Characterization of two abundant mRNAs of Autographa californica nuclear polyhedrosis virus present late in infection

VIROLOGY

124,3X-365

(1983)

Characterization of Two Abundant mRNAs of Autographa californica Nuclear Polyhedrosis Virus Present Late in Infection DENNIS Department

Z. ROHEL, of Microbiology Received

MARK

A. COCHRAN,

and Immunology, July

C&x-n’s

8, 1982; accepted

AND University,

Septembe-r

PETER Kingston,

FAULKNER’ K7L

SN6 Canada

29, 1982

Cytoplasmic poly(A)+ RNA isolated from Spodoptera frugipedu cells late after infection with Autograph culzfornicu nuclear polyhedrosis virus (30-40 hr pi) was fractionated according to size on denaturing methyl mercury gels. Two major RNA species (1.4 kb and 0.75 kb) and several minor RNA species were detected by ethidium bromide staining. The predominant RNA species of about 1.4 kb was considered to be polyhedrin mRNA because (1) in vitro translation of the RNA, which was eluted from methyl mercury gels, yielded a polypeptide of MW 33K, which comigrated with polyhedrin. (2) When poly(A)+ RNA was fractionated on a sucrose column and then translated in vitro, the distribution and abundance profiles of a 33K polypeptide product and of 1.4-kb RNA were similar. (3) The 33K polypeptide made in vitro and purified polyhedrin gave rise to similar patterns of peptides when digested with S. aureus V8 protease. The polyhedrin mRNA (1.4 kb) hybridized to BornHI-F and HiudIII-V AcNPV DNA fragments and hybridization selection with BarnHI-F AcNPV DNA yielded a 33K polypeptide, which comigrated with polyhedrin. The second RNA species (0.75 kb in size) hybridized to overlapping EcoRI-P and HindIII-Q regions of the AcNPV genome and translated into a methionine deficient polypeptide of MW = 8K. It was synthesized in large quantities late in the infection and appeared to be coordinately expressed with polyhedrin in infected cells. The 8K polypeptide was detected as early as 15 hr pi and was still synthesized at 60 hr pi. INTRODUCTION

al., 1953). A second form of the virus, consisting of a single enveloped nucleocapsid (nonoccluded virus, NOV), is responsible for systemic spread of infection within the insect and for cell to cell spread in tissue culture (Faulkner, 1981). AcNPV possesses a double-stranded closed circular DNA genome of approximately 80 X lo6 daltons (128 kb) (Miller and Dawes, 1978; Smith and Summers, 1978; Vlak and Odink, 1979). Recently, physical maps of several AcNPV variants have been constructed (Miller and Dawes, 1979; Smith and Summers, 1979,198O; Vlak, 1980; Lubbert et al., 1981; Cochran et al., 1982) and a consensus map for the orientation of the genome has been published (Vlak and Smith, 1982). The genome of AcNPV could encode as many as 50-75 proteins (Summers et al., 1980) and up to 30 polypeptides have been detected in purified nonoccluded viruses (NOV) using SDS-polyacrylamide gel electrophoresis (PAGE) (Summers and

Autographa cal$brnica (alfalfa looper) nuclear polyhedrosis virus (AcNPV) is a baculovirus (Matthews, 1979) and, in the past few years, has served as a model for biochemical and molecular genetic studies of insect baculoviruses. A major characteristic of nuclear polyhedrosis viruses is that crystalline occlusion bodies form in nuclei of infected cells. These contain occluded virus particles (OV) embedded in the proteinaceous matrix of the occlusion bodies. The major nonvirion protein of occlusion bodies, polyhedrin (Summers and Smith, 1975), is considered to be an octomer of a polypeptide of molecular weight about 33,000 (Rohrmann, 19’77) and accounts for approximately 95% of the protein mass of occlusion bodies (Bergold et ‘To whom dressed.

