Journal of Biotechnology, 15 (1990) 169-186

169

Elsevier

BIOTEC 00534

D N A distribution and respiratory activity of Spodopterafrugiperda populations infected with wild-type and recombinant Autographa californica nuclear polyhedrosis virus B. Schopf, M.W. Howaldt * and J.E. Bailey Department of Chemical Engineering, California Institute of Technology, Pasadena, California, U.S.A. (Received 5 August 1989; accepted 9 February 1990)

Summary

Spodoptera frugiperda cells were infected with a wild-type Autographa californica nuclear polyhedrosis virus and with a recombinant Autographa californica nuclear polyhedrosis virus. The recombinant virus was derived from the wild-type virus and produced fl-galactosidase instead of polyhedrin. The changes in cell size, cell growth, viability, D N A distribution, and respiratory activity were followed through the time course of the infection. The DNA content as measured by flow cytometry of infected cells increased to approximately 1.8 times the value of uninfected cells and the distributions of single-cell DNA content of the infected cells were strongly deformed. Early in the infection the resp!ratory activity passed through a maximum. The mitochondrial activity based on Rhodamine 123 labelling of cells infected with the recombinant virus, as determined by flow cytometry, also passed through a maximum at 24 h post infection while the mitochondrial activity of cells infected with the wild-type virus continued to increase. Evolution of single-cell mitochondrial Correspondence to: J.E. Bailey, Dept. of Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, U.S.A. * Present address: Lehrstuhl f'tir Bioprozesstechnik, Universit~it Stuttgart, Nobelstr. 12, D-7000 Stuttgart 80, F.R.G. Abbreoiations: AcNPV = Autographa californica nuclear polyhedrosis virus; flgal-AcNPV = recombinant Autographa californica nuclear polyhedrosis virus which contains the E. coli lacZ gene (encodes fl-galactosidase); fll = infected with flgal-AcNPV at a multiplicity of infection of 1 plaque forming unit per cell; h.p.i. = hours post infection; MOI = multiplicity of infection; P F U = plaque forming unit; S f 9 = Spodoptera frugiperda insect cells; wt-AcNPV = wild-type .4utographa californica nuclear polyhedrosis virus; wtl = infected with wt-AcNPV at a multiplicity of infection of 1 plaque forming unit per cell. 0168-1656/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

170 activity was different in uninfected populations and in populations infected with wild-type and with recombinant virus. In all experiments performed, the recombinant virus influenced cell behavior and the measured parameters earlier than the wild-type virus. The influence of the multiplicity of infection was stronger for the wild-type virus than for the recombinant virus. Baculovirus; AcNPV; Spodopterafrugiperda insect cell line; fl-Galactosidase; D N A distribution; Respiratory activity

Introduction

The baculovirus-insect cell system has recently received increased interest both for the production of insecticides and for the production of recombinant proteins (Miller et al., 1983; Tramper et al., 1986; Angelo et al., 1987; Carbonell et al., 1985; Herrera et al., 1988; Kuroda et al., 1986; Miyamoto et al., 1985; Smith et al., 1985). The fife cycle of baculoviruses is biphasic. In nature, the virus is packed in occlusion bodies made from polyhedrin, the so-called polyhedra. These occlusion bodies are taken up by other insects. The polyhedra dissolve in the alkaline p H of the midgut, the virions are released and initiate the infection. In the second phase of the infection, the nonoccluded form of the virus spreads the infection within the animal and, at the end of this stage, occluded virus is produced and released from lysed cells. In cell culture, infection occurs only through the nonoccluded form. In recombinant viruses one takes advantage of the fact that proteins which are expressed late in the infection are not essential for virus replication. The cloned gene of interest is usually placed under the control of the strong polyhedrin promoter which is temporally controlled. Instead of the polyhedrin gene the recombinant gene is expressed late in the infection; i.e., such recombinant viruses lack polyhedra. In this study we investigate the differences in Spodoptera frugiperda cells which are infected with a wild-type Autographa californica nuclear polyhedrosis virus and a recombinant virus. The recombinant virus was constructed using the wild-type Autographa californica nuclear polyhedrosis virus and produces fl-galactosidase instead of polyhedrin. We compare several parameters which are influenced by the infection; in particular cell growth, cell size, viability, the single-cell D N A distribution and the respiratory activity of the cells as determined by oxygen consumption and single-cell mitochondrial activity.

