Infection of Spodoptera frugiperda cells with Autographa californica nuclear polyhedrosis virus I. Synthesis of intracellular proteins after virus infection

VIROLOGY

99, 386-398

Infection

(197%

of Spodoptera

frugiperda Cells with Autographa Nuclear Polyhedrosis Virus

I. Synthesis

of intracellular

Proteins

Californica

after Virus Infection

ERIC B. CARSTENS, SIAN T. TJIA, AND WALTER DOERFLER’ Institute

of Genetics, University

of Cologne, Cologne, Germany

Accepted July 23, 1979

The replication of Autograph califomica nuclear polyhedrosis virus (AcNPV) in frugiperda cells in culture has been studied with different methods. The first virus-induced polypeptides (with molecular weights of 46K, 30K, 29K) in infected cells appeared at 3 hr postinfection. Viral DNA synthesis started at about 5 hr postinfection. By electron microscopy, intranuclear nucleocapsids were detected at 10 hr postinfection and at about the same time, the titer of intracellular infectious particles began to rise. The pattern of viral protein synthesis was rather complex; within the first 24 hr postinfection, some 30-35 different polypeptides appeared sequentially in infected cells. Some of these polypeptides seemed to be structural proteins of the vii-ion. The 28K polyhedrin polypeptide was synthesized originally as a precursor and was modified posttranscriptionally. Polyhedrin was synthesized until late in infection. Two distinct stages exist in AcNPV replication: (i) the rapid synthesis of AcNPV-specific nucleic acids and proteins and the assembly of nucleocapsids, some of which develop by budding to extracellular virus; (ii) intranuclear membrane synthesis, polyhedra formation, and occlusion of intranuclear, enveloped virions.

Spodoptem

INTRODUCTION

Nuclear polyhedrosis viruses (NPVs), as members of the family Baculoviridae, have been considered as valuable biological insecticides (Falcon, 1976). In the insect host, the virus is synthesized in the nucleus of the infected midgut columnar cells. The ensuing generalized infection involves tracheal cells, hemocytes, and fat body and leads to the formation of polyhedral inclusion bodies which are released from the dead insect. The virions are released from the polyhedra when they are ingested by other insects and reach the larval gut. From there, they initiate an infection in the gut epithelium. Evidence for this pathway has largely come from electron microscope analysis of infected insects (Bergold, 1963; Summers and Arnot, 1969; Harrap, 1972a, b, c; Stoltz et al., 1973). Several continuous insect cell lines are ’ To whom reprint requests should be addressed. 0042~6822/79/160386-13$02.00/O Copyright All tights

Q 19’79 by Aoademic Press, Ine of reproduction in any form reserved.

now available allowing for the analysis of the molecular biology and morphogenesis of these viruses (Hink, 1970; Goodwin et al., 1970; Granados, 1976; Knudson and Harrap, 1976; Knudson and Buckley, 1977). Development of a plaque assay has facilitated the study of the infectious cycle in insect cell lines (Wood, 1977; Brown and Faulkner, 1978). Isolated polyhedra and virus liberated from polyhedra are relatively noninfectious to cells in culture, virus-containing hemolymph and cell-free extracts of infected cells can lead to an infectious cycle in insect cells (Granados, 1976; Knudson and Buckley, 1977; Volkman and Summers, 1977). Although several reports have appeared describing the polypeptide composition of purified baculovirions (Young and Lovell, 1973; Padhi et al., 1974; Payne et al., 1977; Harrap et al., 1977; Summers and Smith, 1978), there are no reports on the synthesis of proteins in infected insect cells. We have

386

PROTEIN

SYNTHESIS

IN AcNPV-INFECTED

chosen to study Autograph californica nuclear polyhedrosis virus (AcNPV) in the cell line derived fromSpodopteraj?-ugiperda. In this paper, we report results obtained from electron microscopic analyses of infected cells. The sequence of appearance of newly synthesized proteins in infected cells is presented. There is evidence that certain proteins are modified by post-translational cleavage. In addition, some correlation is made between proteins present in purified virions and those present in infected cells. Finally, data are given which indicate that three different plaque isolates-shown to exhibit different restriction endonuclease cleavage patterns of their DNAs (Tjiaet al., 1979)-synthesize identical polypeptides in infected cells. MATERIALS