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be ad-

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0042-6822/83/020357-09$03.00/O Copyright All rights

0 1993 by Academic Press, Inc. of reproduction in any form reserved

ROHEL,

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COCHRAN,

Smith, 1978; Smith and Summers, 1978; Vlak, 1979). By analysis of genetic recombinants between Rachiplusia ou and AcNPV it was established that the structural gene for polyhedrin lay within the EcoRI-I fragment of AcNPV (Smith and Summers, 1980). This was directly confirmed by hybridization selection with 7.3kb EcoRI-I fragment (Vlak et al., 1981). In this paper we describe the isolation and characterization of the two major poly(A)+ RNAs in AcNPV-infected S. frugiperda cells late in infection. The results show that (1) a 1.4-kb mRNA codes for polyhedrin and hybridizes to the BamHIF and HindIII-V regions of the AcNPV DNA genome and (2) a 0.75-kb mRNA codes for a methionine-deficient 8K polypeptide similar to the 7.5K polypeptide described by Vlak et al. (1981) and hybridizes to EcoRI-P and HindIII-Q regions of viral DNA. MATERIALS

AND

METHODS

Cells and virus. Spodoptera

fmgiperda

(SF) cells (Vaughn et al., 1977) were grown at 28” in BML-TC/lO insect tissue culture medium (Gardiner and Stockdale, 1975) supplemented with 10% heat-inactivated fetal bovine serum (FCS) and 50 pg/ml of gentamicin. The virus was the HR-3 strain of AcNPV (Brown et al., 1979). Cells were infected at a multiplicity of 5-10 PFU/cell in complete medium. Protein labeling. Intracellular proteins were labeled by incubating infected cells for varying periods of time with 20 &i/ ml of either [?S]methionine (1193 Ci/ mmol) or [4,5-3H]leucine (59.8 Ci/mmol) in either L-methionine or L-leucine-free medium, respectively. Following a 1,6, or 12hr pulse, the cells were washed with icecold TClOO medium, scraped from the surface, and pelleted at 700 g for 5 min. They were resuspended in electrophoresis sample buffer (ESB) (Brown et al., 1980) and heated in a boiling water bath for 5 min. To prepare [35S]methionine-labeled polyhedrin, AcNPV-infected SF cells were labeled from 24 to 40 hr postinfection (pi) with 20 &i/ml of [?S]methionine and the polyhedrin polypeptide was isolated and

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FAULKNER

purified by preparative gel electrophoresis as described by Brown et al. (1980). RNA isolation. AcNPV-infected SF cells were scraped from the flasks, pelleted at 700 g for 5 min at O”, and resuspended in 30 mM Tris-HCl (pH 8.3), 50 mM NaCl, 5 mM MgClz, 250 mM sucrose, and 200 pg/ ml heparin. Nonidet P-40 (NP-40) was added to 0.5% and the cells were incubated for 5 min at 0’ with occasional vortexing. Nuclear material was removed by sedimentation first at 1500g for 5 min, and then at 2500 g for 10 min. The supernatant was then extracted with Tris-saturated phenol - chloroform - isoamyl alcohol (25:24:1). Poly(A)+ RNA was selected from cytoplasmic RNA by the method of Aviv and Leder (1972). RNA fractionation. (a) Sucrose gradients: Cytoplasmic poly(A)+ RNA was heat denatured at 65” for 5 min, layered onto a linear sucrose gradient (15-30% w/v in 50 mMTris-HCl, pH 7.4,0.1 MNaCl, and 0.5% SDS), and centrifuged at 25,000 rpm in a Beckman SW 41 rotor for 17 hr at 20”. (b) Methyl mercury gels: 1% agarose gels containing 5 mMmethy1 mercury hydroxide (Alfa Ventron Div. of Thiokol Corp., Danvers, Mass.) were prepared according to Bailey and Davidson (1976) with minor modifications and run in a submarine gel apparatus. RNA samples were denatured with 12 mM CH3HgOH for 20 min and glycerol (final concentration 10%) and bromophenol blue were added prior to electrophoresis. The agarose gels were run at 100 V for 5-7 hr. After electrophoresis, the gel was detoxified by washing for 40 min in either 0.5 M ammonium acetate or 50 mM 2-ME and then stained with ethidium bromide (2 pg/ml). DNA preparation. The construction, isolation, purification, and characterization of pBR 322 plasmids containing BamHI and EcoRI fragments of AcNPV DNA have been described (Cochran et al., 1982). HindIII-V AcNPV DNA fragment was subcloned from Hind111 endonuclease digest of pAcEcoIl DNA into Hind111 site of plasmid vector pUC 8 (Messing strain). Blot hybridization. Poly(A)+ RNA was fractionated on methyl mercury gels and transferred to diazobenzyloxymethyl