Materials and Methods

Cells and viruses A continuous cell-fine of Spodoptera frugiperda IPL-Sf9 obtained from ATCC was used. The cell cultures were maintained at 28°C in modified T N M - F H medium containing 90 ml of Grace's medium (Gibco), 0.3 g lactalbumin, 0.3 g yeastolate, 10

171 ml fetal bovine serum (Hyclone) and 5 000 U penicillin and 5 mg streptomycin (Sigma). Cells were removed from the surface of tissue culture flasks for passaging by flushing media over the surface with a Pasteur pipette. Cell counts and cell size were determined using a Coulter Counter (Coulter Electronics, Inc.). The percent of viable cells was determined by trypan blue exclusion. The wild-type virus used was the E2-clone of an Autographa californica nuclear polyhedrosis virus (wt-AcNPV), which was first isolated by Smith and Summers (1978). The recombinant virus (flgal-AcNPV), which produces fl-galactosidase (flgal), was obtained from Michael Lochrie (California Institute of Technology). This flgal-AcNPV was constructed by homologous recombination using the E2 wild-type virus and the pAc360 plasmid containing the fl-galactosidase gene (Luckow and Summers, 1988). All experiments with the wt-AcNPV and the recombinant virus were done with fourth and fifth passage virus. For the infection the virus was left on the cells for 2 h at 28°C, aspirated off and replaced with fresh medium. Control flasks were treated in a similar manner. The virus titers for the wild-type and the flgal-AcNPV were determined using the end point dilution method (Summers and Smith, 1987). For the wild-type titer the serial dilutions were screened for the occurrence of polyhedra. The recombinant virus titer was determined by the addition of X-gal to the culture medium; flgal synthesis was then monitored based on the developing blue color (Summers and Smith, 1987).

fl-Galactosidase determination Cells were grown and infected in T25-flasks. The extracellular concentration of flgal was determined by removing a sample of the culture medium. Extraneous debris was removed by centrifugation (200 × g for 5 min). Total enzyme levels were determined by harvesting cells and subsequently disrupting cells via ultrasound sonication (Heat Systems - Ultrasonics, Inc.) operated at 25% power and 50% pulsed energy for 3 rain. fl-Galactosidase (flgal) concentrations were determined using the o-nitrophenyl-galactopyranoside (ONPG) assay. To 2.4 ml of Z-buffer (60 m M N a 2 H P O 4 • 7 H20, 40 mM Na2HPO4, 10 mM KC1, 1 mM MgSO 4 • 7 H 2 0 and 0.05 mM Mercaptoethanol, pH 7.0) were added 0.3 ml of sample and 0.3 ml of O N P G solution (10 mg ml-1). After a lag time of 3 rain for temperature equilibration, the change in optical density with time was recorded at 420 nm and 37°C on a Shimadzu UV260 Spectrophotometer. Activities were determined using an extinction coefficient of ( = 4 500 1 m o l - 1 cm-1. Flow cytometric measurements Experiments were performed on a cytofluorograph H50 (Ortho Instruments) using an argon laser (Lexel Corporation) operated at 488 nm and 200 mW. The propidium iodide fluorescence was measured after passing through a 580 nm highpass filter. The fluorescence of Rhodamine 123-stained mitochondria was determined using a 515-555 nm bandpass interference filter. DNA analysis of intact cells Cells were stained using a modification of the procedure of Hillwig (Hillwig and Eipel, 1979). Cells were harvested, washed once with phosphate buffered saline

172 (PBS) and fixed in 70% methanol at 106 cells per ml. After 0.5 h at 25°C, cells were maintained at 4°C until assayed. The fixed cells were stable at 4°C for at least 10 days. Prior to measuring, 10 6 cells were washed with PBS, then resuspended in 2 ml of the staining solution containing 50 mg 1-1 propidium iodide (Sigma) in 180 m M Tris buffer, 180 m M NaC1 and 70 m M MgCI 2 • 6 H20, p H 7.2. RNAse (Sigma) was added to a final concentration of 1 mg m1-1 to eliminate interference with double-stranded RNA. After 15 min of incubation with RNAse at 25°C the fluorescence of the D N A was measured. T o assure that a single-cell suspension was present, the peak height versus the area of the fluorescence was determined. In addition, the cell suspension was observed under a microscope. Unless mentioned otherwise, all D N A distributions were obtained with whole cells and propidium iodide.

DNA analysis of nuclei (Hugues and Osborne, 1981) 106 cells were washed twice with 15 m M HEPES, 135 m M NaC1 at p H 7.4 and centrifuged (200 x g for 5 rain at 4°C). The pellet was resuspended in 100 /~1 of ice-cold lysis buffer, 10 m M Tris, 4 m M MgCI 2, 2 m M CaC12, 600 m M sucrose, 5 m M E D T A and 0.005% Triton X-100 at p H 7.4. After 5 min on ice, the nuclei were collected by centrifugation (360 x g for 7 min at 4°C), washed with 500/~1 of lysis buffer without Triton and resuspended in 10 m M Tris, 4 m M MgC12 and 2 m M CaC12 at p H 7.4. The nuclear R N A was digested for 5 min at 25°C with 1.3 mg ml-~ RNAse. The D N A was stained with 50 mg 1-~ propidium iodide and analyzed as described previously.

