Infection with Langat Flavivirus or Expression of the Envelope Protein Induces Apoptotic Cell Death

Virology 286, 328–335 (2001) doi:10.1006/viro.2001.0980, available online at http://www.idealibrary.com on

Infection with Langat Flavivirus or Expression of the Envelope Protein Induces Apoptotic Cell Death Grigori G. Prikhod’ko,* ,1 Elena A. Prikhod’ko,† Jeffrey I. Cohen,† and Alexander G. Pletnev* *Laboratory of Infectious Diseases and †Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892 Received February 23, 2001; returned to author for revision April 3, 2001; accepted April 22, 2001; published online July 9, 2001 Langat (LGT) flavivirus, derived from infectious full-length cDNA clone 636, was investigated for its apoptotic activities in mouse neuroblastoma (Neuro-2a) and simian kidney (Vero and LLC-MK 2) cells. The hallmark of apoptosis, cleavage of cellular DNA, was observed 48 h after infection of Vero, LLC-MK 2, and Neuro-2a cells by electrophoresis analysis. Apoptosis in infected cells was also confirmed by TUNEL assay. LGT-infected Neuro-2a cells showed an increase in caspase-3-like protease (DEVDase) activity. Expression of the major envelope glycoprotein (E) alone reduced cell viability in both Vero and Neuro-2a cells, and the baculovirus P35 protein, which inhibits multiple caspases, completely blocked this effect. Cleavage of cellular DNA was observed in E gene-transfected Vero cells by TUNEL assay. Expression of E protein or caspase-9 resulted in activation of caspase-3-like proteases in Neuro-2a cells. The caspase-3-like protease specific inhibitor, Ac-DEVDCHO peptide, partially inhibited E protein- or caspase-9-induced apoptosis in Neuro-2a cells. These observations indicate that infection of cells with LGT virus or expression of LGT virus E protein induces apoptosis through a caspase-3-like protease pathway. Key Words: flaviviruses; Langat virus and envelope glycoprotein E; apoptosis; DEVDase activity; Neuro-2a and Vero cells; caspases and inhibitors.

INTRODUCTION

by viruses of the TBE complex. For this purpose, several partially attenuated LGT virus strains were investigated in the United States, Russia, and Czechoslovakia (Mayer et al., 1976; Nathanson et al., 1968; Price et al., 1970; Smorodincev and Dubov, 1986). One such strain, Yelantsev, was used in a human trial in Russia between 1969 and 1973. Vaccination with Yelantsev virus induced a low rate of encephalitis; 35 of 649,479 volunteers were diagnosed with meningoencephalitis or meningoencephalomyelitis (Smorodincev and Dubov, 1986). During this time, the E5 strain of LGT virus was selected by passage in embryonated chicken eggs (Nathanson et al., 1968; Thind and Price, 1966) and subsequently was found to have reduced neurovirulence for mice compared to wildtype or Yelantsev LGT virus. Immunization of animals and human volunteers showed that the E5 virus induces high levels of neutralizing antibodies to Powassan and Kyasanur forest disease viruses, as well as to TBE virus (Price et al., 1970; Price and Thind, 1973). The neuroinvasiveness or “peripheral virulence” of the E5 virus was also reduced at least 2000-fold compared to the wild-type virus (Pletnev and Men, 1998). Recently several LGT virus full-length cDNA clones have become available and one of them, clone 636, exhibits an attenuation phenotype in mice similar to that observed with the E5 LGT virus (Campbell and Pletnev, 2000). The neurovirulence of LGT virus is not well understood. However, during the last few years, studies of

The Flavivirus genus includes mostly arthropod-borne pathogens, many of which are widely distributed throughout the world. Mosquito-borne flaviviruses such as to dengue (DEN), Japanese encephalitis (JE), and yellow fever (YF) viruses cause disease in millions of persons annually in the tropics and subtropics. Tickborne flaviviruses grouped within the TBE complex (Tickborne encephalitis, Kyasanur forest disease, Langat, Louping ill, Negishi, Omsk hemorrhagic fever, and Powassan viruses) are mainly found in the Northern Hemisphere and, except for Langat virus, cause human disease of varying severity that can have a mortality as high as 20–30% (Calisher et al., 1989; Monath and Heinz, 1996). Tick-borne encephalitis remains a pressing public health problem in Eastern Europe and Russia, where 9000–12,000 people are diagnosed with the disease annually (Gaidamovich, 1995). Langat (LGT) virus is the only virus in the TBE complex which does not produce disease in humans under natural conditions, and therefore it has been proposed as a candidate live virus vaccine against encephalitis caused

1 To whom correspondence and reprint requests should be addressed at Plasma Derivatives Department, American Red Cross Holland Laboratory, 15601 Crabbs Branch Way, Rockville, MD 20855-2743. Fax: (301) 738-0708. E-mail: [email protected].