AND

METHODS

Virus and cells. Autographa

califomica

nuclear polyhedrosis virus (AcNPV) was kindly supplied by Dr. Arthur McIntosh, formerly at the Institute of Microbiology, Rutgers University. Spodoptera fmgiperda cells were a gift from Dr. Keith Harrap, Oxford University. The AcNPV was plaque purified three times, then passaged once in cell culture before use. The infectious tissue culture fluid was used directly as inoculum for the reported experiments. The cells were passaged twice weekly using TC100 medium (Gardiner and Stockdale, 1975) in the modification of Langridge (personal communication) supplemented with 0.26% tryptose broth, 10% inactivated fetal calf serum, and 50 pglml of gentamycin. The incubation temperature was 2’7”. Plaque assay. Virus samples and tissue culture media used as inocula were titered by plaque assay on confluent monolayers of S. frugiperda cells. Multidish plates (Falcon No. 3008) were used, and the method of Wood (1977) was followed in the modification described by Tjia et al. (1979). Single plaques were isolated with a Pasteur pipet and resuspended in TC-100 medium containing 10% fetal bovine serum. Purijkation of virus. AcNPV was purified from infected tissue culture medium by the method of Summers and Smith (1978). In brief, S. fmgiperda cells were infected

CELLS

387

with 50 PFU/cell of AcNPV. After a 1.5-hr adsorption period, the virus was removed and replaced with TC-100 medium containing 10% calf serum. At 22 hr postinfection, the medium was removed and centrifuged at 2000 rpm for 15 min to remove cells. The supernatant was carefully layered onto 10 ml of 20% sucrose (w/w) in TE (0.01 M Tris-hydrochloride, pH 7.5, 0.001 IM EDTA) and centrifuged in the SW 27 rotor of a Beckman ultracentrifuge at 26,000 rpm for 2 hr. The pelleted virus was suspended in 0.1 ml of TE, pH 7.5 and carefully layered onto linear gradients of 20 to 52% sucrose. After centrifugation at 39,000 rpm in the SW 41 rotor for 4 hr, the opalescent band of virus particles was collected, diluted 1:5 with TE, and the virions were repelleted by centrifugation for 1 hr at 39,000 rpm in the SW 41 rotor. In some experiments, virions were purified as described by Tjia et al. (1979). Kinetics of replication of extracellular and intracellular viruses. S. frugiperda cells growing in monolayers were inoculated with 10 PFU per cell of AcNPV. Adsorption at 27” proceeded for 1 hr; subsequently, the inoculum was removed, the cells were washed with phosphate buffered saline (PBS) three times, and fresh medium was added. At various times after inoculation, the amount of infectious virions present in the medium was determined by plaque titration after the medium had been freed of cells by low speed centrifugation. The cells from the same plate were washed three times with PBS, the cells were resuspended in 1 ml of TC-100 medium containing 10% fetal bovine serum, and frozen and thawed five to six times. The extracts were clarified by centrifugation in a GLC-2B Sorvall centrifuge at 1000 rpm for 10 min, and infectious virions were titrated by plaque assay. Virus samples were stored at +4” without appreciable loss in infectivity. Electron microscopy. Cells on 60-mm plastic petri dishes were fixed in situ with 3.5% glutaraldehyde in Millonig’s buffer (Millonig, 1962) and prepared for embedding and thin sectioning as previously described (Weber et al., 1977). Purified virions were negatively stained with 2% phosphotungstic acid (PTA), pH 7.2.

CARSTENS,

388

TJIA,

Protein gel electrophoresis. The sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis (SDS-PAGE) system as described previously (Carstens and Weber, 1977) was used. Whole cell extracts were prepared for electrophoresis by removing the cell culture medium from the cell monolayers and adding 0.075 ml of the stock sample buffer (0.05 M Tris-HCl, pH 6.8, 1% SDS, 1% p-mercaptoethanol, 10% glycerol) per 10’ cells. Washing of the monolayers was avoided to minimize the loss of extracellular virus at late times after infection. Electron microscope studies showed that washing did not remove a lot of the extracellular virus. When the intracellular proteins were subsequently used for SDS-PAGE analysis, the serum concentration was reduced to 5% to lower the amount of serum albumin in the samples. Higher levels of serum tended to interfere with the mobility of polypeptides in the range of 68,000 daltons on the slab gels. Whole cell extracts of adenovirus type 2infected Hep-2 cells, labeled with [35S]methionine, were used as molecular weight markers using the molecular weights reported for the adenovirus polypeptides as standards (Weber et al., 1977). S. fmcgiperda cells were seeded into 16mm-diameter wells of a 24-well plate (lo7

