Superinfection of baculovirus-infected gypsy moth cells with the nuclear polyhedrosis viruses of Autographa californica and Lymantria dispar

Vwus Research, Elsevier

351

7 (1987) 351-364

VRR 00339

Superinfection of baculovirus-infected gypsy moth cells with the nuclear polyhedrosis viruses of Autographa californica and Lymantria dispar J. Thomas

McClintock

Insect Pathology Laboratory,

Agricultural (Accepted

and Edward

Research Sewice,

for publication

M. Dougherty

USDA, Beltsudle, MD 20705. LJ.S.A

5 March 1987)

Summary The ability of the multiple-embedded nuclear polyhedrosis viruses (NPVs) of Autographa cali’ornica (AcMNPV) or Lymantria &par (LdMNPV) to replicate in either a permissive system, LdMNPV-infected gypsy moth cells, or a semipermissive system, AcMNPV-infected gypsy moth cells, upon superinfection, was studied. Inoculation of LdMNPY-infected cells with AcMNPV up to 8 h postinfection (p.i.) resulted in significantly reduced LdMNPV non-occluded virus (NOV) production, suggesting viral interference. In these same experiments, AcMNPV NOV production occurred with maximal titers at 48 h p.i. in cells superinfected by 6 h p.i. In the converse experiment, superinfection of AcMNPV-infected gypsy moth cells by LdMNPV during the early phases of replication (up to 8 h) resulted in the production of polyhedral inclusion bodies, unlike gypsy moth cells infected only with AcMNPV where virus particles are not produced. In both systems, DNA-DNA dot hybridizations detected simultaneous viral DNA replication of both viruses. Analysis of [ 35S]methionine-labeled fractions from superinfected cells by SDS-polyacrylamide gel electrophoresis revealed polypeptides similar to those produced by AcMNPV and LdMNPV during solitary infections. These results suggest that both the primary and superinfecting viruses adsorbed, penetrated, and initiated macromolecular synthesis. Moreover, viral progeny were produced in the semipermissive system upon superinfection with LdMNPV during the early (Yphase of replication. Superinfection;

Baculovirus;

Correspondenceto: J. Thomas BARC-W,

Beltsville,

0168-1702/87/$03.50

Gypsy

McClintock, MD 20705, U.S.A.

moth cell; Nuclear

USDA,

ARS,

0 1987 Elsevier Science Publishers

Insect

polyhedrosis

Pathology

B.V. (Biomedical

Lab..

Division)

virus

Rm 214, Bldg OllA,

352 Introduction Multiple infections of invertebrate cell lines have been demonstrated with various insect viruses (Kimura and McIntosh, 197.5; Kurstak and Garzon. 1975; McIntosh et al.. 1979). McIntosh et al. (1979) reported a loo-fold decrease in polyhedral inclusion body (PIB) production when Trichopiusia ni TN-368 cells. inoculated initially with a low passage strain of ~~tu~~~~~~ ~ffi~forn~~unuclear polyhedr~s~s virus (AcMNPV), were superinfected with a high passage strain of the same virus. In an earlier report, Kimura and McIntosh (1975) demonstrated the inhibition of PIB production in Chile iridescent virus-infected TN-368 cells superinfected with AcMNPV. In both studies the decrease in FIB production, as assessed by microscopy and virus titrations. was attributed to viral interference. Other studies using phase and electron microscopy have shown that individual cells of the greater wax moth, Gulieriu trlel~~neliu, could support the replication of multiple DNA viruses during dual infections (Kurstak and Garzon. 1975). In that simultaneous replication occurred resulting in the synthesis of a instance, densonucleosis virus or a nuclear polyhedrosis virus (NPV) in. the nucleus and a tipula iridescent virus in the cytoplasm. Previously we described the semipermissive infection of a gypsy moth cell line with AcMNPV in which the infection cycle is restricted during the early phase of viral replication (McClintock et al., 1986b}. Although the rate of AcMNPV DNA synthesis approximated that observed in the permissive TN-368 cell line, protein synthesis was aborted during the early fy phase of replication. Thereafter. both host and viral protein synthesis were completely suppressed. Because of this drastic inhibitory effect on cellular and viral macromolecular synthesis following single infection, we examined the ability of the semipermissive system to support a superinfecting nonhomologous virus. Since previous studies on multiple infections were based entirely on microscopic and bioassay data no information was provided on the molecular events occurring during supe~nfection of baculovirus-infected cells. Here we report on the effect of superinfecting either the semipermissive system. AcMNPV-infected gypsy tnoth cells. or the permissive system, L.ymuntria dispur NPV (LdMNPVf-infected gypsy moth cells. with a second NPV inoculum. We show that viral interference occurs in LdMNPV-infected gypsy moth cells superinfected with AcMNPV, and, based on DNA-DNA dot hybridizations, we demonstrate simultaneous viral DNA replication in both systems. Moreover, when AcMNPV-infected gypsy moth cells are superinfected with LdMNPV during the early cy phase of replication, the block in resulting in the production of viral the se~pern~issive infection is overcome, progeny.