Mitochondrial uptake of Rhodarnine 123 10 6 cells were harvested, washed once with complete Grace's medium and resuspended in the staining solution (10/~g ml-~ Rhodamine 123 (Eastman Organic Chemicals) in medium). After 45 min incubation at 28°C the cells were washed once, resuspended in medium and analyzed immediately by flow cytometry.

Arresting cells in metaphase Cells were inoculated at 2 x 10 6 cells per ml. After 3 d, demecolcine (Sigma) was added to 5 ~g ml-~ and left on the cells for 36 h. The cells were then washed and fixed in 70% methanol as described above.

Respiratory activity The oxygen consumption of the cells was determined in a Yellow Springs biological oxygen monitor equipped with a microchamber of 600 /tl volume (YSI model 5300). 2 x 10 6 cells were centrifuged (100 x g for 5 min at 4°C) and resuspended in 1 ml of medium. After 5 min for temperature equilibration, the oxygen consumption was recorded. A constant temperature of 28°C was maintained during the experiment.

173 Results

Cell growth Cells, which were not infected, were washed in fresh m e d i u m so as to subject them to a c o m p a r a b l e treatment as the infected cells (see Materials and Methods), which is sometimes called m o c k infected in the literature. The growth curves of uninfected and infected cells are shown in Fig. 1. Uninfected cells reach a stationary phase after 120 h.p.i. (hours post infection). The cells infected with unity multiplicity of infection ( M O I ) b y the wild-type virus (designated w t l ; see Abbreviations for explanations) grow at the same rate as the uninfected cells up to 24 h.p.i.; however, growth ceases at 24 h.p.i. Wild-type infection with an M O I of 10 results in decreased growth rates and the subsequent cessation of growth at 24 h.p.i. Cells infected with flgal-AcNPV show little growth after infection with no a p p a r e n t difference between ill- and fll0-infected cells (ill0, for example, denotes cells infected by the r e c o m b i n a n t virus at an M O I of 10).

Viabifity I n Fig. 2 the cell viability is given as a function of time. The f l g a l - A c N P V affects cell viability earlier than the wt-AcNPV. Viability of flgal-AcNPV-infected cells at an M O I of 1 and 10 decreases sharply at = 48 h.p.i. W t l - and wtl0-infected cells behave differently from one another as well as from the flgal-AcNPV-infected cells.

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Fig. 1. Relative growth of uninfected, of wt-infected, and of flgal-AcNPV-infected Sf9 cells. Cell numbers are normalized to the 4-h value. At this point in time, the individual cell numbers per ml were 1.62 × 105, 8.56× 105, 7.1 ×105, 6.56× 105 and 9.52× 105 for the uninfected, wtl-infected, wtl0-infected, ill-infected, and fll0-infected cells, respectively.

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Fig. 2. Viability of uninfected, of wt-infected, and of flgal-AcNPV-infected Sf9 cells as determined by trypan blue exclusion. At an MOI of 10, cell viability rapidly drops at = 72 h.p.i., while cell viability for an M O I of 1 decreases at 96 h.p,i.

Cell size Uninfected cells show a small range in cell size of 14-15 /tm (Fig. 3). The size reported here is the mode of the volume distribution as determined with the Coulter counter. The volume is then converted into a diameter assuming that all particles are spherical. The size of the flgal-AcNPV-infected cells increases rapidly upon infection. At 4 h.p.i, an increase in cell diameter is observed. After 48 h.p.i, the flgal-AcNPV-infected cells reach their m a x i m u m diameter of approximately 1.5 times the diameter of uninfected cells. Cells infected with wt-AcNPV at an M O I of 10 also reach a m a x i m u m size at 48 h.p.i., with the increase in diameter being more gradual than with the recombinant virus. The wtl-infected cells do not enlarge in size until after 24 h and they reach their m a x i m u m cell size after 72 h.p.i. At later times the cells are rapidly lysing and accurate size determinations cannot be made.

fl-Galactosidase activity Total fl-galactosidase activity (determined after cell lysis using sonication - see Materials and Methods) first appears between 18-22 h.p.i, at the M O I ' s examined (Fig. 4). The activity increases strongly after 24 h.p.i. At 96 h.p.i, the flgal concentration is highest (0.0002 U per cell) for/3gal-AcNPV-infected cells with an M O I of 10. The maximum activity of the ill-infected cells occurs approximately 24 h later. The extracellular fl-galactosidase activity begins to increase at 48 h.p.i, for an M O I of 1 and 10. The increase begins gradually and is more rapid after 72 h.p.i.