CHARACTERIZATION

(DBM) paper (Alwine et al., 1977). Plasmid DNA was nick translated (Rigby et al., 197’7) and used as a probe. In the case of BumHI-F, EcoRI-D, and HindHI-V probes, the complete pBR 322 plasmids pAcBamF2, pAcEcoRID19 and pAcHindIIIV (Cochran et al., 1982) were nick translated, since they contained a single vDNA fragment, but in order to obtain the EcoRI-P probe, it was necessary to excise the fragment from plasmid pAcEcoHP, which contained both EcoRI-H and EcoRI-P AcNPV DNA fragments. Hybridization was for 16 hr in 20-30 ml of hybridization buffer containing 10% dextran sulfate (Wahl et al., 1979). DBM paper was washed for 5 hr with continuous rocking at 42’ as described by Alwine et al. (1977). The filters were then blotted dry, covered in Saran wrap, and exposed to Kodak XAR-5 X-ray film at -70” using DuPont Cronex Lightning Plus intensifying screens. Hybridization-selection. (a) DBM-cellulose: The BumHI-F fragment of AcNPV DNA genome was covalently linked to DBM-cellulose as described by Noyes and Stark (1975). The cytoplasmic poly(A)+ RNA (40 hr pi) was hybridized to and eluted from BumHI-F DNA-cellulose following the procedure of Noyes and Stark (1975). (b) Nitrocellulose filters: Cytoplasmic RNA (40 hr pi) was hybridized to BumHI-F DNA fragment immobilized on nitrocellulose filters following the procedure of Belle Isle et al. (1981). Cell-free translation of proteins. RNA was translated in vitro using the wheat germ cell-free protein synthesizing system (Roberts and Paterson, 1973), obtained from Bethesda Research Laboratories, Inc. (BRL), Bethesda, Md. Each 30 ~1 translation assay contained 20-30 &i of [35S]methionine (1193 Ci/mmol) and the incubation was at 25” for 75 min. The micrococcal nuclease-treated rabbit reticulocyte lysate system (Pelham and Jackson, 1976) was obtained from NEN. Each 25 ~1 translation assay contained 8.5 &i of L[3,4,5-3H]leucine (110 Ci/mmol) (NEN) and the incubation was at 37” for 60 min. The products of in vitro translation assays were incubated for 15 min at 30” with 100 pg/ ml ribonuclease A and 10 mM EDTA. Prior

OF

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to electrophoresis, samples were mixed with an equal volume of 2 X ESB and heated at 100” for 5 min. Polyacrglamide gel analysis. Discontinuous sodium dodecyl-sulfate-polyacrylamide slab gel electrophoresis (SDSPAGE) (Laemmli, 1970) was used for the analysis of the translation products and the intracellular proteins from infected cells. Radioactive proteins were detected by fluorography at -70” using Kodak XAR5 X-ray film. Approximate molecular weights were determined from the migration of proteins relative to ‘*C-labeled molecular weight markers as indicated in the legends. In vitro synthesized 33K protein was compared with [?S]methionine-labeled polyhedrin polypeptide (33K) by limited proteolysis (Cleveland et al., 1977) with S. uureus V8 protease. RESULTS

Poly(A)+ RNA in AcNPV-Infected giperdu Cells

S. fru-

Cytoplasmic, poly(A)+ RNA was prepared from AcNPV-infected S, frugiperdu cells late in infection (40 hr pi) and analyzed on denaturing methyl mercury gels (Fig. 1). Two major RNA species, estimated to be about 1.4 and 0.75 kb in size, and several minor species, the most prominent being 2.8, 3.2, 0.9, and 1.15 kb, were detected by ethidium bromide staining. Many other poly(A)+ RNA species were observed at earlier times after infection (18 and 24 hr pi). Among those detected by ethidium bromide staining at 24 hr pi were RNAs estimated to be 5.6,4.8,3.8,3.2, 2.8, 2.4, 1.9, 1.4, 1.15, 1.05, 0.9, 0.75, and 0.6 kb (data not shown). The size estimates were made using E. coli ribosomal RNA (23 S and 16 S) and globin mRNA (10 S) as markers. However, when the AcNPV RNAs were separated on glyoxal denaturing gels (McMaster and Carmichael, 1977), the sizes for the two major poly(A)+ RNA species were determined to be approximately 1.15 and 0.64 kb, using the same markers (data not shown). A final decision on the size of the two late AcNPV mRNA species probably awaits nucleotide sequence analysis. The two bands comigrat-

360

ROHEL,

COCHRAN,

kb

3.2 2.8:

(28s

(18s 1.4, 1150.90.75,

FIG. 1. Analysis of cytoplasmic poly(A)+ RNAs from infected cells on 1% agarose gels in the presence of 5 mM methyl mercury hydroxide. IC, poly(A)+ RNA from AcNPV-infected SF cells; CC, poly(A)+ RNA from uninfected cells; MK, markers (ribosomal RNA from mouse L cells). Estimated sizes in kilobases of poly(A)+ RNAs from virus-infected cells are indicated on the left.