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FIG. 1. LGT virus induces apoptosis. (A) Vero cells infected with LGT virus clone 636 were fixed 24 and 48 h after infection (indicated at the bottom) and processed for TUNEL assay according to the manufacture protocol. The same field of cells was observed by fluorescence (bottom row) and by phase-contrast (top row) microscope. Staurosporine-treated and mock-infected cells were served as positive and negative controls of apoptosis. Magnification, ⫻200. (B) TUNEL-positive, LGT virus-infected cells are shown by high-resolution digital fluorescence microscopy at 48 h after infection. Magnification, ⫻1000.

other flaviviruses (TBE, JE, DEN, and Murray Valley encephalitis viruses) have shown that neurovirulence is associated with the level of apoptosis induced by viruses (Andrews et al., 1999; Despres et al., 1996, 1998; Isaeva et al., 1998; Jan et al., 2000; Kamulov et al., 1998; Liao et al., 1997; McMinn, 1997), and that determinants of neurovirulence of DEN flavivirus are located within the envelope (E) and nonstructural (NS3) genes (Duarte dos Santos et al., 2000). Since LGT virus clone 636 is attenuated in mice, we determined whether the virus can induce apoptosis in both neural and nonneural cells. In addition, we investigated the ability of the E and NS2BNS3 to induce apoptosis in these cells. RESULTS LGT virus infection induces apoptosis and causes cleavage of cellular DNA To determine whether LGT virus provokes an apoptotic response of host cells during infection, Vero cells inoculated at a multiplicity of infection (m.o.i.) of 10 were subjected to the terminal deoxynucleotidyl-transferasemediated dUTP nick end labeling (TUNEL) assay for detecting the presence of chromosomal DNA cleavage, an event indicative of apoptosis. Forty-eight hours after infection with LGT virus, approximately 12% cells were TUNEL-positive. Similarly, about 9% of cells treated with 100 nM staurosporine, a mitochondria-dependent apo-

ptotic agent, were TUNEL-positive (Fig. 1A). Furthermore, nuclear condensation and invagination were also detected in LGT virus-infected cells by fluorescence microscopy (Fig. 1B). No TUNEL-positive cells were observed at 24 h after infection or in mock-infected cells, which served as a negative control of apoptosis. To further confirm that LGT virus induces apoptosis in Vero cells, cells were infected with LGT virus at an m.o.i. of 10 and maintained for 48 h, and total DNA was extracted from both adherent and floating cells and analyzed by electrophoresis. Low-molecular-weight apoptotic DNA was detected in LGT virus—but not mock-infected cells (Fig. 2). To determine whether LGT virus induces apoptosis in other cell lines, total DNA was extracted from other nonneural (LLC-MK 2) and neural (Neuro-2a) cells at 48 h after infection. Apoptotic DNA fragments were observed in both cell lines (Fig. 2). LGT virus induces activation of caspase-3-like proteases in Neuro-2a cells Caspases play an important role in cell death. Caspase-3-like proteases cleave and inactivate proteins that protect cells from apoptosis. For example, cleavage of I CAD, an inhibitor of caspase-activated deoxyribonuclease (CAD), leads to chromosomal DNA degradation (Nicholson and Thornberry, 1997). To detect activation of caspase-3-like proteases, we used a colorimetric assay

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maximum of 50.0 nmol/h during the next 24 h. Virus release was detected later, at 24 h after infection, suggesting that infection of Neuro-2a cells with LGT virus induces apoptosis during the replication cycle. In contrast, DEVDase activity in Vero cells was barely detectable 36 h after infection (Fig. 3B). These results suggest that Vero cells may lack inducible DEVDase activity. LGT virus E protein induces apoptosis and cleavage of cellular DNA