Materials

and Methods

Cells and virus The experiments reported here utilized the L.. d&par cell subline (IPLB-LD-652Y) maintained in IPL-52B medium (KC Biological, Inc., Lenexa. KS) (Goodwin and

353 Adams, 1980). The T. ni cell line (TN-368) was maintained in modified TNM-FH medium containing 5% fetal calf serum (Hink et al., 1977) and used for the propagation and assay of AcMNPV. Both cell lines were maintained at 27 o C. A plaque-purified clone of AcMNPV (6R) and one isolated from the Hamden LdMNPV isolate (5-7D) were propagated in TN-368 and IPLB-LD-652Y, respectively, as previously described (McClintock et al., 1986a,b). Virus inocula were prepared by centrifuging (20,000 X g) the supernatants from NPV-infected cell cultures and resuspending the extracellular NOV pellet in complete media. Superinfection Log phase IPLB-LD-652Y cells were inoculated with NOV of LdMNPV or AcMNPV at a multiplicity of infection of 100 and 75 tissue culture infective doses (TCID,,) per cell, respectively, for 1 h, rinsed with sterile Locke’s solution and incubated in complete media (McClintock et al., 1986b). At various times postinfection (p.i.) the culture medium was removed and the infected cells were superinfected with the alternate virus for 1 h. After adsorption, the superinfecting virus inoculum was removed, the cells rinsed, and incubated with complete medium. Cultures were observed daily by phase microscopy for cytopathic effects (CPE) and PIB production. Unless stated otherwise, the times that cells were harvested are reported relative to inoculation of the superinfecting virus. Endpoint

dilution assay

Endpoint dilution assays (McClintock et al., 1986b) utilizing TN-368 AcMNPV and IPLB-LD-652Y cells for LdMNPV were used for selective of NOV production. Isolation

cells for titration

of viral DNA

Viral DNA was isolated from purified PIBs as previously described (McClintock et al., 1986a). Approximately 10” PIBs per ml we re dissolved in an equal volume of 0.1 M Na,CO, at room temperature for 1 h. The alkali-dissolved virus was treated with SDS at a final concentration of 0.5%, extracted with buffered phenol and dialyzed against TE buffer (10 mM Tris hydrochloride and 1 mM EDTA [pH 7.61). The viral DNAs were characterized by agarose gel electrophoresis and the concentration determined spectrophotometrically. Nick-translation

and hybridization

Viral DNA was labeled in vitro by nick-translation (Rigby et al., 1977) using [a3’P]dCTP (New England Nuclear, Boston, MA). The nick-translated probe was extracted with phenol, centrifuged in a Sephadex G-50 column, denatured with NaOH, boiled for 5 rnin, and neutralized with HCl.

