Rina Hamajima

Baculovirus genes modulating intracellular innate antiviral immunity of lepidopteran insect cells.

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.

Baculoviruses: diversity, evolution and manipulation of insects

Baculoviruses, members of the family Baculoviridae, are large, enveloped viruses that contain a double‐stranded circular DNA genome of 80–180 kbp, encoding 90–180 putative proteins. These viruses are exclusively pathogenic for arthropods, particularly insects, and have been developed, or are being developed, as environmentally sound pesticides and eukaryotic vectors for foreign protein expression, surface display, gene delivery for gene therapy, vaccine production and drug screening. The baculoviruses contain a set of approximately 30 core genes that are conserved among all baculovirus genomes sequenced to date. Individual baculoviruses also contain a number of lineage‐ or species‐specific genes that have greatly impacted the diversification and evolution of baculoviruses. In this review, we first describe the general properties and biology of baculoviruses and then focus on the baculovirus genes and mechanisms involved in the replication, spread and survival of baculoviruses within the context of their diversity, evolution and insect manipulation.

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

ABSTRACT We previously identified a novel baculovirus-encoded apoptosis suppressor, Apsup, from the baculovirus Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV). Apsup inhibits the apoptosis of L. dispar Ld652Y cells triggered by infection with p35-defective Autographa californica MNPV (vAcΔp35) and exposure to actinomycin D or UV light. Here, we examined the functional role of Apsup in apoptosis regulation in insect cells. Apsup prevented apoptosis and the proteolytic processing of L. dispar initiator caspase Dronc (Ld-Dronc) in Ld652Y cells triggered by overexpression of Ld-Dronc, LdMNPV inhibitor-of-apoptosis 3 (IAP3), or Hyphantria cunea MNPV IAP1. In vAcΔp35-infected apoptotic Ld652Y cells, Apsup restricted apoptosis induction and prevented processing of endogenous Ld-Dronc. Conversely, upon RNA interference (RNAi)-mediated silencing of apsup, LdMNPV-infected Ld652Y cells, which typically support high-titer virus replication, underwent apoptosis, accompanied by the processing of endogenous Ld-Dronc. Furthermore, endogenous Ld-Dronc coimmunoprecipitated with transiently expressed Apsup, indicating that Apsup physically interacts with Ld-Dronc. Apsup prevented the apoptosis of Sf9 cells triggered by vAcΔp35 infection but did not inhibit apoptosis or activation of caspase-3-like protease in vAcΔp35-infected Drosophila melanogaster S2 cells. Apsup also inhibited the proteolytic processing of L. dispar effector caspase Ld-caspase-1 in the transient expression assay but did not physically interact with Ld-caspase-1. These results demonstrate that Apsup inhibits apoptosis in Ld652Y cells by preventing the proteolytic processing of Ld-Dronc. Together with our previous findings showing that Apsup prevents the processing of both overexpressed Ld-Dronc and Bombyx mori Dronc, these results also demonstrate that Apsup functions as an effective apoptotic suppressor in various lepidopteran, but not dipteran, insect cells.

Degradation of rRNA in BM-N cells from the silkworm Bombyx mori during abortive infection with heterologous nucleopolyhedroviruses

Cell lines derived from the silkworm, Bombyx mori, are only permissive for B. mori nucleopolyhedrovirus (NPV), with other NPVs generally resulting in abortive infection. Here, we demonstrate that rRNA of B. mori BM-N cells undergoes rapid degradation through site-specific cleavage upon infection with NPVs from Autographa californica (AcMNPV), Hyphantria cunea (HycuMNPV), Spodoptera exigua (SeMNPV) and Spodoptera litura (SpltMNPV). No significant decreases in cellular RNA were observed in Ld652Y, Se301, Sf9, SpIm and S2 cells infected with AcMNPV or HycuMNPV, indicating the response is unique to BM-N cells. A transient expression assay using a cosmid library of the HycuMNPV genome demonstrated that HycuMNPV P143 is responsible for rRNA degradation, which was also detected in BM-N cells transfected with plasmids expressing the P143 proteins from AcMNPV, SeMNPV and SpltMNPV. These results indicate that B. mori evolved to acquire a unique antiviral immune mechanism that is activated by P143 proteins from heterologous NPVs.

Identification of amino acid residues of AcMNPV P143 protein involved in rRNA degradation and restricted viral replication in BM-N cells from the silkworm Bombyx mori.

We previously demonstrated that rRNA undergoes rapid and extensive degradation in Bombyx mori BM-N cells upon infection with AcMNPV, which is triggered by AcMNPV P143 (Ac-P143) protein. Here, we showed that six amino acid residues of Ac-P143 protein, distributing between positions 514 and 599, are involved in rRNA degradation in BM-N cells. The six residues are highly conserved among P143 proteins from AcMNPV, HycuMNPV, SeMNPV and SpltMNPV, which trigger rRNA degradation in BM-N cells upon infection, but are only partially conserved in Bm-P143 protein, which does not induce rRNA degradation in BM-N cells. We also demonstrated that substitution of only two selected residues (N565S/L578F) of Bm-P143 protein with the corresponding Ac-P143 protein residues generates a mutant Bm-P143 protein that is capable of triggering rRNA degradation in BM-N cells. These results indicate that BmNPV evolved a unique P143 protein to evade the antiviral response and allow replication in B. mori cells.

Cloning and functional characterization of the Lymantria dispar initiator caspase dronc.

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 1329bp that was predicted to encode a 443 amino-acid polypeptide with a molecular mass of 50,706Da and 54-57% 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 proteinsare responsible, at least in part, for the differing sensitivity of Ld652Y and BM-N to apoptosis induction upon NPV infection.

