-
[show abstract]
[hide abstract]
ABSTRACT: Ld652Y cells from the gypsy moth, Lymantria dispar, are extremely sensitive to various apoptotic stimuli, whereas BM-N cells from the silkworm, Bombyx mori, are relatively resistant to apoptotic stimuli. We previously cloned and characterized a B. mori homologue (bm-dronc) of Drosophila melanogaster dronc. In the present study, we cloned and characterized an L. dispar homologue of dronc (ld-dronc) comparatively with Bm-Dronc. The open reading frame of ld-dronc consisted of 1329 bp that was predicted to encode a 443 amino-acid polypeptide with a molecular mass of 50,706 Da and 54% to 57% amino acid sequence identity with Dronc homologues from other lepidopteran insects identified to date. Ld-Dronc had a long prodomain, large p20 domain, and small p10 domain, and a catalytic site composed of (308)QTCRG(312), which was distinct from the sites QACRG in Bm-Dronc and QMCRG in Dronc homologues of several other lepidopteran insects. Transiently expressed Ld-Dronc underwent proteolytic processing in the lepidopteran cell lines L. dispar Ld652Y, Spodoptera frugiperda Sf9, and B. mori BM-N, and dipteran D. melanogaster S2, but only triggered apoptosis in the lepidopteran cell lines. Endogenous Ld-Dronc underwent processing in Ld652Y cells upon infection with vAcΔp35, but not in mock-infected Ld652Y cells, supporting the involvement of Ld-Dronc in apoptosis induction. In vAcΔp35-infected apoptotic cells, Ld-Dronc underwent proteolytic processing more rapidly and extensively than Bm-Dronc. Similar results were obtained for Ld-Dronc and Bm-Dronc expressed transiently in S2, Ld652Y, Sf9, and BM-N cells. Taken together, these findings suggest that the intrinsic properties of Dronc proteins are responsible, at least in part, for the differing sensitivity of Ld652Y and BM-N to apoptosis induction upon NPV infection.
Biochemical and Biophysical Research Communications 06/2013; · 2.48 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Innate immunity is essential for insects to survive infectious pathogens. In baculovirus-infected lepidopteran cells, apoptosis and global protein synthesis shutdown are major mechanisms of intracellular innate immunity that inhibit viral replication. In contrast, baculoviruses have evolved diverse genes and mechanisms to counter the antiviral immunity activated in infected cells. In this review, we summarize the current knowledge of the cellular antiviral pathways and the baculovirus genes that modulate antiviral immunity. The studies highlighted illustrate a high degree of diversity in both the cellular responses against viral infections and viral responses against intracellular antiviral immunity, providing an important basis of further studies in this field.
Virology 01/2013; 435(1):1-13. · 3.35 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Ld652Y cells derived from the gypsy moth, Lymantria dispar, are permissive for productive infection with L. dispar multiple nucleopolyhedrovirus (LdMNPV), but undergo apoptosis upon infection with various other NPVs, including those isolated from Bombyx mori, Hyphantria cunea, Spodoptera exigua, Orgyia pseudotsugata, and Spodoptera litura. In this study, we examined whether LdMNPV-encoded inhibitor of apoptosis 2 (Ld-IAP2) and 3 (Ld-IAP3) are involved in apoptosis suppression in LdMNPV-infected Ld652Y cells. We found that neither Ld-IAP2 nor Ld-IAP3 was able to suppress the apoptosis of Ld652Y cells induced by p35-defective Autographa californica MNPV (vAcΔp35). However, both Ld-IAP2 and Ld-IAP3 induced apoptosis in Ld652Y cells in a transient expression assay. The apoptosis induced by Ld-IAP3 was accompanied by the stimulation of caspase-3-like protease activity and cleavage of the B. mori homolog of the initiator caspase Dronc, and was precluded by the LdMNPV-encoded apoptosis suppressor protein Apsup and H. cunea MNPV IAP3. Inconsistent with the results obtained previously in SpIm, Ld652Y and High Five cells infected with NPVs from H. cunea, O. pseudotsugata, and A. californica, respectively, considerable stimulation of caspase-3-like protease activity was not observed in LdMNPV-infected Ld652Y cells, likely due to the strong apoptosis suppression activity of Apsup. These results, together with the previous finding that RNAi-mediated silencing of apsup induces apoptosis of LdMNPV-infected Ld652Y cells, indicate that Apsup, but not Ld-IAP2 or Ld-IAP3, is primarily responsible for the suppression of apoptosis in LdMNPV-infected Ld652Y cells. However, it remains inconclusive whether Ld-IAP2 and Ld-IAP3 function as pro-apoptotic proteins in LdMNPV-infected Ld652Y cells.
