Autographa californica nuclear polyhedrosis virus phosphoproteins and synthesis of intracellular proteins after virus infection

VIROLOGY

109, 25-34

(1981)

Autographa californica Nuclear Polyhedrosis Virus Phosphoproteins and Synthesis of Intracellular Proteins after Virus Infection JAMES

E. MARUNIAK*.’

‘-Depurtment of Biology, Unhersity Texas Agm’cultural Experiment

AND

MAX D. SUMMERS?

of Texas, Austin, Texas 78710. and Department Texas A CC M University, College Station, Station, Accepted

August

of Entom,ology.

Texas

778!+3

27. 1980

Polypeptides and phosphoproteins isolated from nuclear and cytoplasmic fractions of TN-368-10 cells infected with Au.to~upha califowica nuclear polyhedrosis virus (Ac.WNPV) were analyzed by polyacrylamide gel electrophoresis. Likewise, AcMNPV extracellular virus and alkali-released virus were compared. AcMNPV extracellular virus possessed at least nine phosphoproteins while the alkali-released virus had about 14 phosphoproteins. The major structural polypeptide of AcMNPV, polyhedrin, was phosphorylatrd. Intracellular proteins of infected TN-368-10 cells were pulse-labeled with [%]methionine and separated into nuclear and cytoplasmic fractions. At least 36 virus-induced polypeptides were detected in infected cells, and several polypeptides were found as early as 5 hr postinfection. The patterns of synthesis and appearance of infected cell polypeptides were complex, but polypeptide differences between nuclear and cytoplasmic fractions were detected. Twenty phosphoproteins labeled with a*P were also detected in the two fractions, and at least six had electrophoretic mobilities similar to virus-associated phosphoproteins. INTRODUCTION

Autographa califomica nuclear polyhedrosis virus (AcMNPV), an insect baculovirus, produces two infectious forms during infection and replication in Lepidopteran cell culture. Investigations with AcMNPV and other baculoviruses show that early in infection, nucleocapsids assemble in the nucleus, and then bud through the plasma membrane (Volkman et al., 19%). This form of the virus is referred to as extracellular virus (EV), and it is distinct from the other infectious form because it is not occluded. Later, nucleocapsids are enveloped either singly or in multiple numbers by viral membranes which are apparently synthesized de novo (Stoltz et crl., 1973; Volkman et al., 1976). In the nucleus, the enveloped nucleocapsids are occluded by protein into paracrystalline structures called polyhedra. The polyhedra ’ Present address: Department and Public Health, Yale Arbovirus Yale University School of Medicine, New Haven, Connecticut 06510.

of Epidemiology Research Unit, P.O. Box 3333,

can be disrupted with alkali to release the virus, and this form of the virus is called alkali-released virus. Although the EV has a low physical to infectious particle ratio of 120:1, it is 2 x lo3 times more infectious than alkali-released virus as assayed in TN368 cells (Volkman et al., 1976). The EV envelope reveals some surface modification or peplomers, whereas envelopes from alkali-released virus do not (Kawamota et al., 1977; Knudson and Harrap, 1976; Summers and Volkman, 1976). Antigenic properties of EV and alkali-released virus are different as determined by crossneutralization tests (Volkman et al., 1976). Qualitative and quantitative differences in structural polypeptide composition of the EV and alkali-released virus have been demonstrated (Smith and Summers, 1978; Summers and Smith, 1978). Since plaquepurified isolates of EV or alkalireleased virus produced progeny typical of a normal infection, these differences were phenotypic. The sequence of the NPV infection process in cell culture has been examined