AND DOERFLER

cells per well). After an attachment period of 30 min, the medium was replaced with infectious tissue culture fluid (100 PFU/ cell). After a 1-hr adsorption period, the inoculum was replaced with TC-100 medium containing 5% fetal calf serum. At time intervals after infection as indicated, the medium was replaced with fresh TC-100 medium without serum or methionine and 25 @Z/ml of [35S]methionine. After a 1-hr pulse, the radioactive medium was removed, and either the cells were immediately harvested in 0.075 ml of stock sample buffer or were chased for various lengths of time in 0.5 ml of complete TC-100 medium containing 5% fetal calf serum. The solubilized cells were stored at -20” until they were analyzed. RESULTS

Time Course of Infection

S. fmgiperda cells were infected with AcNPV as described. At various time intervals postinfection, the extra- and intracellular virus titers were determined by plaque assay. The data presented in Fig. 1 demonstrate that the titer of extracellular (intracellular) virus started to increase at 7 hr (10 hr) postinfection (p.i.) and reached a maximum between 24 and 36 hr (20 and 22 hr). Similar kinetics of replication have been described for AcNPV in Trichoplusia ni 368 cells (Volkman et al., 1976). It is concluded that the AcNPV exhibits a relatively short cycle of replication in S. fmgiperda cells. It is conceivable that the virus titer continues to increase beyond 24 to 36 hr postinfection, but that this titer rise is masked by the simultaneously occurring occlusion of virions in polyhedra. Electron

IO

20

30

40

Hours

50 after

so

70

so

90

100 110

mfection

FIG. 1. Growth curve of Autographa californica NPV in S. frwgiperda cells. Cells were infected with 10 PFU/cell. Titer of virus was determined by plaque assay at various times after infection. Extracellular virus (0); intracellular virus (0).

of S. fmgiperda

Cells by AcNPV

Microscopy

of PurQied Virions

Samples of sucrose gradient purified virus were stained with phosphotungstic acid and examined in the electron microscope. Most of the particles were intact nucleocapsids with varying amounts of envelope material present around the particles. Some aberrant forms were also seen, such as hairpin nucleocapsids and empty particles.

PROTEIN

SYNTHESIS

IN AcNPV-INFECTED

CELLS

389

extracts were analyzed by SDS-gel electrophoresis. Each track shows the polypeptide Samples of S. frugiperda cells infected pattern obtained when the cells were pulsewith AcNPV were harvested at various labeled at the indicated times following times after infection, and the cells were mock infection with PBS. All time points prepared for thin sectioning and examina- revealed an identical polypeptide pattern. tion in the electron microscope. At 2 hr after The same relative level of radioactivity was infection, a few virus particles were seen in seen between all cell polypeptides and no some of the cells. These particles were additional bands were observed at any of present in both the cytoplasm and the the time points studied. These results nucleus of the infected cells. There was no indicated that protein synthesis was virevidence of release of the viral DNA into tually unchanged for at least 32 hr at 27” the nucleus via nuclear pores. Between and implied that the cells remained in the 2 and 8 hr after infection, no virus particles logarithmic growth phase. Minor changes were observed in the cells, although some in polypeptide patterns may be revealed by ultrastructural changes could be detected more elaborate methods. in the appearance of the cytoplasmic and/or The polypeptide pattern seen with nuclear contents. The first newly assembled AcNPV-infected cells was in marked conparticles were noted at 10 hr postinfection trast to that from mock-infected cells (Fig. 2a). These particles were confined to (Fig. 4). The amount of incorporation of the nucleus and appeared to be non-envel- [35S]methionine into the host cell polyoped nucleocapsids. There was no evidence peptides gradually decreased starting 6of any intranuclear membrane formation or 10 hr postinfection (Fig. 4, tracks 6-10) of polyhedra formation at this time. By 20 hr such that at 32 hr after infection, very few after infection, large numbers of extracel- of the labeled polypeptides comigrated with lular virus particles were evident (Fig. 2b) polypeptides in the mock-infected cells. which were all enveloped single nucleo- This result suggested that host protein capsids. They appeared to gain their enve- synthesis was gradually shut down follope while budding through the cytoplasmic lowing infection with AcNPV. However, it membrane. The intracytoplasmic particles must be noted that not all of the hostwere not enveloped. Large numbers of specific polypeptides were affected to the particles were also apparent in the nucleus same extent or at the same time after inat 20 hr postinfection. These particles were fection. In fact, some bands which coeither nonenveloped nucleocapsids or were migrated with host cell polypeptides were enveloped by membranes which might have stimulated during the course of the inbeen synthesized in the nucleus. Small fection, e.g., polypeptides with molecular polyhedra were observed at this time, con- weights of 25K (Fig. 4, track 6) and 15K taining several enveloped virus particles. (Fig. 4, track 12). While host-specific Many multi-nucleocapsids were seen sur- protein synthesis appeared to gradually rounded by a single membrane. Similar ob- decrease starting at 6- 10 hr after infection, servations were reported by Granados et al. there was a concomitant increase in the syn(1978) for Heliothis xea NPV. By 48 hr thesis of new polypeptides appearing at postinfection, most of the intranuclear various times after infection and continuing virus particles were enveloped and many for various lengths of time. The first virusvirions were occluded into large polyhedra specific polypeptides having apparent mo(Fig. 2~). lecular weights of 46K, 30K, and 29K could be detected in this experiment at 6 hr postTime Course of Polypeptide Synthesis infection (Fig. 4, track 6). The 46K and 29K Plaque-purified virus was used as inoculum polypeptides behaved in a similar fashion with respect to their synthesis pattern. to study the time course of virus-induced Both were detected at 6 hr, reached a maxipolypeptide synthesis in S. frugiperda cells. Figure 3 shows a typical autoradio- mum rate of synthesis between 8 and 14 hr gram obtained when mock-infected cell postinfection and were synthesized in lesser Electron