FIG. 2. LGT virus infection results in cleavage of cellular DNA. Vero, LLC-MK 2, or Neuro-2a cells (indicated at the bottom) were infected with LGT virus. Floating and attached cells were harvested 48 h after infection and treated with lysis buffer containing detergent Triton X-100 and protease K. Total DNA was isolated from lysates by phenolchloroform extraction and analyzed by electrophoresis in a 1.2% agarose gel. Cellular DNA isolated from staurosporine-treated Neuro-2a cells or mock-infected cells were used as control samples. Positions of DNA molecular weight markers (1-kb DNA ladder) are indicated at the right.

based on the spectrophotometric detection of the chromophore p-nitroanilide (pNA) after cleavage from the synthetic oligopeptide substrate, Ac-DEVD-pNA. The kinetics of both virus production and enzyme activity were studied in Neuro-2a and Vero cells infected with LGT virus (Fig. 3). In Neuro-2a cells, DEVDase activity was detected approximately 12 h after infection, reaching a

Comparative analysis of sequences of flaviviral parental and neuroadapted strains generated during adaptation in mice and monkeys has shown that while mutations are distributed through the genomes of these viruses, most mutations cluster in the regions encoding E and NS2-NS3 proteins (Figs. 4A and 4B). Since LGT virus induces apoptosis, it was of interest to determine whether the E and/or NS2B-NS3 proteins possess apoptotic activities. We cloned LGT virus genes, E and NS2B-NS3, under the transcriptional control of cytomegalovirus IE promoter, providing high level expression in a variety of cell types (Fig. 4C). LGT virus E and NS2B-NS3 were expressed in the cytoplasm of transfected Neuro-2a cells. The E protein was present as discrete irregular foci in the cytoplasm and the NS2B-NS3 protein showed a pattern of staining similar to the E protein (Fig. 5, bottom row). Vector-transfected or virus-infected cells served as negative or positive controls for viral protein expression, respectively. Propidium iodide staining served as a control for nuclear location (Fig. 5, top row). To determine whether LGT virus proteins are able to affect viability of cells, Vero and Neuro-2a cells were cotransfected with plasmid expressing ␤-galactosidase to visualize transfected cells and either vector DNA or plasmids expressing E, NS2B-NS3, or caspase-9. Since dying cells detach from the tissue culture plate and are removed prior to fixation, the loss of blue cells is a

FIG. 3. LGT virus infection activates caspase-3-like proteases. (A) Growth of LGT virus in Vero (solid circles) and Neuro-2a (open circles) cells. (B) Caspase activation in infected cells. At various hours postinfection (hpi), the medium was collected and titered on Vero cells by plaque assay, and cells were harvested and processed for detection of DEVDase activity by colorimetric assay. Enzyme activity of infected cells harvested at 0 hpi was set at 0 nmol/h. Results are means ⫾ SD for duplicate experiments.

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FIG. 4. The distribution of mutations associated with neuroadaptation in flaviviruses, and plasmid subclones used in analysis of the apoptotic activities of the LGT virus proteins. (A) Schematic representation of the flavivirus genome showing 5⬘ and 3⬘ noncoding regions (NCR) and the open reading frame. Structural (shaded boxes) or nonstructural (unshaded boxes) proteins are shown. (B) Schematic representation of amino acid mutations that occurred during neuroadaptation of flaviviruses (shown by bars). The identity of parental and neuroadapted strains of flaviviruses is indicated on the right. The amino acid sequences of DEN, JE, YF, TBE, and LGT viruses were reported earlier (Duarte dos Santos et al., 2000; Nitayaphan et al., 1990; Pletnev and Men, 1998; Santos et al., 1995; Wallner et al., 1996). Vertical lines indicate amino acid differences, and variable regions are boxed. (C) Schematic representation of plasmids encoding LGT virus E and NS2B-NS3 proteins. The transcriptional start site is shown by a flag and the putative polyadenylation signal is indicated by a wavy line.