“6

MARUNIAKANDSUMMERS

by electron microscopy. At l-2 hr pi, the virus enters the cell by viropexis, and some unenveloped particles are present in both the nucleus and cytoplasm. From 4-8 hr pi, virus particles are not observed. Virus morphogenesis begins and by 12-15 hr pi unenveloped virions can be frequently found in the cytoplasm. These nucleocapsids then aquire an envelope by budding through the plasma membrane. Polyhedra formation is observed by 18 hr pi and increases up to 48 hr (Knudson and Harrap, 1976; Carstens et al., 1979). Infectious EV is released from cells as early as 7 to 12 hr pi, and a maximum titer is reached between 24 and 36 hr. Observation by light microscopy shows polyhedra at 14-18 hr pi and increasing numbers to 48 hr. Concomitant with an increase in polyhedra is a decrease in EV (Knudson and Tinsley, 1974; Volkman et al., 1976; Carstens et crl., 1979). The synthesis of AcMNPV and Spodoptera fmgiperda MNPV DNA begins abut 6 hr pi and reaches a maximal rate of replication at 12 to 18 hr pi (Knudson and Tinsley, 1978; Brown et al., 1979; Tjia et al., 1979). AcMNPV antigens can be detected at 6-8 hr pi by the immunoperoxidase technique. Polyhedrin is detected as early as 12 hr pi in some cells (Summers et al., 1978). Carstens et al. (1979), reported on the synthesis of AcMNPV polypeptides in S. fmgiperda cells. There are no reports on the synthesis of baculovirus proteins and phosphoproteins in Trichoplnsia ni cells and on the separation of the protein synthesis products into nuclear and cytoplasmic fractions. In this report, polyacrylamide gel electrophcresis was used to analyze protein and phosphoprotein synthesis and processing in nuclear and cytoplasmic fractions of AcMNPV-infected cells. Virus-induced intracellular polypeptides were detected relative to host background as infection progressed. The nuclear and cytoplasmic fractions of infected cells showed both qualitative and quantitative differences in intracellular proteins and phosphoproteins. The phosphoproteins of AcMNPV extracellular virus and alkalireleased virus were also compared and significant qualitative and quantitative

differences between the two forms of infectious virus were found. MATERIAIS ANI) METHODS Vimses, cell lines, trrld imects. AcMNPV was obtained from Dr. P. V. Vail (USDASEA, Fresno, California) and routinely grown in Trichoplusia ?ri larvae. The AcMNPV was plaque purified, the DNA physically mapped, and the virus designated AcMNPV-E2 (Smith and Summers, 1978; Smith and Summers, 1979). Original and cloned cell lines of T. pi (TN-368 and TN-368-10) were maintained in 2%cm” flasks in TNM-FH medium containing 10% fetal calf serum (FCS) as described previously (Hink, 1970; Volkman and Summers, 1975). Iufectiotl of’ cells. Experimental conditions for infection have been described previously (Volkman and Summers, 1975: Volkman et al., 1976). The virus proteins were radioactively labeled using 2 x 10’ cells. The cells were allowed to attach in serumless medium in 150~cm’ flasks, the medium was removed, and cells \vere infected with EV at a multiplicity of infection (m.0.i.) of ,5. After an adsorption period of 1 hr, the inoculum was removed. The isotope in medium deficient for the specific labeled precursor was added for the appropriate time interval, and complete medium was added. Pwi$catio~w of ertracellular cinls. pelf h,edra, avd rrlkali-rrlcased cirus. EV which was obtained from culture media at 40 hr pi was purified by equilibrium density centrifugation (1.17- 1.18 g/ml) in sucrose gradients as described by Smith and Summers (1978). Polyhedra were purified from infected TN-368 cells 5-7 clays pi. The cells Lvere pelleted by centrifugation at 12,100 g for 10 min at 4” (JA-20 rotor, J21C Beckman), and the cell pellet was disrupted in TE buffer (0.01 M Tris, 0.001 M EDTA, pH 7.8) using a Brinkman polytron (PT 1OST generator) operating at full speed for 2 min. Polyhedra were pelleted from the cell lyzate by centrifugation at 12,100 g for 10 min. The pellet was resuspended in TE buffer and polyhedra were purified by centrifugation in sucrose gradients (Summers and Smith, 1976).

BACLJLOVIRUS-INFECTED

Enveloped nucleocapsids were released from purified polyhedra by the dilute alkaline saline treatment (0.1 M Na,CO,, 0.01 M EDTA, 0.17 M NaCl, pH 10.9) and purified in sucrose gradients (Summers and Smith, 1976). The multiple bands at densities of 1.20-1.25 g/ml were removed, and the alkaline soluble polyhedrin was recovered from the top of the gradients and stored at -70” for further studies. Sodium dodecyl sulfate-polyacrylamide gel electroph,oresis (SDS-PAGE). Vertical

CELL

POLYPEPTIDES

“7

dried with a stream of N,. Samples were analyzed by SDS-PAGE in slab gels and exposed to Kodak RP X-Omat X-ray film for 1 to 2 weeks. Alkali cellular

treatment of 3ZP-labeled virus. The effect of alkali

estra-

on covalently associated RZP was examined by treating :J2P-labeled EV under the same alkaline conditions as alkali-released virus. The EV was incubated at 4” for 1 hr in dilute alkaline saline, and the preparation was extracted with phenol and precipitated with ethanol. The alkaline-treated sample and an untreated EV sample were compared by SDS-PAGE and autoradiography.

