NOTES. In Vivo Induction of Apoptosis Correlating with Reduced Infectivity during Baculovirus Infection. Thomas E. Clarke and Rollie J.

Transcription

1 JOURNAL OF VIROLOGY, Feb. 2003, p Vol. 77, No X/03/$ DOI: /JVI Copyright 2003, American Society for Microbiology. All Rights Reserved. NOTES In Vivo Induction of Apoptosis Correlating with Reduced Infectivity during Baculovirus Infection Thomas E. Clarke and Rollie J. Clem* Molecular, Cellular, and Developmental Biology Program, Division of Biology, Kansas State University, Manhattan, Kansas Received 5 August 2002/Accepted 24 October 2002 Spodoptera frugiperda caterpillars were infected with a mutant of Autographa californica M nucleopolyhedrovirus lacking the antiapoptotic p35 gene. Viral infectivity, replication, and spread were substantially reduced compared to that of a control revertant virus. Terminal deoxynucleotidyltransferase-mediated dutp-biotin nick end labeling confirmed that apoptosis occurred in mutant-infected caterpillars, thus directly correlating reduced infectivity and in vivo induction of apoptosis. * Corresponding author. Mailing address: Division of Biology, 232 Ackert Hall, Kansas State University, Manhattan, KS Phone: (785) Fax: (785) rclem@ksu.edu. Contribution number J from the Kansas Agricultural Experiment Station. Apoptosis is a mechanism of programmed cell death in which key proteins are cleaved by cysteine proteases, known as caspases, the cellular DNA is fragmented by apoptosis-activated endonucleases, and the contents of the cell are packaged into membrane-bound vesicles, known as apoptotic bodies, and phagocytosed by neighboring cells (11). Many viruses have been shown to induce apoptosis as a natural consequence of infection, and apoptosis often prematurely terminates the replication cycle of the virus and results in a reduction in the overall yield of viral progeny. In order to prevent this, viruses frequently carry antiapoptotic genes that either mimic naturally occurring host antiapoptotic genes or which interfere with the activity of host proapoptotic factors (15, 17). Baculoviruses are known to possess at least two different types of antiapoptotic genes, namely, the p35 and iap (inhibitor of apoptosis) genes (4). The baculovirus Autographa californica M nucleopolyhedrovirus (AcMNPV) is capable of preventing apoptosis through the action of the antiapoptotic protein P35, which blocks apoptosis by binding to and inactivating caspases (1). Infection of Sf21 cells, derived from the fall armyworm caterpillar Spodoptera frugiperda, with AcMNPV mutants lacking the p35 gene results in the apoptotic death of the infected cells within 24 h postinfection (6). Apoptotic Sf21 cells show very little production of late and very late viral transcripts and proteins, and they yield significantly less progeny than cells infected with wild-type AcMNPV (7, 12). In addition, S. frugiperda caterpillars are highly resistant to infection by AcMNPV mutants lacking the p35 gene, requiring 1,000-fold more mutant virus than the wild type to achieve the same degree of lethality by intrahemocoelic injection, although only 25-fold more p35 mutant virus is required to orally infect S. frugiperda neonate larva (7, 8). In contrast, cells derived from the caterpillar Trichoplusia ni are resistant to a variety of apoptotic stimuli, including infection with p35 deletion strains of AcMNPV, and the T. ni caterpillar itself is equally susceptible to infection by the p35 deletion strains and the wild-type strains of AcMNPV (7, 8). Finally, a role for apoptosis in the attenuated behavior of p35 mutant viruses in S. frugiperda is further suggested by the observation that the infectivity of a p35 mutant virus was completely restored by expression of Cp-iap, an unrelated antiapoptotic iap gene from a different baculovirus (8). Homologues of the AcMNPV p35 gene have been found in the related viruses Bombyx mori NPV (13) and Spodoptera littoralis NPV (9). Although the B. mori NPV p35 gene is significantly less capable of blocking caspase activity than the AcMNPV p35 gene (14) and does not appear to play a substantial role in inhibiting apoptosis during infection (13), the S. littoralis NPV p35 homolog, p49, is able to replace the AcMNPV p35 gene in AcMNPV, allowing productive infection of Sf9 cells (9). In addition, wild-type AcMNPV has been shown to induce apoptosis in cultured cells from S. littoralis (2) and Choristoneura fumiferana (16). However, to date there have been no reports