354 DNA samples for dot hybridization were prepared by the method of Meinkoth and Wahl (1984) as modified by McClintock et al. (1986b). The DNA samples were immobilized on nitrocellulose filters (Kafatos et al., 1979) while under vacuum using a Hybri Dot manifold (Bethesda Research Laboratories, Inc., Gaithersburg, MD). Filters were baked, placed into a Seal-a-Bag (Dazey Corp, Industrial Airport, KS) and saturated with a solution containing 10 x Denhardt’s (0.1% Ficoll, 0.1% polyvinylpyrrolidone, and 0.1% bovine serum albumin), 4 X SET buffer (0.60 M NaCl, 0.12 M Tris hydrochloride [pH 8.01, and 4 mM EDTA), 0.2% SDS, 150 pg of tRNA, and 50% deionized formamide. The filters were prehybridized at 37 o C for AcMNPV and 42°C for LdMNPV for 2 to 4 h. Following prehybridization, the “P-labeled viral DNA probe was added to the hybridization solution and incubated for 24 to 48 h at the same temperature as prehybridizations. The filters were rinsed. dried, and processed for autoradiography. After autoradiography the DNA dots were excised and analyzed by liquid scintillation spectrophotometry. Radiolabeling (SDS-PAGE)

of superinfected-cell

proteins and SDS-polyacrylamide

gel electrophoresis

Superinfected IPLB-LD-652Y cells were radiolabeled with 20 PCi of [35S]methionine per ml in amino acid-free medium. Pulse-labeled cells were centrifuged (12,500 x g), rinsed with Locke’s solution, resuspended in disruption buffer (2% SDS, 2% 2-mercaptoethanol, 20% sucrose, 0.05% bromophenol blue, and 5 mM Tris hydrochloride [pH 6.81) boiled for 5 min, and analyzed by SDS-PAGE (Laemmli, 1970) and autoradiography. The molecular weights of the virus-induced proteins were determined by comparing electrophoretic mobilities to structural proteins of AcMNPV and LdMNPV and to standard molecular weight markers (BRL, Gaithersburg, MD).

Results Virus production

in superinfected

cells

Assay of culture medium from LdMNPV-infected gypsy moth cells superinfected with AcMNPV in permissive cell lines showed infectious progeny of both viruses (Table 1). LdMNPV NOV production was reduced up to 8 h p.i. (although no effect at 16 h) compared to cells infected only with LdMNPV. Surprisingly, infectious AcMNPV NOV was produced in these cells when superinfection occurred by 6 h p.i., with the maximal titers at 48 h p.i. Since our previous study demonstrated the absence of viral progeny during the semipermissive replicate cycle of AcMNPV in a gypsy moth cell line (McClintock et al., 1986b), viral interference by superinfection with LdMNPV could not be determined. Instead, we asked if the semipermissive system could support viral replication upon superinfection with LdMNPV. Superinfection of AcMNPV-infected gypsy moth cells with LdMNPV at 2, 4, 6, and 8 h p.i. resulted in the

Harvest (h p.i.) ‘: 1.70x104 5.62 x 10’ 2.02 x 104 3.98 x 107

2.42 x 10” 3.16X103 2.15 x IO3 3.45 x 102 _

24 1.61 x 2.74 x 1.70x 3.98 x _ _

AcMN PV 12

4 10’ IO4 105 103

_ 1.08X10’ 4.95 x 102 2.10 x 10’ 5.34x104 1.39x 102

_c 7.18x 102 3.57 x 102 1.39x102 3.43x10” 2.02 x 102

5.62 x lo5 7.18 x 103 3.98X10’ 4.64 x lo3 _ _

24 _ 2.18 x 7.18 x 1.61 x 1.42x 8.49 x

LdMNPV I2

4

a

48

NOV Titer (TCID,,,/ml)

GYPSY MOTH CELLS SUPERINI~~CTED WITH AcMNPV.

103 lo2 10’ IO5 103

7.18 x 102 3.16 x lo2 2.02x10’ 3.51 x 10s 3.50 x 106

48

a NOV production in superinfected cells was determined for each virus in their respective cell lines by the endpoint dilution assay. Virus titers are expressed as tissue culture infective doses at 50% infection per ml (TCID,,/ml). ’ Harvest times p.i. to the superinfecting virus. c -, not determined. d NOV production in LdMNPV-infected cells.

