VIROLOGY 233, 292–301 (1997) ARTICLE NO. VY978612

A Role for Baculovirus in Budded Production

Julie Olszewski* and Lois K. Miller*,†,1

*Department of Genetics and †Department of Entomology, University of Georgia, Athens, Georgia 30602 Received February 12, 1997; returned to author for revision March 25, 1997; accepted April 22, 1997

Insect cells infected with tsB1074, a temperature-sensitive mutant of Autographa californica nuclear polyhedrosis virus, exhibit a ‘‘single-cell-infection’’ phenotype whereby the infection progresses through the very late phase culminating in occlusion body formation, but neighboring cells do not become infected. Marker rescue mapping and DNA sequencing correlated a single nucleotide substitution within the baculovirus gp41 gene with the temperature-sensitive phenotype of tsB1074. The product of the gp41 gene, GP41, is an O-glycosylated found in occluded but not budded virions [M. Whitford and P. Faulkner (1992) J. Virol. 66, 3324–3329]. However, budded virus was not produced in tsB1074-infected cells at the nonpermissive temperature of 33Њ, indicating an additional role for GP41 in budded virus formation. Electron microscopy revealed that nucleocapsids were produced but retained in the nucleus of tsB1074-infected cells at 33Њ. Thus, GP41 was required for the egress of nucleocapsids from the nucleus in the pathway of budded virus synthesis. ᭧ 1997 Academic Press

INTRODUCTION cleus (Braunagel and Summers, 1994; Harrap, 1972; Stoltz et al., 1973). The nuclear enveloped virions are belonging to the family are then embedded within a paracrystalline matrix, com- large, enveloped, double-stranded DNA viruses that pri- posed primarily of the protein polyhedrin, to form OV. OV marily infect insects. Autographa californica nuclear poly- are released from infected cell nuclei when the nuclear hedrosis virus (AcMNPV), the most extensively character- and plasma membranes disintegrate beginning around ized baculovirus to date, has a genome of 134 kb (Ayres 72 hr p.i. et al., 1994) with the potential to encode at least 150 BV and OV, although genetically identical, have enve- genes. The virus life cycle leads to the production of two lopes of differing lipid and protein composition (Brauna- virion forms which have distinct roles in viral pathogene- gel and Summers, 1994; Rohrmann, 1992), which are sis. Occluded virus (OV) initiates infection within the in- important for their respective tissue tropisms (Granados sect midgut. From this primary infection, budded virus and Lawler, 1981; Monsma et al., 1996; Volkman and (BV) is produced which disseminates the infection Summers, 1977). Nucleocapsids appear to be similar in throughout the insect host (for reviews see Blissard and structure in both budded and occlusion-derived virions Rohrmann, 1990; Miller, 1996). (ODV). unique for each of the virion forms are The synthesis of the two virion forms is temporally localized primarily to their envelopes (Braunagel et al., regulated during infection. BV is produced during the late 1996a, 1996b; Hong et al., 1994; Rohrmann, 1992; Theil- phase, following viral DNA replication. Newly replicated mann et al., 1996; Volkman et al., 1984). One notable viral DNA is condensed as a nucleoprotein complex, exception to this is the baculovirus , GP41. which is packaged into structures to form nucleo- GP41, expressed during the late and very late phases of . During the late phase, these nucleocapsids infection, contains single N-acetylglucosamine (GlcNAc) leave the nucleus, travel through the cytoplasm, and bud residues O-glycosidically linked to the polypeptide chain through a modified plasma membrane to acquire an en- (Whitford and Faulkner, 1992b). A monoclonal antibody velope (Adams et al., 1977; Fraser, 1986). BV production begins around 12 hr postinfection (hr p.i.) and increases to GP41 detected the protein in ODV but not BV (Whitford logarithmically until about 20 hr p.i. (Knudson and Harrap, and Faulkner, 1992b). Fractionation of ODV indicated that 1976; Lee and Miller, 1979) when there is a transition to GP41 was neither capsid-associated nor envelope-asso- the very late phase of infection. During the very late ciated. Instead, GP41 was proposed to reside within the phase, nucleocapsids remain in the nucleus and acquire tegument (Whitford and Faulkner, 1992b), defined as the an envelope from membranes elaborated within the nu- region between the nucleocapsids and the envelope. We report here the mapping and characterization of a temperature-sensitive (ts) mutant of AcMNPV, tsB1074, 1 To whom correspondence and reprint requests should be ad- dressed at the Department of Entomology, 413 Biological Sciences which has a mutation in the gp41 gene. TsB1074 was Bldg., The University of Georgia, Athens, GA 30602. Fax: (706) 542- originally isolated by mutagenesis of wild-type (wt) 2279. E-mail: [email protected]. AcMNPV with 5-bromodeoxyuridine (BrdU) followed by a

0042-6822/97 $25.00 292 Copyright ᭧ 1997 by Academic Press All rights of reproduction in any form reserved.