of in vivo apoptosis induced by baculovirus infection. Virus construction. In order to study in detail the role of apoptosis as an antiviral mechanism in the insect immune system, we performed a series of experiments in which S. frugiperda and T. ni larvae were injected with measured doses of a p35 deletion virus and a revertant virus, both expressing enhanced green fluorescent protein (egfp). The p35 deletion virus vhsgfp/p35del was constructed in TN-368 cells by using a p35 deletion mutant of the L1 strain of AcMNPV, vp35delbsu36igus (obtained from Lois Miller), which contained the -glucuronidase (GUS) gene under control of the Drosophila hsp70 promoter at a site immediately upstream of, and in opposite orientation to, the wild-type polyhedrin gene. The vp35delbsu36igus virus itself was constructed by inserting the hsp70-gus cassette into the previously described virus vp35del (8). In vhsgfp/p35del, the GUS gene was replaced 2227

2 2228 NOTES J. VIROL. FIG. 1. The progress of infection following injection with vhsgfp/p35del (closed symbols) or vp35delrev (open symbols). Larvae of T. ni and S. frugiperda were injected with virus, and hemolymph from cohorts of 10 caterpillars was collected every 24 h. (A) Infection of hemocytes as assessed by egfp fluorescence. (B) Levels of budded virus in the hemolymph. with the egfp gene. Construction of the revertant virus vp35delrev involved replacing the p35 gene back into the vhsgfp/p35del virus by cotransfecting DNA extracted from vhsgfp/p35del with the plasmid pecori-s (10), containing wild-type p35, into Sf21 cells and isolating an occlusion-positive, egfp-positive plaque. Both viruses were subjected to three rounds of plaque purification, and their correct genomic structure was verified by restriction enzyme analysis. Virus stocks were grown and the titers were determined on TN-368 cells by plaque assay. Viral infectivity. These recombinant viruses were used to infect late-instar S. frugiperda (obtained from Agripest, Zebulon, N.C.) and T. ni (obtained from Entopath Inc., Easton, Pa.) larvae by intrahemocoelic injection. The caterpillars were injected with measured doses of budded virus diluted in complete TC-100 medium within 12 h of molting to the fifth instar (the penultimate instar in S. frugiperda and the final instar in T. ni). Injection-related mortality was less than 1% in each species. Intrahemocoelic injections were performed with a Hamilton 26-gauge syringe by inserting the needle between the first and second abdominal segment of the caterpillar and depositing between 1 and 4 l of virus-containing medium at the rear of the abdomen to avoid excessive bleeding. Caterpillars that survived to pupation were considered to have survived viral infection. Caterpillars that died exhibited egfp expression, confirming viral infection. In both species of caterpillars, initial experiments demonstrated that the egfp-expressing viruses each had 50% lethal dose values that were similar to those previously reported for a p35 mutant and revertant viruses lacking egfp (7), with 50% lethal dose values of 0.1 PFU for vp35delrev and 1 PFU for vhsgfp/p35del in T. ni and 20 PFU for vp35delrev and 10 5 PFU for vhsgfp/p35del in S. frugiperda (data not shown). Thus, similar to previously published results, deletion of p35 results in greatly decreased infectivity in S. frugiperda larvae. Hemolymph assays. In order to better assess the pathogenesis of virus infection, T. ni and S. frugiperda caterpillars were injected with either vhsgfp/p35del or vp35delrev ( PFU in T. ni and PFU in S. frugiperda; the dose injected was chosen on the basis of the lethality of vhsgfp/ P35del to each caterpillar species), and hemolymph was extracted daily from cohorts of 10 caterpillars into ice-cold anticoagulant buffer [4 mm NaCL, 40 mm KCl, 1.7 mm piperazine-n,n -bis(2-ethanesulfonic acid), 146 mm sucrose, 0.1% polyvinyl pyrrolidine, 8 mm EDTA, 9.5 mm citric acid, 27 mm sodium citrate]. Hemocytes were separated from the hemolymph by centrifugation and were examined for egfp expression. A minimum of 100 cells were scored from each sample, and the means 95% confidence limits are shown in Fig. 1A. Levels of virus in the hemolymph were assessed by a modified end point dilution assay. Cell-free hemolymph was serially diluted in anticoagulant buffer and was added to TN- 368 cells, and for each set of wells the lowest dilution displaying egfp expression from infected cells was recorded. For the purposes of calculating the