Autographa Californica Multiple Nuclear Polyhedrosis Virus Research Articles

TLR9 activation is required for cytotoxic response elicited by baculovirus capsid display.

Although the baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) infects lepidopteran invertebrates as natural hosts, represents an efficient vector for vaccine development. Baculovirus surface display induces strong humoral responses against viruses and parasites. A novel strategy based on capsid display carrying foreign antigens in the AcMNPV particle further improved the immune response by eliciting CD8+ T cell activation. In this study, we analyze the intracellular mechanisms and signalling pathways involved in CD8+ T cell activation by capsid display. Our results show that baculovirus can attach to the cell surface, enter dendritic cells (DCs), transit within endocytic vesicles and escape to the cytosol for further degradation by the proteasome. We found that the availability of viral proteins, endosomal acidification, and proteasome activity are needed for efficient Major Histocompatibility Complex class-I presentation by baculovirus carrying Ovalbumin in the viral capsid. Importantly, we demonstrated with this strategy that the induction of cytotoxic T cells and IL-12 production by DCs are TLR9-dependent and STING-independent. Finally, our study shows differential intracellular processing for capsid and surface baculovirus proteins in DCs and highlights the role of different danger receptors during cytotoxic T cell priming through the capsid display delivery system, which could lead to improved baculovirus-based vaccines development.

Baculovirus Surface Display of Zika Virus Envelope Protein Protects against Virus Challenge in Mouse Model.

Zika virus (ZIKV) is emerging as a significant pathogen worldwide and may cause severe neurological disorders such as fetal microcephaly and Guillain-Barre syndrome. No drug or listed vaccines are currently available for preventing ZIKV infection. As a major target of neutralizing, ZIKV envelop (E) protein usually used for vaccine development. Nevertheless, the immunogenicity of ZIKV envelop (E) protein expressed by baculovirus display system has never been assessed. In this study, we reported a new strategy for surface display of ZIKV E protein by a recombinant baculovirus vector derived from Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) and assessed its immunogenicity in mice. We produced recombinant fusion ZIKV E protein linked with signal peptide (SP) and transmembrane domain (TM) of AcMNPV GP64. The results showed that the recombinant protein was easy to produce by baculovirus display system. BALB/c mice immunized with this recombinant E protein developed ZIKV specific serum antibodies. The anti-E protein sera from the mice were able to effectively neutralize ZIKV in vitro. More importantly, AG6 (IFN-α/β and IFN-γ receptor deficient) mice immunized with recombinant E protein were protected against lethal ZIKV challenge. Together, these findings demonstrated that the recombinant E protein displayed by baculovirus can be conveniently prepared and displayed good immunogenicity in immunized mice. It is a promising practical approach for prompting the development of vaccine and related immunology research.Electronic supplementary materialThe online version of this article (10.1007/s12250-020-00238-x) contains supplementary material, which is available to authorized users.

Ac34 protein of AcMNPV promoted progeny virus production and induced the apoptosis in host Sf9 cells.

To analyze the function of Ac34 in Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) and elucidate the JNK apoptotic signaling pathway activation in host Spodoptera frugiperda 9 (Sf9) cells induced by the recombinant virus AcMNPV-Ac34-EGFP. AcMNPV is an important species of baculoviruses. First, viral propagation assay indicated that overexpression of Ac34 protein promoted replication of AcMNPV. Quantitative RT-PCR analysis showed that Ac34 increased the transcriptional level of late genes 38k and vp39, which suggested that Ac34 promoted the production of progeny virus by upregulating transcription of late genes. Second, AcMNPV-Ac34-EGFP inhibited the proliferation of Sf9 cells. Moreover, Sf9 cells infected with AcMNPV-Ac34-EGFP resulted in abundant expression of SfP53 and its accumulation in the nucleus. c-Jun N-terminal kinase (JNK) activation requires MKK4 and MKK7 mediated phosphorylation at Thr183 and Tyr185. We found increased levels of p-JNK1/2 in Sf9 cells infected by AcMNPV-Ac34-EGFP, with concomitant induction of Sf9 cell death. Furthermore, treatment of infected Sf9 cells with SP600125 (an inhibitor of JNK pathway) downregulated p-JNK1/2 and influenced the expression of virus-induced apoptosis protein SfP53, as well as Cytochrome C and Bax. AcMNPV-Ac34-EGFP virus upregulated the progeny virus production and triggered apoptosis via activation of the JNK pathway in Sf9 cells. In this work, we unveiled an effective virus replication factor-Ac34 and more importantly, developed a recombinant virus that can be used as an improved version of biopesticide.