Virus Genes 07/2012; 45(2):370-9. · 1.85 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We cloned and characterized a novel Bombyx mori homologue (bm-dronc) of Drosophila melanogaster dronc (dm-dronc), which could encode a polypeptide of 438 amino acid residues. Bm-Dronc shares relatively low amino acid sequence identities of 25% and 26% with Dm-Dronc and Aedes aegypti Dronc (Aa-Dronc), respectively. Bm-Dronc has the sequence QACRG surrounding the catalytic site (C), which is consistent with the QAC(R/Q/G)(G/E) consensus sequence in most caspases but distinct from the sequences PFCRG and SICRG of Dm-Dronc and Aa-Dronc, respectively. Bm-Dronc possesses a long N-terminal prodomain containing a caspase recruitment domain (CARD), a p20 domain and a p10 domain, exhibiting cleavage activities on synthetic substrates Ac-VDVAD-AMC, Ac-IETD-AMC and Ac-LEHD-AMC, which are preferred by human initiator caspases-2, -8 and -9, respectively. Bm-Dronc transiently expressed in insect cells and Escherichia coli cells underwent spontaneous cleavage and caused apoptosis and stimulation of caspase-3-like protease activity in various lepidopteran cell lines, but not in the dipteran cell line D. melanogaster S2. The apoptosis and the stimulation of caspase-3-like protease activity induced by Bm-Dronc overexpression were abrogated upon transfection with either a double-stranded RNA against bm-dronc or a plasmid expressing functional anti-apoptotic protein Hycu-IAP3 encoded by the baculovirus Hyphantria cunea multiple nucleopolyhedrovirus (MNPV). Apoptosis induction in BM-N cells by infection with a p35-defective Autographa californica MNPV or exposure to actinomycin D and UV promoted the cleavage of Bm-Dronc. These results indicate that Bm-Dronc serves as the initiator caspase responsible for the induction of caspase-dependent apoptosis.
Insect biochemistry and molecular biology 09/2011; 41(11):909-21. · 3.25 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Baculoviruses encode inhibitors of apoptosis (IAPs), which are classified into five groups, IAP1-5, based on their sequence homology. Most of the baculovirus IAPs with anti-apoptotic functions belong to the IAP3 group, with certain exceptions. The functional roles of IAPs from other groups during virus infection have not been well established. We have previously shown that Hyphantria cunea multiple nucleopolyhedrovirus (HycuMNPV) encodes three iap genes, hycu-iap1, hycu-iap2 and hycu-iap3, and that only Hycu-IAP3 has anti-apoptotic activity against actinomycin D-induced apoptosis of Spodoptera frugiperda Sf9 cells. In the present study, we demonstrate that transient expression of Hycu-IAP1 is capable of inducing apoptosis and/or stimulating caspase-3-like protease activity in various lepidopteran and dipteran cell lines. Transient-expression assay analysis also demonstrates that not only Hycu-IAP1 but also IAP1s from Autographa californica MNPV, Bombyx mori NPV and Orgyia pseudotsugata MNPV (OpMNPV) are capable of inducing apoptosis, and that apoptosis induced by Hycu-IAP1 is precluded by the functional anti-apoptotic baculovirus protein Hycu-IAP3. In HycuMNPV-infected Spilosoma imparilis (SpIm) cells and OpMNPV-infected Ld652Y cells, caspase-3-like protease activity is markedly stimulated during the late stages of infection, and the caspase-3-like protease activity stimulated in HycuMNPV-infected SpIm cells is repressed by RNA interference-mediated silencing of hycu-iap1. In addition, initiator caspase Bm-Dronc, the B. mori homologue of Dronc, is cleaved upon transfection of BM-N cells with a plasmid expressing Hycu-IAP1. These results provide the first evidence that baculovirus IAP1s act to induce caspase-dependent apoptosis, possibly by replacing the cellular IAP1 that prevents Dronc activation.