Microscopy

of Infected Cells

390

CARSTENS, TJIA, AND DOERFLER

FIG. 2. Electron micrographs of thin sections of S. frug@erda cells infected with Autographa cal~~oornicaNPV. (a) Ten hours postinfection; (b) 20 hr postinfection; (c) 48 hr postinfection. Bar equals 0.5 firn.

amounts up to 32 hr postinfection. The 30K polypeptide was synthesized at a constant rate throughout the course of infection. Between 6 and 8 hr postinfection, several more virus-induced polypeptides became visible. These included bands with molecular weights of 67K, 65K, 54K, 39K, 24K,

22-23K, and 22K (Fig. 4, track 3). The 67K polypeptide could be first detected at this time, but the peak of its synthesis occurred at 12 to 16 hr after infection. Very little, if any, of this polypeptide could be detected after 24 hr postinfection. The 65K polypeptide was stimulated in its synthesis

PROTEIN

SYNTHESIS

IN AcNPV-INFECTED

CELLS

391

FIG. 2. -Continued.

at 8 to 12 hr after infection, and from then until 32 hr postinfection, its synthesis remained at a relatively high rate. A very faint band of 54K was observed at 8 hr postinfection. It was very weak at all late times after infection, but nevertheless, it was present in about constant amounts up to 32 hr postinfection. Both the 24K and 22K polypeptides seemed to be synthesized up until 32 hr postinfection, while the 22-23K polypeptide was only apparent between 6 and 22 hr after infection and was not visible on the gels at later times. Between 8 and 10 hr after infection, polypeptides of 125K, 112K, 108K, 98K, 93K, 88K, 52K, 50K, and 28K became visible (Fig. 4, track 10). The 125K, 112K, and 108K bands were very weak and difficult to assess as to the exact time of their appearance and disappearance. The 112K band seemed to be present up until about 22 hr after infection, but it may continue to be synthesized later than that. The 98K polypeptide appeared to comigrate with a polypeptide from mock-infected cells, but it was stimulated in its synthesis between 8 and 10 hr after infection and from then on, was synthesized until at least 32 hr postinfection. The 93K band also corn&rated with a cellular polypeptide but its syn-

thesis was not shut down at the same rate as that of other cellular bands, still being made at 26 hr after infection. The 88K polypeptide was synthesized at the same relative rate at late times after infection as the 93K and 98K polypeptides. The 50K and 52K proteins behaved very similarly, being synthesized at the same general level up to 32 hr postinfection in the case of the 52K polypeptide and 26 hr postinfection in the easeof the 50K polypeptide. The 28K polypeptide became visible at about 10 hr after infection and from that time until at least 65 hr after infection, increased in its incorporation of [3sS]methionine. This polypeptide probably corresponds to the polyhedrin protein reported by others to have a molecular weight of 28K (Summers and Smith, 1978). At 10 hr postinfection, additional virusinduced bands could be observed. These included bands of molecular weights of 36K, 33K, 21K, 16K, 15-13K, and 12K. The 36K and 33K polypeptides seemed to be synthesized at a maximum rate between 12 and 22 hr and could still be detected at 32 hr after infection. The 21K polypeptide migrated with a mobility very similar to that of a cellular polypeptide, but appeared to be stimulated in its synthesis between 10 and 22 hr. A group of polypeptides having a

