus (BmNPV) is the most damaging virus for the production of silkworm cocoons. Antivirus research continues to be an important aspect of the silkworm industry. Two-dimensional electrophoresis and mass spectrometry have been applied for analyzing the midgut proteome of BmNPV-infected silkworms. In recent years, the isobaric tags for relative and absolute quantitation (iTRAQ) method has frequently been used when studying interaction between BmNPV and Bombyx mori, and useful information has been obtained. In this study, midgut proteins of BmNPV-infected silkworms were extracted from silkworm variety NIL·LVR with anti-BmNPV activity at 48 h, and proteome analysis was carried out using the label-free method. 2196 proteins were identified. Among them, there were 85 differentially expressed proteins, 45 upregulated proteins (immune-activated proteins), 28 downregulated proteins, and six proteins were specific for the BmNPV group and another six specific for control group. Many of the immune-activated proteins have been reported to have innate immune functions, and the downregulated proteins are involved in apoptosis or abnormal cell viability. In conclusion, this study provides evidence for host defense against BmNPV infection by both innate immunity and apoptosis, revealing the potential function of the midgut after oral infection of BmNPV in Bombyx mori. SignificanceBombyx mori nuclear polyhedrosis virus (BmNPV) has a great impact on the sericulture industry. However, the mechanism of resistance to BmNPV has not been fully elucidated. The silkworm midgut is not only the major organ for food digestion and nutrient absorption but also an immune organ serving as the first line of defense against microbial invasion and proliferation. Here we combined label-free quantitative proteomic, bioinformatics, quantitative real-time PCR and SDS-PAGE analyses and found that BmNPV invasion causes complex protein alterations in the larval midgut of NIL·LVR with anti-BmNPV activity. The results showed that many upregulated differentially expressed proteins have been reported to have innate immune functions and the downregulation proteins are involved in apoptosis or abnormal cell viability. These findings provide evidence for host defense against BmNPV infection by both innate immunity and apoptosis, and reveals the potential function of the midgut after infection of BmNPV in Bombyx mori.

Bombyx mori Nuclear Polyhedrosis Virus (BmNPV) Induces Host Cell Autophagy to Benefit Infection.

Bombyx mori nuclear polyhedrosis virus (BmNPV) is an important pathogen of silkworms. Despite extensive studies in recent decades, the interaction between BmNPV and host cells is still not clearly understood. Autophagy is an intrinsic innate immune mechanism and it controls infection autonomously in virus-infected cells. In this study, we found that BmNPV infection could trigger autophagy, as demonstrated by the formation of autophagosomes, fluorescent Autophagy-related gene 8-Green Fluorescent Protein (ATG8-GFP) punctate, and lipidated ATG8. Meanwhile, autophagic flux increased significantly when monitored by the ATG8-GFP-Red Fluorescent Protein (RFP) autophagy tandem sensor and protein degradation of p62. In addition, almost all of the identified autophagy-related genes (Atgs) had been up-regulated post infection in mRNA levels. Then, we screened Atgs with the greatest fold-change during virus infection. Interestingly, all of the screened Atgs positively regulated the expression of virus genes. Further studies showed that Atg7 and Atg9 could contribute to the level of autophagy caused by viral infection. Our results demonstrated that BmNPV induced host cell autophagy to benefit its infection. These results offer insight into the complex interactions between virus and host cell, and viral pathogenesis.

Transformed lepidopteran cells expressing a protein of the silkmoth fat body display enhanced susceptibility to baculovirus infection and produce high titers of budded virus in serum-free media

Baculovirus vectors constitute important tools for therapeutic protein production and mammalian cell transduction for gene therapy applications. A prerequisite for such applications is that the cell lines in which baculoviruses are propagated be maintained in serum-free media that are devoid of potential human pathogens. However, in serum-free media, the performance of baculovirus-based systems can be significantly reduced. In this report, we show that silkmoth-derived host cell lines for the Bombyx mori-nuclear polyhedrosis virus (BmNPV) that are transformed with the gene for the promoting protein (PP), a silkmoth-derived secreted factor containing a lipid-binding domain, display enhanced susceptibility to BmNPV infection and enhanced budded virus productivity in serum-free media. For transformed silkmoth cells maintained in serum-free media, the rate of BmNPV entry is enhanced by two orders of magnitude relative to the untransformed cells, while the rate of budded virus production is increased five-fold. The infectivity-enhancing effect can be also conferred to normal cells grown in serum-free media by addition of conditioned media from the transformed cells, which contain the secreted recombinant PP. Thus, PP substitutes for serum factors whose presence facilitates baculovirus entry into the cells. However, the effects of silkmoth-derived PP may be specific to the BmNPV–silkmoth system since little or no changes in viral infectivity are obtained by PP expression in Trichoplusia ni-derived High-Five™ cells grown in serum-free media and infected with a different baculovirus (AcNPV).

Fluorescent differential display analysis of gene expression for NPV resistance in Bombyx mori L.