2 4 6 8 16 control d

%me of super-infection (h)

NOV PRODUCTION IN Ldh~NPV-INFECTED

TABLE 1

3% TABLE

2

THE EFFECT Time of superinfection 2 4 6 8 16 24 48 control

OF LdMNPV

SUPERINFECTI~N PlBs ”

(h)

’

+ 69 49 36 30 0 0 0 0

131 151 164 I70 200 200 200 200

a

( -)

2

3

“,

PIBs

polyhedral

inclusion

bodies (PIBs) at 7 days p.i.

c ‘-.

2

3

MOTH CELLS. --___

35 2s 18 15 0 0 0 0

b 1

GYPSY

% Cells containing

-

’ No. of gypsy moth cells with (+ ) or without h PIB production in AcMNPV-infected cells.

Hr

ON AcMNPV-INFECTED

Hr

d ’

2

WI

3

1

2

3

174

612 1143

2 hr

4 hr

6 hr

e kir

8 hr

f ’

2

3

68

4

Hr

g ’

2

3

HI

’

2

3

4

53

4

42

12

53

$2

201

12

47

24

1764

24

63

24

65

48

2534

48

69

48

66

AcMNPV

LdMNPV

652’1

A Fig. l(A). Viral DNA synthesis in LdMNPV-infected iPLB-LD-652Y cells superinfected with AcMNPV. The kinetics of viral DNA synthesis was followed by DNA-DNA dot hybridization of nibs-transIated AcMNPV DNA to total DNA extracted from LdMNPV-infected gypsy moth cells superinfected with AcMNPV at (a) 2 (b) 4 (c) 6 and (d) 8 h pi. Viral DNA synthesis in AcMNPV-infected cells (e) and in LdMNPV-infected cells (f) was also analyzed in parallel with that of uninfected control IPLB-LD-652Y cells (g). Harvest times are indicated to the left of the corresponding DNA spots. Harvest times for superinfected ceils are indicated in h [Hr] p.i. to the superinfecting virus, whereas times for AcMNPV-infected cells and LdMNPV-infected cells are represented in h p.i. Hyb~dization responses of DNA from superinfected cells are shown in lanes 1 (3.0 X IO4 cells) and 2 (1.0~ lo4 cells). After autoradiography, the DNA spots from lane 1 were excised and quantitated by liquid scintillation counting (lane 3).

4

36

4

34

42

4

46

12

66

12

46

12

51

12

24

46

24

63

24

123

24

536

46

67

48

106

48

267

48

640

4

2 hr

4

Hr

1

4

2

hr

6

3 ~~~

Hr

36

_‘L

2

hr

8

9

3-

Hr 42

4

90

~‘~___

4

2

~~

3

43

12

213

12

47

12

62

24

1366

24

51

24

42

2461

48

56

48

50

46

LdMNPV

AcMNPV

hr

6521

B Fig, l(B). Viral DNA synthesis in LdMNPV-infected cells superinfected with AcMNPV and probed LdMNPV DNA. LdMNPV-infected cells were superinfected with AcMNPV at (a) 2 (b) 4 (c) 6 and h pi. and compared to viral DNA synthesis in LdMNPV-infected cells (e) and AcMNPV-infected (f). Uninfected cell DNA is shown in panel g. Harvest times, hybridization responses, and scintillation data are as described in the legend of Fig. IA.

with (d) 8 cells liquid

production of PIBs (Table 2) a phenomenon not observed when LdMNPV-infected cells were superinfected with AcMNPV. The percentage of cells containing PIBs declined with increasing times between inoculations of the primary and superinfecting virus up to 16 h p.i., following which no PIB production was detected. It should be noted that cells infected only with LdMNPV produced PIBs by 48 h p.i. (McClintock et al., 1986a).