Journal of General Virology 07/2011; 92(Pt 11):2654-63. · 3.36 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Ld652Y cells from Lymantria dispar readily undergo apoptosis upon infection with a variety of nucleopolyhedroviruses (NPVs), while L. dispar multicapsid NPV (LdMNPV) infection of Ld652Y cells results in the production of a high titer of progeny viruses. Here, we identify a novel LdMNPV apoptosis suppressor gene, apsup, which functions to suppress apoptosis induced in Ld652Y cells by infection with vAcΔp35, a p35-defective recombinant Autographa californica MNPV. apsup also suppresses apoptosis of Ld652Y cells induced by actinomycin D and UV exposure. Apsup is expressed in LdMNPV-infected Ld652Y cells late in infection, and RNA interference-mediated apsup ablation induces apoptosis of LdMNPV-infected Ld652Y cells.
Journal of Virology 03/2011; 85(10):5237-42. · 5.40 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We previously demonstrated that the Hyphantria cunea multicapsid nucleopolyhedrovirus (HycuMNPV) gp64 gene (hycu-gp64) is uniquely localized on the viral genome with a large homologous region of 1582bp, hycu-hr6, immediately upstream of the hycu-gp64 gene. In the present study, we compared the regulation of gp64 early promoters from HycuMNPV, Autographa californica multicapsid NPV (AcMNPV) and Bombyx mori NPV (BmNPV) by cis-acting hycu-hr6 and trans-acting IE1s in three cell lines (Spodoptera frugiperda Sf9, Bombyx mori BM-N and Spilosoma imparilis SpIm). A transient expression assay with plasmids harboring a reporter luciferase gene demonstrated that the gp64 early promoters are positively regulated by hycu-hr6, independent of virus and cell types. In contrast, gp64 early promoters were regulated positively or negatively by trans-acting IE1s, in a cell- and virus-type dependent manner, indicating that cellular factors, as well as viral factors, are responsible for IE1-dependent regulation of gp64 early promoters. However, hycu-gp64 early promoter activity was consistently suppressed by HycuMNPV IE1 (Hycu-IE1), irrespective of the cell lines used. Analysis of the hycu-gp64 early promoter region revealed two novel sequence elements that were involved in Hycu-IE1-dependent negative regulation of the hycu-gp64 early promoter. These two novel regulatory sequence elements could compensate for each other but could not be substituted with AcMNPV IE1 binding motif (Ac-IBM). These results suggest that IE1 regulates gp64 early promoters to produce the proper amount of GP64 protein, depending upon NPV-insect cell systems.
Virus Research 01/2011; 155(1):83-90. · 2.94 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We have previously shown that budded viruses of Bombyx mori nucleopolyhedrovirus (BmNPV) enter the cell cytoplasm but do not migrate into the nuclei of non-permissive Sf9 cells that support a high titer of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) multiplication. Here we show, using the syncytium formation assay, that low-pH-triggered membrane fusion of BmNPV GP64 protein (Bm-GP64) is significantly lower than that of AcMNPV GP64 protein (Ac-GP64). Mutational analyses of GP64 proteins revealed that a single amino acid substitution between Ac-GP64 H155 and Bm-GP64 Y153 can have significant positive or negative effects on membrane fusion activity. Studies using bacmid-based GP64 recombinant AcMNPV harboring point-mutated ac-gp64 and bm-gp64 genes showed that Ac-GP64 H155Y and Bm-GP64 Y153H substitutions decreased and increased, respectively, the multiplication and cell-to-cell spread of progeny viruses. These results indicate that Ac-GP64 H155 facilitates the low-pH-triggered membrane fusion reaction between virus envelopes and endosomal membranes.
