Persistent Baculovirus Infection Results from Deletion of the Apoptotic Suppressor Gene p35

Infection with the wild-type baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) results in complete death of Spodoptera frugiperda (Sf) cells. However, infection of Sf cells with AcMNPV carrying a mutation or deletion of the apoptotic suppressor gene p35 allowed the cloning of surviving Sf cells that harbored persistent viral genomes. Persistent infection established with the virus with p35 mutated or deleted was blocked by stable transfection of p35 in the host genome or by insertion of the inhibitor of apoptosis (iap) gene into the viral genome. These artificially established persistently virus-infected cells became resistant to subsequent viral challenge, and some of the cell lines carried large quantities of viral DNA capable of early gene expression. Continuous release of viral progenies was evident in some of the persistently virus-infected cells, and transfection of p35 further stimulated viral activation of the persistent cells, including the reactivation of viruses in those cell lines without original continuous virus release. These results have demonstrated the successful establishment of persistent baculovirus infections under laboratory conditions and that their establishment may provide a novel continuous, nonlytic baculovirus expression system in the future.     

Anti- and pro-apoptotic activities of baculovirus and Drosophila IAPs in an insect cell line

The anti-apoptotic activities of two baculovirus IAPs, OpIAP and CpIAP, were directly compared with that of two Drosophila IAPs, DIAP1 and DIAP2, in the same insect cell line, SF-21 cells. Like OpIAP and CpIAP, DIAP1 inhibited actinomycin D-induced apoptosis and apoptosis induced by Doom. Removal of the RING finger of DIAP1 reduced but did not eliminate its anti-apoptotic activity. DIAP2 was unable to inhibit actinomycin-D induced apoptosis but was able to partially inhibit Doom-induced apoptosis. The baculoviral BIR and RING finger regions, when separated, were unable to block apoptosis induced by actinomycin D or Doom. Instead, the BIR regions of OpIAP and CpIAP as well as the RING finger regions of CpIAP and DIAP1 induced apoptosis. Thus, there were significant differences in the manner in which the different domains of the viral and cellular homologues of IAPs interacted with the components of the pathways regulating apoptosis in SF-21 cells.     

Localization of a Baculovirus-Induced Chitinase in the Insect Cell Endoplasmic Reticulum

Confocal immunofluorescence microscopy was used to demonstrate that the Autographa californica nucleopolyhedrovirus (AcMNPV) chitinase was localized within the endoplasmic reticulum (ER) of virus-infected insect cells. This was consistent with removal of the signal peptide from the chitinase and an ER localization motif (KDEL) at the carboxyl end of the protein. Chitinase release from cells, a prerequisite for liquefaction of virus-infected insect larvae, appears to be aided by synthesis of the p10 protein. Deletion of p10 from the AcMNPV genome delayed the appearance of chitinase activity in the medium of virus-infected cells by 24 h and also delayed liquefaction of virus-infected Trichoplusia ni larvae by the same period.     

Characterization of the antiapoptotic (p35) gene homologue of Spodoptera litura nucleopolyhedrosis virus (SlNPV)

Antiapoptotic genes of baculoviruses have been shown to prevent virus induced apoptosis in insect cells. Dot blot and Southern hybridizations of EcoRI genomic library and genomic digests of Spodoptera litura nucleopolyhedrosis virus (SlNPV) respectively give strong hybridization signals with antiapoptotic DNA (p35 gene) probe of the prototype Autographa californica nucleopolyhedrosis virus (AcNPV). Both the hybridizations indicate the presence of a homologous gene in the 1.8 kb EcoRI-Y fragment of SlNPV. The sequence of 1.244 kb region of this fragment encompasses an open reading frame coding for a polypeptide of 296 amino acids under sequential early (TATA) and late (TAAG) promoter motifs like that in other baculovirus p35 genes. The putative SlNPV p35 ORF expresses abundantly as a 35 kDa protein in Spodoptera frugiperda (Sf9) cells when allowed to express under the polyhedrin promoter of AcNPV.     

