Search

Multiplication of the invertebrate DNA baculoviruses activates the host DNA damage response (DDR), which promotes virus DNA replication. DDR signaling is initiated by the host insect’s phosphatidylinositol-3 kinase-related kinases (PIKKs), including ataxia telangiectasia-mutated kinase (ATM). Like other PIKKs, ATM phosphorylates an array of host DDR proteins at serine/threonine glutamine (S/TQ) motifs, the result of which leads to cell cycle arrest, DNA repair, or apoptosis. To define the role of host PIKKs in baculovirus replication, we compared replication levels of the baculovirus prototype species Autographa californica multiple nucleopolyhedrovirus in permissive Spodoptera frugiperda (SF21) cells with and without ATM function. Caffeine, which inhibits multiple DDR kinases, and the ATM-specific inhibitors KU-55933 and KU-60019 each prevented phosphorylation of Spodoptera histone H2AX (SfH2AX), a recognized indicator of ATM activity. However, only caffeine reduced autographa californica multiple nucleopolyhedrovirus (AcMNPV)-induced bulk phosphorylation of S/TQ protein motifs. Furthermore, only caffeine, not KU-55933 or KU-60019, reduced AcMNPV yields, suggesting a limited role for ATM. To investigate further, we identified and edited the Spodoptera ATM gene (sfatm). Consistent with ATM’s known functions, CRISPR/Cas9-mediated knockout of sfatm eliminated DNA damage-induced phosphorylation of DDR marker SfH2AX in SF21 cells. However, loss of sfatm failed to affect the levels of AcMNPV multiplication. These findings suggested that in the absence of the kinase SfATM, another caffeine-sensitive host DDR kinase promotes S/TQ phosphorylation and baculovirus multiplication. Thus, baculoviruses activate and utilize the host insect DDR in an ATM-independent manner. IMPORTANCE The DDR, while necessary for the maintenance and fidelity of the host genome, represents an important cellular response to viral infection. The prolific DNA baculoviruses activate and manipulate the invertebrate DDR by using mechanisms that positively impact virus multiplication, including virus DNA replication. As the key DDR initiator kinase, ATM was suspected to play a critical role in this host response. However, we show here that baculovirus AcMNPV activates an ATM-independent DDR. By identifying the insect host ATM ortholog (Spodoptera frugiperda SfATM) and evaluating genetic knockouts, we show that SfATM is dispensable for AcMNPV activation of the DDR and for virus replication. Thus, another PIKK, possibly the closely related kinase ATR (ATM- and Rad3-related kinase), is responsible for efficient baculovirus multiplication. These findings better define the host pathways used by invertebrates to engage viral pathogens, including DNA viruses.

Baculovirus-encoded inhibitor of apoptosis (IAP) proteins likely evolved from their host cell IAP homologs, which function as critical regulators of cell death. Despite their striking relatedness to cellular IAPs, including the conservation of two baculovirus IAP repeat (BIR) domains and a C-terminal RING, viral IAPs use an unresolved mechanism to suppress apoptosis in insects. To define this mechanism, we investigated Op-IAP3, the prototypical IAP from baculovirus OpMNPV. We found that Op-IAP3 forms a stable complex with SfIAP, the native, short-lived IAP of host insect Spodoptera frugiperda . Long-lived Op-IAP3 prevented virus-induced SfIAP degradation, which normally causes caspase activation and apoptosis. In uninfected cells, Op-IAP3 also increased SfIAP steady-state levels and extended SfIAP's half-life. Conversely, SfIAP stabilization was lost or reversed in the presence of mutated Op-IAP3 that was engineered for reduced stability. Thus, Op-IAP3 stabilizes SfIAP and preserves its antiapoptotic function. In contrast to SfIAP, Op-IAP3 failed to bind or inhibit native Spodoptera caspases. Furthermore, BIR mutations that abrogate binding of well-conserved IAP antagonists did not affect Op-IAP3's capacity to prevent virus-induced apoptosis. Remarkably, Op-IAP3 also failed to prevent apoptosis when endogenous SfIAP was ablated by RNA silencing. Thus, Op-IAP3 requires SfIAP as a cofactor. Our findings suggest a new model wherein Op-IAP3 interacts directly with SfIAP to maintain its intracellular level, thereby suppressing virus-induced apoptosis indirectly. Consistent with this model, Op-IAP3 has evolved an intrinsic stability that may serve to repress signal-induced turnover and autoubiquitination when bound to its targeted cellular IAP. IMPORTANCE The IAPs were first discovered in baculoviruses because of their potency for preventing apoptosis. However, the antiapoptotic mechanism of viral IAPs in host insects has been elusive. We show here that the prototypical viral IAP, Op-IAP3, blocks apoptosis indirectly by associating with unstable, autoubiquitinating host IAP in such a way that cellular IAP levels and antiapoptotic activities are maintained. This mechanism explains Op-IAP3's requirement for native cellular IAP as a cofactor and the dispensability of caspase inhibition. Viral IAP-mediated preservation of the host IAP homolog capitalizes on normal IAP-IAP interactions and is likely the result of viral IAP evolution in which degron-mediated destabilization and ubiquitination potential have been reduced. This mechanism illustrates another novel means by which DNA viruses incorporate host death regulators that are modified for resistance to host regulatory controls for the purpose of suppressing host cell apoptosis and acquiring replication advantages.

Inhibitor-of-apoptosis (IAP) proteins are key regulators of the innate antiviral response by virtue of their capacity to respond to signals affecting cell survival. In insects, wherein the host IAP provides a primary restriction to apoptosis, diverse viruses trigger rapid IAP depletion that initiates caspase-mediated apoptosis, thereby limiting virus multiplication. We report here that the N-terminal leader of two insect IAPs, Spodoptera frugiperda SfIAP and Drosophila melanogaster DIAP1, contain distinct instability motifs that regulate IAP turnover and apoptotic consequences. Functioning as a protein degron, the cellular IAP leader dramatically shortened the life span of a long-lived viral IAP (Op-IAP3) when fused to its N terminus. The SfIAP degron contains mitogen-activated kinase (MAPK)-like regulatory sites, responsible for MAPK inhibitor-sensitive phosphorylation of SfIAP. Hyperphosphorylation correlated with increased SfIAP turnover independent of the E3 ubiquitin-ligase activity of the SfIAP RING, which also regulated IAP stability. Together, our findings suggest that the SfIAP phospho-degron responds rapidly to a signal-activated kinase cascade, which regulates SfIAP levels and thus apoptosis. The N-terminal leader of dipteran DIAP1 also conferred virus-induced IAP depletion by a caspase-independent mechanism. DIAP1 instability mapped to previously unrecognized motifs that are not found in lepidopteran IAPs. Thus, the leaders of cellular IAPs from diverse insects carry unique signal-responsive degrons that control IAP turnover. Rapid response pathways that trigger IAP degradation and initiate apoptosis independent of canonical prodeath gene (Reaper-Grim-Hid) expression may provide important innate immune advantages. Furthermore, the elimination of these response motifs within viral IAPs, including those of baculoviruses, explains their unusual stability and their potent antiapoptotic activity. IMPORTANCE Apoptosis is an effective means by which a host controls virus infection. In insects, inhibitor-of-apoptosis (IAP) proteins act as regulatory sentinels by responding to cellular signals that determine the fate of infected cells. We discovered that lepidopteran (moth and butterfly) IAPs, which are degraded upon baculovirus infection, are controlled by a conserved phosphorylation-sensitive degron within the IAP N-terminal leader. The degron likely responds to virus-induced kinase-specific signals for degradation through SKP1/Cullin/F-box complex-mediated ubiquitination. Such signal-induced destruction of cellular IAPs is distinct from degradation caused by well-known IAP antagonists, which act to expel IAP-bound caspases. The major implication of this study is that insects have multiple signal-responsive mechanisms by which the sentinel IAPs are actively degraded to initiate host apoptosis. Such diversity of pathways likely provides insects with rapid and efficient strategies for pathogen control. Furthermore, the absence of analogous degrons in virus-encoded IAPs explains their relative stability and antiapoptotic potency.

The baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) initiates apoptosis in diverse insects through events triggered by virus DNA (vDNA) replication. To define the proapoptotic pathway and its role in antivirus defense, we investigated the link between the host's DNA damage response (DDR) and apoptosis. We report here that AcMNPV elicits a DDR in the model insect Drosophila melanogaster . Replication of vDNA activated DDR kinases, as evidenced by ATM-driven phosphorylation of the Drosophila histone H2AX homolog (H2Av), a critical regulator of the DDR. Ablation or inhibition of ATM repressed H2Av phosphorylation and blocked virus-induced apoptosis. The DDR kinase inhibitors caffeine and KU55933 also prevented virus-induced apoptosis in cells derived from the permissive AcMNPV host, Spodoptera frugiperda . This block occurred at a step upstream of virus-mediated depletion of the cellular inhibitor-of-apoptosis protein, an event that initiates apoptosis in Spodoptera and Drosophila . Thus, the DDR is a conserved, proapoptotic response to baculovirus infection. DDR inhibition also repressed vDNA replication and reduced virus yields 100,000-fold, demonstrating that the DDR contributes to virus production, despite its recognized antivirus role. In contrast to virus-induced phosphorylation of Drosophila H2Av, AcMNPV blocked phosphorylation of the Spodoptera H2AX homolog (SfH2AX). Remarkably, AcMNPV also suppressed SfH2AX phosphorylation following pharmacologically induced DNA damage. These findings indicate that AcMNPV alters canonical DDR signaling in permissive cells. We conclude that AcMNPV triggers a proapoptotic DDR that is subsequently modified, presumably to stimulate vDNA replication. Thus, manipulation of the DDR to facilitate multiplication is an evolutionarily conserved strategy among DNA viruses of insects and mammals.

IE1 is the principal transcriptional regulator of the baculoviruses. Like multifunctional transcription factors of other large DNA viruses, IE1 is an essential, site-specific DNA-binding phosphoprotein that activates virus gene expression and promotes genome replication. To define the poorly understood mechanisms by which IE1 achieves its diverse functions, we identified IE1 domains that contribute to productive infection of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), the baculovirus prototype. Site-directed mutagenesis revealed that the N-terminal 23 residues of IE1 are required for origin-specific DNA replication and AcMNPV propagation, but not for DNA-binding-dependent transcriptional activation. Within this defined replication domain, we identified an invariant TPXR/H motif that resembles a consensus cyclin-dependent kinase phosphorylation site. Amino acid substitutions of potential phosphorylation sites within or near this motif caused loss of IE1-mediated DNA replication activity. Remarkably, substitution of the single threonine (residue 15) within the TPXR/H motif caused complete loss of AcMNPV multiplication. The replication domain was required for IE1 phosphorylation. It was also sufficient for conferring phosphorylation of a heterologous protein. Importantly, IE1 hyperphosphorylation coincided exclusively with AcMNPV DNA replication. The temporal regulation of IE1 phosphorylation and the essential nature of the TPXR/H motif suggest that phosphorylation critically alters and possibly activates DNA replication activity of IE1 during infection. The striking conservation of the TPXR/H motif among IE1 proteins further suggests that this molecular switch may be a common mechanism by which the alphabaculoviruses coordinate DNA replication and gene expression by using a single regulator.

Apoptosis is an important antivirus defense by virtue of its impact on virus multiplication and pathogenesis. To define molecular mechanisms by which viruses are detected and the apoptotic response is initiated, we examined the antiviral role of host inhibitor-of-apoptosis (IAP) proteins in insect cells. We report here that the principal IAPs, DIAP1 and SfIAP, of the model insects Drosophila melanogaster and Spodoptera frugiperda , respectively, are rapidly depleted and thereby inactivated upon infection with the apoptosis-inducing baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). Virus-induced loss of these host IAPs triggered caspase activation and apoptotic death. Elevation of IAP levels by ectopic expression repressed caspase activation. Loss of host IAP in both species was triggered by AcMNPV DNA replication. By using selected inhibitors, we found that virus-induced IAP depletion was mediated in part by the proteasome but not by caspase cleavage. Consistent with this conclusion, mutagenic disruption of the SfIAP RING motif, which acts as an E3 ubiquitin ligase, stabilized SfIAP during infection. Importantly, SfIAP was also stabilized upon the removal of its 99-residue N-terminal leader, which serves as a critical determinant of IAP turnover. These data indicated that a host pathway initiated by virus DNA replication and acting through instability motifs embedded within IAP triggers IAP depletion and thereby causes apoptosis. Taken together, the results of our study suggest that host modulation of cellular IAP levels is a conserved mechanism by which insects mount an apoptotic antiviral response. Thus, host IAPs may function as critical sentinels of virus invasion in insects.

The inhibitor-of-apoptosis (IAP) proteins encoded by baculoviruses bear a striking resemblance to the cellular IAP homologs of their invertebrate hosts. By virtue of the acquired selective advantage of blocking virus-induced apoptosis, baculoviruses may have captured cellular IAP genes that subsequently evolved for virus-specific objectives. To compare viral and host IAPs, we defined antiapoptotic properties of SfIAP, the principal cellular IAP of the lepidopteran host Spodoptera frugiperda . We report here that SfIAP prevented virus-induced apoptosis as well as viral Op-IAP3 (which is encoded by the Orgyia pseudotsugata nucleopolyhedrovirus) when overexpressed from the baculovirus genome. Like Op-IAP3, SfIAP blocked apoptosis at a step prior to caspase activation. Both of the baculovirus IAP repeats (BIRs) were required for SfIAP function. Moreover, deletion of the C-terminal RING motif generated a loss-of-function SfIAP that interacted and dominantly interfered with wild-type SfIAP. Like Op-IAP3, wild-type SfIAP formed intracellular homodimers, suggesting that oligomerization is a functional requirement for both cellular and viral IAPs. SfIAP possesses a ∼100-residue N-terminal leader domain, which is absent among all viral IAPs. Remarkably, deletion of the leader yielded a fully functional SfIAP with dramatically increased protein stability. Thus, the SfIAP leader contains an instability motif that may confer regulatory options for cellular IAPs that baculovirus IAPs have evolved to bypass for maximal stability and antiapoptotic potency. Our findings that SfIAP and viral IAPs have common motifs, share multiple biochemical properties including oligomerization, and act at the same step to block apoptosis support the hypothesis that baculoviral IAPs were derived by acquisition of host insect IAPs.

Apoptosis is an important antivirus defense. To define the poorly understood pathways by which invertebrates respond to viruses by inducing apoptosis, we have identified replication events that trigger apoptosis in baculovirus-infected cells. We used RNA silencing to ablate factors required for multiplication of Autographa californica multicapsid nucleopolyhedrovirus (Ac M NPV). Transfection with double-stranded RNA (dsRNA) complementary to the Ac M NPV late expression factors ( lef s) that are designated as replicative lef s ( lef-1 , lef-2 , lef-3 , lef-11 , p143 , dnapol , and ie-1 / ie-0 ) blocked virus DNA synthesis and late gene expression in permissive Spodoptera frugiperda cells. dsRNAs specific to designated nonreplicative lef s ( lef-8 , lef-9 , p47 , and pp31 ) blocked late gene expression without affecting virus DNA replication. Thus, both classes of lef s functioned during infection as defined. Silencing the replicative lef s prevented Ac M NPV-induced apoptosis of Spodoptera cells, whereas silencing the nonreplicative lef s did not. Thus, the activity of replicative lef s or virus DNA replication is sufficient to trigger apoptosis. Confirming this conclusion, Ac M NPV-induced apoptosis was suppressed by silencing the replicative lef s in cells from a divergent species, Drosophila melanogaster . Silencing replicative but not nonreplicative lef s also abrogated Ac M NPV-induced shutdown of host protein synthesis, suggesting that virus DNA replication triggers inhibition of host biosynthetic processes and that apoptosis and translational arrest are linked. Our findings suggest that baculovirus DNA replication triggers a host cell response similar to the DNA damage response in vertebrates, which causes translational arrest and apoptosis. Pathways for detecting virus invasion and triggering apoptosis may therefore be conserved between insects and mammals.