Viral DNA synthesis

in superinfected

cells

Results from DNA-DNA dot hybridization of LdMNPV-infected gypsy moth cells superinfected with AcMNPV at 2, 4, 6, and 8 h p.i. revealed increasing amounts of DNA for both viruses. Using 32P-labeled AcMNPV or LdMNPV DNA to probe DNA from superinfected cells, AcMNPV DNA synthesis was detected between 4 and 12 h p.i. regardless of time of superinfection (Fig. 1A). However, the initiation of LdMNPV DNA synthesis occurred between 24 and 48 h pi. and from 12 to 24 h p.i. when superinfected with AcMNPV at 2 and 4 h p.i. and 6 and 8 h pi. respectively (Fig. 1B). After these times total intensity of both viral DNA-DNA dots increased, as measured by autoradiography and liquid scintillation counting,

358 We concluded from these data that the kinetics of AcMNPV DNA synthesis in LdMNPV-infected cells, superinfected with AcMNPV, was temporally similar to that observed in AcMNPV-infected TN-348 cells, the permissive system (unpublished observations); however, the quantity of AcMNPV DNA was significantly reduced. Similarly, LdMNPV DNA synthesis was delayed upon superinfection and the quantity appeared to be reduced when compared to that observed in LdMNPVinfected gypsy moth cells. AcMNPV-infected cells superinfected at 2, 4, 6, and 8 h p.i. with LdMNPV and probed with radiolabeled AcMNPV DNA revealed increased rates of viral DNA synthesis regardless of the time of superinfection (Fig. 2A). However. when such superinfected cells were probed with LdMNPV DNA, it appeared that AcMNPV

b

a WI

.__’

4

3

2

H,

276

24

’

2

3

4

2hr

280

4

t2

1954

12

2352

24

8669

24

3748

48

9336

48

4708

2

AcMNPV

3

515

8hr

f

Hr ’

1

6 hr

4hr

e Hr

3

H,

2

4

5421

2

3

’

Hr

494

24

4453

d

C

g ’

2

LdMNPV

’

2

3

3

Hr

57

4

22

64

12

43

72

24

57

75

48

47

652Y

A Fig. 2(A). Viral DNA synthesis in AcMNPV-infected IPLB-LD-652Y cells superinfected with LdMNPV. The kinetics of viral DNA synthesis was followed by DNA-DNA dot hybridization of nick-translated AcMNPV DNA (A) and LdMNPV DNA (B) to total DNA extracted from AcMNPV-infected gypsy moth cells superinfected with LdMNPV at (a) 2 (b) 4 (c) 6 and (d) 8 h pi. DNA synthesis in AcMNPV-infected cells (e), LdMNPV-infected cells (f), and uninfected control IPLB-LD-652Y cells (g) were also analyzed in parallel with that of superinfected cells. Harvest times, hybridization responses, and liquid scintiIlatiol1 data are as described in the legend of Fig. 1.

359 a nr

b 2

1

3

.,

2

3

Hr_-1.

2

3

79

4

: 192

12

125

227

24

303

'265 2

d

C t

Hr

72

46

hr

4

e

2

Hr p’

hr

714

6 hr

0

f

3

Hr_l

--2_

3

9 H,

4

36

4

12

213

12

24

1386

24

46

2491

48

LdYNPV

AcMNPV

hr

1----2

3

n

30

35

6521

B Fig. 2(B) Viral DNA synthesis in LdMNPV-infected cells (e) and analyzed in parallel with that of superinfected cells (a-d).