quantitative parameter of viability of cells (Joe et al., 1998). The number of viable (flat blue) cells was reduced in both Vero and Neuro-2a cells transfected with the E gene or the caspase-9 gene, which served as a positive control for apoptosis. At 18 h after transfection, there were 41% fewer viable Vero cells expressing E protein and ␤-galactosidase than cells transfected with empty vector and the ␤-galactosidase plasmid. The loss of viability of Neuro-2a cells transfected with the E gene was 32% (Fig. 6A). Unlike E protein, expression of the NS2B-NS3 protein did not affect the viability of both Vero and Neuro-2a cells (Fig. 6A). To verify that the reduction in viability of Vero and Neuro-2a cells was due to apoptosis and not due to death by a nonapoptotic pathway, LGT virus E protein

was coexpressed with baculovirus P35, a stoichiometric inhibitor of active caspases. P35 is cleaved by caspases at the aspartate residue within the sequence DQMD2G and the cleavage products form a stable complex with the caspase. Baculovirus P35 inhibits caspases 1, 3, 6, 8, and 10. Coexpression of P35 with caspase-9 completely blocked apoptotic cell death in both Neuro-2a and Vero cells (Fig. 7). P35 was also able to inhibit E proteininduced cell death in both Neuro-2a- and Vero-cotransfected cells (Fig. 7). Thus, E protein induces apoptosis and coexpression of E protein with a baculovirus protein, P35, blocks apoptosis. To further verify that LGT virus E protein induces apoptosis in Vero cells, Vero cells were transfected with plasmid expressing E protein or empty vector DNA and

FIG. 5. Subcellular localization of LGT virus E and NS2B-NS3 proteins expressed in Neuro-2a cells. Cells were transfected with pCIE, pCINS2B-NS3, or, as a negative control, with vector pCI. As a positive control of viral protein expression, cells were infected with LGT virus. At 18 h after transfection or infection, cells were fixed in formaldehyde and doubly stained with propidium iodide (PI) and rabbit anti-LGT polyclonal antibody followed by FITC-conjugated goat anti-rabbit antibody. The same field of cells was photographed for PI (top row) and anti-LGT virus proteins (lower row).

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FIG. 6. LGT virus E protein induces apoptosis. (A) Vero or Neuro-2a cells were cotransfected with plasmids pCMV␤-gal and pCIE, pCINS2B-NS3, and pCIcasp9 and the number of viable, blue-stained flat cells was determined relative to that observed in cells cotransfected with plasmid pCMV␤-gal and empty vector DNA, pCI. Cells transfected with pCIcasp9 served as a control for cells undergoing cell death. The cells were fixed, stained, and counted 18 h after transfection. Error bars indicate the standard deviation. (B) Vero cells transfected with plasmids pCI and pCIE were fixed 18 h after transfection and analyzed by TUNEL assay. Magnification, ⫻200.

examined by TUNEL assay. Increased number of TUNELpositive cells was observed at 18 h after transfection with the E gene (Fig. 6B). LGT virus E protein induces activation of caspase-3like proteases in Neuro-2a cells To determine whether expression of E protein induces activation of caspase-3-like proteases, both Neuro-2a and Vero cells were transfected with vector DNA, or plasmids expressing E protein or caspase-9, and lysates of transfected cells were assayed for DEVDase activity. E protein induced an activation of caspase-3-like proteases in Neuro-2a, but not in Vero cells. DEVDase activity in lysates from Neuro-2a cells transfected with E and caspase-9 genes was 4.7 and 6.5 nmol/h, respectively (Fig. 8). There was no significant DEVDase activity in Vero cells expressing E protein or caspase-9. This is consistent with the previous observations that little or no DEVDase activity can be induced in this cell line after infection with LGT virus (Fig. 3B). To further study the effect of LGT virus E protein on DEVDase activity in Neuro-2a cells, cells were transfected with E or caspase-9 genes and treated with Ac-DEVD-CHO, a peptide inhibitor of DEVDase activity. The peptide inhibition resulted in reduction of E protein- or caspase-9-induced apoptosis. In contrast, treatment of transfected Vero cells

FIG. 7. Inhibition of LGT virus E protein-induced apoptosis. Cells were cotransfected with plasmids expressing P35 and E protein or caspase-9 and the percentage of cells undergoing apoptosis was determined by the ␤-gal transient-expression assay for apoptosis 18 h after transfection (see Materials and Methods). In addition, cells transfected with pCIE or pCIcasp9 were treated with Ac-DEVD-CHO. The empty vector, pCI, was used as a negative control and to maintain equal plasmid DNA concentrations for each of the transfections. The percentage of apoptotic cells was calculated relative to the pCI control, set at 0% (Prikhod’ko et al., 1999), which was similar to the level for mock-transfected controls.