mean, samples that yielded no infection even when undiluted were assigned a dilution value of Three replicates were scored for each sample, and the mean dilutions ( 95% confidence limits) at which virus could be detected in the hemolymph were calculated and are shown in Fig. 1B. The pathogenesis of wild-type AcMNPV in both T. ni and S. frugiperda has been described previously (3), and the revertant virus vp35delrev did not differ significantly from the wild type in buildup of infected hemocytes or viral hemolymph titers in either species (Fig. 1). In addition, the progression of vhsgfp/p35del infection was similar to that of vp35delrev in T. ni. However, in S. frugiperda the pattern of infection with

3 VOL. 77, 2003 NOTES 2229 vhsgfp/p35del virus differed dramatically from that of vp35delrev (Fig. 1). S. frugiperda caterpillars injected with PFU of vhsgfp/p35del showed very little increase in the levels of infected hemocytes or the titers of virus in the hemolymph, with many caterpillars having levels of virus in the hemolymph below the threshold of detection in the assay used. External fluorescence. Throughout the course of these experiments the external pattern of infection, including egfp fluorescence, observed for T. ni injected with either virus or for S. frugiperda injected with vp35delrev did not differ significantly from the pattern of infection observed in T. ni or S. frugiperda injected with wild-type AcMNPV expressing egfp (3). S. frugiperda injected with vhsgfp/p35del, however, displayed greatly decreased external egfp fluorescence. In order to further examine this phenomenon, 240 S. frugiperda caterpillars were injected with PFU of vhsgfp/p35del, and cohorts of 40 randomly selected caterpillars were sacrificed every 24 h. External examinations were performed on each remaining caterpillar daily to determine the extent of infection, and the titers of hemolymph extracted from each caterpillar was determined by plaque assay on TN-368 cells. Three distinct patterns of external egfp fluorescence were observed: none (no evident external fluorescence), weak (small scattered foci of external fluorescence), or strong (extensive, spreading external fluorescence). After 120 h, 68% of the caterpillars displayed no external fluorescence, 22% displayed weak fluorescence, and 10% displayed strong fluorescence (for images of infected caterpillars, see _lab/supplemental_figures/). Fluorescence in both the weak and strong categories tended to appear first in the ventral surface of the caterpillar, especially in the appendages. Differences in the degree of melanization of the dorsal and ventral surface of the caterpillars were taken into account in assessing infection status. In individuals displaying the strong phenotype, the infection typically spread from the appendages to form dorsoventral bands around the circumference of each segment before eventually spreading throughout the epidermis of the caterpillar. The level of virus observed in the hemolymph from 48 h onwards correlated strongly with the infection status of the caterpillar at the time of sacrifice, with strong-phenotype caterpillars having levels of virus approximately 100-fold higher than that of weak-phenotype caterpillars but still approximately 100-fold below that of vp35delrev-infected caterpillars. Internal fluorescence. Dissection and internal assessment of infection was also performed on S. frugiperda caterpillars injected with vhsgfp/p35del. Despite a large degree of variation in the extent of the infection among the caterpillars, some general observations could be made. At the 24-h time point, caterpillars showing external signs of infection (n 13) showed a distribution of egfp expression and individual foci of infection concentrated in the ventral and lateral peripheral fat body adjacent to the body wall, with fewer foci in the peripheral fat body of the dorsal midline and very little egfp expression in the perivisceral fat body within the hemocoel. In addition, approximately half of the caterpillars showing no external sign of infection (n 12) contained several small egfp-positive foci of infection in the fat body. By 48 h, all examined caterpillars lacking external egfp expression were also free of egfp expression internally. The strong phenotype. Over the course of the infection, strong-phenotype caterpillars showed a pattern of increasing egfp expression in the peripheral, and to a lesser extent the perivisceral, fat body coupled with very large reduction