Virology 09/2010; 404(2):204-14. · 3.35 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We previously demonstrated that Bombyx mori nucleopolyhedrovirus (BmNPV) multiplication is restricted in permissive BmN-4 cells upon coinfection with Hyphantria cunea NPV (HycuNPV). Here, we show that HycuNPV-encoded hycu-ep32 gene is responsible for the restricted BmNPV multiplication in HycuNPV-coinfected BmN-4 cells. The only homologue for hycu-ep32 is in Orgyia pseudotsugata NPV. hycu-ep32 could encode a polypeptide of 312 amino acids, and it contains no characteristic domains or motifs to suggest its possible functions. hycu-ep32 is an early gene, and Hycu-EP32 expression reaches a maximum by 6 h postinfection. hycu-ep32-defective HycuNPV, vHycuDeltaep32, was generated, indicating that hycu-ep32 is nonessential in permissive SpIm cells. In BmN-4 cells, HycuNPV infection resulted in a severe global protein synthesis shutdown, while vHycuDeltaep32 did not cause any specific protein synthesis shutdown. These results indicate that the restriction of BmNPV multiplication by HycuNPV is caused by a global protein synthesis shutdown induced by hycu-ep32 upon coinfection with HycuNPV.
Virology 03/2010; 398(2):149-57. · 3.35 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Lepidopteran baculovirus-specific protein FP25K performs many roles during the infection cycle, including functions in the production of occlusion bodies (OBs) and budded viruses (BVs), oral infection, and postmortem host degradation. To explore the common and specific functions of FP25K proteins among lepidopteran baculoviruses, we performed comparative analyses of FP25K proteins from group I and group II nucleopolyhedroviruses (NPVs) and granulovirus (GV). Using recombinant Bombyx mori NPVs (BmNPVs), we showed that the FP25Ks from NPVs were able to eliminate all the phenotypic defects observed in an infection with a BmNPV mutant lacking functional fp25K but that FP25K from GV did not show abilities to recover oral infectivity and postmortem host degradation. We also observed that introduction of Autographa californica multiple NPV (AcMNPV) fp25K into the BmNPV genome enhanced OB and BV production. According to these results, we generated a novel BmNPV-based expression vector with AcMNPV fp25K and examined its potential in BmN cells and B. mori larvae. Our results showed that the introduction of AcMNPV fp25K significantly increases the expression of foreign gene products in cultured cells and shortens the time for obtaining the secreted recombinant proteins from larval hemolymph.
Journal of Virology 03/2010; 84(10):5191-200. · 5.40 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We previously identified a large homologous region (hr), hycu-hr6, in the genome of the Hyphantria cunea nucleopolyhedrovirus (HycuNPV) and suggested that hycu-hr6 was the largest baculovirus promoter enhancer hr identified so far. In this study, we examined the enhancement activity of hycu-hr6 against two promoters from constitutive baculovirus immediate early genes, the HycuNPV ie1 (hycu-ie1) and the Orgyia pseudotsugata multicapsid (M) NPV ie2 (op-ie2), and against a promoter from the inducible Drosophila heat shock protein 70 gene (hsp70) in five lepidopteran (BmN-4, Ld652Y, Sf9, SpIm, and TN368) and one dipteran (S2) insect cell lines. Comparative analyses of transient expression assays using the firefly luciferase gene (luc) as a reporter showed that hycu-hr6 enhanced the activity of all three promoters in all tested cell lines. Comparison of the enhancement efficiency of hycu-hr6 with that of Autographa californica MNPV (AcMNPV) hr5, an enhancer commonly used to improve the activity of AcMNPV ie1 promoter in many insect expression vectors, established that hycu-hr6 was a more efficient enhancer. These results indicate that hycu-hr6 is a versatile and superior promoter enhancer in several insect cell lines and favor the incorporation of hycu-hr6 into insect expression vectors to maximize promoter activity.