with the peptide inhibitor had no effect on apoptosis induced by E protein or caspase-9 (Fig. 7). DISCUSSION In the present study we investigated the apoptotic properties of LGT flavivirus and the genes encoding its major envelope glycoprotein, E, and the NS2B-NS3 pro-

FIG. 8. LGT virus E protein induces activation of caspase-3-like proteases in Neuro-2a cells, but not in Vero cells. Cells were transfected with plasmids pCI, pCIE, and pCIcasp9. Cell lysates were harvested 18 h after transfection and assayed for DEVDase activity using the colorimetric substrate Ac-DEVD-pNA. DEVDase activity was calculated relative to the control vector-transfected cells, set at 0 nmol/h. Cells expressing caspase-9 served as a positive control for induction of apoptosis. Results are means ⫾ SD for duplicate experiments.

LGT VIRUS AND E PROTEIN INDUCE APOPTOSIS

tein. We found that LGT virus clone 636 induces apoptosis in both neural and nonneural cell lines. LGT virus induces activation of caspase-3-like proteases in Neuro2a-infected cells, and DNA cleavage and loss of cell viability in both neural and kidney cell lines. To localize possible triggers of apoptosis, we focused our research on two LGT virus genes, E and NS2B-NS3, because these genes are more frequently mutated during neuroadaptation of flaviviruses. NS3 protein of flaviviruses is a trifunctional protein, which has protease, NTPase, and helicase activities. The proteolytic activity of protease NS3 is dependent on the presence of its cofactor, NS2B, and is associated with the membrane fraction (Clum et al., 1997). These two proteins together are required for the cleavage of the polyprotein of flaviviruses between nonstructural proteins (NS) 2A and 2B, 2B and 3, 3 and 4A, and 4B and 5. Transient-expression of the NS2B-NS3 gene encoding the LGT virus protease and its cofactor did not induce apoptosis. The E protein is the major envelope protein of the virion of flaviviruses and is important for virion assembly, receptor binding, and membrane fusion (reviewed in Monath and Heinz, 1996). The envelope proteins of several RNA viruses are known to induce apoptosis. The avian leukosis-sarcoma virus envelope fusion protein complexed to immunoglobulin mediates apoptosis by binding to the cellular receptor, CAR1, a member of TNFR family (Brojatsch et al., 1996). Expression of Sindbis (SIN) virus envelope glycoproteins, E1 and E2, and the human immunodeficiency virus (HIV) envelope glycoprotein complex, gp120-gp41, both trigger apoptosis in transfected cells (Joe et al., 1998; Koga et al., 1990). The mechanism by which SIN virus envelope proteins induce apoptosis is unknown. However, virus infection of AT-3 rat prostatic adenocarcinoma cells activates the transcription factor, NF-␬B, which triggers cell death by transcription-dependent induction of ICE protease, p53, and TNF (reviewed in Griffin and Hardwick, 1998). HIV gp120gp41-mediated apoptosis is induced by binding to CD4 receptor molecules (Koga et al., 1990). Transient expression of the major envelope protein gene of LGT virus induces apoptosis in two cell lines, Neuro-2a and Vero cells. Expression of E protein induced activation of caspase-3-like proteases in Neuro-2a cells, and cleavage of chromosomal DNA with reduction of viability in both Neuro-2a and Vero cells. Interestingly, while baculovirus P35 completely blocked E proteininduced apoptosis in both cell lines, a DEVDase peptide inhibitor reduced E protein-induced apoptosis in Neuro-2a cells, but not in Vero cells. Similarly, a DEVDase peptide inhibitor partially blocked caspase-9-induced apoptosis in Neuro-2a cells, but not in Vero cells. In addition, little or no DEVDase activity was detectable in Vero cells infected with LGT virus or transfected with plasmids expressing E protein or caspase-9. In contrast, DEVDase activity was easily detectable in Neuro-2a cells