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TIME POST INFECTION [h] Fig. 3. Cell size of uninfected, of wt-infected, and of flgal-AcNPV-infected Sf9 cells. Cell size reported here is the mode of the volume distribution as determined with the Coulter counter. The volume is then converted into a diameter assuming that all particles are spherical.

The extracellular level of flgal for the ill-infected cells increases more rapidly than for fll0-infected cells. At 144 h.p.i., when flgal synthesis has stopped, cells infected using the lower MOI have produced higher extracellular fl-galactosidase activity.

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176

Influence of fl-galactosidase on growth In order to determine whether the different infection behavior of cells infected with the wild-type virus and the recombinant virus was caused by fl-galactosidase, Sf9 cells were infected with wt-AcNPV at an MOI of 10. To the virus solution two different activities of bacterial fl-galactosidase (2.5 U m l - 1 and 25 U ml-1 fl-galactosidase) were added. Sf9 cells were also mock infected with fl-galactosidase solution. After 2 h the fl-galactosidase-containing solution was removed from all samples and replaced with fresh medium. Forty-eight hours later growth rate and viability were compared, fl-Galactosidase did not have any effect on the growth and viability of mock infected Sf9 cells. Also, the growth and viability of wtl0-infected cells did not show any influence of fl-galactosidase activities. DNA distribution of intact cells In Fig. 5a the single-ceU D N A distribution of uninfected Sf9 cells is shown. Interpretation of these peaks is facilitated by the examination of Fig. 5b which shows the D N A distribution of Sf9 cells which were arrested in metaphase through demecolcine addition. The peak labelled "2" corresponds to cells with two genome equivalents, and the one labelled "4" are metaphase cells of a second population which has double the ploidy of the first. Thus, the two major peaks and small feature marked '1', '2', and '4' in Fig. 5a correspond, respectively, to one-genome content cells (Gl-phase cells of the lower ploidy population), two-genome cells (G2-phase cells of the lower ploidy population and G r p h a s e cells of the higher ploidy population), and four-genome cells of the higher ploidy population. In Fig. 6 the D N A distributions of uninfected and infected Sf9 cells are shown as a function of time. The times in the figure correspond to time post infection. Before infection, cells were grown for 48 h. Fig. 6a gives the sequence of D N A distributions obtained for uninfected Sf9 cells. As the cells approach stationary phase (72 h.p.i. corresponds to 120 h culture time), the proportion of cells with large DNA content is-higher than during exponential growth. In Fig. 6b and c the time course of the D N A distributions of wt-AcNPV-infected cells is shown. Fig. 6d and e are the corresponding histograms for the ill- and fll0-infected cells. While the D N A patterns of the uninfected populations remain

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Fig. 5. DNA distributions of single-cell DNA contents of uninfected Sf9 cells in exponentialgrowth (a) and of Sf9 cells which were arrested in G2-phaseby demecolcineaddition (b).

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Fig. 6. D N A distributions of uninfected (a) and of infected (b-e) Sf9 cells as a function of time post infection; b and c were o b t a i n e d from w t l - and w t l 0 - i n f e c t e d cells, respectively; d and e were o b t a i n e d f r o m i l l - and fll0-infected cells, respectively.

similar, those of the infected cells change significantly over time. The two major peaks decrease in size and become broader. Late in infection, one is unable to resolve the peaks. For the w t - A c N P V a greater influence of MOI is demonstrated. A change in the D N A distribution occurs earlier with the high infection ratio. The illand fll0-infected cells, however, show a similar behavior with time, i.e., the MOI does not have an apparent influence. In Fig. 7 the means of fluorescence of the histograms from Fig. 6 are displayed. The mean fluorescence of the uninfected cells is rather constant although the proportion of higher ploidy cells increases as the cells approach stationary phase. However, the mean of the fluorescence of the infected cells increases with both viruses to about twice the initial value, indicating a significant increase of D N A per cell. The wtl0-infected cells begin to display a different D N A distribution at 24 h.p.i. and a corresponding increase in the mean of the fluorescence. Wtl-infected cells show a deformed histogram with an increase of the mean at 48 h.p.i. At 72 h the wtl- and wtl0-infected ceils have reached the same mean D N A fluorescence value of approximately 1.8 times the mean of uninfected Sf9 cells. In contrast to the wt-infected cells that show a change in D N A distribution at 24 h.p.i., the distribution of the flgal-AcNPV-infected cells begins to change at 8 h.p.i.