Immediate early viral protein IE1 is a potent transcriptional activator encoded by baculoviruses. Although the requirement of IE1 for multiplication of Autographa californica multicapsid nucleopolyhedrovirus (Ac M NPV) is well established, the functional roles of IE1 during infection are unclear. Here, we used RNA interference to ablate IE1, plus its splice variant IE0, and thereby define in vivo activities of these early proteins, including gene-specific regulation and induction of host cell apoptosis. Confirming an essential replicative role, simultaneous ablation of IE1 and IE0 by gene-specific double-stranded RNAs inhibited Ac M NPV late gene expression, reduced yields of budded virus by more than 1,000-fold, and blocked production of occluded virus particles. Depletion of IE1 and IE0 had no effect on early expression of the envelope fusion protein gene gp64 but abolished early expression of the caspase inhibitor gene p35 , which is required for prevention of virus-induced apoptosis. Thus, IE1 is a positive, gene-specific transactivator. Whereas an Ac M NPV p35 deletion mutant caused widespread apoptosis in permissive Spodoptera frugiperda cells, ablation of IE1 and IE0 prevented this apoptosis. Silencing of ie-1 also prevented Ac M NPV-induced apoptosis in nonpermissive Drosophila melanogaster cells. Thus, de novo synthesis of IE1 is required for virus-induced apoptosis. We concluded that IE1 causes apoptosis directly or contributes indirectly by promoting virus replication events that subsequently trigger cell death. This study reveals that IE1 is a gene-selective transcriptional activator which is required not only for expedition of virus multiplication but also for blocking of its own proapoptotic activity by upregulation of baculovirus apoptotic suppressors.

The baculoviruses are large double-stranded DNA viruses of insects that trigger widespread apoptosis. To block host cell apoptosis and thereby enhance multiplication, these viruses encode diverse suppressors of apoptosis (reviewed in references 5, 6, and 14). P49 and P35 are two baculovirus-encoded apoptotic suppressors that function by inhibiting a broad range of the cell death proteases known as caspases (2, 3, 19, 30, 40, 45). Although related to one another, P49 and P35 display different caspase specificities in the infected insect cell (21, 22, 45). Because selective inhibition of caspases may be advantageous in therapeutics for apoptosis-associated diseases, the molecular basis of target specificity by P49 and P35 is of considerable interest. The caspases are a family of cysteinyl aspartate-specific proteases that are critical effectors of apoptosis in metazoans (reviewed in references 15, 23, 32, and 33). Caspase-mediated proteolysis promotes cellular disassembly that includes chromatin condensation, nuclear DNA cleavage, membrane blebbing, and cell fragmentation. Thus, these death proteases are subject to regulation by diverse cellular and viral mechanisms (4, 6, 15, 33, 34, 36). Upon apoptotic signaling, initiator caspases are autoactivated through interactions of their N-terminal prodomain with specific adaptor proteins (1, 31, 35, 42). Subsequently, the effector caspases are activated by initiator caspase-mediated cleavage of their inactive zymogen (procaspase) form. Procaspase cleavage occurs between the large and small subunit domains, allowing for the assembly of the active protease, consisting of a dimer of heterodimers with two active sites (15, 23, 36). The substrate specificity of initiator caspases differs from that of effector caspases, due in part to their unique functions during execution of apoptosis (37). Not surprisingly, because of their central role in cell death, both types of caspases are important therapeutic targets for the treatment of apoptosis-associated diseases (11, 23). The novel selectivity of P49 and P35 for different caspases in insects provides a unique opportunity to define the in vivo determinants of caspase specificity in the experimentally advantageous system provided by the baculovirus-infected cell. Studying baculovirus caspase inhibitors has led to important insights into the role and regulation of caspases in apoptosis (4, 5, 7, 14). In general, a single baculovirus species encodes only one functional inhibitor, either a substrate inhibitor or an inhibitor of apoptosis protein (IAP). P35 from the baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) is the founding member of the family of substrate inhibitors that includes baculovirus P49 and entomopoxvirus P33 (3, 9, 27). Sequence comparisons have also revealed P35 homologs in Spodoptera littoralis NPV (SlNPV), Spodoptera litura NPV, Leucania separata NPV, and Bombyx mori NPV (reviewed in reference 5). The potency of P35 as a caspase inhibitor is attributed to its novel solvent-exposed reactive-site loop (RSL), which is readily recognized and cleaved at Asp87 by the target caspase (2, 3, 24, 30, 40, 43). Cleavage of the P4-P1 recognition motif DQMD87↓G (see Fig. ​Fig.1A),1A), located at the apex of the RSL, triggers a conformational change that positions the P35 N terminus in the caspase active site to prevent peptide hydrolysis and thereby form a stable complex (8, 10, 12, 24, 40). The inhibited complex consists of the caspase homodimer with each of the two active sites occupied by a separate monomer of P35. FIG. 1. Requirement of Cys2 for caspase inhibition by P49. (A) Comparison of P49 and P35. P49 (446 residues) and P35 (299 residues) share significant amino acid sequence identity. Cleavage of P49 and P35 occurs at Asp94 and Asp87, respectively, within the caspase ... P49 is a stoichiometric substrate inhibitor with P35-like properties. First discovered in SlNPV (9), P49 has the capacity to inhibit both effector and initiator caspases (19, 29, 45). Cleavage of P49 at an aspartate residue (Asp94) within the caspase recognition motif TVTD94↓G (see Fig. ​Fig.1A)1A) is necessary for formation of an inhibitory complex with the target caspase (19, 29, 45). Although the structure of P49 is unknown, sequence alignments suggest that it resembles that of P35, including the presence of a prominent RSL that presents Asp94 for cleavage (29, 45). Thus, we predicted that P49 functions by using a P35-like mechanism for caspase inhibition. P49 prevents proteolytic processing of effector caspases Sf-caspase-1 and Sf-caspase-2 during baculovirus infection of the moth Spodoptera frugiperda (order Lepidoptera) (45). Caspase cleavage of P49 at TVTD94↓G is required for P49-mediated suppression of virus-induced apoptosis. Thus, P49 is a substrate inhibitor of the initiator caspase designated Sf-caspase-X, which is responsible for the proteolysis and activation of Sf-caspase-1 and -2 (45). In a cellular context, Sf-caspase-X is also inhibited by baculovirus Op-IAP, but not P35, which fails to inactivate other initiator caspases, including those from invertebrates (16, 21, 28, 39, 45). Because P49's TVTD recognition motif resembles the caspase processing sites TETD↓G and AETD↓G of pro-Sf-caspase-1 and -2, respectively, we hypothesized that P49's in vivo specificity is determined by its caspase recognition motif. To test this possibility, we altered the recognition motifs of P49 and P35, delivered the modified caspase inhibitors to Spodoptera cells by using recombinant baculoviruses, and monitored proteolytic processing of Sf-caspase-1 and -2 during infection. We report here that, when P49's TVTD motif was swapped with P35's DQMD motif, P49 was impaired as an initiator caspase inhibitor and instead functioned downstream as an effector caspase inhibitor. In contrast, when P35's DQMD motif was swapped with TVTD, P35's selectivity for effector caspases was unaltered. Thus, P49 requires a TVTD motif for Spodoptera initiator caspase inhibition, but this motif alone is insufficient to confer the same activity upon P35. To search for additional determinants of caspase selectivity, we compared the biochemical properties of P49 and P35. By using recombinant P49, produced and purified from Escherichia coli for the first time, we determined that P49 and P35 use comparable mechanisms for caspase inhibition. In contrast to P35, which acts as a monomer, P49 functions as a homodimer with the capacity to form a stable complex with two individual caspase dimers. Thus, P49 is the first example of a divalent caspase inhibitor in which caspase targeting can be altered by sequence alterations in the caspase recognition motif.