CARSTENS,

392

TJIA,

AND DOERFLER

the 13K polypeptide continued to incorporate significant amounts of radioactivity. The 12K polypeptide which appeared at this time also seemed to be synthesized mainly between 10 and 22 hr after infection. Two faint bands of 16K and 15.5K could also be detected at 10 hr postinfection (Fig. 4, track 10). The 15.5K band seemed to be synthesized at relatively constant rates up to at least 32 hr postinfection. The 16K polypeptide disappeared after 22 hr postinfection. Other polypeptides which appeared at 12 hr after infection had molecular weights of 20K and 19K (Fig. 4, track 12). These polypeptides continued to be present until 32 hr postinfection. Only three other new polypeptides were FIG. 3. SDS-polyacrylamide slab gel autoradiogram of S. fmgiperda cells pulse-labeled with [WIseen at later times after infection. One was methionine at the indicated times after mock infection a band of 62K appearing at about 16 hr postwith PBS. The numbers across the top of the electroinfection (Fig. 4, track 16). It was present pherogram refer to the hour postinfection when the until 32 hr after infection. The other two cells were pulse-labeled while the numbers along the polypeptides appeared very late after inside indicate the molecular weights of polypeptides fection, that is at about 22-24 hr (Fig. 4, in kilodaltons. track 22). These bands had molecular weights of about 1’7K and 1’7.5K, and apmolecular weight range of 15K to 13K were peared to increase their levels of incorporaalso seen. All of these polypeptides seemed tion of [35S]methionine from 22 to 32 hr to incorporate approximately the same postinfection. The polypeptide pattern amounts of radioactive label up until about seen following the 1-hr pulse at 32 hr post22 hr postinfection. After that time, only infection remained relatively constant in N

Mock Infected frugiperda

SoodoDtera

Cells ’

cv

M Infected Ad/ 212 6 8 10 12 1416181

Infected M /mZ426283036232hd * Lc,-

c-26 p-20

FIG.4. SDS-polyacrylamide slab gel autoradiogram of S. fmgiperda cells pulse-labeled with [YG]methionine for 1 hr at the indicated times after infection with AcNPV. Adenovirus type 2-infected cell extracts (Ad) were included as molecular weight markers. Mock-infected cells pulse-labeled at 2 and 32 hr p.i. were also included (M). The numbers across the top of the electropherogram refer to the hour postinfection when the cells were pulse-labeled while the numbers along the sides of the electropherogram refer to the molecular weights in kilodaltons of the various polypeptides.

PROTEIN SYNTHESIS abcdefghi

j

IN AcNPV-INFECTED

k

replication starts at about 5 hr postinfection (Tjia et al., 1979). This would, therefore, indicate that the three polypeptides 46K, 30K, and 29K were synthesized before the initiation of viral DNA. The scheme in Fig. 6 summarizes our findings on the newly synthesized polypeptides in AcNPV-infected cells up to 32 hr postinfection. Post-Translational

FIG. 5. SDS-polyacrylamide slab gel autoradiogram of S. f?ugiperda cells pulse-labeled for 1 hr at various times after infection with [Wlmethionine. (a) Adenovirus type e-infected Hep-2 cell extract; (b) mock-infected cells pulse-labeled for 1 hr at 1 hr after mock-infection; (c-i) infected cells pulse labeled for 1 hr at 1, 2, 3, 4, 5, 6, and 8 hr postinfection, respectively; (j) infected cells pulse-labeled for 1 hr at 8 hr post-infection and then chased in the absence of [WImethionine for 15 hr; (k) mock-infected cells labeled with [3sS]methionine. The black dots indicate the polypeptides referred to by the molecular weight designations along the right hand side of the electropherogram. The molecular weights of polypeptides are expressed in kilodaltons.

other 1-hr pulses until at least 65 hr postinfection (results not shown). Virus-Induced Polypeptides Early after Infection

393

CELLS

ModQications

To determine whether any of the virus induced polypeptides were post-translationally modified, samples were pulselabeled with [35S]methionine for 1 hr and the labeled methionine was subsequently chased in the absence of radioactive methionine for various lengths of time. One sample was pulse-labeled for 1 hr at 8 hr postinfection (Fig. 7, track b) and chased for the next 15.5 hr (Fig. 5, track j; Fig. 7, track c), a second sample was pulselabeled for 1 hr at 18 hr postinfection (Fig. 7, track f) and chased for the following 22 hr (Fig. 7, track g); and a third sample was continuously labeled from 11 to 33.5 hr postinfection (Fig. 7, track e). At the end of the case period, the intracellular proteins were analyzed. The results are shown in Figs. 5 and 7. Tracks j (Fig. 5)