slab SDS-PAGE was performed according to Laemmli (1970), as modified for baculovirus structural proteins (Summers and Smith, 1978). Ph.osphorylated amino acids in polyLabeling of virus phosphoproteirLs. TN- hedrin. Purified “‘P-labeled polyhedrin was 368 cells were infected in 150-cm? flasks as extracted with phenol and precipitated with described above. After adsorption, the ethanol. Acid hydrolysis of the preparation inoculum was removed and the attached and high-voltage electrophoresis of the hydrolyzed products were performed cells were rinsed in phosphate-free TNMFH containing 10% dialyzed FCS and 10 according to the procedure of Pal et nl. the phosmu HEPES, pH 6.1. Twenty milliliters of (1975). After electrophoresis, amino acids were detected phosphate-free TNM-FH containing 1 mCi phorylated by autoradiography. of carrier-free [“ZP]monosodium phosphate Labeliwg of intracellular viral proteins (New England Nuclear, 500 mCi/mmol) was added. At 48 hr pi, the cell supernatant with [‘35S]Tnethionirle. Wells of a Falcon (3008) multicluster plate were seeded with was removed and the labeled EV was purified from the supernatant. Phosphate-free 2 x lo” TN-368-10 cells in 0.5 ml TNM-FH TNM-FH plus 10% FCS was then added with 10% FCS. The cells were allowed to to the cells, and the polyhedra were purified attach for 1 hr and infected with Aci%INPVat 5 days pi. Labeled alkali-released virus E2 at an m.o.i. of either 1 or 200 in 0.1 ml and polyhedrin were subsequently purified of TNM-FH with 10% FCS. After rocking as described above. the cells every 15 min for 1 hr, the inoculum Virus and polyhedrin preparations were was removed and complete TNM-FH extracted with phenol and precipitated with medium with FCS was added at time zero. ethanol (Lai, 1976) to remove DNA and At 4, 9, 14, and 23 hr pi, the medium was lipids from the preparations. Sixty microremoved. The cells were rinsed with TNMgrams of unlabeled alkali-released virus FH methionine-deficient medium, and they was added to provide carrier protein for were maintained for 1 hr in 0.5 ml of the labeled EV. The labeled EV, alkali-released methionine-deficient medium (Smith and virus, and polyhedrin were disrupted in Summers, 1978). The deficient medium was 1.0% SDS and 1.0% 2-mercaptoethanol by removed and 50 PCi of [:YS]methionine heating at 100” for 5 min, and they were (Amersham, 561 Ci/mmol) in 0.5 ml of TNMextracted two times with phenol. Phenol FH methionine-deficient medium ~vas soluble proteins and proteins at the phenoladded for a 1 hr pulse. aqueous interface were precipitated with After labeling, the cells were fractionated 5 volumes of 95% ethanol at -20” overnight. into nuclear and cytoplasmic fractions using Labeled polyhedrin was precipitated from a modification of published techniques (Chin the phenol phase by adding 5 volumes of and Maizel, 1976; Penman, 1969; Wechsler acetone at -20”. The samples were cen- and Fields, 1978). The labeled TN-368-10 trifuged at 2000 g for 1 hr at 4”, the cells were suspended with a Pasteur pipet, supernatant decanted, and the precipitates and they were rinsed in hypotonic TMN

MARUNIAKANDSUMMERS

28

buffer (0.01 M Tris, 0.0015 M MgC12, 0.014 M NaCl, pH 7.2). The cells were transferred to tubes and pelleted by centrifugation at 250 g for 10 min (IEC-HNSII Centrifuge with a 958 rotor). The cells were resuspended in ice cold TMN buffer containing 0.5% (v/v) NP-40, 0.3% (v/v) Tween-40, and 0.1% (w/v) sodium deoxycholate and incubated on ice for 30 min with a vortex treatment for 4 see at 15 min incubation. The preparation was centrifuged at 1000 g for 1 min to pellet the nuclei leaving the cytoplasmic fraction in the supernatant. The nuclei were recovered, rinsed once with 1 ml TMN buffer, and suspended in 1 ml TMN buffer. The proteins in the cytoplasmic-rich fraction and in the nuclear-rich fraction were precipitated with 10 volumes of acetone overnight at -20”. The precipitates were recovered by pelleting at 2500 g (Beckman Model TJ-6, TH-4 rotor) for 2 hr at 4”, and the supernatants were decanted. The precipitates were air dried, the entire precipitate of each sample was analyzed by SDS-PAGE, and the dried gel was autoradiographed for 3 days. Labeling