The molecular mechanisms by which RNA viruses induce apoptosis and apoptosis-associated pathology are not fully understood. Here we show that flock house virus (FHV), one of the simplest RNA viruses (family, Nodaviridae ), induces robust apoptosis of permissive Drosophila Line-1 (DL-1) cells. To define the pathway by which FHV triggers apoptosis in this model invertebrate system, we investigated the potential role of Drosophila apoptotic effectors during infection. Suggesting the involvement of host caspases, the pancaspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluromethylketone ( z -VAD-fmk) prevented FHV-induced cytopathology and prolonged cell survival. RNA interference-mediated ablation of the principal Drosophila effector caspase DrICE or its upstream initiator caspase DRONC prevented FHV-induced apoptosis and demonstrated direct participation of this intrinsic caspase pathway. Prior to the FHV-induced activation of DrICE, the intracellular level of inhibitor-of-apoptosis (IAP) protein DIAP1, the principal caspase regulator in Drosophila melanogaster , was dramatically reduced. DIAP1 was depleted despite z -VAD-fmk-mediated caspase inhibition during infection, suggesting that the loss of DIAP1 was caused by an upstream FHV-induced signal. The RNA interference-mediated knockdown of DIAP1 caused rapid and uniform apoptosis of DL-1 cells and thus indicated that DIAP1 depletion is sufficient to trigger apoptosis. Confirming this conclusion, the elevation of intracellular DIAP1 levels in stable diap1 -transfected cells blocked caspase activation and prevented FHV-induced apoptosis. Collectively, our findings suggest that DIAP1 is a critical sensor of virus infection, which upon virus-signaled depletion relieves caspase inhibition, which subsequently executes apoptotic death. Thus, our study supports the hypothesis that altering the level or the activity of cellular IAP proteins is a general mechanism by which RNA viruses trigger apoptosis.

Immediate-early protein IE1 is a principal regulator of viral transcription and a contributor to origin-specific DNA replication of the baculovirus Autographa californica multicapsid nucleopolyhedrovirus (Ac M NPV). Since these viral functions involve interaction of dimeric IE1 with palindromic homologous region ( hr ) enhancer-origin elements of the Ac M NPV genome within the nucleus, it is presumed that proper nuclear transport of IE1 is essential for productive infection. To investigate the mechanisms of IE1 nuclear import, we analyzed the effect of site-directed mutations on IE1 subcellular distribution. As demonstrated by fluorescence microscopy and biochemical fractionation of plasmid-transfected cells, wild-type IE1 localized predominantly to the nucleus. Substitution or deletion of amino acid residues within a positively charged domain (residues 534 to 538) adjacent to IE1's oligomerization motif impaired nuclear import and caused loss of transactivation. Moreover, upon coexpression, these import-defective mutations prevented nuclear entry of wild-type IE1. In contrast, double-mutated IE1 defective for both nuclear import and dimerization failed to block nuclear entry or transactivation by wild-type IE1. Thus, import-defective IE1 dominantly interfered with wild-type IE1 by direct interaction and cytosolic trapping. Collectively, our data indicate that the small basic domain encompassing residues R 537 and R 538 constitutes a novel nuclear localization element that functions only upon IE1 dimerization. These findings support a model wherein IE1 oligomerizes within the cytosol as a prerequisite for nuclear entry and subsequent high-affinity interaction with the symmetrical binding sites comprising Ac M NPV hr enhancer-origin elements.

IE1 is a principal transcriptional regulator of Autographa californica multicapsid nucleopolyhedrovirus (Ac M NPV). Transactivation by IE1 is stimulated when early viral promoters are cis linked to homologous-region ( hr ) enhancer sequences of Ac M NPV. This transcriptional enhancement is correlated with the binding of IE1 as a dimer to the 28-bp palindromic repeats comprising the hr enhancer. To define the role of homophilic interactions in IE1 transactivation, we have mapped the IE1 domains required for oligomerization. We report here that IE1 oligomerizes by a mechanism independent of enhancer binding, as demonstrated by in vitro pull-down assays using fusions of IE1 (582 residues) to the C terminus of glutathione S -transferase. In vivo oligomerization of IE1 was verified by immunoprecipitation of IE1 complexes from extracts of plasmid-transfected SF21 cells. Analyses of a series of site-directed IE1 insertion mutations indicated that a helix-loop-helix (HLH)-like domain extending from residue 543 to residue 568 is the primary determinant of oligomerization. Replacement of residues within the hydrophobic face of the putative dimerization domain disrupted IE1 homophilic interactions and caused loss of IE1 transactivation of hr -dependent promoters in plasmid transfection assays. Thus, oligomerization is required for IE1 transcriptional stimulation. HLH mutations also reduced IE1 stability and abrogated transactivation of non- hr -dependent promoters. These data support a model wherein IE1 oligomerizes prior to DNA binding to facilitate proper interaction with the symmetrical recognition sites within the hr enhancer and thereby promote the transcription of early viral genes.

TED (transposable element D) is a 7.5-kb, middle-repetitive retrotransposon of the moth Trichoplusia ni. It was identified as a single-copy insertion (TEDFP-D) within the DNA genome of Autographa californica nucleopolyhedrovirus (AcMNPV), having transposed during infection of cultured T. ni cells with this baculovirus (33). Comprised of gag, pol, and env genes that are flanked by long terminal repeats (Fig. ​(Fig.1A),1A), TEDFP-D was the first example of spontaneous, retroelement-mediated transfer of functional host genes to an animal virus (11, 30, 33). On the basis of sequence similarity within pol, TED is most closely related to the Drosophila retrotransposons 17.6, 297, tom, and gypsy (11, 18, 31, 37, 46) and thus is classified as a member of the gypsy family of retroelements (42). Recent studies have indicated that TED, tom, and gypsy possess env-like genes that encode membrane-associated glycoproteins with properties expected of vertebrate retroviral envelope proteins (35, 40, 45). These findings, combined with evidence that gypsy produces enveloped viruslike particles (VLP) that are infectious in Drosophila larvae (19, 40, 41), suggest that the gypsy family transposons may be facultative retroviruses of insects (9, 38). FIG. 1 (A) TED genetic organization. TED (7.5 kb) contains gag, pol, and env genes flanked by 270-bp long terminal repeats (solid boxes). PR, RT, and IN are conserved retroviruslike domains within pol. (B) TED sequences expressed by baculovirus vectors. TED ... The assembly of VLP containing genomic RNA and the components necessary for reverse transcription is critical to the movement of retrotransposons and retroviruses (for reviews, see references 4, 17, and 38). In the case of retrotransposons Ty1 and Ty3 of Saccharomyces cerevisiae, synthesis of gag-encoded structural proteins is required for transposition, suggesting that VLP assembly is an important intermediate step in this process (2, 13). VLP may have multiple functions, including compartmentalization of genomic RNA, along with reverse transcriptase (RT) and integrase (IN), protection of the genome from intracellular nucleases, and genome transport to the nucleus, among others. In vertebrate retroviruses, distinct domains of the Gag precursor that are separated by proteolytic cleavage into matrix (MA), capsid (CA), and nucleocapsid (NC) proteins accomplish these functions (reviewed in references 5, 7, 17, 49 and 50). Despite the importance of gag in transposition, little is known about Gag protein processing and VLP assembly by the env-containing retrotransposons. Due to their striking resemblance to the vertebrate retroproviruses, these simple elements may provide useful models for retrovirus CA assembly, morphogenesis, and evolution. An effective means for investigation of TED Gag structure and function has been the overexpression of TED by using baculovirus vectors (10). Infection of cultured lepidopteran cells with TED-containing AcMNPV recombinants demonstrated that the primary translation product of TED gag is Pr55gag (30). Consistent with Gag function, Pr55gag assembles to produce 60-nm-diameter VLP. Expression of TED gag-pol, which requires a −1 ribosomal frameshift (Fig. ​(Fig.1A),1A), yields VLP composed of the major CA protein p37gag, traces of Pr55gag, and RT activity. The appearance of p37gag correlated with expression of the 5′ portion of TED pol with sequence similarity to retroviral proteases (PR). Thus, it was proposed that p37gag is derived by PR-mediated processing of Pr55gag (30). Moreover, PR cleavage of the TED Gag-Pol polyprotein Pr195gag-pol was predicted to yield RT and other functions necessary for transposition. To examine the processing and assembly of TED Gag proteins, we have extended the use of baculovirus vectors to overexpress gag and pol in cultured Spodoptera frugiperda (SF21) cells that lack endogenous copies of TED. By using polyclonal antisera raised against TED Gag proteins, we confirmed that Pr55gag is cleaved to produce the major VLP protein p37gag. Moreover, we report here that cleavage is mediated by the TED pol-encoded PR, which is generated from the frameshifted polyprotein Pr195gag-pol. The Gag cleavage site was mapped to the junction of a central NC-like domain and a C-terminal acidic domain within Pr55gag. Analogous acidic domains were identified at the C termini of Drosophila retroelements 17.6, 297, and tom, suggesting that the function of the acidic domain is conserved. Nonetheless, the acidic domain was dispensable for VLP assembly, whereas the adjacent NC-like domain was required. These findings suggested that p37gag provides both CA and NC functions and that TED incorporates multiple functions into a single Gag protein. This feature distinguishes TED from those vertebrate retroviruses in which the gag products are proteolytically separated.