AND

FAULKNER

by fluorography of methyl mercury gel, translated into a 33K polypeptide, which comigrated with the polyhedrin. Furthermore, the distribution and abundance profile of a 33K translation product followed a sucrose gradient profile of 1.4-kb RNA (data not shown). A partial peptide digest pattern of the translated 33K peptide was similar to that of polyhedrin (Fig. 2). Further information that 1.4-kb poly (A)+ RNA was polyhedrin mRNA was obtained by translation of RNA cut out from a denaturing methyl mercury gel. The band containing 1.4-kb RNA was cut out, crushed into small pieces, and mixed with oligo(dT)-cellulose suspended in the binding buffer. The cellulose was washed several times with binding buffer and the RNA was eluted with the low molarity buffer. When the RNA was translated in the wheat germ cell-free translation system using [35S]methionine as the tracer, a polypeptide of MW 33K (p33), which comigrated

B

A 0

0.1

0.5

2.5

G

0.1

0.5

2.5

ing with 18 S ribosomal RNA from L cells are the ribosomal RNAs of S. frmg-iperdu cells. No 26 S ribosomal RNA was detected on denaturing methyl mercury gels. S.fmgiperda cells thus resemble other insect cells in that following denaturation, the 26 S ribosomal RNA splits into two pieces similar in molecular weight to 18 S ribosomal RNA (Stollar et al., 1971; Dalgarno et al., 1972; Jordan, 1975). Translation Product of l.g-kb RNA Cytoplasmic poly(A)+ RNA isolated at 36 hr pi was centrifuged in sucrose gradients, following which, half of each fraction was run on a denaturing methyl mercury gel and the other half was translated in a wheat germ cell-free translation system. Gradient fraction which contained only the 1.4-kb RNA species, as detected

FIG. 2. SDS-PAGE analysis of the products of partial proteolytic digestion of p33 made in vitro and polyhedrin protein purified from infected cells. [56S]Methionine-labeled polypeptides were excised individually from 12.5% polyacrylamide gels, partially digested with 0.1,0.5, and 2.5 fig V8 protease, and the resulting fragments were resolved by electrophoresis on a 15% polyacrylamide gel. (A) Proteolytic digest of polyhedrin from infected cells (3 day exposure). (B) Proteolytic digest of p33 made in vitro (8 week exposure).

CHARACTERIZATION

OF

mRNAs

with the monomer of polyhedrin protein, was detected (Fig. 3, lane D). Data in Fig. 3 also show the translation product of cytoplasmic poly(A)+ RNA, which had been further selected by hybridization to BarnHI-F fragment of AcNPV DNA bound to DBM-activated cellulose. A faint band in a 33K region comigrating with AcNPV polyhedrin was detected (lane B). Using

A

BCD

OF

AcNPV

361

ABC

D 1)

ah

443

EF

-184 (13.3

FIG. 3. SDS-PAGE analysis of the [%]methioninelabeled polypeptides synthesized in the wheat germ cell-free translation system. Cytoplasmic poly(A)+ RNA from infected cells (40 hr pi) was either directly translated (C), or further separated by methylmercury gel electrophoresis, following which the 1.4-kb RNA was eluted from the gel as described in the text and translated (D). Slot A shows the translation product of cytoplasmic RNA (40 hr pi) which had been selected by hybridization to the BarnHI-F AcNPV DNA fragment immobilized on nitrocellulose. Slot B shows the translation product of a cytoplasmic poly(A)+ RNA (40 hr pi), which had been further selected by hybridization to the BumHI-F fragment of AcNPV DNA bound to DBM-cellulose. Slot E shows a translation product of globin mRNA, while slot F shows separation of ‘%-labeled marker polypeptides obtained from NEN. MW (X10e3) are indicated on the right.