Persistence of an Occlusion-Negative Recombinant Nucleopolyhedrovirus in Trichoplusia ni Indicates High Multiplicity of Cellular Infection

We use data from the serial passage of co-occluded recombinant Autographa californica nuclear polyhedrosis virus (AcMNPV) to estimate the viral multiplicity of infection of cells within infected insects. Co-occlusion, the incorporation of wild-type and mutant virus genomes in the same occlusion body, has been proposed as a strategy to deliver genetically modified viruses as insecticides in a way that contains their spread in the environment. It may also serve as a means whereby naturally occurring mutant forms of NPVs can be maintained in a stable polymorphism. Here, a recombinant strain of AcMNPV was constructed with a deletion of its polyhedrin gene, rendering it incapable of producing occlusion bodies (i.e., occlusion negative). This was co-occluded with wild-type AcMNPV and used to infect fifth-instar Trichoplusia ni larvae. The fate of both genotypes was monitored over several rounds of insect infection. Levels of the occlusion-negative virus genome declined slowly over successive rounds of infection. We applied these data to a model of NPV population genetics to derive an estimate of 4.3 ± 0.3 viral genomes per occlusion body-producing cell.     

4-Hydroxynonenal induces p53-mediated apoptosis in retinal pigment epithelial cells

4-Hydroxynonenal (4-HNE) has been suggested to be involved in stress-induced signaling for apoptosis. In present studies, we have examined the effects of 4-HNE on the intrinsic apoptotic pathway associated with p53 in human retinal pigment epithelial (RPE and ARPE-19) cells. Our results show that 4-HNE causes induction, phosphorylation, and nuclear accumulation of p53 which is accompanied with down regulation of MDM2, activation of the pro-apoptotic p53 target genes viz. p21 and Bax, JNK, caspase3, and onset of apoptosis in treated RPE cells. Reduced expression of p53 by an efficient silencing of the p53 gene resulted in a significant resistance of these cells to 4-HNE-induced cell death. The effects of 4-HNE on the expression and functions of p53 are blocked in GSTA4-4 over expressing cells indicating that 4-HNE-induced, p53-mediated signaling for apoptosis is regulated by GSTs. Our results also show that the induction of p53 in tissues of mGsta4 (−/−) mice correlate with elevated levels of 4-HNE due to its impaired metabolism. Together, these studies suggest that 4-HNE is involved in p53-mediated signaling in in vitro cell cultures as well as in vivo that can be regulated by GSTs.     

Cancer-derived p53 mutants suppress p53-target gene expression--potential mechanism for gain of function of mutant p53

Tumour-derived p53 mutants are thought to have acquired ‘gain-of-function’ properties that contribute to oncogenicity. We have tested the hypothesis that p53 mutants suppress p53-target gene expression, leading to enhanced cellular growth. Silencing of mutant p53 expression in several human cell lines was found to lead to the upregulation of wild-type p53-target genes such as p21, gadd45, PERP and PTEN. The expression of these genes was also suppressed in H1299-based isogenic cell lines expressing various hot-spot p53 mutants, and silencing of mutant p53, but not TAp73, abrogated the suppression. Consistently, these hot-spot p53 mutants were able to suppress a variety of p53-target gene promoters. Analysis using the proto-type p21 promoter construct indicated that the p53-binding sites are dispensable for mutant p53-mediated suppression. However, treatment with the histone deacetylase inhibitor trichostatin-A resulted in relief of mutant p53-mediated suppression, suggesting that mutant p53 may induce hypo-acetylation of target gene promoters leading to the suppressive effects. Finally, we show that stable down-regulation of mutant p53 expression resulted in reduced cellular colony growth in human cancer cells, which was found to be due to the induction of apoptosis. Together, the results demonstrate another mechanism through which p53 mutants could promote cellular growth.     

Studies of the Nucleopolyhedrovirus Infection Process in Insects by Using the Green Fluorescence Protein as a Reporter

A recombinant Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) expressing the green fluorescence protein (GFP) under the control of the AcMNPV polyhedrin promoter was constructed to study the spatial and temporal regulation of baculovirus infection in a permissive host. Larvae that ingested AcMNPV-GFP showed localized expression of GFP in the midgut epithelial cells, as well as hemocytes, at 24 h postinfection. The presence of fluorescence in these tissues indicated not only that the virus was replicating but also that the very late viral proteins were being synthesized. Secondary infection occurred within the tracheal cells throughout the body cavity, confirming earlier reports, and these foci of infection allowed entry of the virus into other tissues, such as the epidermis and the fat body.     