in fat body volume. Infection of the tracheal epithelia in strongphenotype caterpillars was first observed at 48 h as widely scattered individual egfp-positive cells, in a pattern inconsistent with direct cell-to-cell transmission of the virus through the extent of the trachea. The number of egfp-positive cells increased as the infection progressed, with bare regions of chitin appearing along the major trachea in most strong-phenotype caterpillars by 96 h postinfection. With the exception of the epithelia of the appendages and surrounding the spiracles (in which infection in the former could be detected as early as 24 h), body wall epithelia did not typically show signs of infection in these caterpillars until 96 h and then did so only in regions immediately adjacent to infected fat body. Caterpillars displaying the strong phenotype lived between 6 and 8 days following injection, at which point they died and melanized, although they did not undergo the melting process commonly associated with fatal baculovirus infection, as was previously reported (7). The weak phenotype. In contrast to the strong phenotype, weak-phenotype caterpillars showed a pattern of decreasing egfp expression in all tissues with the eventual reduction of egfp expression to a limited number of persistent foci. The numbers and distribution of egfp-positive foci of infection in the fat body and tracheal epithelia of weak-phenotype caterpillars were low and widely scattered by 48 h, and by 96 h these tissues were free of egfp expression in almost all of the weak-phenotype caterpillars examined. In addition, fat body and tracheal epithelia in weak-phenotype caterpillars did not show any of the reduction in volume or tissue destruction observed in the strong-phenotype caterpillars. At time points 96 h and beyond, weak-phenotype caterpillars showed egfp expression localized in the appendages and spiracles, with the frequent appearance of egfp fluorescence in the brain, ganglia, and in isolated muscle bands. Caterpillars displaying the weak-infection phenotype survived up to 14 days after injection, during which time they did not molt. The actual cause of death of these caterpillars is uncertain; however, the reduction in the fat body tissue observed very late in the infection suggested that the caterpillars had ceased feeding and had died of starvation. Weak-phenotype caterpillars that did not die of starvation attempted a precocial molt to pupa despite being in the penultimate instar and died as partially melanized prepupa. Histology and terminal deoxynucleotidyltransferase-mediated dutp-biotin nick end labeling (TUNEL) assay. Tissue from vp35delrev-infected and vhsgfp/p35del-infected (strong phenotype) S. frugiperda caterpillars at 96 h postinjection was fixed in 4% paraformaldehyde, paraffin-embedded, and sectioned, and the sections were stained with hematoxylin and eosin (Fig. 2). Uninfected tissue showed normal fat body and epithelial morphology, while vp35delrev-infected epithelia displayed typical baculovirus pathology, with the tissues being distinctly swollen and the nuclei of both epithelial and fat body cells having increased in diameter (occlusion bodies present in the nuclei of vp35delrev-infected cells before staining were dissolved by alkaline conditions during the staining

4 2230 NOTES J. VIROL. FIG. 2. Hematoxylin-and-eosin-stained peripheral tissue sections from abdomens of uninfected (A), vp35delrev-infected (B), or vhs- GFP/P35del-infected (strong phenotype) (C) S. frugiperda caterpillars sacrificed 96 h postinjection. m, muscle; fb, fat body; cu, cuticle; ep, epidermis. Additional images can be viewed at /virology/clem_lab/supplemental_figures/. procedure). In contrast, vhsgfp/p35del-infected fat body from caterpillars displaying the strong phenotype showed a highly disrupted morphology consisting of both condensed regions of darkly staining tissue and the presence of cavities in the normally contiguous fat body (Fig. 2C). Epithelia from vhsgfp/p35del-infected insects had a similar disrupted morphology (not pictured). To determine whether apoptosis was occurring in the tissues of the caterpillars, sections were subjected to TUNEL staining by using the ApopTag Red kit (Serologicals Corp., Norcross, Ga.), which labels DNA having free 3 hydroxyl groups with rhodamine-dutp and thus efficiently labels apoptotic cells. In sections taken from vhsgfp/p35del-infected S. frugiperda strong-phenotype caterpillars at 48 h postinjection, the majority