of

intracellular

phosphopro-

Intracellular phosphoproteins were labeled in TN-368-10 cells by infecting with AcMNPV at an m.o.i. of 200, and the cells treated as described above for [““Slmethionine incorporation. Infected cells were washed once with phosphate-free TNM-FH and maintained 1 hr in the latter medium at 9, 14, and 23 hr pi. They were pulsed 1 hr with 100 $.Zi of carrier-free :jZP in 0.5 ml phosphate-free TNM-FH, and the cells were fractionated into cytoplasmic and nuclear fractions which were analyzed by SDS-PAGE. teins.

RESULTS Intracellular

Polypeptides during Infection,

Treatment of infected and uninfected TN368-10 cells with 0.5% NP-40, 0.3% Tween40, and 0.1% deoxycholate in hypotonic saline was effective for preparing nuclear and cytoplasmic cell fractions. The treatment produced about a 95% yield of nuclei from the starting material when observed by phase microscopy. Contamination of the nuclear fraction by the cytoplasmic com-

ponents was checked by a reconstruction experiment in which [3H]leucine-labeled extracts were added to unlabeled nuclei for 30 min at 4”. Isolation of the nuclei showed that only 1% of the [“Hlleucinelabeled cytoplasmic extracts was assorted with nuclei. Effect of m.o.i. on Intracellular ms Polypeptide Synthesis

Baculovi-

A comparison of mock-infected cellular protein synthesis to synthesis when cells were infected with AclMNPV-E2 at an m.o.i. of 1 showed that cellular polypeptides and background were prominent as late as 24 hr pi in the infected cell extracts (Fig. 1). However, virus-induced polypeptides were readily detected in TN-368-10 cells infected with AcMNPV using an m.o.i. of 200. The effect of high m.o.i. was evident because intracellular polypeptides IP32 (Intracellular Protein 32,000 daltons) at 5 hr pi and IP30 at 24 hr pi were detected earlier when compared with the low m.o.i. samples. At 15 hr pi, there was also a significant reduction of background, hostcell protein synthesis in the high m.o.i. Kinetics of Virus-Induced Polypeptide Synthesis at the High m.o.i.

At 5 hr pi, a 1 hr labeling with [““S]methionine showed that an early protein, IP32, was detected in both the nucleus and cytoplasm and it remained a prominent protein at the 10, 15, and 24 hr pi pulse intervals (Fig. 1). Another early virusinduced protein, IP45-46, appeared in higher concentrations relative to mockinfected cells. At 10 hr pi, major differences between infected and uninfected cells were detected. For example, a greater number of newly synthesized virus-induced polypeptides were differentiated from uninfected host cell proteins, and differences in polypeptide composition in the cytoplasmic and nuclear fractions existed (Fig. l).Virus-induced polypeptides corresponding to molecular weights IP125, IP73, IP60.6, IP45-46, IP32, and IP19 were found in both cytoplasmic and nuclear fractions, while IP34 was found only in the cytoplasm. The syn-

BACULOVIRUS-INFECTED 5h IO h u I 200 I 200 -t--ii-?-i-E-t-iiCNCNCNCNCN

I

CELL POLYPEPTIDES

15 h 200

I

24h 200 AMN AR

FV

FIG. 1. Intracellular labeling of cellular and A&fNPV-E2 polypeptides with [“YS]methionine. Cells were uninfected, U, or infected with either an m.o.i. of 1 or 200, labeled with ,50 /.~Lci of [Y?]methionine for 1 hr at the indicated times and fractionated into nuclear, N, and cytoplasmic, C, components, then electrophoresed on 10.8% SDS-polyacrylamide gels and subjected to autoradiography as described in Materials and Methods. Intracellular virus polypeptides detected by autoradiography are indicated with lines and numbered in kilodaltons. The Coomassie blue stain of AcMNPV alkali-released virus co-run with the labeled samples is to the right with a preparation of extracellular virus.