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Fig. 7. M e a n of the D N A fluorescence of uninfected, of wt-infected, a n d of f l g a l - A c N P V - i n f e c t e d Sf9 cells as a function of t i m e post infection.

After 16 h.p.i, the histograms have changed dramatically and the mean has increased by approximately 60% (Figs. 6d, e and 7). The cells with an MOI of 10 have a lower fluorescence at 72 h.p.i, than at 48 h.p.i., which is probably caused by lysis of the cells. Due to cell fragility and the subsequent lysis that occurred during the harvesting and staining, the histograms could not be analyzed at later times since the noise levels were too high.

DNA distribution of nuclei The cells were infected with wt-AcNPV and flgal-AcNPV with an MOI of 1 and 10. After 48 h.p.i., the nuclei were isolated, stained, and analyzed by flow cytometry. Fig. 8 shows the mean of the fluorescence of infected cells relative to the mean D N A fluorescence of uninfected cells. The open bars correspond to data obtained from intact cells, while the shaded bars indicate data from isolated nuclei. The amount of D N A in the nuclei of wt-AcNPV-infected cells is nearly the same as that of uninfected cells. However, the nuclei of flgal-AcNPV-infected cells have a significantly lower fluorescence, indicating a smaller D N A content. Increase in whole cell D N A while nuclear D N A declines for infected cells relative to uninfected cells presumably indicates viral D N A in the infected cells.

Respiration activity The reference value at 0 h.p.i, corresponds to the respiration rate of uninfected cells, whose oxygen consumption was measured immediately after dislodging. The uninfected cells show a sharp initial increase in respiratory activity at 5 and 8 h.p.i., followed by a slow decrease in oxygen consumption with time (Fig. 9). flgalAcNPV-infected cells show first differences from the noninfected cells at 8 h.p.i.,

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180 where, relative to the noninfected cells, the respiration of ill-infected cells has increased and that of fil0-infected cells has decreased. For both of these MOIs the m a x i m u m respiratory activity occurred at 16 h.p.i., followed by a sharp decrease. The wt-AcNPV-infected cells behave differently. The wtl0-infected cells show a higher respiratory activity at 16 h.p.i, followed by a gradual decrease in respiratory activity. The wtl-infected cells begin at the same activity level as the uninfected cells. Their oxygen consumption rate declines at 16 h.p.i, and then increases to approximately the 8 h value. At 72 h.p.i, all infected cells exhibit slightly less respiratory activity than the uninfected cells. Additional experiments with an M O I of 1 demonstrate a further decrease in oxygen consumption at later times post infection (data not shown).

Mitochondrial fluorescence The cationic fluorochrome Rhodamine 123 binds specifically to mitochondria of living cells (Johnson et al., 1980) and the incorporation depends on mitochondrial transmembrane potential. In a flow cytometric assay in which the total fluorescence per cell is measured, a change in fluorescence may be caused either by a change in the transmembrane potential or by a change in the mitochondrial mass or by a combination of these factors. Fig. 10 shows the sequence of single-cell distributions of mitochondrial fluorescence of uninfected, of wtl0- and of fll0-infected Sf9 cells. In Fig. 11 the corresponding mean fluorescence values are given. The distribution of mitochondrial fluorescence of uninfected Sf9 cells does not change significantly from 0 to 72 h.p.i. At 8 h.p.i, the distribution of fll0-infected cells has shifted towards higher fluorescence, and the mean fluorescence has increased to roughly 145% of the value of uninfected Sf9 cells. As the infection proceeds, the single-cell fluorescence continues to increase and the distribution becomes broader. The mean of the fluorescence of

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Fig. 10. Distributions of single-cell Rhodamine 123-fluorescenceof uninfected (a), fll0-infected (b), and wl0-infected (c) Sf9 cells.

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the ill- and fll0-infected cells passes through a m a x i m u m at 24 h.p.i.. At 48 h.p.i. the noise level is highly increased, the mode of the fluorescence distribution has shifted towards lower fluorescence and the mean fluorescence has dropped sharply. Late in infection (72 h.p.i.), the histograms are strongly deformed and the noise has increased to such a degree that the mean fluorescence cannot be determined accurately anymore. The distribution of mitochondrial fluorescence of wtl0-infected cells also changes at 8 h.p.i. However, the change is not as pronounced as with the fll0-infected cells, and the resulting increase in the mean of the fluorescence is small. The distribution continues to broaden and the mean of the fluorescence increases and reaches a plateau value at 72 h.p.i. The noise level at 72 h.p.i is higher than at 48 h.p.i. In contrast to the fll0-infected cells, a decrease in mean fluorescence does not occur, and, even at 72 h.p.i., the distribution shows a peak which is clearly separated from the noise. The single-cell distribution of mitochondrial fluorescence of ill- and wtl-infected cells follows the same pattern as the ill0- and wtl0-infected cells, respectively (data not shown). The change of single-cell mitochondrial activity of wtl-infected cells, however, was significantly slower than the change of wtl0-infected cells. Interestingly, the wtl-infected cells never reach the high mean of the wtl0-infected cells.