Baculovirus capsid display in vaccination schemes: effect of a previous immunity against the vector on the cytotoxic response to delivered antigens

The baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) infects lepidopteran invertebrates as natural hosts, although it also has been used as display vector for vaccine development. In this work, we evaluated the effectiveness of repetitive doses of AcMNPV-based vectors on the cytotoxic immune response specific to the capsid-displayed heterologous antigen ovalbumin (OVA). Our results demonstrate that baculovirus vectors induce a boosting effect in the cytotoxic immune response to OVA, making possible to recover the levels obtained in the primary response. Moreover, mice preimmunized with wild-type baculovirus showed a complete lack of antigen-specific CD8 cytotoxic T lymphocytes (CTLs) that may be related to the presence of antibodies directed to baculoviral surface proteins, particularly to GP64. However, baculovirus was able to induce the innate immune response in spite of a previous response against this vector, although some quantitative differences reflect a distinct activation of the immune cells in prime and boost. This is the first report in which the novel capsid display strategy is evaluated in prime-boost schemes to improve efficient CTL responses.

Improving promiscuous mammalian cell entry by the baculovirus Autographa californica multiple nuclear polyhedrosis virus.

The insect baculovirus AcMNPV (Autographa californica multiple nuclear polyhedrosis virus) enters many mammalian cell lines, prompting its application as a general eukaryotic gene delivery agent, but the basis of entry is poorly understood. For adherent mammalian cells, we show that entry is favoured by low pH and by increasing the available cell-surface area through a transient release from the substratum. Low pH also stimulated baculovirus entry into mammalian cells grown in suspension which, optimally, could reach 90% of the transduced population. The basic loop, residues 268–281, of the viral surface glycoprotein gp64 was required for entry and a tetra mutant with increasing basicity increased entry into a range of mammalian cells. The same mutant failed to plaque in Sf9 cells, instead showing individual cell entry and minimal cell-to-cell spread, consistent with an altered fusion phenotype. Viruses grown in different insect cells showed different mammalian cell entry efficiencies, suggesting that additional factors also govern entry.

ウイルス感染による宿主自然免疫応答の解析と感染制御への応用

The baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) has been widely used not only to achieve a high level of foreign gene expression in insect cells but also for efficient gene transduction into mammalian cells without any replication. In addition to the efficient gene delivery, baculovirus has been shown to induce host innate immune responses in various mammalian cells and in mice. The baculovirus has abundant CpG motifs in the viral genome and is capable of inducing pro-inflammatory cytokines and interferons (IFNs) through Toll-like receptor (TLR)-dependent and -independent signaling pathways in a cell-type-specific manner. The cytoplasmic helicase proteins RIG-I (retinoic-acid-inducible protein I) and MDA5 (melanoma-differentiation-associated gene 5) have been identified as viral dsRNA detectors and the adaptor IPS-1 (IFN-beta promoter stimulator-1) interacts with RIG-I and MDA5 to facilitate type-I IFN production mediated interferon regulatory factor 3 (IRF3) and 7 (IRF7). These helicases and IPS-1, however, were not essential for the type-I IFN and inflammatory cytokine responses to baculovirus. The baculovirus also has a strong adjuvant activity, and recombinant baculoviruses encoding neutralization epitopes elicit protective immunity in mice. This review deals with the current status of our knowledge of the induction of host innate immune responses by baculovirus and discusses the future prospects for baculovirus vectors.