of TUNEL-positive cells were alone or were in small clusters located predominantly in the fat body but also in the body wall epithelia (data not shown). Extensive TUNEL staining was observed in the peripheral fat body in all strong-phenotype vhsgfp/p35del-infected caterpillars sacrificed at 96 h postinjection (Fig. 3). Counterstaining of the sections with 4,6 diamidino-2-phenylindole (DAPI) demonstrated that the majority of TUNEL-positive nuclei were condensed and fragmented, confirming apoptotic cell death. TUNEL staining correlated well with egfp expression, confirming that apoptosis was stimulated by virus infection. Hematoxylin and eosin staining of tissue sections adjacent to TUNEL-assayed sections also revealed that damage to the peripheral fat body was in regions containing high proportions of TUNEL-positive cells, indicating that the tissue damage was due to extensive apoptosis (data not shown). We were unable to reliably detect egfp expression or TUNEL labeling in sections from weak-phenotype caterpillars infected with vhsgfp/p35del (data not shown). TUNEL-positive cells were occasionally observed in sections taken from vp35delrev-infected caterpillars at 96 h postinjection; however, TUNEL staining in these cells was significantly weaker in intensity than that observed in vhsgfp/p35delinfected caterpillars, and the nuclei that were TUNEL-positive invariably contained large quantities of occlusion bodies, indicating a lack of apoptosis (Fig. 3). Neither TUNEL-positive nuclei nor occlusion bodies were observed in sections obtained from vp35delrev-infected caterpillars sacrificed 48 h postinjection (data not shown), suggesting that TUNEL-positive results with vp35delrev-infected tissues were due to free ends of DNA resulting from either the late stages of viral replication or nonapoptotic DNA fragmentation occurring after the completion of the viral replication cycle. Sections from uninfected caterpillars sacrificed in the penultimate and ultimate instar contained small numbers of scattered isolated TUNEL-positive cells (3 to 5 per section), indicative of a low level of background apoptosis occurring in the caterpillar tissues (data not shown). Larger numbers of TUNEL-positive nuclei could be observed in uninfected caterpillars in the tonofibrillae cells attaching muscles to the body wall; however, this is an expected result of the process by which these epidermal cells become transformed into connective tissue (19). Conclusions. It was previously hypothesized that apoptosis was responsible for the decrease that is seen in the infectivity of p35 mutant viruses in S. frugiperda larvae, based on the following correlative pieces of evidence (reviewed in reference 5): (i) the induction of apoptosis in Sf21 cells by p35 mutant viruses and the decreased infectivity of these mutant viruses in S. frugiperda larvae; (ii) the lack of apoptosis in TN-368 cells infected with p35 mutant viruses and the normal infectivity of these mutant viruses in T. ni larvae; (iii) the reduced levels of viral replication associated with apoptosis in cultured cells; and (iv) the ability of an unrelated antiapoptotic gene, Cp-iap, to rescue both the in vitro replication and in vivo infectivity de-

5 VOL. 77, 2003 NOTES 2231 FIG. 3. Tissue sections from peripheral regions of S. frugiperda caterpillar abdomens. Caterpillars were either uninfected (A to D), infected with vp35delrev (E to H), or infected with vhsgfp/p35del (strong phenotype) (I to L) and were sacrificed 96 h postinjection. Sections were stained with DAPI to visualize nuclei, while apoptotic cells were identified by TUNEL assay with rhodamine-labeled dutp. Photographs were taken under normal light (A, E, and I), DAPI fluorescence (B, F, and J), egfp fluorescence (C, G, and K), or rhodamine fluorescence for TUNEL (D, H, and L). fb, fat body; tr, trachea; bw, body wall (cuticle and epidermis); ob, occlusion bodies. Additional images can be viewed at virology/clem_lab/supplemental_figures/. fects of p35 mutant viruses. However, up to now apoptosis occurring in the cells of p35 mutant-infected larvae had not actually been documented. In this study, we have demonstrated for the first time a strong correlation between viral induction of apoptosis in vivo and a deficiency in viral infectivity following injection of virus directly into the hemocoel. In S. frugiperda caterpillars infected with vhsgfp/p35del and displaying the strong phenotype, we observed large numbers of apoptotic cells corresponding to regions of high egfp expression in caterpillars that became strongly infected. Extensive