Discussion

Polyploidy in continuous Lepidoptera insect cell lines has previously been reported (Hillwig and Eipel, 1978/79). These authors noted that ploidy levels of the same cell line were not fixed but were dependent on culture conditions of the cell

182 lines studied. Monolayer cultures had a lower ploidy level than suspension cultures (spinner flasks). Our results using cells arrested in metaphase (Fig. 5) indicate that Sf9 cells grown as a monolayer are primarily of low ploidy. In addition there exists a small fraction of cells with twice the amount of D N A of the first population. Late in exponential growth (Fig. 6a, 120 h culture time), the fraction of two-genome-equivalent cells in the population becomes larger. This might be due to an increase in G2-phase cells. However, this explanation is unlikely since cells in stationary phase are often arrested in the G0-phase. A more plausible explanation is that the proportion of higher ploidy cells increases as the cells approach stationary phase. The D N A content of Sf9 cells infected with the wild-type or the recombinant virus increased as the infection progressed. Early in the infection the two distinct peaks became broader. At later times, these separate peaks could not be resolved and only one peak with a broad distribution existed, indicating that the D N A content of the cells varied considerably which is probably due to different numbers of virus genomes per cell. In the experiments with isolated nuclei reported here an infection time of 48 h.p.i, was chosen. At this time the mean of the DNA-fluorescence of the infected cells as measured with the whole cell assay had increased significantly (Fig. 7). The majority of the cells had not lysed at 48 h.p.i., i.e., the fluorescence which was measured in intact cells (Figs. 6 and 7) resulted from the D N A present in the nucleus and the cytoplasm. This increase in DNA-fluorescence was not observed with nuclei at 48 h.p.i. The flgal-AcNPV-infected cells exhibited a decreased fluorescence relative to uninfected cells. Other investigators have reported the existence of an endonuclease of viral origin which selectively degrades cellular D N A but not viral D N A (Brown et al., 1979; Faulkner and Carstens, 1986). The comparison of propidium iodide fluorescence from nuclei and intact cells infected with flgal-AcNPV suggests that a significant proportion of the virus is present in the cytoplasm but not in the nucleus. These data may indicate the presence of such a specific endonuclease responsible for cellular D N A degradation. However, interpretation of these data must be made with caution. It is not clear why the fluorescence of wt-AcNPV-infected cells did not differ from that of the uninfected Sf9 cells, since at 48 h.p.i, the wt-AcNPV-infected cells already contained many polyhedra in the nucleus. The increase in respiration of uninfected Sf9 cells during the first 8 h after the infection may have been caused by the conditioning of the cells to fresh medium twice; i.e., during the 2-h-infection period and then after the replacement with fresh medium. Part of the increase in respiratory activity which was observed with infected cells was probably due to the same effect. However, the peak-value of the ill-, ill0- and wtl0-infected cells occurred at 16 h.p.i. At this time, the respiratory activity of the uninfected cells already decreased, indicating that the increase in respiration was caused by the infection. The single-cell distributions of mitochondrial fluorescence of the flgal-AcNPVinfected cells and the corresponding mean of the fluorescence followed a pattern similar to the respiratory activity. However, while the highest respiration occurred at 16 h.p.i., the highest mean mitochondrial fluorescence was detected at 24 h.p.i. For