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infected with LGT virus or expressing E protein or caspase-9. Further experiments are necessary to better delineate the mechanism(s) by which LGT virus E protein induces apoptosis. The observations that flaviviral protein synthesis activates transcriptional factor NF-␬B before the appearance of apoptosis (Marianneau et al., 1997), and that Bcl-2 blocks flavivirus-induced apoptosis (Liao et al., 1997, 1998), suggest that NF-␬B, p53, Bid, and Bax may have important roles in induction of apoptosis by LGT virus. MATERIALS AND METHODS Cell cultures and virus The mouse neuroblastoma Neuro-2a cell line was maintained in antibiotic-free minimum essential medium (MEM) with Earle’s salts supplemented with 2 mM L-glutamine and 10% fetal bovine serum. The monkey kidney Vero and LLC-MK 2 cell lines were maintained in MEM supplemented with 10% fetal bovine serum, 1% glutamine, 50 ␮g/ml gentamicin, and 25 ␮g/ml fungizone. Medium, serum, and antibiotics were obtained from GIBCO BRL (Gaithersburg, MD). Amplification of LGT virus clone 636 was previously described (Campbell and Pletnev, 2000). Virus was titered in Vero cells. Plasmid constructs A PCR fragment containing the LGT virus E gene was generated by using pTP21-636 (Campbell and Pletnev, 2000) as a template and two primers as follows: (1) 5⬘-GGTGCGAGATCTAGAATGTCGAGATGCACCCACC, corresponding to the E gene 5⬘end at nt 971–986 and extended by 18 nucleotides to include BglII/XbaI restriction sites and a start codon, and (2) 5⬘-GGTCTTGCGGCCGCTCAGGCTCCAACCCCCAG, corresponding to the E gene 3⬘ end at nt 2444–2458 and extended by 17 nucleotides to include a NotI restriction site and a stop codon. The PCR product was digested with XbaI and NotI and cloned into the XbaI–NotI-digested pCI expression vector (Promega, Madison, WI) to construct pCIE. To construct pCINS2B-NS3, the LGT virus NS2B-NS3 gene was amplified by PCR using pTP21–636 and two primers as follows: (1) 5⬘-GGTGCGAGATCTAGAATGTCCTTTAATGAGCCTATG, corresponding to the NS2B gene 5⬘ end at nt 4205–4222 and extended by 18 nucleotides to include BglII/XbaI restriction sites and a start codon, and (2) 5⬘-GGTCTTGCGGCCGCTCAGCGTCTTCCAGAAGC, corresponding to the NS3 gene 3⬘ end at 6446–6460 and extended by 17 nucleotides to include a NotI site and a stop codon. The PCR product was digested with XbaI and NotI and cloned into pCI resulting in pCINS2BNS3. Plasmid expressing caspase-9 (pCIcasp9) was a gift from Charles M. Zacharchuk (National Cancer Institute,

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NIH, Bethesda, MD). Plasmid expressing p35, pCIP35, was previously described (Bertin et al., 1997). pCMV␤-gal was purchased from Stratagene (La Jolla, CA). TUNEL assay Vero (6 ⫻ 10 5) or Neuro-2a (8 ⫻ 10 5) cells were seeded in 35-mm-diameter culture dishes containing glass coverslips and inoculated at a multiplicity of infection of 10 the following day. In other experiments, uninfected cells were transfected with 1 ␮g of plasmid DNA using LipofectAmine Plus (GIBCO BRL) according to the manufacturer’s instructions. At various time postinfection or transfection, cells were fixed with 3% paraformaldehyde in PBS, permeabilized in 0.1% Triton X-100 dissolved in 0.1% sodium citrate, and labeled in a TUNEL reaction mixture using the Fluorescein In Situ Cell Death Detection Kit (Boehringer Mannheim Biochemicals, Gaithersburg, MD). Cells stained with fluorescein isothiocyanate (FITC) were examined by fluorescence microscopy. Mock-infected cells were treated for 3 h prior to fixation with 100 nM staurosporine (Sigma-Aldrich, Inc., St. Louis, MO), as a control for apoptotic cells. Internucleosomal DNA fragmentation For nucleosomal ladder preparation (Prikhod’ko et al., 1999), infected or transfected cells (⬃2 ⫻ 10 6) were detached with a mixture of trypsin and versene (BioWhittaker, Walkersville, MD) and mixed with the previously removed cell culture medium containing floating cells. The combined (attached and floating) cells were pelleted at 5000 g for 2 min and incubated in 20 mM Tris–HCl (pH 7.6), 10 mM EDTA, 0.2% Triton X-100, and protease K (200 ␮g/ml) overnight at room temperature (RT). DNA was extracted with phenol-chloroform and precipitated with 2 vol of ethanol and 0.1 vol of 5 M NaCl, treated with RNase A, and then subjected to electrophoresis through 1.2% agarose gels.