IE1 is the principal early transregulator of Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV). The 582-residue protein stimulates viral transcription and binds as a dimer to 28-bp palindromic repeats (28-mers) comprising the AcMNPV homologous region (hr) transcription enhancers. To define IE1 domains responsible for hr-dependent transactivation, we first constructed a series of IE1 fusions to the DNA binding domain of the yeast GAL4 transactivator. In transfection assays, GAL4-IE1 fusions stimulated transcription from a TATA-containing AcMNPV promoter only upon cis linkage to GAL4 DNA binding sites. IE1 N-terminal residues 8 to 118 were sufficient for GAL4-binding-site-dependent transactivation. To identify IE1 residues required for hr interaction, we tested a series of IE1 mutations for 28-mer binding by using electrophoretic mobility shift assays. Deletion of IE1 residues other than the N-terminal transactivation domain eliminated 28-mer binding. Of 14 insertion mutations, only IE1(I425) and IE1(I553) failed to bind the 28-mer either as homodimers or as heterodimers with functional IE1. In contrast to insertion IE1(I425), IE1(I553) also failed to compete with wild-type IE1 for DNA binding and suggested a defect in oligomerization. Consistent with loss of oligomerization, substitutions within a hydrophobic repeat (residues 543 to 568) at the IE1 C terminus abolished 28-mer binding and demonstrated that this helix-loop-helix-like domain is required for DNA interaction. These data confirm that IE1 contains separable domains for transactivation and oligomerization-dependent DNA binding. Furthermore, they support a model wherein hr-mediated transactivation by IE1 involves sequence-specific DNA binding that contributes to transcriptional stimulation by interaction with components of the basal transcription complex.

The DNA damage response (DDR) of a host organism represents an effective antiviral defense that is frequently manipulated and exploited by viruses to promote multiplication. We report here that the large DNA baculoviruses, which require host DDR activation for optimal replication, encode a conserved replication factor, LEF-7, that manipulates the DDR via a novel mechanism. LEF-7 suppresses DDR-induced accumulation of phosphorylated host histone variant H2AX (γ-H2AX), a critical regulator of the DDR. LEF-7 was necessary and sufficient to block γ-H2AX accumulation caused by baculovirus infection or DNA damage induced by means of pharmacological agents. Deletion of LEF-7 from the baculovirus genome allowed γ-H2AX accumulation during virus DNA synthesis and impaired both very late viral gene expression and production of infectious progeny. Thus, LEF-7 is essential for efficient baculovirus replication. We determined that LEF-7 is a nuclear F-box protein that interacts with host S-phase kinase-associated protein 1 (SKP1), suggesting that LEF-7 acts as a substrate recognition component of SKP1/Cullin/F-box (SCF) complexes for targeted protein polyubiquitination. Site-directed mutagenesis demonstrated that LEF-7's N-terminal F-box is necessary for γ-H2AX repression and Autographa californica multiple nucleopolyhedrovirus (AcMNPV) replication events. We concluded that LEF-7 expedites virus replication most likely by selective manipulation of one or more host factors regulating the DDR, including γ-H2AX. Thus, our findings indicate that baculoviruses utilize a unique strategy among viruses for hijacking the host DDR by using a newly recognized F-box protein.

Baculovirus p35 prevents programmed cell death in diverse organisms and encodes a protein inhibitor (P35) of the CED-3/interleukin-1 beta-converting enzyme (ICE)-related proteases. By using site-directed mutagenesis, we have identified P35 domains necessary for suppression of virus-induced apoptosis in insect cells, the context in which P35 evolved. During infection, P35 was cleaved within an essential domain at or near the site DQMD-87G required for cleavage by CED-3/ICE family proteases. Cleavage site substitution of alanine for aspartic acid at position 87 (D87A) of the P1 residue abolished P35 cleavage and antiapoptotic activity. Although the P4 residue substitution D84A also caused loss of apoptotic suppression, it did not eliminate cleavage and suggested that P35 cleavage is not sufficient for antiapoptotic activity. Apoptotic insect cells contained a CED-3/ICE-like activity that cleaved in vitro-translated P35 and was inhibited by recombinant wild-type P35 but not P1- or P4-mutated P35. Thus, baculovirus infection directly or indirectly activates a novel CED-3/ICE-like protease that is inhibited by P35, thereby preventing virus-induced apoptosis. Our findings confirmed the inhibitory activity of P35 towards the CED-3/ICE protease, including recombinant mammalian enzymes, and were consistent with a mechanism involving P35 stoichiometric interaction and cleavage. P35's inhibition of phylogenetically diverse proteases accounts for its general effectiveness as an apoptotic suppressor.

The hr5 enhancer element stimulates early viral transcription and may function as an origin of DNA replication for Autographa californica nuclear polyhedrosis virus (AcMNPV). The smallest functional unit of hr5 is a 28-bp repeat consisting of an imperfect palindrome (28-mer). To identify essential sequences and examine the molecular basis of hr5 activity, the effects of site-directed mutations on transcriptional enhancement by the 28-mer and binding of the AcMNPV transregulator IE1 were investigated. In transfection assays and infections with AcMNPV recombinants, activation of a basal viral promoter required sequences within both halves of the 28-mer. Basal promoter activation also required a critical spacing between these half sites. Mobility shift assays indicated that hr5 probes containing a single 28-mer were bound by in vitro-synthesized IE1. Competition assays using DNA fragments that contained mutated 28-mers demonstrated that both half sites were required for optimal binding of IE1. Similar assays using mutated 28-mer DNAs and nuclear extracts indicated that the relative affinity with which AcMNPV infection-specific proteins bound to the 28-mer was similar to that of in vitro-synthesized IE1. By using a combination of DNA binding and antibody supershift assays, it was demonstrated that IE1 binds to the 28-mer as a dimer. Collectively, these findings support a model in which symmetrical IE1 binding and simultaneous interaction with each half site are required for IE1-mediated transcriptional enhancement by hr5. Thus, sequence-specific binding may be one of the mechanisms by which IE1 directly or indirectly transregulates baculovirus gene expression.