FIG. 4. SDS-PAGE analysis of the [‘Hlleucineand [%]methionine-labeled products of in vitro translation of 0.75kb cytoplasmic poly(A)+ RNA from infected cells. The 0.‘75-kb cytoplasmic poly(A)* RNA from infected cells was isolated on a sucrose gradient and translated in nuclease-treated reticulocyte lysate cell-free systems using either [sH]leucine (lane B) or [%S]methionine (lane C) as the tracer. Lanes A and D were [%]methionine and [3H]1eucine-1abe1ed intracellular polypeptides, respectively, from infected cells (45 hr pi). The polypeptide migrating slightly slower than ovalbumin (43K) in lane C is an endogenous polypeptide in reticulocyte lysate which heavily incorporates [%]methionine. MW (X10m3) of ‘“C-labeled protein markers (BRL) are indicated on the right.

BarnHI-F DNA bound to nitrocellulose filters (Belle Isle et al., 1981), a much more pronounced 33K polypeptide was observed following the translation of selected RNA in the [3H]leucine reticulocyte lysate system (Fig. 3, lane A). Translation

Product

of 0.75kb RNA

Cytoplasmic poly(A)+ RNA from fected cells (36 hr pi) was fractionated

inac-

ROHEL,

362

0 9

12

15 18 24364660

COCHRAN,

MU

-43

- 25.7

-184

-13.3

p8-

'

-6.2

AND

FAULKNER

peptide (Fig. 4, lane B) was detected which comigrated with an intracellular polypeptide present in large quantities late in infection (lane D). This polypeptide could not be labeled with [?S]methionine (Fig. 4, lane C). The small-molecular-weight translation product comigrated with bovine trypsin inhibitor (6.2K) close to the front on Laemmli SDS gels (12.5%). However its molecular weight, as estimated on 6 M urea-15% acrylamide gels to resolve small polypeptides, was 8K (~8) (data not shown). Polypeptide p8 was first observed at about 15 hr pi and continued to be synthesized until late in infection (up to 60 hr pi) (Fig. 5). At 36-48 hr pi, p8 was produced in large amounts, similar to those of polyhedrin, as indicated by Coomassie blue staining of Laemmli SDS gel (data not shown). The pattern of synthesis of p8 and polyhedrin (~33) appeared to follow similar time scale (Fig. 5).

Mapping RNA Transcripts Infected Cells

from AcNPV-

-3 FIG. 5. Intracellular proteins labeled with [3H]leutine at different times postinfection. S. frug&?rdo cells (7.5 X lO’/well of 4.5 cm’) in 12-well plates were infected with AcNPV and at times postinfection the cells were labeled for 1 hr with 20 &i/ml of [3HJleucine (59.8 Ci/mmol) in 1 ml of leucine-free BML-TC/lO medium. The cells were harvested, heated in a boiling water bath for 5 min in ESB, and stored at -20°C. The proteins were analyzed by electrophoresis on 14% polyacrylamide gel (28 X 14.5 X 0.3 cm). The times postinfection are indicated on top of the figure and the molecular weights (X10m3) of “C-labeled protein markers (MK) (BRL) are on the right hand side.

cording to size on a 1530% (w/v) sucrose gradient and one-third of each fraction was analyzed by methyl mercury gel electrophoresis. Two of the gradient fractions contained 0.75-kb RNA as the only species detectable by ethidium bromide staining. The remaining portions of these fractions were translated in nuclease-treated reticulocyte lysate cell-free systems using either [3H]leucine or [35S]methionine as the label. When the 0.75kb RNA was translated in a reticulocyte lysate cell-free system, containing [3H]leucine, a single poly-

Poly(A)+ RNA from cells infected with AcNPV (30 hr pi) was first heat denatured and then separated on a nondenaturing SDS-sucrose gradient (15-30%), following which the RNA in the individual fractions was further separated by methyl mercury gel electrophoresis. The RNA was then transferred by blotting to diazotized paper and hybridized with individual 32P-labeled probes. The BamHI-F, HindIII-V, EcoRIP, and EcoRI-D AcNPV DNA fragments were used as probes on the basis of RNA:DNA “criss-cross” hybridization according to the procedure of Bachenheimer (1980) (manuscript in preparation). The 1.4-kb RNA species hybridized to the BarnHI-F and HindIII-V DNA (Fig. 6) and the 0.75-kb species hybridized to the EcoRIP (Fig. 6) and HindIII-Q (data not shown) region of the genome. There was also a smaller (0.6-kb) RNA species at the top of the gradient which hybridized to the EcoRI-D region (Fig. 6). DISCUSSION