interfered with (Fig. 2B). Protein synthesis

LdMNPV

DNA

in superinfected

replication

AcMNPV-infected

cells (f) was

at each time used for superinfection

cells

LdMNPV-infected cells superinfected with AcMNPV at 4 h p.i. and radiolabeled from 24 to 28 h p.i. revealed several viral-induced proteins with apparent molecular weights of 59.5, 46.5, 39.0, 35.0, and 30.5K (Fig. 3). Also during this time the synthesis of one host protein with a molecular weight of 42.OK and six virus-induced proteins with molecular weights of 36.3, 35.0, 29.2, 27.5, 23.5, and 18.5K were suppressed. The identity of these virus-induced proteins was determined by comparing their electrophoretic mobilities to those proteins from gypsy moth cells infected with LdMNPV (McClintock et al., 1986a) and AcMNPV (Fig. 4) (McClintock et al., 1986b). When LdMNPV-infected cells were superinfected with AcMNPV at 6 and 8 h p.i., followed by pulse-labeling from 24 to 28 h p.i., protein profiles similar to those found when such infected cells were superinfected at 4 h p.i. were observed (Fig. 3). However, two additional proteins with molecular weights of 22.4 and 20.8K were detected in LdMNPV-infected cells superinfected at 6 h p.i. which were not observed in singly-infected cells. These proteins may have been present in LdMNPV-infected cells but too diffuse to accurately resolve and determine the

360

MW u 94(76.0

68-59.5

k

(59.5

-46.5 -42.0

43-

439.0 ‘36.3 *35.0 -30.5 ‘29.2

31-

-27.5

” -22.4 420.8

Zl-

-18.5

-18.5 14.3

-23.5

~23.5

-23.5

-

4 hr

6 hr

8

hr

Fig. 3. SDS-polyacrylamide slab gel sutoradiogram of [ 35S]methionine-labeled polypeptides from LdMNPV-infected IPLB-LD-652Y ceils superinfected with AcMNPV. Uninfected (C). LdMNPV-infected (V), and superinfected gypsy moth cells (S) were pulse-labeled for 4 h at various time intervals pi. The numbers above the wells indicate the time in h following the primary (V) or superinf~~tin~ (S) inoculum at which the pulses were initiated (first number) and terminated (second number). The time at which cells were superinfected is indicated at the bottom of each panel. The numbers on the right indicate the apparent molecular weights ( x 10’) of virus-induced polypeptides. The numbera along the left of the autoradiogram refer to the standard molecular weight (MW) markers (X 10’).

corresponding molecular weights. Pulse-labeling from 48 to 52 h revealed one additional protein with a molecular weight of 76.OK. Also during this time the 46.5, 39.0, 36.3, 35.0 and 30SK proteins were suppressed, whereas the synthesis of the 29.2K protein increased. Similar protein profiles were observed when LdMNPV-infected cells were superinfected with AcMNPV at 16 h p.i. (data not shown). AcMNPV-infected gypsy moth cells superinfected with LdMNPV revealed several proteins unique to both parental inocula (Fig. 4). Ten polypeptides with apparent molecular weights of 110.0, 76.0, 57.0, 53.0, 50.0, 45.0. 36.3. 34.0, 31.0, and 19.4K were detected in AcMNPV-infected cells superinfected with LdMNPV at 2 h p.i. Three proteins with molecular weights of 46.5, 34.0, and 31.OK corresponded to three of four early Q: proteins observed in AcMNPV-infected gypsy moth cells. the

361

476.0

4 76.0

453.0 4 50.0 -46.5 -36.3

~46.5

-46.5

-36.3 .34.0 ‘31.0

31-

-34.0 *31.0

2114.3

.

-19.4

C

AcMNPV

2 hr

4 hr

6 hr

8

hr

Fig. 4. Autoradiographic comparison of electrophoretically separated [s5S]methionine-labeled polypeptides from AcMNPV-infected IPLB-LD-652Y cells superinfected with LdMNPV. Pulse-labeling times and the apparent MW ( X 103) for both viral-induced polypeptides and the standard MW markers are as described in the legend of Fig. 3. Compare the absence of proteins, either of cell or viral origin, in gypsy moth cells infected only with AcMNPV and pulse-labeled from 12 to 16 h and from 24 to 2X h p.i. with those proteins observed in superinfected cells.

semipermissive system. One unique protein with a molecular weight of 53.OK was also observed. The remaining six polypeptides had molecular weights corresponding to those previously reported for LdMNPV (McClintock et al., 1986a). No additional polypeptides were detected in AcMNPV-infected cells superinfected at later time intervals; however, the synthesis of the early polypeptides decreased as the time of superinfection increased. These results suggested that both viruses absorbed, penetrated, and initiated viral protein synthesis in AcMNPV-infected gypsy moth cells superinfected with LdMNPV.