Synthesized

Since the induction of virus-specific protein synthesis seemed to occur very soon after infection, the infected cells were pulselabeled for 1 hr every hour up to 8 hr after infection. The cells were then harvested into stock sample buffer immediately after the pulse and stored at -20” until being analyzed later by SDS-gel electrophoresis. The cell extracts were subjected to electrophoresis and the resulting electropherograms are shown in Fig. 5. The 46K, 30K, and 29K polypeptides could be detected as early as 3 hr postinfection (Fig. 5, track e). There was an increase in the radioactive labeling of a polypeptide of 25K between 3 and 8 hr after infection, while the 22K polypeptide could be detected at 6 hr postinfection (Fig. 5, track h). Viral DNA

Hours Post Infection

FIG. 6. A schematic drawing of the time of appearance and disappearance of the virus induced intracellular polypeptides shown in Figs. 4 and 5. A schematic drawing of an electropherogram of purified virion polypeptides labeled with [Wlmethionine is also shown.

CARSTENS, TJIA, ANDDOERFLER

394 abcdefghij pirc”-mb-DD

and c (Fig. 7) contain samples from cells in which the label was chased following a -120 pulse at 8 hr after infection. Several new -100 polypeptides could be seen while at least 85 65-1, D two polypeptides disappeared. Specifically, c-62 = -48.5 the 29K and 22K bands were absent in the chased samples when compared with the err -26 pulsed samples (Fig. 5, track i, and Fig. 7, track b). After the chase, a prominent band of 28K comigrating with the tentative polyhedrin protein appeared. Minor species of 22.5K and 18.5K were also observed. Track f representing the pulsed, and FIG. 7. SDS-polyacrylamide slab gel autoradio- track g representing the chased samples gram of S. fmLgiperda cells pulse-labeled for 1 hr with at 18 hr postinfection also provided evi[Wlmethionine. (a and j) Adenovirus type 2-infected dence for the appearance of new polyHep-2 cells; (b) infected S. fmgiperda cells pulse- peptides and for the disappearance of some labeled for 1 hr at 8 hr p.i.; (c) infected S. frugiperda of the pulse-labeled polypeptides (Fig. 7). cells pulse-labeled for 1 hr at 8 hr pi. and then chased The weak 29K and 22K bands present after for 15.5 hr; (d) infected S. frugiperda cells pulsea pulse at 18 hr seemed to be greatly relabeled for 1 hr at 11 hr p.i.; (e) infected S. fmgiperda cells continuously labeled from 11 to 33.5 hr pi.; (f) duced following the chase. Similarly, the infected S. jhqiperdu cells pulse-labeled for 1 hr at 67K, 20K, and 19K bands also seemed to 18 hr p.i.; (g) infected S. frugiperdu cells pulse-labeled disappear, while new bands of 22.5K and for 1 hr at 18 hr p.i. and then chased for 22 h; (h) mock- 18.5K appeared after the chase (Fig. 7, infected S. frugiperdu cells pulse-labeled for 1 hr at track g). There was also a great increase 12 hr p.i.; (i) mock-infected S. frugiperdu cells in the amount of radioactivity in the 28K pulse-labeled for 1 hr at 12 hr pi. and chased for 20 hr. and 15K polypeptides relative to the amount The black dots designate polypeptides referred to by found in the same polypeptides following the molecular weight designations along the lefta l-hr pulse. The post-translational cleavage hand side of the electropherogram. The molecular weights of polypeptides are expressed in kilodaltons. occurring in the infected cells resulted in 17 h Pulse AdtMO

6

D

E SiM

17hChase 0

B

0 E

27h Pulse SiViM

0

S D

E Sl

-125

FIG. 8. SDS-polyacrylamide slab gel autoradiogram of S. fmgiperda cells infected with various plaque-purified stocks of Autograph califomica NPV. The infected cells were pulse-labeled with [%]methionine for 1 hr at 17 hr (17-hr pulse) and 27 hr (27-hr pulse) p.i. A duplicate set of infected cells, also pulse-labeled at 17 hr p.i., was chased in the absence of radioactive precursor for 10 hr (17-hr chase). Ad, adenovirus type 2-infected cell extract; M, mock infected; 0, original virus stock before plaque purification; B, D, E, three different plaque-purified virus stocks (Tjia et al., 1979); S, plaque-purified Autogruphu califomicu NPV obtained from Dr. M. D. Summers, Texas A & M; V, purified extracellular AcNPV labeled with [Y5S]methionine from 2 to 22 hr p.i.