Poly(A)+ RNA, extracted late in the infectious cycle (36 hr or more postinfec-

CHARACTERIZATION

FIG. 6. Hybridization of RNA on DBM-paper poly(A)+ RNA from infected cells was separated peak-containing fractions were further analyzed transferred to a DBM filter by blotting and then F, and (4) HindIII-V AcNPV DNA fragments. the figure and the positions and sizes (in kb) of

tion), was found in two predominating species. Their sizes based on migration in denaturing methyl mercury gels were 1.4 and 0.75 kb if E. coli rRNAs were used as markers (Fig. l), but smaller sizes (1.15 and 0.64 kb, respectively) could be computed based on migration in glyoxal gels. Several lines of evidence indicate that the 1.4-kb RNA contained polyhedrin mRNA as the principle component. (1) When translated in the wheat germ cellfree system, a single 33K polypeptide which comigrated with polyhedrin was detected. (2) When cytoplasmic poly(A)+ RNA from AcNPV-infected cells was separated on nondenaturing sucrose gradients, and the fractions were translated, the distribution of 33K protein in the translation product followed the amounts of 1.4-kb RNA observed in gels. (3) Limited proteolysis with S. aureus V8 protease of the 33K translation product revealed a pattern similar to that of purified polyhedrin (Fig. 2). Blot hybridization analysis and hybridization selection demonstrated that polyhedrin mRNA annealed to BarnHI-F (1.9 kb) AcNPV DNA, which lies within the EcoRI-I fragment (Figs. 3 and 6). HindIIIV (0.93 kb) which lies within the BamHIF fragment also annealed to 1.4-kb RNA (Fig. 6). Taking into account that the max-

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to vDNA probes. [3HjIJridine-labeled, cytoplasmic on linear sucrose gradients (10-309~ w/v), and on methylmercury agarose gels. The RNA was probed with (1) EcoRI-P, (2) EcoRI-D, (3) BarnHIThe sucrose fractions are indicated at the top of viral mRNAs are indicated on the left.

imum size of polyhedrin mRNA is 1.4 kb, the present studies indicate that the gene locus lies within units 1.9-5.4 of the map of Cochran et al. (1982). The second most abundant poly(A)+ RNA, characterized from cell extracts, was a 0.75-kb RNA which is transcribed from the EcoRI-P locus of AcNPV DNA (Fig. 6). The binding data with EcoRI-P and HindIII-Q AcNPV DNA probes locate the structural gene between 87.35 and 89.55 map units. The 0.75-kb RNA translated into a small methionine-deficient polypeptide which comigrated with a polypeptide (MW 8K) synthesized in large amounts late in infection. This polypeptide (designated p8) was detected as early as 15 hr pi and was still synthesized at 60 hr pi. p8 polypeptide labeled readily with [3H]leucine, but we were not able to demonstrate incorporation of [?S]methionine. A similar polypeptide has been found by Vlak et al. (1981) who described an abundant 7.5K polypeptide in AcNPV-infected cells which comigrated with a minor virion polypeptide. However, comigration does not indicate identity and further evidence is required to show whether p8 is a minor NOV protein. p8 is synthesized in large quantities late in the infection at times similar to those observed with polyhedrin. Maxi-