183 the wt-AcNPV-infected cells, the development of the mean mitochondrial fluorescence differed appreciably from the respiratory activity. In particular, the mean fluorescence of the wt-infected cells continued to increase until 48 h.p.i, and began to level off at 72 h.p.i., while the respiratory activity of the wtl0-infected cells passed through a m a x i m u m at 16 h.p.i, and the activity of the wtl-infected cells did not change significantly at all. Rhodamine-induced fluorescence as measured by flow cytometry reflects the overall mitochondrial activity of the cell and depends on the membrane potential and on the total mitochondrial surface in the cell. The use of the fluorescent dye nonyl-acridine-orange which binds to mitochondria in a membrane-potential independent manner (Ratinaud et al., 1988) should enable one to distinguish between these factors. Apparently the increase in respiration early in infection is characteristic of A c N P V and is not dependent on the cell line, since similar behavior was observed in Trichopulsia ni cells infected with an AcNPV (Streett and Hink, 1978). These authors hypothesized that the initiation of virus replication caused an increase in respiratory activity. Tjia et al. (1979) found that the m a x i m u m replication rate of wild-type virus occurred between 14-22 h.p.i. The data indicating the increase in mean D N A content of infected cells reported here support this result. The greatest increase in the mean D N A fluorescence occurred between 8 - 2 4 h.p.i, for flgalAcNPV-infected cells. Wtl0-infected cells showed a slower increase in their mean fluorescence than flgal-AcNPV-infected cells. Although the time course of D N A synthesis differed, oxygen consumption for ill- and fll0-infected cells and wtl0-infected cells peaked at 16 h.p.i. The wt-AcNPV-infection with an M O I of 1 appears to be less efficient than the flgal-AcNPV-infection at an M O I of 1. Effects observed at later times with the wtl-infected cells appear to be due to secondary infection by budded virus. This explains the delayed increase in D N A content of the cells (Fig. 7) and the respiration behavior, showing a minor peak at 48 h.p.i (Fig. 9), as well as the delayed increase in mitochondrial fluorescence (Fig. 11). If one normalizes the oxygen consumption rate to the volume of the cell, the wt-AcNPV-infected cells showed a slight increase followed by a decrease in oxygen consumption while the oxygen consumption of the flgal-AcNPV-infected cells decreased continuously. Infection with recombinant AcNPV differed from that with wild-type virus. The recombinant virus stopped cell growth completely while wild-type infected cells continued to grow somewhat through the first 24 h of infection. The viability of flgal-AcNPV-infected cells began to decrease about 24 h earlier than that for the corresponding wild-type infected cells. At 72 h.p.i, m a n y flgal-AcNPV-infected cells had already lysed. Cell diameter and mean of D N A content increased more rapidly with the recombinant virus. The demonstrably different behavior between wild-type AcNPV and flgal-AcNPV could be due to a toxic effect of fl-galactosidase. Since the addition of fl-galactosidase to uninfected Sf9 cells and wtl0-infected cells showed no significant effect on growth or infection behavior, fl-galactosidase does not seem to be toxic to the cells. It may be synthesis rather than presence of fl-galactosidase, or the absence of polyhedrin synthesis, which contributes to these differences. However, this does not

184 e x p l a i n the different b e h a v i o r early in the infection, w h e n flgal-synthesis has n o t yet started. S m i t h et al. (1983) c o n s t r u c t e d m u t a n t s of the E 2 - w i l d - t y p e A c N P V which c a r r i e d deletions in the p o l y h e d r i n gene a n d d i d n o t p r o d u c e occlusion bodies. Cells infected with these m u t a n t s were lysed from 1 to 3 d p o s t infection while cells i n f e c t e d with the u n m o d i f i e d w i l d - t y p e A c N P V were n o t lysed b e f o r e 4 to 5 d p o s t infection. These a u t h o r s suggested that the m u t a t i o n s m a y have an influence on the s t a b i l i t y of the p l a s m a m e m b r a n e late in the infection. A n o t h e r p o s s i b i l i t y is that the two viruses m a y have different activities within the cell. K e l l y a n d W a n g (1981) o b s e r v e d that the infectivity of viral D N A i n c r e a s e d after t r e a t m e n t with D N A relaxing enzymes which r e d u c e d the degree of supercoiling. T h e i n t r o d u c t i o n of the r e c o m b i n a n t gene m a y have c h a n g e d the degree of supercoiling of the viral D N A a n d t h e r e b y i n c r e a s e d the infectivity. This different infection b e h a v i o r is i m p o r t a n t for a n u m b e r of reasons. If the p r o d u c t i o n of a r e c o m b i n a n t p r o t e i n is the goal, then a less toxic (lethal) r e c o m b i n a n t virus m a y b e a d v a n t a g e o u s since p r o d u c t f o r m a t i o n a p p e a r s for a longer t i m e b e f o r e lysis of the cells occurs. If, however, a m o r e p o t e n t virus for use as an insecticide is desired, then the faster lysis of the cells m a y reduce the t i m e s p a n b e t w e e n u p t a k e o f the virus b y the insect a n d death. In such c o n s t r u c t s the r e c o m b i n a n t gene w o u l d have to be inserted at a different p o i n t in the b a c u l o v i r u s g e n o m e so that p o l y h e d r i n is p r o d u c e d a n d virus is occluded.