Prorenin Processing Enzyme (PPE) Produced by Baculovirus-Infected Sf-9 Insect Cells: PPE Is the Cysteine Protease Encoded in the AcMNPV Gene

In infection cultures of Spodoptera frugiperda (Sf-9) insect cells with a recombinant baculovirus, vhpR, carrying human preprorenin cDNA in the polyhedrin locus of Autographa californica multiple nuclear polyhedrosis virus (AcMNPV), the expressed inactive recombinant human (rh)-prorenin is reported to be proteolytically processed to yield active rh-renin in the very late phase of culture (Takahashi et al., Biosci. Biotechnol. Biochem., 71, 2610-2613 (2007)). To identify the enzyme that catalyzes the processing of rh-prorenin, referred to as prorenin processing enzyme (PPE), we purified potential PPE from virus-infected Sf-9 culture supernatant by the use of an internally quenched fluorescent (IQF) substrate for PPE. The 32-kDa protein band agreed well with PPE activity on the final Mono Q FPLC. By N-terminal amino acid sequence analysis, the protein was revealed to be a cysteine protease encoded by the AcMNPV gene. Enzyme activity was inhibited by cysteine protease inhibitors but not by other protease inhibitors. When the purified rh-prorenin was incubated with the 32-kDa protein, renin activity appeared concomitant with the disappearance of rh-prorenin. The N-terminal amino acid sequence of the activated product was identical to that of the rh-renin that had accumulated in the infection cultures. These results indicate that the 32-kDa cysteine protease derived from the AcMNPV gene is the enzyme PPE of virus-infected Sf-9 cells.

A suspended cell line from Trichoplusia ni (Lepidoptera): Characterization and expression of recombinant proteins

AbstractA suspended cell line from Trichoplusia ni embryos was established, and its susceptibility to Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) infection was investigated. This cell line had characteristics distinct from the BTI‐Tn5Bl‐4 cell line (Tn5Bl‐4) from T. ni in growth, and showed approximately the same responses to AcMNPV infection, production of occlusion bodies, and levels of recombinant protein expression. No clumps were observed at maximum cell density at late‐log phase in shake‐flask or T‐flask cultures, and thus the cells represent a useful new contribution for baculovirus research. The cells consist of two major morphological types: approximately 70% spindle‐shaped cells and 30% round cells. The cell line was highly susceptible to virus infection and produced around 107 AcMNPV occlusion bodies per cell, on average. Production of β‐galactosidase and secreted alkaline phosphatase was high with 3.97 ± 0.13 × 104IU/mL and 3.48 ± 0.40 IU/mL, respectively. This cell line may be applicable for studies of scale‐up production of viruses or baculovirus‐insect cell expression. We also believe the new line can be a source for cell clones with higher production of virus and recombinant proteins compared to the parent or other existing cell lines such as Tn5Bl‐4.

Baculovirus-mediated interferon alleviates dimethylnitrosamine-induced liver cirrhosis symptoms in a murine model

The wild-type baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) infects a range of mammalian cell types in vitro but does not replicate in these cells. The current study investigated the in vivo effect of AcMNPV in the mouse model of liver cirrhosis induced by the mutagen dimethylnitrosamine. Intraperitoneal injection of AcMNPV induced an immune response. The baculovirus was taken up by the liver and spleen where it suppressed liver injury and fibrosis through the induction of interferons. This study presents the first evidence of the feasibility of using baculovirus to treat liver cirrhosis.

Induction of Natural Killer Cell-dependent Antitumor Immunity by the Autographa californica Multiple Nuclear Polyhedrosis Virus

Wild-type Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) infects a variety of mammalian cell types in vitro, but does not replicate in these cells. We investigated the effects of AcMNPV in the induction of the immune response and tumor metastasis in mice. After intravenous injection, AcMNPV was taken up by the liver and spleen, and preferentially infected dendritic cells (DCs) and B cells in the spleen; costimulatory molecules CD40, CD80, and CD86 were upregulated in the DCs. The hepatic mononuclear cells (MNCs) in these animals were highly cytotoxic to natural killer (NK)-sensitive YAC-1 and B16 melanoma cells, but not to NK-resistant EL4 cells. Intravenous injection of AcMNPV-induced NK cell proliferation in the liver and spleen, and enhanced antitumor immunity in mice with B16 liver metastases. Furthermore, such treatment increased the survival of C57BL/6, J alpha 281 (-/-), and interferon (IFN)-gamma (-/-) mice that were previously injected with B16 tumor cells. AcMNPV injection did not enhance the survival of NK cell-depleted mice. Moreover, one AcMNPV treatment effectively prolonged survival in a B16 liver metastasis model, and was equivalent to five treatments with recombinant interleukin-12 (IL-12) protein. These findings suggest that AcMNPV efficiently stimulates NK cell-mediated antitumor immunity.