Expression of p35 from the DNA genome of Autographa californica nuclear polyhedrosis virus (AcMNPV) suppresses virus-induced apoptosis and promotes virus replication in Spodoptera frugiperda (SF21) cells. To examine the molecular mechanism by which p35 prevents apoptosis in insects, SF21 cells were stably transfected with p35. Neomycin-resistant cell lines that synthesized protein P35 were identified. Stable transfection with p35 protected SF21 cells from apoptosis induced by actinomycin D concentrations that caused apoptotic death of untransfected cells. Cellular expression of p35 also blocked apoptosis induced by infection with p35 null mutants and restored mutant replication to levels comparable to those of wild-type virus. In contrast, stable expression of the mammalian death suppressor bcl-2 failed to block actinomycin D- or AcMNPV-induced apoptosis. Thus, p35 was sufficient to prevent apoptosis, whereas bcl-2 was not, suggesting that the activities of the two nonhomologous death regulators are functionally distinct. Stable expression of the truncation mutant p35(1-76), containing the N terminus of p35, failed to block apoptosis. However, p35(1-76) interfered with p35 antiapoptotic activity, since stably transfected cells underwent apoptosis upon infection with wild-type AcMNPV. Despite normal levels of viral p35 transcription, P35 levels were selectively reduced during infection. Thus, p35(1-76) acted as a dominant inhibitor by directly or indirectly affecting the synthesis or stability of viral P35. These results suggested that the N terminus of P35 constitutes a functional domain which is required to interact with other proteins, possibly host invertebrate death regulators or P35 itself.

Autographa californica nuclear polyhedrosis virus (AcMNPV) contains five homologous regions (hr1 through hr5) interspersed throughout its genome. Analysis of plasmid transfections indicates that the hrs function as transcriptional enhancers and possible origins of viral DNA replication. The role of these repetitive elements in regulating expression from the AcMNPV genome was examined by constructing a series of recombinant viruses that tested the effect of hr5 on expression of the adjacent p35 gene (p35). When embedded within the viral genome, hr5 stimulated transcription from the early p35 promoter in a position- and orientation-independent manner. Moreover, hr5 and the upstream activating region of p35 were functionally interchangeable. A 28-bp imperfect palindrome, repeated six times within hr5, was the minimal sequence required for p35 promoter activation. hr5 also stimulated another early AcMNPV promoter but not a late promoter or a host-derived promoter, suggesting that enhancement is promoter specific during infection. To investigate its role during AcMNPV replication, hr5 was deleted from its normal position within the viral genome. The resulting hr5 mutants exhibited no apparent defects in replication, as judged by production of budded virus and levels of very late gene expression, even though steady-state levels of p35 RNA were reduced. These results indicated for the first time that hr5 functions as a transcriptional enhancer within the viral genome. However, the element is not required for AcMNPV replication in cultured cells. Thus, loss of one of five possible origins of DNA replication is not deleterious to viral growth. Since p26 was removed from the hr5 deletion mutants, this gene is also nonessential for viral replication.

Positive-strand RNA [(+)RNA] viruses invariably replicate their RNA genomes on modified intracellular membranes. In infected Drosophila cells, Flock House nodavirus (FHV) RNA replication complexes form on outer mitochondrial membranes inside ∼50-nm, virus-induced spherular invaginations similar to RNA replication-linked spherules induced by many (+)RNA viruses at various membranes. To better understand replication complex assembly, we studied the mechanisms of FHV spherule formation. FHV has two genomic RNAs; RNA1 encodes multifunctional RNA replication protein A and RNA interference suppressor protein B2, while RNA2 encodes the capsid proteins. Expressing genomic RNA1 without RNA2 induced mitochondrial spherules indistinguishable from those in FHV infection. RNA1 mutation showed that protein B2 was dispensable and that protein A was the only FHV protein required for spherule formation. However, expressing protein A alone only “zippered” together the surfaces of adjacent mitochondria, without inducing spherules. Thus, protein A is necessary but not sufficient for spherule formation. Coexpressing protein A plus a replication-competent FHV RNA template induced RNA replication in trans and membrane spherules. Moreover, spherules were not formed when replicatable FHV RNA templates were expressed with protein A bearing a single, polymerase-inactivating amino acid change or when wild-type protein A was expressed with a nonreplicatable FHV RNA template. Thus, unlike many (+)RNA viruses, the membrane-bounded compartments in which FHV RNA replication occurs are not induced solely by viral protein(s) but require viral RNA synthesis. In addition to replication complex assembly, the results have implications for nodavirus interaction with cell RNA silencing pathways and other aspects of virus control.

TED is a lepidopteran retrotransposon found inserted within the DNA genome of the Autographa californica nuclear polyhedrosis virus mutant, FP-D. To examine the proteins and functions encoded by this representative of the gypsy family of retrotransposons, the gag- and pol-like open reading frames (ORFs 1 and 2) were expressed in homologous lepidopteran cells by using recombinant baculovirus vectors. Expression of ORF 1 resulted in synthesis of an abundant TED-specific protein (Pr55gag) that assembled into viruslike particles with a diameter of 55 to 60 nm. Expression of ORF 2, requiring a -1 translational frameshift, resulted in synthesis of a protease that mediated cleavage of Pr55gag to generate p37, the major protein component of the resulting particles. Expression of ORF 2 also produced reverse transcriptase that associated with these particles. Both protease and reverse transcriptase activities mapped to domains within ORF 2 that contain sequence similarities with the corresponding functional domains of the pol gene of the vertebrate retroviruses. These results indicated that TED ORFs 1 and 2 functionally resemble the retrovirus gag and pol genes and demonstrated for the first time that an invertebrate member of the gypsy family of elements encodes active forms of the structural and enzymatic functions necessary for transposition via an RNA intermediate. TED integration within the baculovirus genome thus represents one of the first examples of transposon-mediated transfer of host-derived genes to an eukaryotic virus.

Site-directed mutagenesis was used to examine the organization of cis-acting regulatory elements that comprise the promoter of the early 35,000-molecular-weight protein gene (35K protein gene) encoded by the EcoRI-S region of the baculovirus Autographa californica nuclear polyhedrosis virus. The promoter fragment, extending from positions -226 to +12 relative to the early RNA start site (position +1), was fused to the reporter gene encoding chloramphenicol acetyltransferase (CAT) and then inserted into the genome of recombinant viruses (3.96 map units) in order to ascertain the role of regulatory elements in the context of a normal infection. A combination of deletions and linker insertions revealed that early transcription was mediated by a basal (minimum) promoter, consisting of the TATA element (positions -30 to -25), that was in turn responsive to an upstream activating region located between -90 and -30. The TATA element exerted the single greatest influence on the level of early promoter activity and contained all information necessary to direct transcription from a site located 30 nucleotides downstream. The upstream activating region provided a 10- to 15-fold stimulation of transcription from the early +1 start site that was mediated by distinct DNA elements. These regulatory elements included two GC motifs (centered at positions -81 and -54, respectively), composed of alternating G and C residues, and a CGT motif (position -40) that contained the core sequence A(A/T)CGT(G/T). Each motif was required for full promoter activity during the early phase of infection. This organization that employs diverse cis-acting stimulatory elements is typical of promoters responsive to RNA polymerase II and may facilitate increased expression of A. californica nuclear polyhedrosis virus genes early in infection when the level of viral DNA for transcription is critically low.

