Caspases are a family of cysteine proteases implicated in the biochemical and morphological changes that occur during apoptosis (programmed cell death). The loop domain of Bcl-2 is cleaved at Asp34 by caspase-3 (CPP32) in vitro, in cells overexpressing caspase-3, and after induction of apoptosis by Fas ligation and interleukin-3 withdrawal. The carboxyl-terminal Bcl-2 cleavage product triggered cell death and accelerated Sindbis virus-induced apoptosis, which was dependent on the BH3 homology and transmembrane domains of Bcl-2. Inhibitor studies indicated that cleavage of Bcl-2 may further activate downstream caspases and contribute to amplification of the caspase cascade. Cleavage-resistant mutants of Bcl-2 had increased protection from interleukin-3 withdrawal and Sindbis virus-induced apoptosis. Thus, cleavage of Bcl-2 by caspases may ensure the inevitability of cell death.

Programmed cell death is an active process of self destruction that is important in both the development and maintenance of multicellular animals. The molecular mechanisms controlling activation or suppression of programmed cell death are largely unknown. Apoptosis, a morphologically and biochemically defined type of programmed cell death commonly seen in vertebrates, was found to be initiated during baculovirus replication in insect cells. A specific viral gene product, p35, was identified as being responsible for blocking the apoptotic response. Identification of the function of this gene will allow further definition of the molecular pathways involved in the regulation of programmed cell death and may identify the role of apoptosis in invertebrate viral defense systems.

The SF-21 insect cell line undergoes rapid and widespread apoptosis when treated with actinomycin D or when infected with a mutant of the baculovirus Autographa californica nuclear polyhedrosis virus lacking a p35 gene or a functionally active iap (inhibitor of apoptosis) gene. Here we provide evidence that the basis for the induction of apoptosis by these two different stimuli is the cessation of RNA synthesis. We also show that expression of eitherp35 or two different functional iap homologs blocks apoptosis independently of other viral genes, indicating that these gene products act directly on the cellular apoptotic pathway. The rus Autographa califomica nuclear polyhedrosis virus (AcM-NPV), which triggers but does not block apoptosis in the cell line SF-21, derived from Spodoptera frugiperda (6). However, this mutant and other mutants with site-specific mutations in p35 do not induce apoptosis in another cell line, 7,18). Although derived from different lepidopteran insect species, both the SF-21 and the TN-368 cell lines are fully permissive for wild-type AcMNPV infection. Disruption ofp35 in AcMNPV results in significantly reduced viral replication and gene expression in SF-21 cells (7, 18) and decreased infectivity in S. frugiperda larvae (7), indicating that apoptosis provides an antiviral defense mechanism in invertebrates. The response to p35-mutants appears to be species specific, since the infectivity of p35-mutants is normal in larvae (7) of Trichoplusia ni, from which the TN-368 cell line was derived. AcMNPV p35-mutants also induce apoptosis in a cell line derived from the silkworm Bombyx moni (6), and a functional p35 homolog is present in the silkworm baculovirus B. mon nuclear polyhedrosis virus (22), a close relative of AcMNPV.Expression ofp35 in mammalian neuronal cells has recently been shown to block apoptosis triggered by diverse stimuli (34). Thus, the pathway or the point(s) at which both P35 and Bcl-2 act is conserved between invertebrates and vertebrates (17a, 34, 41). However, p35 appears to be unrelated to other known genes, including bcl-2, in available databases (10).The second baculovirus gene found to prevent apoptosis, iap, was identified by its ability to functionally replace p35 in a genetic complementation assay (8). To date, homologs of iap have been found in three baculoviruses: Cydia pomonella granulosis virus (Cp-iap), Orgyia pseudotsugata nuclear polyhedrosis virus (Op-iap), and AcMNPV (Ac-iap). The Ac-iap gene was identified only by its homology to the other two iaps, Cp-iap and Op-iap, which are functionally able to replace p35 in the complementation assay (3,8)

Arthropod-borne viruses (arboviruses) circulate in nature between arthropod vectors and vertebrate hosts. Arboviruses often cause devastating diseases in vertebrate hosts, but they typically do not cause significant pathology in their arthropod vectors. Following oral acquisition of a viremic bloodmeal from a vertebrate host, the arbovirus disease cycle requires replication in the cellular environment of the arthropod vector. Once the vector has become systemically and persistently infected, the vector is able to transmit the virus to an uninfected vertebrate host. In order to systemically infect the vector, the virus must cope with innate immune responses and overcome several tissue barriers associated with the midgut and the salivary glands. In this review we describe, in detail, the typical arbovirus infection route in competent mosquito vectors. Based on what is known from the literature, we explain the nature of the tissue barriers that arboviruses are confronted with in a mosquito vector and how arboviruses might surmount these barriers. We also point out controversial findings to highlight particular areas that are not well understood and require further research efforts.

The baculovirus inhibitor of apoptosis gene, iap, can impede cell death in insect cells. Here we show that iap can also prevent cell death in mammalian cells. The ability of iap to regulate programmed cell death in widely divergent species raised the possibility that cellular homologs of iap might exist. Consistent with this hypothesis, we have isolated Drosophila and human genes which encode IAP‐like proteins (dILP and hILP). Like IAP, both dILP and hILP contain amino‐terminal baculovirus IAP repeats (BIRs) and carboxy‐terminal RING finger domains. Human ilp encodes a widely expressed cytoplasmic protein that can suppress apoptosis in transfected cells. An analysis of the expressed sequence tag database suggests that hilp is one of several human genes related to iap. Together these data suggest that iap and related cellular genes play an evolutionarily conserved role in the regulation of apoptosis.

The caspases are cysteine proteases that have been implicated in the execution of programmed cell death in organisms ranging from nematodes to humans.

No abstract

Spodoptera frugiperda SF-21 cells infected with Autographa californica nuclear polyhedrosis virus mutants which lack a functional p35 gene undergo apoptosis, a type of programmed cell death. To identify p35-homologous genes in other baculoviruses, A. californica nuclear polyhedrosis virus DNA containing a deletion in p35 was cotransfected into SF-21 cells along with genomic DNAs from other baculoviruses. One of the viral DNAs which were able to rescue wild-type infection was from Cydia pomoneUla granulosis virus (CpGV). The CpGV gene responsible for the effect was mapped to a 1.6-kb Saii-SstI subclone of the SaiI B fragment of CpGV. The sequence of the SalI-SstI subclone revealed an open reading frame capable of encoding a polypeptide of 31 kDa which was sufficient to rescue wild-type infection; this gene was thus called iap