Discussion Previous studies on multiple virus infections with invertebrate cells have been based on recognition of viral progeny or occlusion bodies by phase or electron microscopy and bioassay data. Such reports did not attempt to examine the molecular events occurring during superinfection of baculovirus-infected insect

362 cells. In this report we have shown viral interference, simultaneous viral DNA and protein synthesis, and the ability of the semipermissive system to support viral replication upon LdMNPV superinfection. We show that AcMNPV is capable of interfering with the replication of LdMNPV in a gypsy moth cell line and that a significant decrease in LdMNPV production occurs if AcMNPV superinfection occurs within the first 8 h after LdMNPV infection. However, AcMNPV replication occurred and appeared, at least qualitatively, to undergo normal replication if superinfection occurred by 6 h p.i. This may be due to the initiation of LdMNPV DNA synthesis at 12 h p.i. whereas AcMNPV DNA synthesis occurs at 8 h p.i. in the permissive or semipermissive system (McClintock et al.. 1986a,b). When AcMNPV-infected cells were superinfected with LdMNPV during the early phase of the replicative cycle, viral progeny were produced. Because of this, we investigated the kinetics of viral DNA replication in superinfected cells. Based on DNA-DNA dot hybridizations we demonstrated that both AcMNPV and LdMNPV DNA replication occurred in AcMNPV-infected cells superinfected with LdMNPV. In contrast, when LdMNPV-infected cells were superinfected with AcMNPV during the early (Y phase of LdMNPV replication, LdMNPV DNA synthesis was delayed by several hours. However, similar rates of AcMNPV DNA synthesis were observed regardless of the time of superinfection. The delay in the appearance of LdMNPV DNA may indicate a decline in available host cell functions that participate in viral replication as the time of superinfection increases, or that the synthesis of LdMNPV-induced early enzymes was retarded by the presence of AcMNPV. Using SDS-PAGE, the protein profiles of LdMNPV-infected cells superinfected with AcMNPV were compared to cells infected only with AcMNPV or LdMNPV. Three proteins with apparent molecular weights of 46.5, 39.0, and 35.OK corresponded to those of AcMNPV, while several other proteins, with molecular weights of 59.5, 36.3, 30.5, 29.2, 27.5. 23.5, and 18.5K, were similar to those observed for LdMNPV. However, one protein with an apparent molecular weight of 76.OK appeared to be modified (i.e. glycosylated, phosphorylated, etc) since only one protein from LdMNPV had a similar molecular weight of 73.3K (McClintock et al.. 1986a). Protein profiles of AcMNPV-infected cells superinfected with LdMNPV also revealed polypeptides similar to those of AcMNPV and LdMNPV. Viral progeny were produced in these studies but were not evident by SDS-PAGE and autoradiography. This might suggest that virus-directed protein synthesis was significantly delayed and not observed, or that the structural proteins occurred at undetectable levels. Alternatively, these proteins were modified in the superinfected cell line. Although viral DNA and protein synthesis occurred in LdMNPV-infected cells superinfected with AcMNPV, no occlusion bodies were observed by phase microscopy. However, upon assaying supernatant from such superinfected cells our data indicate that AcMNPV NOV replication occurred only if superinfection occurred during the early (Y phase of LdMNPV replication. Two lines of evidence suggest that AcMNPV NOV replication occurred. The endpoint dilution data detected a substantial increase in virus titers, and these values exceeded titers of the residual virus inoculum. This enhancement of AcMNPV NOV titers by LdMNPV