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mum synthesis was observed at 24-60 hr pi, whereas the maximum NOV production occurs earlier (15-24 hr pi). The function of p8 in the replication cycle is as yet undetermined. It does not appear to be a major structural component of the virion but it may be necessary for morphogenesis of occlusion bodies since pulse labeling with amino acids showed coordinate synthesis of polyhedrin and p8. Thus far, p8 has not been identified as a component of occlusion bodies. However, their morphogenesis is complex and ultrastructural studies have demonstrated that major structural modifications occur in nuclei of infected cells, for example de novo synthesis of membranes (Stoltz et al., 1973; Chung et al., 1980). These events probably require interaction of virus nonstructural proteins yet to be identified which may be present in large amounts within ceils. ACKNOWLEDGMENTS We thank D. Agnew for technical assistance. This study was supported by a grant from the Medical Research Council of Canada. REFERENCES ALWINE, J. C., KEMP, D. J., and STARK, G. R. (1977). Method for detection of specific RNAs in agarose gels analyzed by diazobenzyloxymethyl-paper and hybridization with DNA probes. Proc. Nat. Acad. Sci USA 74,5350-5354. AVIV, H., and LEDER, P. (1972). Purification of biologically active globin mRNA by chromatography on oligothymidylic acid-cellulose. Proc Nat. Ad Sci. USA 69,1408-1412. BACHENHEIMER, S. L. (1980). Physical mapping of complex mRNA populations by “criss-cross” DNARNA hybridization. And. B&hem, 106,486-491. BAILEY, J. M., and DAVIDSON, N. (1976). Methylmercury as a reversible denaturing agent for agarose gel electrophoresis. Anal Biochmz. 70, 75-85. BELLE ISLE, H., VENKATESAN, S., and Moss, B. (1981). Cell-free translation of early and late mRNAs selected by hybridization to cloned DNA fragments derived from the left 14 million to 72 million daltons of the vaccinia virus genome. Virology 112, 306-317. BERGOLD, G. H. (1953). Insect viruses. Ao!v. Vims Res. 1.91-139. BROWN, M., CRAWFORD, A. M., and FAULKNER, P. (1979). Genetic analysis of a baculovirus, Autographa califbrnica nuclear polyhedrosis virus. I. Isolation of temperature sensitive mutants and as-

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sortment into complementation groups. J. ViroZ. 31, 190-198. BROWN, M., FAULKNER, P., COCHRAN, M. A., and CHUNG, K. L. (1980). Characterization of two morphology mutants of AutoQrapha cal&rnica nuclear polyhedrosis virus with large cuboidal inclusion bodies. J. Gen Viral. 50, 309-316. CHUNG, K. L., BROWN, M., and FAULKNER, P. (1980). Studies on the morphogenesis of polyhedral inclusion bodies of a baculovirus Autographa califmica NPV. J. Gen. Viral. 46, 335-347. CLEVELAND, D. W., FISCHER, S. G., KIRSCHNER, M. W.,’ and LAEMMLI, U. K. (1977). Peptide mapping by limited proteolysis in SDS and analysis by gel electrophoresis. J. B&hem. 252, 1102-1106. COCHRAN, M. A., CARSTENS, E. B., EATON, B. T., and FAULKNER, P. (1982). Molecular cloning and physical mapping of restriction endonuclease fragments of Autographa californica nuclear polyhedrosis virus DNA. J. Viral 41, 940-946. DALGARNO, L., HOSKING, D. M., and SHEN, C. H. (1972). Steps in the biosynthesis of ribosomal RNA in cultured Aeaks aegypti cells. Eur. J. B&hem 24,498506. FAULKNER, P. (1981). Baculoviruses. In “Pathogenesis of Invertebrate Microbial Diseases” (E. A. Davidson, ed.), pp. 3-37. Allenheld, Osmun, Montclair, New Jersey. GARDINER, G. R., and STOCKDALE, H. (1975). Two tissue culture media for production of lepidopteran cells and nuclear polyhedrosis viruses. J. Inwdebr. Pathol. 25,363-370. JORDAN, B. R. (1975). Demonstration of intact 26s ribosomal RNA molecules in Drosophila cells. J. Mel Bid 98, 277-280. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lo&m) 227, 680-685. LUBBERT, H., KRUCZEK, I., TJIA, S., and DOERFLER, W. (1981). The cloned Eco RI fragments of Autograph califiica nuclear polyhedrosis virus DNA. Gene 16, 343-345. MATTHEWS, R. E. F. (1979). Classification and nomenclature of viruses. Intervirology 12,150-296. MCMASTER, G. K., and CARMICHAEL, G. C. (1977). Analysis of single- and double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange. Proc. Nat. Acad. Sci. USA 74, 4835-4838. MILLER, L. K., and DAWES, K. P. (1978). Restriction endonuclease analysis to distinguish two closely related nuclear polyhedrosis viruses: Autographa californica MNPV and Trichoplusia ni MNPV. App. Env. Micro. 35,1206-1210. MILLER, L. K., and DAWES, K. P. (1979). Physical map of the DNA genome of Autographa c&fm-nica nuclear polyhedrosis virus. J. Viral 29. 1044-1055. NOYES, B. E., and STARK, G. R. (1975). Nucleic acid