Using the fluorescent differential display (FDD) technique, we analysed the differential expression of genes related to BmNPV (Bombyx mori nuclear polyhedrosis virus) resistance. Silkworm strains used included a highly resistant strain named NB, a highly susceptible strain named 306 and a near-isogenic line named 306NNZZ. Two differential bands, G12 532 and G13 313 , were found linked to BmNPV resistance and were further identified. Band G12 532 was found in the midguts of the eighth generation of backcross (BC 8 ) of strain 306NNZZ but was not present in those of strain 306. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis confirmed that the band existed in BC 8 of strains 306NNZZ and NB while not in strain 306. Northern blot analysis showed that G12 532 expressed actively in BC 8 of strains 306NNZZ and NB while expressed inactively in strain 306. These results suggest that the G12 532 is a fragment of the gene related to silkworm's resistance to BmNPV disease. The sequence of G12 532 has an 85% similarity to sequences with GenBank accession numbers of AV398077 and AV398034 that are relative to BmNPV infection demonstrated by previous studies. Its coding region (37 amino acids) has 54% similarity with B. mori endonuclease and reverse transcriptase-like protein. Band G13 313 was appeared in the hemolymph of highly resistant strain NB and near isogenic line 306NNZZ in FDD, but was not found in highly susceptible strain 306, all of which were confirmed by RT-PCR and Northern blot analyses. All the experimental results obtained suggest that G13 313 is also a fragment of the gene related to BmNPV resistance. The sequence of band G13 313 does not have a high similarity to the above relative sequences in GenBank. It might be a new RNA fragment related to NPV resistance. Our study also showed that the FDD technique could be a promising approach in identifying novel genes while appropriate silkworm strains are used.

Use of an N-terminal half truncated IE1 as an antagonist of IE1, an essential regulatory protein in baculovirus

An immediate-early gene product of baculovirus, IE1, is essential for viral gene expression and for viral DNA replication. It has been demonstrated for Autographa californica nuclear polyhedrosis virus (AcNPV) that the C-terminal region of IE1 is required for dimerization. And the acidic N-terminal region of IE1 has been identified as the activation domain. We constructed an N-terminal 267 amino acid (a.a.) truncated mutant of Bombyx mori nuclear polyhedrosis virus (BmNPV) IE1, which was defective as a transactivator of a viral early gene ( p35) promoter. We then examined possible IE1 antagonistic functions of this defective IE1, IE1TN, in BmNPV-infected cells. A transient expression experiment demonstrated that IE1TN strongly repressed the activation of the hr5-dependent p35 promoter derived from BmNPV infection. In addition, DpnI assay elucidated an inhibitory effect of IE1TN on the hr5-dependent replication of plasmid in BmN cells induced by NPV infection. A marked reduction in the production of virus was observed when the BmN cells were infected with BmNPV after transfection with IE1TN-expression plasmids. These results suggested that IE1TN could act as an IE1 antagonist in silkworm cells infected with BmNPV. We then analyzed the ability of IE1TN to inhibit the multiplication of BmNPV using transgenic silkworms. The BmNPV-resistance of the transgenic silkworms was very weak, suggesting insufficient expression of the transgene product, IE1TN.

Effect of alkalinity and protease in the digestive juice of silkworm, Bombyx mori on BmNPV infection

Two breeds of Bombyx mori, one resistant (Nistari) to BmNPV (Bombyx mori nuclear polyhedrosis virus) and the other sensitive (Hosa Mysore), were inoculated with the virus, and changes in the levels of alkalinity and the level of alkaline protease activity were estimated separately at different time intervals. The midgut epithelial cells and fat bodies were also examined at transmission electron microscope at different intervals after inoculation with BmNPV to understand the mode of entry and multiplication of the virus. The alkaline protease activity in the midgut of the tolerant breed showed a sharp increase up to three days after inoculation, which was significantly higher than the increase in the fore‐gut and hindgut and than that of the sensitive breed. The ultra‐structural studies showed that the invasion and multiplication of the virus take place only in the sensitive breed. The study indicated that the tolerance to BmNPV infection is positively correlated with alkaline protease activity, and that the enzyme is more active in the midgut of the tolerant breed.

Protein synthesis in BM-N cells infected with Bombyx mori nuclear polyhedrosis virus

Polypeptide synthesis in the BM-N cells infected with Bombyx mori nuclear polyhedrosis virus (BmNPV) was examined. At least 26 virus-induced polypeptides were sequentially detected during BmNPV infection when cell extracts pulse-labeled with [ 35S]methionine for 3 hr were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by fluorography. Three virus-induced polypeptides were first detected at 6 hr postinfection (pi) and the synthesis of the majority of virus-induced polypeptides (18 of 26 polypeptides) commenced at 12–18 hr pi. No additional virus-induced polypeptides were detected at 30 hr pi or later. Immunoprecipitation experiments showed that at least 20 virus-induced polypeptides, two of which being unidentified in the pulse labeling experiment without immunoprecipitation, were related to the structural polypeptides of BmNPV virions. Pulse-chase experimets initiated at various times pi showed that at least 12 virus-induced polypeptides disappeared or reduced their radioactivity and at least 16 polypeptides appeared or increased their radioactivity during the chase periods up to 48 hr, suggesting that a number of virus-induced polypeptides underwent post-translational modification. Pulse labeling with N-acetyl-[ 14C]glucosamine and [ 32P]orthophosphate revealed that 6 virus-induced polypeptides were glycosylated and 10 virus-induced polypeptides phosphorylated. In agreement with the results in other NPV species, the polyhedrin of BmNPV were characterized to be a glycophosphoprotein.