A novel method for in vitro radiolabeling and testing enveloped viruses by phosphatidylethanolamine N‐methyltransferase and host cell‐specific binding

The present study developed a novel virus labeling and testing method, referred to as an envelope-labeled virus assay (ELVA), in which virus envelope is labeled in vitro by the action of phosphatidylethanolamine N-methyltransferase (PEMT) and tested through a host cell-specific binding. A recombinant strain (vGFPuv) of Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) and Spodoptera frugiperda (Sf-9) insect cells were used as a model of viruses and host cells, respectively. The labeling mixture, which contained PEMT, [methyl-3H]S-adenosylmethionine (SAM), and a trace amount of detergent Triton X-100, brought about little change in virus titer of vGFPuv on a 1-h incubation, but was so toxic to Sf-9 cells as to immediately cause cell death. After being incubated with vGFPuv, therefore, the labeling mixture was neutralized by adsorptive removal of PEMT and Triton X-100 before Sf-9 cells were contacted with the mixture to extract the virus. The Sf-9 cells were then washed with a phosphate buffered saline (PBS), and lipid extracts with a 1% SDS solution were subjected to a liquid scintillation analysis for the determination of labeling efficiency. As a result, a significant amount of radioactivity was determined in the extracts, demonstrating the validity of ELVA for labeling and testing enveloped viruses. The conditions for the PEMT reaction and cell-virus binding were examined, and the lower detection limit of AcMNPV by ELVA was found to lie in the order of 10(3) plaque forming unit (pfu) per milliliter. Since the labeling reaction and detection of virus are based on neither immunological nor genetic characteristics of virus, ELVA is also expected to be a convenient and comprehensive test of other enveloped viruses.

Characterization of baculovirus Autographa californica multiple nuclear polyhedrosis virus infection in mammalian cells

The baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) is used as a vector in many gene therapy studies. Wild-type AcMNPV infects many mammalian cell types in vitro, but does not replicate. We investigated the dynamics of AcMNPV genomic DNA in infected mammalian cells and used flow cytometric analysis to demonstrate that recombinant baculovirus containing a cytomegalovirus immediate early promoter/enhancer with green fluorescent protein (GFP) expressed high levels of GFP in Huh-7 cells, but not B16, Raw264.7, or YAC-1 cells. The addition of butyrate, a deacetylase inhibitor, markedly enhanced the percentage of GFP-expressing Huh-7 and B16 cells, but not Raw264.7 and YAC-1 cells. The addition of 5-aza-2′-deoxycytidine, a DNA methylation inhibitor, had no enhancing effect. Polymerase chain reaction analysis using AcMNPV- gp64-specific primers indicated that AcMNPV infected not only Huh-7 and B16 cells, but also Raw264.7 and YAC-1 cells in vitro. The genomic DNA was detected in Huh-7 and B16 cells 96 h after infection. Genomic AcMNPV DNA in YAC-1 cells was not transported to the nucleus. Luciferase assay indicated that AcMNPV p35 gene mRNA and p35 promoter activity were clearly expressed only in Huh-7 and B16 cells. These results suggest that viral genomic DNA expression is restricted by different host cell factors, such as degradation, deacetylation, and inhibition of nuclear transport, depending on the mammalian cell type.

Baculovirus ETL promoter acts as a shuttle promoter between insect cells and mammalian cells1

To identify a shuttle promoter that can mediate gene expression in both insect cells and mammalian cells to facilitate the development of a baculovirus vector-based mammalian cell gene delivery vehicle. Recombinant baculoviruses carrying the beta-galactosidase reporter gene under the control of an early to late (ETL) promoter of the Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) or a cytomegalovirus immediate early promoter (CMV promoter) were constructed. COS1, HeLa, CHO-K1, hFob1.19, and MCF-7 mammalian cells were tested for the expression of b-galactosidase. ETL promoter activity was higher in bone-derived hFob1.19 than in COS1, HeLa, CHO-K1, or MCF-7 mammalian cells. The transient plasmid transfection assay indicated that ETL promoter activity in mammalian cells was dependent on baculovirus gene expression. ETL promoter activity in mammalian cells is baculovirus gene expression-dependent, and the shuttle promoter will facilitate the application of baculovirus expression vectors in mammalian cell expression systems and for gene therapy.