thesis of IP54 and IP51 was greater than in the corresponding polypeptides which comigrated in uninfected cells. Other polypeptides were present in the nucleus and cytoplasm, but they were labeled/ to a lesser extent. Thirty-six polypeptides were detected at 15 hr pi in cells infected at an m.o.i. of 200 (Fig. 1). Few polypeptides had similar electrophoretic mobilities relative to those in mock-infected cells, and differences in virus-induced polypeptides in the cytoplasm as compared to the nucleus were apparent. For example, polypeptides IP64, IP62, IP34, IP25, IP23, and IP18 appeared exclusively in the cytoplasm. Other virus-induced polypeptides, IP71, IP70, IP54, IP40, and IP39, were detected in greater levels in the cytoplasm than in the nucleus. As late as 24 hr pi, cellular polypeptide synthesis was still evident in cells infected with an m.o.i. of 1, and IP32 was still synthesized in large amounts (Fig. 1).

However, the detection of most virusinduced proteins has decreased in cells infected with an m.o.i. of ZOO. In particular, IP32 had decreased in the nucleus relative to the concentration in the cytoplasm. The amount of IP68, IP64, IP62, IP19, and IP18.5 remained about the same as at 15 hr pi, while the concentration of IP30 increased dramatically. IP64, IP62, and IP34 were detected only in the cytoplasm. Intracellular fection

Phosphoproteins during In-

At least 20 phosphorylated proteins were detected in infected cell extracts (Fig. 2). Most of the phosphoproteins labeled in infected cell extracts were also present in uninfected cells. However, virusinduced phosphoproteins IP75, IP64, IP27, IP25, IP23, and IP22 were detected predominantly in the cytoplasm, while IP32 and IP18 were observed predominantly in

MARUNIAK

U C

AND

h

N

SUMMERS

15 h C

24h N

C

N

AMN s.me-

FIG. 2. Intracellular labeling of phosphoproteins of uninfected cells, 11. or infected at 10. 13, and 24 hr pi u-ith AcMNPV-E:! at an m.o.i. of 200. (‘ells \verr fractionated into cytoplasmic. c’, or nuclear, N, components, subjected to SDS-PAGE, and autoradiography as described in Materials and Methods. The phosphoproteins are indicated by the lines with the molecular wright of the polypeptides indicated in kilodaltons. AcJ4YPV (i\MN) co-run and stained with Coomaasie blue is to the right.

the nucleus. Most virus-induced intracellular phosphoproteins were detected at each time interval tested. An exception was IP30 which appeared in the cytoplasm at 24 hr pi. A comparison of intracellular phosphoproteins (Fig. 2) to the [YS]methionine-labeled intracellular polypeptides (Fig. 1) revealed IP64, IP27, and IP22 were phosphoproteins predominantly in the cytoplasm, and they were also significant methionine-labeled polypeptides in the cytoplasm (Fig. 1). The early protein, IP32, was a major [““Slmethionine-labeled polypeptide both in the nucleus and in the cytoplasm of infected cells. However, IP32 was phosphorylated at 24 hr to a much greater extent in the nucleus than in the cytoplasm relative to the amount of ]“%Imethionine-labeled protein present. Virus

Phosphoproteius

Gel electrophoresis of the phosphoproteins of the EV, alkali-released virus, and polyhedrin is shown in Fig. 3. A purified preparation of extracellular virus was com-

pared to a preparation which was phenol extracted and ethanol precipitated to remove DNA and lipids. Purified extracellular virus which was not phenol extracted and precipitated (Fig. 3A) had additional R2P associated with protein bands VP73, VP45, VP18.5, VP18, and VP16 with the latter two being more heavily labeled than the phenol extracted preparations (Figs. 3B and C). The EV was treated with the dilute alkaline saline which was used routinely for purification of alkali-released virus, to determine phosphoprotein sensitivity to alkali. The phosphoproteins of the EV treated with alkali and an untreated sample were phenol extracted, precipitated, and analyzed by SDS-PAGE. No differences were detected in the phosphoproteins of the alkali-treated EV (Fig. 3B) and the untreated EV (Fig. 3C). The phosphoproteins included VP75. VP64. VP54. VP37. VP32. VP23, VP18, and VPIG. ’ ’ ’ :‘2P-labeled alkali-released virus was purified from a wild virus isolate, serially passaged virus (passage 16), and a plaque