Restriction Maps of Five Autographa californica MNPV Variants, Trichoplusia ni MNPV, and Galleria mellonella MNPV DNAs with Endonucleases SmaI, KpnI, BamHI, SacI, XhoI, and EcoRI

The restriction sites of Autographa californica nuclear polyhedrosis virus (AcMNPV) E2 DNA were mapped for the endonucleases SmaI, KpnI, BamHI, SacI, XhoI, and EcoRI. The restriction maps of four other AcMNPV variants, Trichoplusia ni (TnMNPV), and Galleria mellonella (GmMNPV) genomes were determined and compared to the endonuclease cleavage maps of AcMNPV E2 DNA. The viral structural polypeptides of AcMNPV variants S3, E2, S1, M3, and R9 were the same when analyzed by polyacrylamide gel electrophoresis. The major structural polypeptides of GmMNPV and TnMNPV had the same pattern in polyacrylamide gels as did AcMNPV structural polypeptides. GmMNPV and TnMNPV had several minor structural protein differences as compared with AcMNPV. AcMNPV variants, TnMNPV, and GmMNPV were distinct but with very similar genomes and protein structures.     

Restriction Map of Rachiplusia ou and Rachiplusia ou-Autographa californica Baculovirus Recombinants

The restriction sites of Rachiplusia ou nuclear polyhedrosis virus (RoMNPV) DNA were mapped for the endonucleases SmaI, KpnI, BamHI, SacI, XhoI, and EcoRI. Of the 60 DNA restriction sites of RoMNPV, 35 mapped in similar positions as compared to the restriction sites of Autographa californica nuclear polyhedrosis virus (AcMNPV) DNA. Two plaque-purified viruses, obtained from randomly picked plaques of a wild-type isolate of RoMNPV, were recombinants of RoMNPV and AcMNPV. The recombinants were shown to have RoMNPV and AcMNPV restriction fragments as well as structural polypeptides from each parental virus. Both recombinant viruses had a major RoMNPV capsid protein but were occluded in the AcMNPV polyhedrin protein.     

Effects of Substituting Granulin or a Granulin-Polyhedrin Chimera for Polyhedrin on Virion Occlusion and Polyhedral Morphology in Autographa californica Multinucleocapsid Nuclear Polyhedrosis Virus

Substitution of granulin from the Trichoplusia ni granulosis virus (TnGV) for polyhedrin of the Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) yielded a few very large (2 to 5 μm) cuboidal inclusions in the cytoplasm and nucleus of infected cells. These polyhedra lacked the beveled edges characteristic of wild-type AcMNPV polyhedra, contained fractures, and occluded few virions. Placing a nuclear localization signal (KRKK) in granulin directed more granulin to the nucleus and resulted in more structurally uniform cuboidal inclusions in which no virions were observed. A granulin-polyhedrin chimera produced tetrahedral occlusions with more virions than granulin inclusions but many fewer than wild-type polyhedra. Despite the unusual structure of the granulin and granulin-polyhedrin inclusions, they interacted with AcMNPV p10 fibrillar structures and electron-dense spacers that are precursors of the polyhedral calyx. The change in inclusion shape obtained with the granulin-polyhedrin chimera demonstrates that the primary amino acid sequence affects occlusion body shape, but the large cuboidal inclusions formed by granulin indicate that the amino acid sequence is not the only determinant. The failure of granulin or the granulin-polyhedrin chimera to properly occlude AcMNPV virions suggests that specific interactions occur between polyhedrin and other viral proteins which facilitate normal virion occlusion and occlusion body assembly and shape in baculoviruses.     