Virus Genes 10/2009; 39(3):403-8. · 1.85 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A newly cloned Helicoverpa armigera nucleopolyhedrovirus (HearNPV) from Kenya, HearNPV-NNg1, has a higher insecticidal activity than HearNPV-G4, which also exhibits lower insecticidal activity than HearNPV-C1. In the search for genes and/or nucleotide sequences that might be involved in the observed virulence differences among Helicoverpa spp. NPVs, the entire genome of NNg1 was sequenced and compared with previously sequenced genomes of G4, C1 and Helicoverpa zea single-nucleocapsid NPV (Hz). The NNg1 genome was 132,425 bp in length, with a total of 143 putative open reading frames (ORFs), and shared high levels of overall amino acid and nucleotide sequence identities with G4, C1 and Hz. Three NNg1 ORFs, ORF5, ORF100 and ORF124, which were shared with C1, were absent in G4 and Hz, while NNg1 and C1 were missing a homologue of G4/Hz ORF5. Another three ORFs, ORF60 (bro-b), ORF119 and ORF120, and one direct repeat sequence (dr) were unique to NNg1. Relative to the overall nucleotide sequence identity, lower sequence identities were observed between NNg1 hrs and the homologous hrs in the other three Helicoverpa spp. NPVs, despite containing the same number of hrs located at essentially the same positions on the genomes. Differences were also observed between NNg1 and each of the other three Helicoverpa spp. NPVs in the diversity of bro genes encoded on the genomes. These results indicate several putative genes and nucleotide sequences that may be responsible for the virulence differences observed among Helicoverpa spp., yet the specific genes and/or nucleotide sequences responsible have not been identified.
Virus Genes 08/2009; 39(2):261-72. · 1.85 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Bombyx mori nucleopolyhedrovirus (BmNPV) fibroblast growth factor (BmFGF) is a glycosylated protein that is efficiently secreted into the medium. Here, we constructed mutant NPVs expressing His-tagged wild-type (wt) or mutant BmFGFs and showed that the two residues, asparagine 44 and 171, are the glycosylation sites of BmFGF. Also, removal of N-linked glycans from BmFGF resulted in almost complete inhibition of the secretion into the medium, suggesting that N-linked glycans of BmFGF are required for its secretion. These residues are not conserved in closely related Autographa californica NPV (AcMNPV)-encoded vFGF (AcFGF). Western blot analysis suggested that AcFGF is not glycosylated and is poorly secreted. A mutant AcFGF possessing two N-linked glycosylation sites was secreted into the medium more abundantly than that which occurred for wt AcFGF. This is the first direct evidence showing the role of N-linked glycans in the secretion process of a baculovirus protein.