6 2232 NOTES J. VIROL. tissue destruction occurred in the fat body and epithelium of these insects, which correlated strongly with areas of TUNEL staining and viral replication. Although we did not find significant numbers of apoptotic cells in caterpillars displaying weak or no egfp expression, this was presumably due to the small number of these cells, their scattered nature, and the propensity for isolated apoptotic cells to be rapidly phagocytosed in vivo (18). Given the available evidence, we conclude that apoptosis can play an important role in insect immunity to baculovirus infection, and thus should be recognized as an arm of the insect immune system. It will be interesting to determine whether a similar effect can occur during oral infection, when the infection must first pass through the bottleneck imposed by the midgut, associated basal lamina, and the tracheal epithelia. While a previous study found that there was less of a reduction in mortality when S. frugiperda larvae were infected orally with p35 mutant AcMNPV than when they were infected by injection (25-fold versus 1,000-fold) (7, 8), it is difficult to directly compare these results, because the feeding studies used neonates while the injection studies used late-instar larvae. Whether apoptosis can be an effective barrier to oral infection is presently under investigation. We gratefully acknowledge Lois Miller for providing the vp35delbsu36igus virus, Angela Iseli and Jamie Thurman for technical support, and Susan Bale for assistance with image processing. We also thank Shelly Christenson of the Kansas Veterinary Diagnostic Laboratory for advice and help with sectioning and histology. This material is based upon work supported by the Cooperative State Research, Education, and Extension Service, U.S. Department of Agriculture, under agreement no Support was also provided by the Kansas Agricultural Experiment Station. ADDENDUM IN PROOF After this paper was accepted for publication, a report describing in vivo apoptosis induction by wild-type ACMNPV in Spodoptera litura larvae was published (P. Zhang, K. Yang, X. Dai, Y. Pang, and D. Su, J. Gen. Virol. 83: ). REFERENCES 1. Bump, N. J., M. Hackett, M. Hugunin, S. Seshagiri, K. Brady, P. Chen, C. Ferenz, S. Franklin, T. Ghayur, P. Li, P. Licari, J. Mankovich, L. Shi, A. H. Greenberg, L. K. Miller, and W. W. Wong Inhibition of ICE family proteases by baculovirus antiapoptotic protein p35. Science 269: Chejanovsky, N., and E. Gershburg The wild-type Autographa californica nuclear polyhedrosis virus induces apoptosis of Spodoptera littoralis cells. Virology 209: Clarke, T. E., and R. J. Clem Lack of involvement of haemocytes in the establishment and spread of infection in Spodoptera frugiperda larvae infected with the baculovirus Autographa californica M nucleopolyhedrovirus by intrahemocoelic injection. J. Gen. Virol. 83: Clem, R. J Baculoviruses and apoptosis: the good, the bad, and the ugly. Cell Death Differ. 8: Clem, R. J Regulation of programmed cell death by baculoviruses, p In L. K. Miller (ed.), The baculoviruses. Plenum Press, New York, N.Y. 6. Clem, R. J., M. Fechheimer, and L. K. Miller Prevention of apoptosis by a baculovirus gene during infection of insect cells. Science 254: Clem, R. J., and L. K. Miller Apoptosis reduces both the in vitro replication and the in vivo infectivity of a baculovirus. J. Virol. 67: Clem, R. J., M. Robson, and L. K. Miller Influence of infection route on the infectivity of baculovirus mutants lacking the apoptosis-inhibiting gene p35 and the adjacent gene p94. J. Virol. 68: Du, Q., D. Lehavi, O. Faktor, Y. Qi, and N. 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Maeda Homologous p35 proteins of baculoviruses show distinctive anti-apoptotic activities which correlate with the apoptosis-inducing activity of each virus. FEBS Lett. 427: O Brien, V Viruses and apoptosis. J. Gen. Virol. 79: Palli, S. R., G. F. Caputo, S. S. Sohi, A. J. Brownwright, T. R. Ladd, B. J. Cook, M. Primavera, B. M. Arif, and A. Retnakaran CfMNPV blocks AcMNPV-induced apoptosis in a continuous midgut cell line. Virology 222: Roulston, A., R. C. Marcellus, and P. E. Branton Viruses and apoptosis. Annu. Rev. Microbiol. 53: Savill, J., and V. Fadok Corpse clearance defines the meaning of cell death. Nature 407: Snodgrass, R. E Principles of insect morphology. Cornell University Press, Ithaca, N.Y.