␤-gal transient-expression assay for apoptosis Neuro-2a (4 ⫻ 10 5) or Vero (3 ⫻ 10 5) cells were seeded in 35-mm-diameter culture dishes. Twenty-four hours later, cells were transfected with 1 ␮g of pCIE, 1 ␮g of pCINS2B-NS3, 0.2 ␮g of pCIcasp9, or 1.5 ␮g of pCIP35 using LipofectAmine Plus (GIBCO BRL). Each DNA sample used for transfection also contained pCI to keep the total amount of plasmid DNA constant and 0.125 ␮g of pCMV␤-gal to detect transfected cells. In inhibition experiments (Fig. 7), 50 ␮M peptide Ac-DEVD-CHO (Alexis Corp., San Diego, CA) was added to the culture medium to prevent apoptosis. Eighteen hours after transfection, cells were rinsed twice with PBS to remove dying, floating cells. The adherent cells were fixed in 2% formaldehyde and 0.2% glutaraldehyde at RT for 5 min and then washed twice with PBS. Fixed cells were stained at RT overnight with PBS containing 0.5 mg/ml X-gal, 5 mM

potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM MgCl 2, and 0.01% SDS. Eight fields of viable bluestained flat cells were counted microscopically at ⫻400 magnification. The percentage of apoptotic cells was calculated relative to the vector control (pCI) transfected cells set at 0% (Prikhod’ko et al., 1999). Photomicroscopy and immunofluorescence Cells were infected with LGT virus at an m.o.i. of 10 or transfected with pCI or pCIE as described above. At 18 h after infection or transfection, cells were fixed in 2% formaldehyde. E or NS2B-NS3 proteins in transfected cells and viral proteins in infected cells were detected with rabbit anti-LGT polyclonal antibody and goat antirabbit FITC-conjugated secondary antibody in 0.2 ␮g/ml propidium iodide. Cells were visualized by fluorescence microscopy at ⫻1000 magnification. Caspase-3-like protease (DEVDase) activity assay Colorimetric assay of caspase-3-like proteolytic activity was performed using an ApoAlert Caspase-3 Colorimetric Assay Kit (Clontech Laboratories, Inc., Palo Alto, CA). This assay uses spectrophotometry to detect chromophore p-nitroanilide, which is cleaved from Ac-DEVDpNA by caspase-3-like proteases. Neuro-2a cells (8 ⫻ 10 5) or Vero cells (6 ⫻ 10 5) were seeded in 60-mm-diameter culture dishes. Twenty-four hours later, cells were transfected with 2 ␮g of pCI or pCIE, or 0.4 ␮g of pCIcasp9. Eighteen hours after transfection, cells were harvested, counted, and pelleted at 2000 g for 5 min. Cells (10 6) were lysed in 100 ␮l of lysis buffer and centrifuged at 12,000 g for 3 min and the supernatant was collected. Supernatant (50 ␮l) was added to an equal volume of assay buffer supplemented with Ac-DEVD-pNA (200 ␮M) and incubated at 37°C for 2 h and the optical density at 405 nm was determined. Values for nanomoles of pNA released expressed per hour were calculated from those observed in A 405 values using a standard curve. To assay DEVDase activity during infection, Neuro-2a or Vero cells were infected with LGT virus at an m.o.i. of 10. At various times, infected cells were harvested, counted, pelleted, and assayed for DEVDase activity, as described above. Enzyme activity was calculated relative to the control cells infected at the time point 0. ACKNOWLEDGMENTS We are grateful to P. D. Friesen (University of Wisconsin, Madison, WI) for p35 gene, and C. M. Zacharchuk (National Cancer Institute, Bethesda, MD) for plasmid expressing caspase-9 gene. We thank J. M. Speicher, R. Fearns, and R. M. Chanock (National Institute of Allergy and Infectious Diseases, Bethesda, MD) for careful review of the manuscript.

LGT VIRUS AND E PROTEIN INDUCE APOPTOSIS

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