363 superinfection may simply be due to the packaging of AcMNPV genomes in LdMNPV capsids/envelopes. Secondly, since extracellular NOV production increased, the suppression of polyhedrin, as evident by SDS-PAGE, may suggest that the transition from NOV to PIB production depends on uninterrupted (Y and/or p phase protein synthesis, and without these events PIB production is inhibited in such superinfected cells. In the converse experiment, AcMNPV-infected cells superinfected with LdMNPV supported viral replication as evident by the production of PIBs. This contrasts with the observations of Kelly (1980) who showed that Spodopteru frugiperdu cells were unable to support the replication of two heterologous viruses upon dual infection. Earlier reports have shown viral replication in cells dually infected with heterologous (Kimura and McIntosh, 1975; Kurstak and Garzon, 1975) or homologous (Summers et al., 1980) virus inocula. These differences may be due to the cell and host type, the inocula size, time of superinfection, and the degree of homology between the viruses. The production of viral progeny in LdMNPV superinfected AcMNPV-infected gypsy moth cells may indicate a “leakthrough” or helper function of the LdMNPV inoculum, phenotypic mixing, or recombination between the parental viruses. Recombinants have been isolated and characterized from insect cells infected with two viruses (Summers et al., 1980). We are currently plaque-purifying those viral progeny produced upon superinfection in the semipermissive system to determine if recombination occurred between AcMNPV and LdMNPV. From our observations it may be possible to obtain baculovirus recombinants between nonhomologous viruses from superinfected cells without resorting to recombinant DNA technology, and that such recombinants could be utilized in an integrated pest management program.

References Goodwin, R.H. and Adams, J.R. (1980) Nutrient factors influencing viral replication in serum-free insect cell line culture. In: Invertebrate systems in vitro (Kurstak, E., Maramorosch. K. and Dubendorfer, A., eds.), pp. 493-509. Elsevier/North Holland Biomedical Press, Amsterdam. Hink, W.F., Strauss, E.M. and Ramoska, W.A. (1977) Propagation of Aufographa _culi/ornicu nuclear polyhedrosis virus in cell culture: Methods for infecting cells. J. Invertebr. Pathol. 30, 785-191. Kafatos, F.C., Jones, C.W. and Efstratiadis, A. (1979) Determination of nucleic acid sequence homologies and relative concentrations by a dot hybridization procedure. Nucleic Acids Res. 7, 1541-1552. Kelly, D.C. (1980) Suppression of baculovirus and iridescent virus replication in dually infected cells. Microbiologica 3, 177-185. Kimura, M. and McIntosh, A.H. (1975) Dual infection of the Trichoplusia ni cell line with the Chllo iridescent virus (CIV) and Autographa californica nuclear polyhedrosis virus. In: Invertebrate tissue culture (Kurstak, E. and Maramorosch, K. eds.), pp. 391-394. Academic Press, Inc., New York, NY. Kurstak, E. and Garzon, S. (1975) Multiple infections of invertebrate cells by viruses. Ann. N.Y. Acad. Sci. 266, 2322240. Laemmli. U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. McClintock, J.T., Dougherty, E.M. and Weiner, R.M. (1986a) Protein synthesis in gypsy moth cells infected with a nuclear polyhedrosis virus of L_vmuntriu dispar. Virus Res. 5, 307-322.

364 McClintock. J.T., Dougherty, E.M. and Weiner, R.M. (1986b) Semiper~ssive replication of a nuclear polyhedrosis virus of A~fo~~~ph~ c~~z~~~~z~c~ in a gypsy moth cell line. J. Virol. 57, 197-204. McIntosh, A.H., Shamy, R. and flsley, C. (1979) Interference with polyhedral inclusion body (PIB) production in Trichoplusia ni cells infected with a high passage strain of Autographa caLi(orni~a nuciear polyhedrosis virus (NPV). Arch. Virol. 60, X3-358. Meinkoth, .I. and Wahl, G. (1984) Hybridization of nucleic acids immobilized on solid supports, Anal. Biochem. 138, 267-284. Rigby. P.W.J., Dieckmann, D.M.. Rhodes, C. and Berg, P. (1977) Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. Biol. 133, 237-25 I. Summers. M.D., Smith, G.E.. Knell, J.D. and Burand, J.P. (1980) Physical maps of Aulogruphu calijomica and Rachiplusia ou nuclear polyhedrosis virus recombinants. J. Viral. 34, 69% 703. (Received

6 January

1987; revision

received

5 March

1987)