Biochemical and Biophysical Research Communications 01/2007; 350(4):1069-75. · 2.48 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The whole-genome sequence of the Hyphantria cunea nucleopolyhedrovirus (HycuNPV) was analysed. The entire nucleotide sequence of the HycuNPV genome was 132 959 bp long, with a G+C content of 45.1 mol%. A total of 148 open reading frames (ORFs) consisting of more than 50 aa were encoded by the genome. HycuNPV shares more than 122 ORFs with other lepidopteran group I NPVs, including Autographa californica MNPV, Bombyx mori NPV, Choristoneura fumiferana MNPV (CfMNPV), Choristoneura fumiferana defective NPV, Epiphyas postvittana MNPV and Orgyia pseudotsugata MNPV (OpMNPV). Six ORFs are identified as being unique to HycuNPV. Most of the HycuNPV ORFs showed higher similarity to CfMNPV and OpMNPV ORFs than to those of the other group I NPVs. HycuNPV encodes two conotoxin-like homologues (ctls), which are observed only in OpMNPV in group I NPVs. HycuNPV encodes three inhibitors of apoptosis (iaps), hycu-iap-1, hycu-iap-2 and hycu-iap-3, a feature that it shares only with CfMNPV. In addition, six homologous regions (hrs) are identified in the HycuNPV genome. These hrs are located in regions similar to those of the OpMNPV hrs, but different from most of the CfMNPV hrs. Based on the close phylogenetic relationship and conservation of group I NPV-specific genes, such as gp64, ie-2 and ptp-1, it is concluded that HycuNPV belongs to the group I NPVs and is most similar to CfMNPV or OpMNPV.
Journal of General Virology 10/2006; 87(Pt 9):2549-62. · 3.36 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Despite close genetic relationship, Bombyx mori nucleopolyhedrovirus (BmNPV) and Autographa californica multicapsid NPV (AcMNPV) display a distinct host range property. Here, BmNPV replication was examined in Sf9 and High Five cells that were nonproductive for BmNPV infection but supported high titers of AcMNPV replication. Recombinant BmNPV, vBm/gfp/lac, containing bm-ie1 promoter-driven egfp showed that few Sf9 and High Five cells infected with vBm/gfp/lac expressed EGFP, while large proportion of EGFP-expressing cells was observed when transfected with vBm/gfp/lac DNA. Immunocytochemical analysis showed that BmNPV was not imported into the nucleus of these two cell lines, while recombinant BmNPV, vBmDelta64/ac-gp64 possessing AcMNPV gp64 was imported into the nucleus, yielding progeny virions in High Five cells, but not Sf9 cells. These results indicate that the defective nuclear import of infected virions due to insufficient BmNPV GP64 function is involved in the restricted BmNPV replication in Sf9 and High Five cells.
Virology 05/2006; 347(2):455-65. · 3.35 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Host range factor 1 (HRF-1) of Lymantria dispar multinucleocapsid nucleopolyhedrovirus promotes Autographa californica MNPV replication in nonpermissive Ld652Y cells derived from L. dispar. Here we demonstrate that restricted Hyphantria cunea NPV replication in Ld652Y cells was not due to apoptosis but was likely due to global protein synthesis arrest that could be restored by HRF-1. Our data also showed that HRF-1 promoted the production of progeny virions for two other baculoviruses, Bombyx mori NPV and Spodoptera exigua MNPV, whose replication in Ld652Y cells is limited to replication of viral DNA without successful production of infectious progeny virions. Thus, HRF-1 is an essential viral factor required for productive infection of NPVs in Ld652Y cells.
Journal of Virology 12/2004; 78(22):12703-8. · 5.40 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The accumulation of cellular proliferating cell nuclear antigen (PCNA) in the nucleus of Sf9 cells has been shown to increase upon infection with Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). Here, analysis by DNase I treatment and chromatin immunoprecipitation revealed that cellular PCNA in the nucleus of Sf9 cells bound AcMNPV DNA. Immunocytochemical analysis showed colocalization of Sf9 cell PCNA and AcMNPV DNA replication sites. Similar colocalization was also observed in BmN-4 cells infected with Bombyx mori NPV, which is inherently missing the pcna gene. The amount of cellular PCNA associated with viral DNA replication sites was greater in cells infected with pcna-defective AcMNPV mutants than in cells infected with wild-type AcMNPV. These results suggest that both cellular and viral PCNAs are involved in AcMNPV DNA replication and that pcna-defective AcMNPV mutants are able to substitute cellular PCNA for viral PCNA.
