Baculovirus IAP1 induces caspase-dependent apoptosis in insect cells

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. Transientexpression 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. INTRODUCTIONInhibitor of apoptosis (iap) genes were first identified in baculoviruses, Cydia pomonella granulovirus (CpGV) and Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV), as genes encoding an anti-apoptotic protein that suppressed virus-induced apoptosis of Spodoptera frugiperda Sf21 cells upon infection with a recombinant Autographa californica MNPV (AcMNPV) having a defective p35 gene (Birnbaum et al., 1994;Crook et al., 1993). Homologues of the baculovirus iap genes were subsequently identified in yeast, nematodes, insects and mammals (Duckett et al., 1996;Fraser et al., 1999;Uren et al., 1998Uren et al., , 1999. However, only a subset of these IAPs function as apoptosis suppressors, and many other IAPs are involved in diverse non-apoptotic roles in cellular survival and differentiation (O'Riordan et al., 2008;Orme & Meier, 2009;Srinivasula & Ashwell, 2008).The IAP proteins are mostly characterized by the presence of one-to-three baculovirus IAP repeat (BIR) motifs in the N-terminal region and most also contain a RING finger domain in the C-terminal region (Mace et al., 2010;O'Riordan et al., 2008;Salvesen & Duckett, 2002;Vaux & Silke, 2005 Hozak et al., 2000;Yan et al., 2010). However, certain trun...

Novel Apoptosis Suppressor Apsup from the Baculovirus Lymantria dispar Multiple Nucleopolyhedrovirus Precludes Apoptosis by Preventing Proteolytic Processing of Initiator Caspase Dronc

“…In vAchrf-1-infected Ld652Y cells, the levels of both full-length and cleaved Ld-Dronc decreased gradually from 24 to 120 h postinfection (Fig. 4B), likely due either to apoptosis induction late in the infection cycle (51,53) or to the shutdown of host protein synthesis, which is often observed during progeny virus replication (54)(55)(56). In contrast, cleaved Ld-Dronc corresponding to prodomain and p20 domain was not detected in LdMNPV-infected Ld652Y cells (Fig.…”

“…We previously found that IAP1s from diverse NPVs and IAP2 and IAP3 from LdMNPV trigger apoptosis when transiently expressed in several lepidopteran insect cells, accompanied by the activation of caspase-3-like protease and proteolytic processing of endogenous Dronc (48,51). These baculovirus IAPs are unable to suppress apoptosis triggered by baculovirus infections and may trigger apoptosis by replacing the cellular IAPs, which presumably bind to Dronc and negatively regulate the activation of Dronc in nonapoptotic cells (48,51,57).…”

“…Transfection of expression plasmids and dsRNAs was performed using Lipofectin (Invitrogen) for Ld652Y and Sf9 cells and Cellfectin II (Invitrogen) for S2 cells as described previously (48,51).…”

Functional characterization of hesp018, a baculovirus-encoded serpin gene

“…Baculoviruses are able to manipulate the cellular environment to enhance their infection (Thiem, 2009), e.g. by inhibiting cell cycle progression (Prikhod'ko & Miller, 1998), inducing DNA damage response (Huang et al, 2011), blocking apoptosis (Ikeda et al, 2011) and inducing shutoff of host gene expression (Ooi & Miller, 1988). They are also able to manipulate host physiology and behaviour through the expression of various viral proteins (Kamita et al, 2005;Katsuma et al, 2012;O'Reilly & Miller, 1989).…”

Function analysis and application of IAP1/2 of Autographa californica multiple nucleopolyhedrovirus

“…5 However, transient expression of Ac-IAP1 was capable of inducing apoptosis and stimulating caspase-3 protease activity in various lepidopteran and dipteran cell lines. 15 So, the function of AcIAPs remained unknown. In this study, Bac-to-Bac baculovirus expression system was used to generate two recombinant baculoviruses, AcMNPV-iap1-egfp and AcMNPV-iap2-egfp, the effects of AcMNPV-iap1/2-egfp on Sf9 (Spodoptera frugiperda 9) cells and Helicoverpa armigera larvae were examined, and the function of BIR2 domain in Ac-IAP2 was analyzed preliminarily, which further conrmed the role of Ac-IAP1/2 during viral infection and offered experimental basis for using AcMNPViap2-egfp as a stronger insecticide and a more effective insect cell expression system.…”