Antibody‐based surface plasmon resonance detection of intact viral pathogen

Surface plasmon resonance (SPR) technique was used to directly detect an intact form of insect pathogen: the baculovirus, Autographa californica multiple nuclear polyhedrosis virus (AcMNPV). An SPR sensor chip with three bio-functional layers was used to detect the intact AcMNPV: amine-reactive crosslinker with a disulfide bond that chemisorbs to gold film, Protein A, and a mouse IgG monoclonal antibody raised against a surface protein of the target viral pathogen. A two-channel (reference & test) micro-fluidic SPR system is used for reliable measurement. Bio-specific response to the AcMNPV is compared with the response for tobacco mosaic virus (TMV) as control. Successive exposure of the sensor chip to both viruses verifies a specific response to AcMNPV. This serves as a prerequisite to the development of a new type of viral pathogen detection sensors.

In vivo analysis of fibroin heavy chain signal peptide of silkworm Bombyx mori using recombinant baculovirus as vector

In order to investigate the functional signal peptide of silkworm fibroin heavy chain (FibH) and the effect of N- and C-terminal parts of FibH on the secretion of FibH in vivo, N- and C-terminal segments of fibh gene were fused with enhanced green fluorescent protein (EGFP) gene. The fused gene was then introduced into silkworm larvae and expressed in silk gland using recombinant AcMNPV ( Autographa californica multiple nuclear polyhedrosis virus) as vector. The fluorescence of EGFP was observed with fluorescence microscope. FibH-EGFP fusion proteins extracted from silk gland were analyzed by Western blot. Results showed that the two alpha helices within N-terminal 163 amino acid residues and the C-terminal 61 amino acid residues were not necessary for cleavage of signal peptide and secretion of the fusion protein into silk gland. Then the C-terminal 61 amino acid residues were substituted with a His-tag in the fusion protein to facilitate the purification. N-terminal sequencing of the purified protein showed that the signal cleavage site is between position 21 and 22 amino acid residues.

The Function of Envelope Protein P74 from Autographa californica Multiple Nucleopolyhedrovirus in Primary Infection to Host

This research investigated the function of envelope protein P74 of Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) in primary infection to host. A p74-inactivation recombinant baculovirus, rAc-gfp(Delta) p74, was constructed by inserting gfp driven by AcMNPV polyhedrin promoter into the p74 locus of AcMNPV genome. Bioassays showed that the P74-null occlusion bodies (OBs) failed to infect its natural host larvae, Spodoptera exigua, per os, while the p74-null budded virus (BVs) could infect host larvae by injection. However, its inability for oral infectivity was rescued by a mixed infection with wild-type OBs or with the purified P74 protein expressed in Spodoptera frugiperda Sf-9 cells, and the P74 protein rescue was in a dosage-dependent manner. The 50% lethal dosage (LD50) value of a P74 overexpression recombinant virus, rAc-p74(++)-polh+, which contained two copies of p74 gene, was not significantly different from that of wild-type virus. One-step growth curve assays of viruses suggested that BV production from cells infected with p74-null virus was similar to that from cells infected with wild-type virus or the P74 overexpression virus. ELISA analysis indicated that P74 protein could bind its host brush border membrane vesicles (BBMV) efficiently with saturation, but it could only bind its sensitive midgut BBMV specifically. In vitro pull-down assay showed that a protein of approximately 35 kDa in the BBMV was involved in the specific binding. These results demonstrated that the P74 protein is essential for oral infectivity of occlusion-derived virus (ODV) and plays a role in midgut attachment and fusion.

Baculovirus as mammalian cell expression vector for gene therapy: an emerging strategy.