BACULOVIRUS-INFECTED

COMPOSITE

CELL

TABLE 1 OF VIRUS PROTEINS” Estracellular virus VP + + + 4 + + + +

Alkalireleased virus PP

-

VP + + I + + + + +

PP

P

P P

31

POLYPEPTIDES

analyzed by SDS-PAGE. The phosphoproteins of the alkali-released viruses derived from each source were identical to those shown (Figs. 3D and E). Although there were fourteen phosphoproteins, VP32 was the most prominent (Table 1). The autoradiograph of :‘“P-labeled polyhedrin (Fig. 3G) revealed the presence of :jZP which migrated at a position that corresponded to the molecular weight of polyhedrin. The results of the high-voltage electrophoresis of acid hydrolyzates of :j2Plabeled polyhedrin demonstrated that phosphoserine was the major phosphoamino acid, while phosphothreonine was a minor one (data not shown).

+ + +

+ +

+ +

+ +

P P

Conzparison of AcMNPV-Infected Cell Polypeptides, Structural Polypeptides, and Phosphoproteim

+ + + + +

P

Table 1 summarizes the YS-labeled polypeptides and phosphoproteins in infected

+

+ +

+

+

+

+

+

+ + + + + + +

+ + + +

73

P

P P P P P P P

” Composite of structural proteins (VP) and phosphoproteins (pp), of extracellular virus and alkalireleased virus are indicated (+). The infected cell proteins (ICP), labeled with [““Slmethionine (see Fig. 1) are numbered 1 to 36. MW is the molecular weight in kilodaltons.

purified isolate, AcMNPV-E2. Each of the :j’P-labeled alkali-released virus preparations was extracted with phenol and

virus

released virus

FIG. 3. Autoradiogram of ‘r”P-labeled extracellular virus, alkali-released virus, and polyhedrin electrophoresed through a 3% stacking gel into 10.8% SDSpolyacrylamide gels. Slot A is purified extracellular virus. Slot B is extracellular virus incubated at 4” for 1 hr in dilute alkaline saline, pH 10.9, then phenol extracted and ethanol precipitated. Slot C is phenol extracted and ethanol precipitated extracellular virus. Slot D is phenol extracted and ethanol precipitated alkali-released virus. Slot E is purified but unextracted alkali-released virus. Slot F is unextracted polyhedrin. Slot G is phenol extracted and acetone precipitated polyhedrin.

32

MARUNIAKANDSUMMERS

cells and in viral structural polypeptides of EV and alkali-released virus. Intracellular polypeptides induced by the viral infection and not detected in virions or uninfected cells were IP71, IP62, IP60.6, IP39, IP38, IP37.5, IP34, IP29, IP27, IP26, and IP25. These were apparently nonstructural polypeptides since we could not detect them in either EV or alkali-released virus (Table 1, Figs. 1 and 3). The alkali-released virus had structural proteins VP46, VP35, VP22, and VP19 which were not detected in the EV at equivalent concentration of protein loaded on the gels. The EV had one protein, VP70, not detected in the alkali-released virus. These qualitative differences do not, however, reflect the quantitative differences in polypeptides. For example, VP64 was a major component of EV whereas it was minor in the alkali-released form (Summers and Smith, 1978; Smith and Summers, 1978). DISCUSSION

The use of a high m.o.i. was an effective means of reducing background host cell protein synthesis so that AcMNPV-induced protein synthesis in TN-368-10 cells was measured by [3%]methionine incorporation. As early as 5 hr postinfection, the AcMNPV-induced synthesis of IP32 was observed. This protein was detected in both the nucleus and cytoplasm and was also highly phosphorylated. This protein, detected at 5 hr, is present before DNA synthesis of AtiNPV in S. fmgiperda cells (Tjia et al., 1979) and also with an NPV of S fmgiperda in S. fmgiperda cells (Knudson and Tinsley, 1978). The 46K and 29K virus-induced polypeptides were better resolved from S. fmgiperda cellular polypeptides (Carstens et al., 1979) since we did not resolve these proteins in T. ni cells at 5 hr pi. The patterns of AcMNPV intracellular polypeptide synthesis in S. fmgiperda cells (Carstens et al., 1979) at 10 hr pi and later time intervals are similar to those we obtained in T. ni cells. Most AciUNPVinduced ““S-labeled proteins were apparent at 10 hr pi except IP19 and IP18.5 which