Baculoviruses induce widespread apoptosis in invertebrates. To better understand the pathways by which these DNA viruses trigger apoptosis, we have used a combination of RNA silencing and overexpression of viral and host apoptotic regulators to identify cell death components in the model system of Drosophila melanogaster . Here we report that the principal effector caspase DrICE is required for baculovirus-induced apoptosis of Drosophila DL-1 cells as demonstrated by RNA silencing. proDrICE was proteolytically cleaved and activated during infection. Activation was blocked by overexpression of the cellular inhibitor-of-apoptosis proteins DIAP1 and SfIAP but not by the baculovirus caspase inhibitor P49 or P35. Rather, the substrate inhibitors P49 and P35 prevented virus-induced apoptosis by arresting active DrICE through formation of stable inhibitory complexes. Consistent with a two-step activation mechanism, proDrICE was cleaved at the large/small subunit junction TETD 230 -G by a DIAP1-inhibitable, P49/P35-resistant protease and then at the prodomain junction DHTD 28 -A by a P49/P35-sensitive protease. Confirming that P49 targeted DrICE and not the initiator caspase DRONC, depletion of DrICE by RNA silencing suppressed virus-induced cleavage of P49. Collectively, our findings indicate that whereas DIAP1 functions upstream to block DrICE activation, P49 and P35 act downstream by inhibiting active DrICE. Given that P49 has the potential to inhibit both upstream initiator caspases and downstream effector caspases, we conclude that P49 is a broad-spectrum caspase inhibitor that likely provides a selective advantage to baculoviruses in different cellular backgrounds.

Members of the inhibitor of apoptosis (iap) gene family prevent programmed cell death induced by multiple signals in diverse organisms, suggesting that they act at a conserved step in the apoptotic pathway. To investigate the molecular mechanism of iap function, we expressed epitope-tagged Op-iap, the prototype viral iap from Orgyia pseudotsugata nuclear polyhedrosis virus, by using novel baculovirus recombinants and stably transfected insect cell lines. Epitope-tagged Op-iap blocked both virus- and UV radiation-induced apoptosis. With or without apoptotic stimuli, Op-IAP protein (31 kDa) cofractionated with cellular membranes and the cytosol, suggesting a cytoplasmic site of action. To identify the step(s) at which Op-iap blocks apoptosis, we monitored the effect of Op-iap expression on in vivo activation of the insect CED-3/ICE death proteases (caspases). Op-iap prevented in vivo caspase-mediated cleavage of the baculovirus substrate inhibitor P35 and blocked caspase activity upon viral infection or UV irradiation. However, unlike the stoichiometric inhibitor P35, Op-IAP failed to affect activated caspase as determined by in vitro protease assays. These findings provide the first biochemical evidence that Op-iap blocks activation of the host caspase or inhibits its activity by a mechanism distinct from P35. Moreover, as suggested by the capacity of Op-iap to block apoptosis induced by diverse signals, including virus infection and UV radiation, iap functions at a central point at or upstream from steps involving the death proteases.

Autographa californica nuclear polyhedrosis virus (AcMNPV) mutants that lack the apoptotic suppressor gene p35 cause apoptosis in Spodoptera frugiperda SF21 cells. To identify a viral signal(s) that induces programmed cell death, we first defined the timing of apoptotic events during infection. Activation of a P35-inhibitable caspase, intracellular fragmentation of host and AcMNPV DNA, and cell membrane blebbing coincided with the initiation of viral DNA synthesis between 9 and 12 h after infection and thus suggested that apoptotic signaling begins at or before this time. Virus entry was required since binding of budded virus to host cell receptors alone was insufficient to induce apoptosis. To therefore determine the contribution of early and late replication events to apoptotic signaling, we used the AcMNPV mutant ts8 with a temperature-sensitive lesion in the putative helicase gene p143. At the nonpermissive temperature at which viral DNA synthesis was conditionally blocked, ts8 caused extensive apoptosis of the SF21 cell line p3576D, which dominantly interferes with anti-apoptotic function of viral P35. Confirming that apoptosis can be induced in the absence of normal viral DNA synthesis, parental SF21 cells also underwent apoptosis when infected with a ts8 p35 deletion mutant at the nonpermissive temperature. However, maximum levels of ts8 p35 deletion mutant-induced apoptosis required a temperature-sensitive event(s) that included the initiation of viral DNA synthesis. Collectively, these data suggested that baculovirus-induced apoptosis can be triggered by distinct early (pre-DNA synthesis) and late replicative events, including viral DNA synthesis or late gene expression.

The p35 gene of Autographa californica nuclear polyhedrosis virus (AcMNPV) is required to block virus-induced apoptosis. The trans-dominant activity of p35 suppresses premature cell death and facilitates AcMNPV replication in a cell line- and host-specific manner. To characterize the p35 gene product (P35), a specific polyclonal antiserum was raised. As revealed by immunoblot analyses of wild-type AcMNPV-infected cells, P35 appeared early (8 to 12 h) and accumulated through the late stages of infection (24 to 36 h). Biochemical fractionation of cells both early and late in infection and indirect immunochemical staining demonstrated that P35 localized predominantly to the cytosol (150,000 x g supernatant); comparatively minor quantities of P35 were associated with intracellular membranes. The cytoplasmic localization of P35 was independent of virus infection. The functional significance of the early and late synthesis of P35 was examined by constructing recombinant viruses in which the timing and level of p35 expression were altered. Delaying P35 synthesis by placing p35 under exclusive control of a strong, very late promoter failed to suppress intracellular DNA fragmentation and apoptotic blebbing in most cells. Thus, earlier expression of p35 was required to block virus-induced apoptosis. Site-specific mutagenesis of the p35 promoter demonstrated that low levels of P35 were sufficient to block apoptosis, whereas higher levels were required to maintain wild-type virus gene expression. Consistent with an early role in infection, P35 was also detected in the budded form of AcMNPV. Because of the lack of sequence similarity and its cytosolic targeting, P35 may function in a manner that is mechanistically distinct from other apoptotic regulators, including Bcl-2 and the adenovirus E1B 19-kDa protein.

The gene encoding the 35-kDa protein (35k gene) located within the EcoRI-S genome fragment of Autographa californica nuclear polyhedrosis virus (AcMNPV) is transcribed early in infection. To examine its function(s) with respect to virus multiplication, we introduced specific mutations of this early gene into the AcMNPV genome. In Spodoptera frugiperda (SF21) culture, deletion of the 35K gene reduced yields of extracellular, budded virus from 200- to 15,000-fold, depending on input multiplicity. Mutant replication was characterized by dramatically diminished levels of late and very late (occlusion-specific) virus gene expression and premature cell lysis. In contrast, 35K gene inactivation had no effect on virus growth in cultured Trichoplusia ni (TN368) cells. Insertion of the 35K gene and its promoter at an alternate site (polyhedrin locus) restored virus replication to wild-type levels in SF21 culture. Subsequent insertion of 4 bp after codon 81 generated a frameshift mutant that exhibited a virus phenotype indistinguishable from that of 35K deletion mutants and demonstrated that the 35K gene product (p35) was required for wild-type replication in SF21 cells. Mutagenesis also indicated that the C terminus of p35, including the last 12 residues, was required for function. In complementation assays, wild-type virus bearing a functional 35K gene allele stimulated all aspects of 35K null mutant replication and suppressed early cell lysis. These findings indicated that p35 is a trans-dominant factor that facilitates AcMNPV growth in a cell line-specific manner.