A putative protein inhibitor of activated STAT (PIASy) interacts with p53 and inhibits p53-mediated transactivation but not apoptosis

The p53 protein has recently been reported to be capable of mediating apoptosis through a pathway that is not dependent on its transactivation function. We report here that the PIASy member of the protein inhibitor of activated STAT family inhibited p53's transactivation function without compromising its ability to induce apoptosis of the H1299 nonsmall cell lung carcinoma cell line. The p53 protein bound to PIASy in yeast two-hybrid assays and coprecipitated in complexes with p53 in immunoprecipitates from mammalian cells. PIASy inhibited the DNA-binding activity of p53 in nuclear extracts and blocked the ability of p53 to induce expression of two of its target genes, Bax and p21^Waf1/Cip1, in H1299 cells. The block in p53-mediated induction of Bax and p21 was determined to be at the level of transactivation, since PIASy inhibited p53's ability to transactivate a p21/luciferase reporter construct. PIASy did not effect the incidence of apoptosis in H1299 cells upregulated for p53. PIASy appears to regulate p53-mediated functions and may direct p53 into a transactivation-independent mode of apoptosis.     

Nuclear FAK promotes cell proliferation and survival through FERM-enhanced p53 degradation

FAK is known as an integrin- and growth factor-associated tyrosine kinase promoting cell motility. Here we show that, during mouse development, FAK inactivation results in p53- and p21-dependent mesodermal cell growth arrest. Reconstitution of primary FAK-/-p21-/- fibroblasts revealed that FAK, in a kinase-independent manner, facilitates p53 turnover via enhanced Mdm2-dependent p53 ubiquitination. p53 inactivation by FAK required FAK FERM F1 lobe binding to p53, FERM F2 lobe-mediated nuclear localization, and FERM F3 lobe for connections to Mdm2 and proteasomal degradation. Staurosporine or loss of cell adhesion enhanced FERM-dependent FAK nuclear accumulation. In primary human cells, FAK knockdown raised p53-p21 levels and slowed cell proliferation but did not cause apoptosis. Notably, FAK knockdown plus cisplatin triggered p53-dependent cell apoptosis, which was rescued by either full-length FAK or FAK FERM re-expression. These studies define a scaffolding role for nuclear FAK in facilitating cell survival through enhanced p53 degradation under conditions of cellular stress.     

Induction of p53-independent apoptosis by simian virus 40 small t antigen

Simian virus 40 small t antigen (st) is required for optimal transformation and replication properties of the virus. We find that in certain cell types, such as the human osteosarcoma cell line U2OS, st is capable of inducing apoptosis, as evidenced by a fragmented nuclear morphology and positive terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling staining of transfected cells. The cell death can be p53 independent, since it also occurs in p53-deficient H1299 cells. Genetic analysis indicates that two specific mutants affect apoptosis induction. One of these (C103S) has been frequently used as a PP2A binding mutant. The second mutant (TR4) lacks the final four amino acids of st, which have been reported to be unimportant for PP2A binding in vitro. However, TR4 unexpectedly fails to bind PP2A in vivo. Furthermore, a long-term colony assay reveals a potent colony inhibition upon st expression, and the behavior of st mutants in this assay reflects the relative frequency of nuclear fragmentation observed in transfections using the same mutants. Notably, either Bcl-2 coexpression or broad caspase inhibitor treatment could restore normal nuclear morphology. Finally, fluorescence-activated cell sorting analysis suggests a correlation between the ability of st to modulate cell cycle progression and apoptosis. Taken together, these observations underscore that st does not always promote proliferation but may, depending on conditions and cell type, effect a cell death response.     

The p33ING1b tumor suppressor cooperates with p53 to induce apoptosis in response to etoposide in human osteosarcoma cells

p33ING1b induces cell cycle arrest and stimulates DNA repair, apoptosis and chemosensitivity. The magnitude of some p33ING1b effects may be due to activation of the tumor suppressor p53. To investigate if the p33ING1b protein affected chemosensitivity of osteosarcoma cells, we overexpressed p33ING1b in p53+/+ U2OS cells or in p53-mutant MG63 cells, and then assessed for growth arrest and apoptosis after treatment with etoposide. p33ING1b increased etoposide-induced growth inhibition and apoptosis to a much greater degree in p53+/+ U2OS cells than in p53-mutant MG63 cells. Moreover, ectopic expression of p33ING1b markedly upregulated p53, p21WAF1 and bax protein levels and activated caspase-3 protein kinase in etoposide-treated U2OS cells. Together, our data indicate that p33ING1b prominently enhances etoposide-induced apoptosis through p53-dependent pathways in human osteosarcoma cells. p33ING1b may be an important marker and/or therapeutic target in the prevention and treatment of metastatic osteosarcoma.