Journal of General Virology 11/2004; 85(Pt 10):2857-62. · 3.36 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Hyphantria cunea nucleopolyhedrovirus (HycuNPV) infection protected SpIm cells from actinomycin D (ActD)-induced apoptosis as early as 4 h postinfection. Analysis by Southern hybridization revealed that the HycuNPV genome possessed three members of inhibitor of apoptosis genes (iaps) that were designated as hycu-iap1, hycu-iap2, and hycu-iap3 because of their amino acid sequence homology with iaps identified in other baculoviruses. Functional analysis of Hycu-IAPs by transient expression assay in Sf9 cells revealed that Hycu-IAP3 blocked apoptosis induced by actinomycin D and rescued replication of p35 deficient-mutant AcMNPV, while Hycu-IAP1 and Hycu-IAP2 did not show any anti-apoptotic functions. Knockdown of hycu-iap3 expression by RNAi during HycuNPV infection in SpIm cells induced apoptosis. These results indicate that Hycu-IAP3 is essential for blockage of apoptosis during HycuNPV infection of permissive SpIm cells.
Virology 05/2004; 321(2):359-71. · 3.35 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Ld652Y cells derived from the gypsy moth, Lymantria dispar, were infected with seven different nucleopolyhedroviruses (NPVs) including those from Autographa californica, Bombyx mori (BmNPV), Hyphantria cunea (HycuNPV), Spodoptera exigua (SeMNPV), L. dispar, Orgyia pseudotsugata (OpMNPV) and Spodoptera litura (SpltMNPV). The results showed that Ld652Y cells infected with BmNPV, HycuNPV, SeMNPV, OpMNPV and SpltMNPV underwent apoptosis, displaying apoptotic bodies, characteristic DNA fragmentation and increased caspase-3-like protease activity; HycuNPV induced the most severe apoptosis. In HycuNPV-infected Ld652Y cells, a considerable amount of viral DNA was synthesized although there was no detectable yield of budded virions and polyhedrin. Northern blot and immunoblot analyses revealed that HycuNPV inhibitor of apoptosis 3 (IAP3), which has been shown to function in Sf9 cells, was expressed in HycuNPV-infected Ld652Y cells at a level higher than or comparable with that in HycuNPV-infected SpIm cells, which produced a high titre of progeny virions without any apoptotic response. These results imply that the relative ease of apoptosis induction in NPV-infected Ld652Y cells is largely dependent on inherent cellular properties rather than functions of the respective NPVs, and indicate that the defect in progeny virion production is not merely due to the virus-induced apoptosis in HycuNPV-infected Ld652Y cells.
Journal of General Virology 04/2003; 84(Pt 3):705-14. · 3.36 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A total of six homologous regions (hycu-hrs1-6) were identified in the genome of Hyphantria cunea nucleopolyhedrovirus (HycuNPV). These hycu-hrs were localized in non-coding regions interspersed throughout the HycuNPV genome and showed structural homology to several other baculovirus hrs. Sequence analyses indicated that hycu-hrs were composed of 65-69-bp direct repeats, each of which contained a 29-31-bp imperfect palindrome embedded within non-palindromes tandemly arranged. hycu-hrs consisted of a variable number of repeats ranging from two for hycu-hr3 to twenty-five for hycu-hr6, and these repeats showed a high degree of identity to each other. The hycu-hr6, which was localized immediately upstream of HycuNPV gp64 gene (hycu-gp64), represented the longest hr among group I NPV hrs reported to date. Transient expression assays demonstrated that the expression of hycu-gp64 promoter-driven luciferase gene (luc) was dramatically enhanced by hycu-hr6 which was placed upstream and downstream of luc in an orientation-independent manner. Moreover, hycu-hr6-dependent enhancement was observed in the absence of any additional viral gene products, although it could be further strengthened in the presence of HycuNPV ie-1 gene product. These results indicate that hycu-hrs function as enhancers of transcription mediated by RNA polymerase II, and suggest for the first time that efficiency of gp64 promoter is dependent on the enhancement function of hrs.
Virus Genes 01/2003; 25(3):281-90. · 1.85 Impact Factor