Transcription of the gene encoding a 35,000-molecular-weight protein (35K protein) from the EcoRI-S region (86.8 to 87.8 map units) of Autographa california nuclear polyhedrosis virus (AcMNPV) occurs early in infection and declines later. The region promoting the gene for the 35K protein, extending from 426 base pairs (bp) upstream to 12 bp downstream from the RNA start site, was linked to the bacterial chloramphenicol acetyltransferase gene (CAT) for analysis. CAT expression was monitored in cells that were transfected with plasmids containing the promoter-CAT fusion as well as cells infected with recombinant viruses containing the chimeric gene inserted into the AcMNPV genome. Mapping of the 5' ends of CAT-specific RNAs indicated that transcription initiated from the proper sites in both assays; moreover, the promoter fragment retained its early activity, despite an alternate location in the viral genome. The 5' boundary of upstream regulatory sequences was determined by constructing deletions of the promoter fragment extending toward the early RNA start site (position +1). In transient assays, a gradual reduction in CAT expression occurred as sequences from positions -426 to -31 were removed. In contrast, promoter deletions from positions -426 to -155 in recombinant viruses exhibited no effect on CAT expression, whereas deletions to position -55 abolished early expression but had no effect on late expression. Late CAT expression was eliminated when deletions to position -4 removed part of the late RNA start site. DNA signals potentiating early transcription were therefore located upstream (between positions -155 and -55) from those involved in late transcription of the gene encoding the 35K protein. Potential consensus sequences for early and late regulatory elements were identified.

It has been shown previously that Drosophila cells infected with black beetle virus synthesize an early viral protein, protein A, a putative element of the viral RNA polymerase. Synthesis of protein A declines sharply by 6 h postinfection, whereas synthesis of viral coat protein alpha continues for at least 14 h. The early shutoff in protein A synthesis occurred despite the presence of equimolar proportions of the mRNAs for proteins A and alpha, RNAs 1 and 2, respectively. We have now been able to mimic this translational discrimination in a cell-free protein-synthesizing system prepared from infected or uninfected Drosophila cells, thus allowing further analysis of the mechanism by which translation of RNA 1 is selectively turned off. The results revealed no evidence for control by virus-encoded proteins or by virus-induced modification of mRNAs by the cell-free system. Rather, with increasing RNA concentration, viral RNA 1 was outcompeted by its genomic partner, RNA 2. This suggests that the early shutoff in intracellular synthesis of protein A is due to decreasing ability of RNA 1 to compete for a rate-controlling translational factor(s) as the concentration of viral RNAs accumulates within the infected cell.

Black beetle virus (BBV), one of a recently discovered class of viruses with a bipartite genome, multiplied readily in Schneider's line 1 of Drosophila cells. Virus yields, on the order of 100 mg per liter of culture, were unusually high and represented some 20% of the total cell protein within 3 days after infection. A derivative subline of these Drosophila cells was found to be resistant to infection by BBV. These resistant cells were also found to carry small amounts of BBV-related particles, possibly a maturation-defective form of BBV.

Black beetle virions contain two RNAs. The smaller one, RNA 2, has previously been shown to be a messenger for viral coat protein. It is shown here, by infecting sensitized Drosophila cells with the individually purified RNAs, that the larger one, RNA 1, carries the viral gene(s) required for RNA polymerase functions. RNA 2 was dispensible for synthesis of viral RNA 1 and subgenomic RNA 3 but was essential for synthesis of RNA 2 and virions. Cells infected with RNA 1 alone produced RNA 3 in proportions 10- to 20-fold greater than cells infected with virions. This overproduction of RNA 3 decreased with increasing proportions of RNA 2 in the infecting RNA 1. We conclude that RNA 1 is the previously unidentified progenitor of subgenomic RNA 3, whereas RNA 2 regulates the amount of RNA 3 produced in the infected cell.

Black beetle virus induces the synthesis of three new proteins, protein A (molecular weight, 104,000), protein α (molecular weight, 47,000), and protein B (molecular weight, 10,000), in infected Drosophila cells. Two of these proteins, A and α, are known to be encoded by black beetle virus RNAs 1 and 2, respectively, extracted from virions. We found that RNA extracted from infected cells directed the synthesis of all three proteins when it was added to a cell-free protein-synthesizing system. When polysomal RNA was fractionated on a sucrose density gradient, the messengers for proteins A and α cosedimented with viral RNAs 1 (22S) and 2 (15S), respectively. However, the messenger for protein B was a 9S RNA (RNA 3) not found in purified virions. Like the synthesis of viral RNAs 1 and 2, intracellular synthesis of RNA 3 was not affected by the drug actinomycin D at concentrations which blocked synthesis of host cell RNA. This indicated that RNA 3 is a virus-specific subgenomic RNA and, therefore, that protein B is a virus-encoded protein.

Analysis of the temporal sequence of RNA transcription within the Autographa californica nuclear polyhedrosis virus genome revealed individual transcription units composed of overlapping early or late RNAs, or both. High-resolution S1 nuclease mapping within the 3.0-kilobase HindIII-K fragment located five overlapping RNAs (1.07, 1.38, 2.63, 3.16, and 3.50 kilobases) transcribed in the same direction and terminated at a common 3' site. The smallest RNAs appeared early but were replaced in time by successively larger RNAs initiated further upstream. Primer extension analysis supported the contention that this temporal regulation involved the sequential activation of upstream promoters and the coordinate deactivation of downstream promoters. As such, transcription from upstream genes may suppress that of downstream genes via transcriptional interference (promoter occlusion) and thereby facilitate sequential expression of different viral functions. In contrast, overlapping RNAs with extended 3' ends were transcribed from the abundantly expressed p10 and polyhedrin genes mapping to the HindIII-Q,P/EcoRI-P and HindIII-V/EcoRI-I fragments, respectively. These RNAs were synthesized maximally during the very late occlusion phase and consisted of a major small transcript and several larger but less abundant transcripts.

The organization of viral genes within the 3.7-kilobase-pair HindIII-K/EcoRI-S region of the Autographa californica nuclear polyhedrosis virus genome (85 to 88 map units) was determined by using a combination of nucleotide sequencing, transcriptional mapping, and in vitro translation of hybrid selected RNA. Two nonoverlapping genes, extending in opposite directions and encoding polypeptides with molecular weights of 35,000 and 94,000 (35K and 94K polypeptides), were identified. Unspliced, messenger-active RNAs were transcribed from both genes early (2 h) after infection. Indicative of immediate-early genes, transcription of the divergent RNAs was unaffected by the protein synthesis inhibitor, cycloheximide. Late in infection, abundant RNAs were transcribed from promoters located at least 2.5 kilobase pairs upstream from the gene encoding the 35K polypeptide. These transcripts completely overlapped both the 35K and 94K polypeptide genes but apparently lacked protein-coding potential, suggesting that the transcripts may play a role in the suppression of early viral gene expression.

Black beetle virus is an insect virus with a split genome consisting of two single-stranded, messenger-active RNA molecules with molecular weights of 1.0 × 10 6 (RNA 1) and 0.5 × 10 6 (RNA 2), respectively. Virions contained two proteins, β with a molecular weight of 43,000 (43K) and γ (5K), and traces of a third protein, α (47K). When translated in cell-free extracts of rabbit reticulocytes, RNA 1 directed the synthesis of protein A (104K), whereas RNA 2 synthesized protein α. The in vitro translation efficiency of the two RNAs was roughly equal. Infection of cultured Drosophila cells induced the synthesis of five new proteins: A , α, β, γ, and B (10K), detected by autoradiography of polyacrylamide gels after electrophoresis of extracts from [ 35 S]methionine-labeled cultures. All but protein γ could also be detected by staining with Coomassie brilliant blue, indicating vigorous synthesis of viral proteins. Pulse-chase experiments in infected cells revealed the disappearance of protein α and the coordinate appearance of proteins β and γ, supporting an earlier proposal that coat protein of mature virions is made by cleavage of precursor α. Proteins A and B were stable in such pulse-chase experiments. The three classes of virus-induced proteins, represented by A, B, and α, were synthesized in markedly different amounts and with different kinetics. Synthesis of proteins A and B peaked early in infection and then declined, whereas synthesis of coat protein precursor α peaked much later. These results suggest that RNA 1 controls early replication functions via protein A (and also possibly protein B), whereas RNA 2 controls synthesis of coat protein required later for virion assembly.