PROTEIN

SYNTHESIS

IN AcNPV-INFECTED

CELLS

395

tracks B, D, E) produced the same polypeptide patterns when compared with the original virus stock (Fig. 8, track 0) at both 17 and 27 hr after infection. No differences could be detected with this method between the samples when they were pulse-labeled at 17 hr and then chased for 10 hr. In addition, all of these stocks of AcNPV synPolypeptides in Puri&ed Virions thesized the same complement of polypepExtracellular virions were purified as tides in infected cells as a stock of AcNPV described under Materials and Methods obtained from Dr. Max D. Summers, Texas from infected cells incubated in the presence A & M University, (Fig. 8, track S). It of 25 pCi/ml of [35S]methionine.These virions would seem that, although some of these were solubilized in SDS stock sample buffer plaque-purified stocks exhibited differences in preparation for SDS-gel electrophoresis. in the base sequence of parts of their DNAs, About 15 bands of radioactivity were seen in these genomes did not code for virusthe autoradiogram, most of which co- induced polypeptides of different sizes in migrated with bands seen in infected cells infected cells. It is conceivable that more (Fig. 8, V). Two major bands of radioactive sensitive methods may reveal minor difmethionine appeared at about 67K and 36K. ferences, if they exist. These bands corresponded to the major Coomassie blue-staining bands. Other bands DISCUSSION could be detected with molecular weights 98K, 93K, 88K, 65K, 52K, 46K, 39K, 30K, This study correlates data obtained from 29.5K, 28.5K, 26K, 23K, 21K, 1’7K, 16K, electron microscopy and SDS-gel electro15-13K, and very low molecular weight phoresis in defining the time course of inmaterial of less than 10K. The major polyfection of S. fmgiperda cells with AcNPV. peptides of 67K and 36K present in the Electron microscope examinations of inparticles appeared only as minor bands in fected cells indicated that penetration and the pulse-labeled infected cell extracts. uncoating of the virus was completed by 4 hr after infection (cf. Knudson and Harrap, Polypeptides Induced by Different Stocks 1976). This result was supported by the apof virus pearance of the first virus-induced polyIt has been reported that plaque purifi- peptides in infected cells at 3 hr p.i. (Fig. 5). cation of virus from a wild-type population It is possible that the 46K, 30K, and 29K polypeptides are synthesized before the can result in the isolation of variants (Miller and Dawes, 1978; Lee and Miller, 1978; initiation of viral DNA replication since Smith and Summers, 1978;Tjia et al., 1979). viral DNA synthesis starts at about 5 These variants contained DNA which, when hr postinfection (Tjia et al., 1979). It is cleaved with various restriction endo- interesting to note that other virus-induced nucleases, gave different DNA fragment polypeptides did not appear until after 6 hr patterns. The possible effect of these of infection. These results indicate that the detectable genotypic variations on virus- three initial proteins may have some funcinduced protein synthesis was examined. tion in priming the infected cells for later Following infection of S. fmgiperda cells stages of virus DNA and protein syntheses. with different plaque isolates, the cells were A great deal of variability was observed pulse-labeled with [35S]methionine for 1 hr in the integrity of negatively stained virions at various times after infection. The cells both in samples purified on metrizamide were either harvested immediately after gradients and sucrose gradients. Quantitathe pulse or chased in absence of label for tion of the actual number of physical par10 hr before being harvested. The samples ticles versus plaque-forming units would reveal whether the nonenveloped, bent, or were analyzed by SDS-gel electrophoresis. All of the plaque purified stocks (Fig. 8, broken particles were as infectious as enthe appearance of at least two polypeptides (22.5K and 18.5K) which were not detected in pulse-labeled cell extracts. No major changes could be detected in mock-infected cells after pulse-labeling and subsequent chase (Fig. ‘7, tracks h, i).