The monopoly of insect cells to host baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) as a eukaryotic gene expression system has been shattered with the growing evidence that it also infects mammalian cells in culture. Although AcMNPV fails to replicate in vertebrate cells, it does express alien genes with levels of expression that are dependent on the strength of the promoter used to drive transcription of the foreign gene. It also has been reported that the recombinant AcMNPV enters human hepatic cells in culture preferentially and specifically in comparison with the other mammalian cells of different origin and sources. This has resulted in the use of AcMNPV as a potent mammalian cell delivery system as a xenovector for gene therapy, more precisely liver-specific gene delivery in vitro and in vivo.

Combination treatment for osteosarcoma with baculoviral vector mediated gene therapy (p53) and chemotherapy (adriamycin).

The insect baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) has been evaluated as a vector for gene delivery to human tumor cells. A human osteogenic sarcoma cell line, Saos-2, was found to be highly susceptible to infection with a baculoviral vector, with nearly 100% of Saos-2 cells being able to express a lacZ reporter gene after a brief exposure to the virus at a m.o.i. of 30 pfu/cell. The production of beta-galactosidase protein was 18-times greater than that in HepG2 cells which were previously thought to be the mammalian cells most susceptible to the baculovirus. The possibility of developing a baculovirus as a cytotoxic vector for p53-defective cancer was tested by destruction of Saos-2 cells (p53-/-) with a recombinant baculovirus containing the wild type p53 gene (BV-p53) in vitro. The p53 baculovirus induced apoptotic cell death in tumor cells in a dose-dependent manner with approximately 60% killing at an m.o.i. of 160 pfu/cell. Combined treatments of gene therapy (p53) and chemotherapy (adriamycin) resulted in synergistic and potent killing of the osteogenic sarcoma cells. For example, greater than 95% of Saos-2 cells were killed by the combination of BV-p53 (m.o.i. of 100) and adriamycin (35 ng/ml), whereas approximately 50% and approximately 55% cells were killed by BV-p53 and adriamycin alone, respectively. These results indicate that a baculoviral gene delivery vector can be used to efficiently target certain types of mammalian cells and the combination treatment of gene-therapy mediated by a baculovirus and chemotherapy may enhance induction of apoptosis in cancer cells.

Expression of highly controllable genes in insect cells using a modified tetracycline-regulated gene expression system

A modified tetracycline-responsive expression system (TRES) for use in insect cells was developed. The TRES contains two components: one encodes a tetracycline-controllable transactivator (tTA) and the other contains a tet operator DNA sequence to drive the luciferase gene. Our results show that the human cytomegalovirus (CMV) promoter, an essential part for strong tTA expression in mammalian system, was not functional in insect cells. Thus further modifications were required. Functional tTA was efficiently expressed in Sf9, Sf21, and TN368 cells by the p10 promoter of Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) in plasmid form with virus co-infection. An increase of up to 258-fold of luciferase activity was detected in these cells when both components in modified TRES were co-transfected. In order to further simplify the experiment, tTA, which is driven by the p10 promoter, was inserted into AcMNPV. Luciferase activity was also strongly stimulated by the infection of this tTA expression-recombinant virus with the transfection of a plasmid containing the second TRES component expressing luciferase. The luciferase expressions in these systems, either in plasmids or the tTA gene in virus and luciferase in plasmid, were significantly suppressed by tetracycline. The time course kinetics of tetracycline action to the TRES were further studied. Within a time span of 50 h, the luciferase activities could be fully suppressed or activated, respectively, corresponding to the addition or removal of tetracycline. These experiments have established a well-regulated gene expression system for further broad applications of molecular biological studies in insect cells.

Baculovirus-based Genetic Screen for Antiapoptotic Genes Identifies a Novel IAP

The prototype baculovirus, Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) expresses p35, a potent anti cell-death gene that promotes the propagation of the virus by blocking host cell apoptosis. Infection of insect Sf-21 cells with AcMNPV lacking p35 induces apoptosis. We have used this pro-apoptotic property of the p35 null virus to screen for genes encoding inhibitors of apoptosis that rescue cells infected with the p35 defective virus. We report here the identification of Tn-IAP1, a novel member of the IAP family of cell death inhibitors. Tn-IAP1 blocks cell death induced by p35 null AcMNPV, actinomycin D, and Drosophila cell-death inducers HID and GRIM. Given the conserved nature of the cell death pathway, this genetic screen can be used for rapid identification of novel inhibitors of apoptosis from diverse sources.