Acknowledgements B. S c h o p f a n d M . W . H o w a l d t were s u p p o r t e d b y grants f r o m the D A A D ( D e u t s c h e r A k a d e m i s c h e r A u s t a u s c h d i e n s t ) . A d d i t i o n a l s u p p o r t for this research was p r o v i d e d b y G r a n t No. EET-8805636 f r o m the N a t i o n a l Science F o u n d a t i o n

(U.S.A). References Angelo, C.St., Smith, G.E., Summers, M.D. and Krug, R.M. (1987) Two of the three influenza viral polymerase proteins expressed by using baculovirus vectors form a complex in insect cells. J. Virol. 61,361-365. Brown, M., Crawford, A.M. and Faulkner, P. (1979) Genetic analysis of a baculovirus Autographa californica nuclear polyhedrosis virus. J. Virol. 31, 190-198. Carbonell, L.F., Klowden, M.J. and Miller, L.K. (1985) Baculovirus-mediated expression of bacterial genes in dipteran and mammalian cells. J. Virol. 56, 153-160. Faulkner, P. and Carstens, E.B. (1986) An overview of structure and replication of baculoviruses. In: Current Topics in Microbiology and Immunology 131, Springer Verlag, Berlin, Heidelberg, pp. 1-19. Herrera, R., Lebwohl, D., Herreros de, A.G., Kallen, R.G. and Rosen, O.M. (1988).Synthesis, purification, and characterization of the cytoplasmic domain of the human insulin receptor using a baculovirus expression system. J. Biol. Chem. 263, 5560-5568. Hillwig, I. and Eipel, H.E. (1978/79) Characterization of insect cell lines by DNA content. Z. Angew. Entomol. 87, 216-220.

185 Hillwig, I. and Eipel, H.E. (1979) Determination of virus production on insect cell lines by flow cytometry. Z. Angew. Entomoi. 88, 225-230. Hugues, B. and Osborne, H.B. (1981) Dexamethasone inhibits a heine-independent event necessary for terminal differention of murine erythroleukemia cells. Biochem. Biophys. Res. Commun. 102, 13421349. Johnson, L.V., Walsh, M.L. and Chen, L.B. (1980) Localization of mitochondria in living cells with rhodamine 123. Proc. Natl. Acad. Sci. USA 77, 990-994. Kelly, D.C. and Wang, X. (1981) The infectivity of nuclear polyhedrosis virus DNA. Ann. Virol. (Inst. Pasteur). 132E, 247-259. Kuroda, K., Hauser, C., Rott, R., Klenk, H.-D. and Doerfler, W. (1986) Expression of the influenza virus haemagglutinin in insect cells by a baculovirus vector. Eur. Mol. Biol. Qrgan. J. 5, 1359-1365. Luckow, V.A. and Summers, M.D. (1988) Trends in the development of Baculovirus expression vectors. Bio/Technology 6, 47-55. Miller, L.K., Lingg, A.J. and Bulla, L.A. (1983) Bacterial, viral, and fungal insecticides. Science 219, 715-721. Miyamoto, C., Smith, G.E., Farell Towt, J., Chizzonite, R., Summers, M.D. and Ju, G. (1985) Production of human c-myc protein in insect cells infected with a baculovirus expression vector. Mol. Cell. Biol. 5, 2860-2865. Ratinaud, M.H., Leprat, P. and Julien, R. (1988) In situ flow cytometric analysis of nonyl acridine-stained mitochondria from splendocytes. Cytometry 9, 206. Smith, G.E. and Summers, M.D. (1978) Analysis of baculovirus genomes with restriction endonucleases. Virology 89, 517-527. Smith G.E., Fraser, M.J. and Summers, M.D. (1983) Molecular engineering of the Autographa californica Nuclear polyhedrosis virus genome: deletion mutations within the polyhedrin gene. J. Virol. 46, 584-593. Smith, G.E., Ju, G., Ericson, B.L., Moschera, J., Lahm, H.-W., Chizzonite, R. and Summers, M.D. (1985) Modification and secretion of human interleukin 2 produced in insect cells by a Baculovirus expression vector. Proc. Natl. Acad. Sci. (U.S.A.) 82, 8404-8408. Streett, D.A. and Hink, W.F. (1978) Oxygen consumption of Trichoplusia ni (TN-368) insect cell line infected with Autographa californica Nuclear Polyhedrosis Virus. J. Invert. Pathol. 32, 112-113. Summers, M.D. and Smith, G.E. (1987) A manual of methods for baculovirus and insect cell culture procedures, Department of Entomology, Texas Agricultural Experiment Station and Texas A&M University, pp. 14-16. Tjia, S.T., Carstens, E.B. and Doerfler, W. (1979) Infection of Spodoptera frugiperda cells with Autographa californica nuclear polyhedrosis virus. Virology 99, 399-409. Tramper, J., Williams, J.B., Joustra, D. and Vlak, J.M. (1986) Shear sensitivity of insect ceils in suspension. Enzyme Microb. Technol. 8, 33-36.