were not detected until 15-24 hr pi. IP60.6 was detected at 10 hr pi but not at 15 and 24 hr pi. By 15 hr pi when host cell background was minimal and most of the virusinduced polypeptides were predominant, differences between nuclear and cytoplasmic polypeptides were more pronounced. IP64 and IP62, unike most other virusinduced intracellular proteins, were detected only in the cytoplasm. A late protein at 24 hr pi, IP30 which corresponded in molecular weight to polyhedrin (Summers and Smith, 1978), was synthesized in large amounts and was present in both the nucleus and cytoplasm. The synthesis of most other virus-induced proteins, with the exception of IP19 and IP18.5, had diminished significantly by 24 hr pi. The pattern of infected cell polypeptide synthesis was consistent with the virus infection cycle. The titer of extracellular virus begins to increase by 10 hr pi when most infected cell proteins are synthesized. At 24 hr pi the formation of polyhedra is observed, and the titer of extracellular virus decreases (Volkman et al., 1976). The 30,000-dalton structural protein of polyhedra was detected in large amounts at this time. IP64 with a similar electrophoretic mobility to a major protein, VP64, of the EV, was found to occur predominantly in the cytoplasm of AcMNPV-infected TN-368-10 cells when labeled with [“Slmethionine. The alkali-released virus and EV both contain VP64, but the EV contained a much higher concentration of this protein (Smith and Summers, 1978). The major capsid protein, VP37, of AcMNPV (Summers and Smith, 1978), was phosphorylated in the EV but apparently not in the alkali-released virus. No intracellular phosphoprotein corresponding to the molecular weight of the capsid protein, VP37, was evident in infected cells. Phosphorylation may occur during the budding process of the EV. However, in infected cells labeled with [3”S]methionine, IP37, which was apparently the capsid protein, VP37, was detected in the cytoplasm. At 15 and 24 hr pi, IP37 was detected predominantly in the nucleus. One virus-induced %-labeled protein,

BACULOVIRUS-INFECTED

IP32, was an early protein synthesized continuously up to 24 hr. It was detected predominantly in the nucleus when infected cells were labeled with 32P. The major phosphoprotein of the alkali-released virus, VP32, had a molecular weight which corresponded to IP32. However, VP32 was a minor phosphoprotein in EV. Qualitative and quantitive differences in structural phosphoproteins were readily apparent when comparing EV and alkalireleased forms of AcMNPV. Alkalireleased virus had VP115, VP85, VP22, VP19, and VP17 phosphoproteins not observed in EV preparations. VP37 was phosphorylated in the EV and apparently not in the alkali-released virus. VP32 was highly phosphorylated in the alkalireleased virus and only slightly labeled in the EV. In conclusion, many infected cell polypeptides had similar mobilities to virus structural proteins and were apparently virus specified. Differences were shown in structural polypeptides and phosphoproteins between AcMNPV extracellular virus and alkali-released virus. These differences are being investigated relative to the infectivity of these two forms of the virus and regulation of virus budding and occlusion in infected cells. These data are necessary for studying the genetics of baculoviruses and the role of gene products in the specificity and regulation of the infection process. ACKNOWLEDGMENTS We thank Gale Smith for help with the illustrations. This work was supported in part by Environmental Protection Agency Grant R80.523201D and Public Health Service Grant AI14755 from the National Institutes of Health. Sole otlded i?l proof. Since the acceptance of this manuscript, there have been two reports on the synthesis of AcMNPV polypeptides in infected cells. H. A. Wood, Virology 102, 21-27 (1980), reported four phases of virus infectivity, replication, and polypeptide synthesis. He detected 18 virus-induced proteins labeled with [““lmethionine in TN-368 cells. P. Dobos and M A. Cochran, Virology 103, 446-464 (1980), detected 22 virus-induced polypeptides in IPLB-SF21 cells. These were compared to virion phosphoproteins and glycoproteins.

CELL

POLYPEPTIDES

33

REFERENCES

BROWN, M., CRAWFORD, A. M., and FAULKNER, P. (1979). Genetic analysis of a baculovirus, Autographa cal~fbrrrica nuclear polyhedrosis virus. 1. Isolation of temperature-sensitive mutants and assortment into complementation groups. b. Vi~ol. 31, 190- 198. CARSTENS, E. B., TJIA, S. T., and DOERFLER, W. (1979). Infection of Spodopfern fkgiperdn cells with Autographa cnl~forrrica nuclear polyhedrosis virus. I. Synthesis of intracellular proteins after virus infection. I’/iro/ogy 99, 386-398. CHIN, W. W., and MAIZEL, J. V., JR. (1976). The polypeptides of Adenovirus. Virology 71, 518-530. HINK, W. F. (1970). Established insect cell line from the cabbage looper, TCchoplztsin ni. Nature (London)

226, 466-467.