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hybridization using DNA covalently coupled to celSUMMERS, M. D., and SMITH, G. E. (1975). T&hoplusia ni granulosis virus granulin: A phenol-soluble, lulose. Cell 5, 301-310. phosphorylated protein. J. Viral 16, 1108-1116. PELHAM, R. B., and JACKSON,R. J. (1976). An efficient mRNA-dependent translation system from reticSUMMERS,M. D., and SMITH, G. E. (1978). Baculovirus ulocytes lysates. Eur. .I Biochem 67,247-256. structural polypeptides. J. Vi& 84, 390-402. RIGBY,P. W. J., DIECKMANN, M., RHODES,C., and BERG, SUMMERS,M. D., SMITH, G. E., KNELL, J. D., and BURP. (1977). Labelling deoxyribonucleic acid to high AND, J. P. (1980). Physical maps of Autographu cdspecific activity in vitro by nick translation with ifwnica and Rachiplusia ou nuclear polyhedrosis DNA polymerase I. J. Mol. Biol. 113, 237-252. virus recombinants. J. Viral 34, 693-703. ROBERTS,B. E., and PATERSON, B. M. (1973). Efficient VAUGHN, J. L., GOODWIN,R. H., TOMPKINS, G. L., and MCCAWLEY, P. (1977). The establishment of two translation of tobacco mosaic virus RNA and rabbit globin 9s RNA in a cell-free system from cominsect cell lines from the insect Spodoptera frugiper& (Lepidoptera:Noctuidae). In vitro 13,213-217. mercial wheat germ. Proc. Nat. Acad Sci. USA 70, 2330-2334. VLAK, J. M. (1979). The proteins of nonoccluded AuROHRMANN, G. F. (1977). Characterization of N-polytographa ca&fornicu nuclear polyhedrosis virus hedrin of two Baculovirus strains pathogenic for produced in an established cell line of Spodsptwa Orgyia pseudotsugata Biochemistry 16,1631-1634. f+-ugipedx J. Invert&r. Path& 34,110-118. SMITH, G. E., and SUMMERS, M. D. (1978). Analysis VLAK, J. M., and ODINK, K. G. (1979). Characterizaof baculovirus genomes with restriction endonution of Autographa califbrnica nuclear polyhedrocleases. Virology 89, 517-527. sis virus deoxyribonucleic acid. J. Gen Viral 44, SMITH, G. E., and SUMMERS, M. D. (1979). Restriction 333-347. maps of five Autographu califbrnica MNPV variVLAK, J. M. (1980). Mapping of Barn HI and Sma I ants, Trichoplusia ni MNPV and GoUeria mellonella DNA restriction sites on the genome of the nuclear MNPV DNAs with endonucleases SmaI, KpnI, polyhedrosis virus of the Alfalfa Looper, AutograBamHI, SacI, Xhol, and EcoRI. J. ViroL 30, 82% pha californica, J. Invert%-. Pathd 36,409-414. 838. VLAK, J. M., SMITH, G. E., and SUMMERS, M. D. (1981). SMITH, G. E., and SUMMERS, M. D. (1986). Restriction Hybridization selection and in vitro translation of map of Rachiplusia ou and Rachiplusia ou-AutoAutographa cal&rnica nuclear polyhedrosis virus. grapha californica baculovirus recombinants. J. ViJ. ViroL 40, 762-771. rol. 33,311-319. VLAK, J. M., and SMITH, G. E. (1982). Orientation of STOLLAR, V., STEVENS, T. M., and SHENK, T. (1971). the genome of Autographa californicu nuclear polyRNA of uninfected and Sindbis virus-infected Aedes hedrosis virus: A proposal. J. Viral. 41, 1118-1121. albopictus cells. Current Twpks Microbial. Immunol. 55,164-169. WAHL, G. M., STERN, M., and STARK, G. R. (1979). STOLTZ, D. B., PAVAN, C., and DA CUNHA, A. B. (1973). Efficient transfer of large DNA fragments from Nuclear polyhedrosis virus: A possible example of agarose to diazobenzoxymethyl-paper and rapid de nor0 intranuclear membrane morphogenesis. J. hybridization with dextran sulfate. Proc. Nat Ad Sci USA 76, 3683-3687. Gen. Viral 19,145-150.