Functional analysis of inhibitor of apoptosis 1 of the silkworm Bombyx mori

Recent advances in genome-wide surveys have revealed a number of lepidopteran insect homologs of mammalian and Drosophila genes that are responsible for apoptosis regulation. However, the underlying molecular mechanisms for apoptosis regulation in lepidopteran insect cells remain poorly understood. In the present study, we demonstrated that the transfection of Bombyx mori BM-N cells with dsRNA against the B. mori cellular iap1 gene (cbm-iap1) induces severe apoptosis that is accompanied by an increase of caspase-3-like protease activity. In these apoptotic cells, the cleaved form of the endogenous initiator caspase Dronc (Bm-Dronc) was detected, indicating that cBm-IAP1 protein depletion by RNAi silencing resulted in the activation of Bm-Dronc. In transient expression assays in BM-N cells, cBm-IAP1 suppressed the apoptosis triggered by Bm-Dronc overexpression and depressed the elevation of caspase-3-like protease activity, but also increased the cleaved form of Bm-Dronc protein. cBm-IAP1 also suppressed the caspase-3-like protease activity stimulated by Bm-caspase-1 overexpression. Co-immunoprecipitation experiments demonstrated that cBm-IAP1 strongly interacts with Bm-Dronc, but only has weak affinity for Bm-caspase-1. Transient expression analyses showed that truncated cBm-IAP1 proteins defective in the BIR1, BIR2 or RING domain were unable to suppress Bm-Dronc-induced apoptosis. In addition, BM-N cells expressing truncated cBm-IAP1 proteins underwent apoptosis, suggesting that intact cBm-IAP1, which has anti-apoptotic activity, was replaced or displaced by the overexpressed truncated cBm-IAP1 proteins, which are incapable of interfering with the apoptotic caspase cascade. Taken together, the present results demonstrate that cBm-IAP1 is a vital negative regulator of apoptosis in BM-N cells and functions by preventing the activation and/or activity of Bm-Dronc and Bm-caspase-1.

HCF-1 encoded by baculovirus AcMNPV is required for productive nucleopolyhedrovirus infection of non-permissive Tn368 cells

Baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) replicates in both Spodoptera frugiperda Sf21 and Trichoplusia ni Tn368 cells, whereas AcMNPV defective in hcf-1 (host cell-factor 1) gene productively infects only Sf21 cells, indicating that HCF-1 is indispensable for the AcMNPV productive infection of Tn368 cells. Here, we demonstrated that HCF-1 protein transiently expressed in Tn368 cells promotes the DNA synthesis of Hyphantria cunea MNPV (HycuMNPV), Orygia pseudotsugata MNPV and Bombyx mori NPV, which are normally unable to replicate in Tn368 cells. We also demonstrated that a recombinant HycuMNPV harboring the hcf-1 gene successfully replicates in Tn368 cells, generating substantial yields of progeny viruses and polyhedra. These results indicate that HCF-1 encoded by AcMNPV is an essential viral factor for productive NPV infection of Tn368 cells. Taken together with the previous findings on HRF-1 (host range factor 1), the present results provide strong evidence that viral genes acquired through horizontal gene transfer play an important role in baculovirus evolution, serving to expand the host range of baculoviruses.

Antiviral immune responses of Bombyx mori cells during abortive infection with Autographa californica multiple nucleopolyhedrovirus

Lepidopteran cells rely on multiple antiviral responses to defend against baculovirus infections, including apoptosis, global protein synthesis shutdown, and rRNA degradation. Here, we characterized apoptosis and rRNA degradation in Autographa californica multiple nucleopolyhedrovirus (AcMNPV)-infected Bombyx mori cells, a system resulting in abortive infection, in relation to viral DNA replication and viral late gene expression. RNAi-mediated silencing of viral DNA replication-related genes prevented apoptosis, but not rRNA degradation, in B. mori cells infected with p35-deficient AcMNPV. Additionally, AcMNPV, but not B. mori nucleopolyhedrovirus (BmNPV), drastically reduced B. mori cellular iap1 transcript levels and p35-deficient AcMNPV induced more prominent apoptosis than did p35-deficient BmNPV. These results, together with previous results that global protein synthesis shutdown follows viral DNA replication, demonstrate that rRNA degradation is the primary antiviral response that abolishes productive AcMNPV infection of B. mori cells. Our results also demonstrate that B. mori cells induce apoptosis to a different extent depending on NPV species.

Bombyx mori homolog of tumor suppressor p53 is involved in apoptosis-mediated antiviral immunity of B. mori cells infected with nucleopolyhedrovirus

&NA; Apoptosis is important in antiviral immunity and affects viral multiplication and pathogenesis. Here, we showed that Bombyx mori cells transiently expressing B. mori homolog of the tumor suppressor p53 (Bm‐p53) protein underwent apoptosis accompanied by elevated caspase‐3‐like protease activity and processing of B. mori Dronc (Bm‐Dronc). RNAi‐mediated silencing of bm‐p53 expression, which significantly diminished accumulation of bm‐p53 transcript and Bm‐p53 protein, prevented apoptosis of B. mori cells infected with a recombinant B. mori nucleopolyhedrovirus defective in the anti‐apoptotic p35 gene (vBm&Dgr;p35) and abolished the activation of caspase‐3‐like protease and processing of Bm‐Dronc. Apoptosis in vBm&Dgr;p35‐infected B. mori cells is associated with viral DNA replication, suggesting involvement of the DNA damage response. The Bm‐p53 pro‐apoptotic function is also found in Spodoptera frugiperda and Lymantria dispar cells. These results indicate that apoptosis induction in vBm&Dgr;p35‐infected B. mori cells is a Bm‐p53‐mediated process promoted by the commencement of viral DNA replication.