KAU’AMOTO, F.. SUTO, C., KUMADA, N.. and KOBAYASHI, M. (1977). Cytoplasmic budding of a nuclear polyhedrosis virus and comparative ultrastructural studies of envelopes. Microbial. Immrtnol. 21, 255-265. KNUDSON, D. L., and HARRAP, K. A. (1976). Replication of a nuclear polyhedrosis virus in a continuous cell culture of Spodoptera ,frugiperdn: Microscopy study of the sequence of events of the virus infection. J. Vim/. 17, 254-268. KNUDSON, D. L., and TINSLEY, T. W. (1978). Replication of a nuclear polyhedrosis virus in a continuous cell line of Spodopfem jkgiprrda: Partial characterization of the viral DNA, comparative DNADNA hybridization, and patterns of DNA synthesis. Virology

87. 42-57.

LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nntltre (London) 227, 680-685. LAI, M. M. c’. (1976). Phosphoproteins of Rous sarcoma viruses. L’irology 74, 287-301. PAL, B. K., MCALIZTER, R. M., GAR~XR, M. B.. BURMAN. ROY, P. (1975). Comparative studies on the structural phosphoproteins of mammalian type ( viruses. d. Viral. 16, 123-131. PENMAN, S. (1969). Preparation of purified nuclei and nucleoli from mammalian cells. Ire “Fundamental Techniques in Virology” (K. Habel and N. P. Salzman, eds.), pp. 35-48. Academic Press, Ne\v York. SMITH, G. E., and SUMMERS, M. D. (1978). Analysis of baculovirus genomes with restriction endonucleases. Virology 89, 517-527. SMITH, G. E.. and SUMMERS, M. D. (1979). Restriction maps of five autogaphu califorrricn MNPV variants, Trichoplusia ni MNPV, and Galleriu mellonrlla MNPV DNAs with endonucleases Sma I, Kpn I. Barn HI, Sac I, Xho I, and Eco RI. J. Viral. 30, 82X-838. STOLTZ, D. B., PAVAN, C., and DACUNHA, A. B. (1973). Nuclear polyhedrosis virus: a possible ex-

34

MARUNIAK

AND SUMMERS

ample of de l~ouo intranuclear membrane morphogenesis. J. Gen. Viral. 19, 145-150. SUMMERS, M. D., and SMITH, G. E. (1976). Comparative studies of baculovirus granulins and polyhedrins. Intervirology 6, 16% 180. SUMMERS, M. D., and SMITH, G. E. (1978). Baculovirus structural polypeptides. Virology 84,390-402. SUMMERS, M. D., and VOLKMAN, L. E. (1976). Comparison of biophysical and morphological properties of occluded and extracellular nonoccluded baculovirus from in viva and i~1 ~ifro host systems. d. Viral. 17, 962-972. SUMMERS, M. D., VOLKMAN, L. E., and HSIEH, C.-H. (19’78). Immunoperoxidase detection of baculovirus antigens in insect cells. J. Gen. Viral. 40, 545-557. TJIA, S. T., CARSTENS, E. G., and DOERFLER, W.

(1979). Infection ofspodoptera frugiperda cells with califomica nuclear polyhedrosis virus. II. The viral DNA and the kinetics of its replication. Virology 99, 399-409. VOLKMAN, L. E., and SUMMERS, M. D. (1975). Nuclear polyhedrosis virus detection: Relative capabilities of clones developed from Z’richoplusia ni ovarian cell line TN-368 to serve as indicator cells in a plaque assay. J. Viral. 16, 1630-1637. VOLKMAN, L. E., SUMMERS, M. D., and HSIEH, C. H. (1976). Occluded and nonoccluded nuclear polyhedrosis virus grown in Trichoplusia ni: Comparative neutralization, comparative infectivity, and i71 vitro growth studies. J. Vim/. 19, 820-832. WECHSLER, S. L., and FIELDS, B. N. (1978). Intracellular synthesis of measles virus-specified polypeptides. J. Viral. 25, 285-297. Autographa