396

CARSTENS,

TJIA,

veloped rod-shaped virions. These experiments have not been done yet. The purification procedures did not seriously affect infectivity. The pattern of protein synthesis in the infected cells is quite complex with the sequential appearance of at least 31 different polypeptides during the first 24 hr after infection (Figs. 4, 6). Most of these polypeptides can be detected by 10 hr after infection (Fig. 4, track lo), i.e., the time of appearance of the first nucleocapsids in the infected cells (Fig. 2a). Many of these polypeptides seem to be structural proteins found in the virus particle. It should be noted, however, that the major polypeptides in the virion (67K and 36K) are relatively minor species in infected cell extracts. In addition, the synthesis of these two proteins appears to be maximal between 8 and 24 hr p.i. Very little of either of these polypeptides is synthesized later after infection. Since the maximum virus yield was obtained at about 24 hr for both extracellular and intracellular viruses, these data would suggest that polypeptides synthesized after this time may be more important in virus occlusion than in the actual synthesis of virus particles. This notion is substantiated by the rather late appearance of the polyhedrin polypeptide (28K) and its increased rate of synthesis until at least 65 hr p.i. This interpretation was supported by electron microscope data which demonstrated that polyhedra formation and occlusion of intranuclear virus particles occurred only after many virus particles had been shed to the outside of the cell. Presumably, some of the other polypeptides appearing later, such as 17K, 17.5K, 62K, as well as 39K and 65K, may be involved in the late process of virus occlusion. The 29K and 22K polypeptides are modified by a post-translational cleavage event (Fig. 7). Some polypeptides in the 20K and 13-14K molecular weight range may also be subject to processing events since all of these proteins become weaker or completely disappear in pulse-chase experiments. The products are polypeptides of 28K, 22.5K, and 18.5K, since these polypeptides increase in radioactivity following

AND DOERFLER

the chase. The observation that the polyhedrin polypeptide was not present in samples which were pulse-labeled for 1 hr at 8 hr p.i., but was present in these samples when they were chased in the absence of radioactive precursor (Fig. 7, track c), suggested that the polyhedrin polypeptide was a cleavage product. There was also a great increase in 28K following the chase of cells pulse-labeled at 18 hr (Fig. 7, track g). Cleavage may require an enzyme which is not present in large enough quantities to cleave all of the precursor molecules. The only major band, which disappears following a chase of samples pulse-labeled at 8 hr postinfection, is the 29K polypeptide. Interestingly, this polypeptide gradually decreased in its radioactivity about 16 hr after infection, while the radioactivity in the polyhedrin polypeptide greatly increased. The presence of a protease in isolated polyhedra has already been demonstrated (Eppstein and Thoma, 1975; Kozlov et al., 1975; Summers and Smith, 1975/76). The fact that identical polypeptide patterns are observed for various plaquepurified stocks (Fig. 8; also Smith and Summers, 1978; Tjia et al., 1979) suggests that the genotypic variations in AcNPV do not noticeably affect the proteins for which these variants code. Detailed physical and genetic maps of the viral DNA are needed before these observations can be explained in molecular terms. The present study provides further evidence that the infectious cycle of AcNPV in S. frugiperda cells mimics the infectious cycle in the insect host (Granados, 1976; Knudson and Harrap, 1976; Knudson and Buckley, 1977). There appear to be two stages involved in the virus replication cycle in both cases. Stage one involves the very rapid synthesis of nucleic acids and proteins, assembly of nucleocapsids, and subsequent budding of particles to the extracellular medium. This is followed by a second slower process of intranuclear membrane synthesis, polyhedra formation, and occlusion of intranuclear enveloped virus particles. This latter stage is characterized by the shut down of host cell protein synthesis.

PROTEIN SYNTHESIS

IN AcNPV-INFECTED

ACKNOWLEDGMENTS We are indebted to Hanna Mansi-Wothke for excellent technical help. This research was supported by a grant from the Federal Ministry of Research and Technology, Bonn, Germany (PTB 8086). REFERENCES BERGOLD, G. H. (1963). The nature of nuclear polyhedrosis viruses. In “Insect Pathology” (E. A. Steinhaus, ed.), Vol. 1, pp. 413-456. Academic Press, New York. BROWN, M., and FAULKNER, P. (1978). Plaque assay of nuclear polyhedrosis viruses in cell culture. Appl. Environ. Microbial. 36, 31-35. CARSTENS, E. B., and WEBER, J. (1977). Genetic analysis of adenovirus type 2. Pleiotropic effects in an assembly mutant. J. Gen. Viral. 37, 453-474. EPPSTEIN, D. A., and THOMA, J. A. (1975). Alkaline protease associated with the matrix protein of a virus infecting the cabbage looper. Biochem. Biophys.

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