AID VY 8612 / 6a39$$$101 06-04-97 14:08:20 vira AP: Virology BACULOVIRUS GP41 IN BUDDED VIRUS PRODUCTION 293 screen for ts phenotypes (Lee and Miller, 1979). At the merase (GIBCO/BRL), and then cut with MscI. This plas- nonpermissive temperature of 33Њ, tsB1074 infection of mid was religated to give pXMX (49.4 to 49.8 m.u.). insect cells exhibits a ‘‘single-cell-infection’’ phenotype, TsB1074 viral DNA was digested with SstII and shot- whereby the infection progresses in individual cells gun cloned into Bluescript II KS/ digested with SstII. through the very late phase of infection, as evidenced by The plasmid pts74 SstIIM, which contains the SstII-M the presence of occlusion bodies in the nuclei of these fragment (49.0 to 50.0 m.u.), was identified by colony infected cells (Lee and Miller, 1979). However, neigh- hybridization using a radioactively labeled probe from boring cells do not become infected and plaques are not pXSS 1.1. Pts74 SstIIM, which had been cut with EcoRV formed. (in the Bluescript MCS) and MscI and religated, was Western analysis of tsB1074-infected cell medium indi- additionally digested with HindIII (Bluescript MCS) and cated that BV was not produced at the nonpermissive SmaI to isolate the 625-bp fragment (49.4 to 49.8 m.u.), temperature. Electron microscopic analysis of wt- and which was inserted into Bluescript II KS/, similarly di- tsB1074-infected cells at 33Њ revealed that nucleocapsids gested, to give pts74 MSma. This clone was constructed remained within the nuclei of tsB1074-infected cells but to be the ts equivalent of the wt clone, pXMX. To create were commonly observed in the cytoplasm and associ- the ts equivalent of the wt pXSIIM, pts74 SstIIM was cut ated with the plasma membranes of wt-infected cells. with SstI (Bluescript MCS), blunted with T4 DNA polymer- Therefore, although GP41 was found to be a component ase, cut with MscI, and religated. This plasmid, pts74 of ODV and not BV with both monoclonal (Whitford and SMsc, contains AcMNPV DNA from 49.0 to 49.4 m.u. Faulkner, 1992b) and polyclonal antisera (this study), mu- To construct a TrpE/GP41 construct, the tation of gp41 affects BV production. This is the first plasmid pXSS 1.1 was digested with SstII, the ends were report of a baculovirus protein that is required for the made blunt by treatment with T4 DNA polymerase, and egress of nucleocapsids from the nucleus. then digested again with SalI (in the Bluescript MCS). A 1.1-kb fragment was gel-isolated and ligated to a pATH- 3 vector (Koerner et al., 1991), which had been digested MATERIALS AND METHODS with HindIII, the ends blunted with T4 DNA polymerase, Cells and virus and then cut again with SalI. The pATH-3 vector contains the N-terminal coding sequence of the Escherichia coli The L1 (wt) strain of AcMNPV (Lee and Miller, 1978) (E. coli) trpE gene, which was fused in frame with gp41 was propagated in the IPLB-SF-21 (Sf-21) insect cell line coding sequence beginning at the SmaI site at 49.8 m.u. (Vaughn et al., 1977) grown at 27Њ in TC-100 medium and ending with the SstII site at 49.0 m.u. (GIBCO/BRL, Gaithersburg, MD) supplemented with 10% fetal bovine serum and 0.26% tryptose broth (O’Reilly et Marker rescue experiments al., 1992). TsB1074 was propagated in Sf-21 cells The tsB1074 mutation was first mapped using a library adapted for growth at 23Њ (O’Reilly et al., 1992), the per- of overlapping AcMNPV genomic clones (Passarelli and missive temperature for tsB1074. Some experiments Miller, 1993) and then mapped further with the plasmid were performed in Sf-21 cells adapted for growth at 33Њ, subclones described above (Fig. 1). Marker rescue ex- which is nonpermissive for tsB1074. periments were performed in Sf-21 cells adapted for growth at 33Њ, which were plated at a density of 2 1 106 Plasmid constructs cells per 60-mm-diameter tissue culture dish. Cotrans- fection of wt genomic clones (5.0 mg/plate) and tsB1074 Construction of the AcMNPV genomic library members viral DNA (0.5 mg/plate) was performed utilizing the lipo- PstHI, PstH5, HC9, and HC10 is described elsewhere fectin method according to the manufacturer’s protocol (Passarelli and Miller, 1993). The plasmid pXma17 was (GIBCO/BRL). Cells were incubated with the mixture of created by cloning the AcMNPV SmaI-D fragment (39.5 DNA, lipofectin, and TC-100 medium without serum at to 49.8 m.u.) into pACYC177 and restriction fragments of 33Њ for 4 hr and then washed with TC-100 medium with- this AcMNPV sequence were subcloned into Bluescript out serum. Cells were then overlayed with agarose as II KS/ (Stratagene, La Jolla, CA) to create pSDPS5, pXA7, previously described (O’Reilly et al., 1992) and main- pXABB 4.5, pXBS 2.1, pXBS 1.0, and pXSS 1.1 as pre- tained at 33Њ. Plates were scored 4 to 5 days later for viously described (McLachlin and Miller, 1994). The plas- the presence of plaques following a subsequent agarose mid pXSS 1.1 (49.0 to 49.8 m.u.) was digested with SstII overlay containing neutral red. and MscI and a 447-bp fragment was isolated and cloned into Bluescript II KS , which had been cut with EcoRV / DNA sequencing and SstII to create pXSIIM (49.0 to 49.4 m.u.). The plasmid pXBS 2.1 (48.2 to 49.8 m.u.) was digested with BamHI, To facilitate DNA sequencing of the smallest plasmid which cuts within the Bluescript multiple cloning site DNA clone which tested positive for marker rescue, pXSS (MCS), the ends blunted by treatment with T4 DNA poly- 1.1 (49.0 to 49.8 m.u.), we subcloned this sequence into

AID VY 8612 / 6a39$$$102 06-04-97 14:08:20 vira AP: Virology 294 OLSZEWSKI AND MILLER two plasmids, pXSIIM (49.0 to 49.4 m.u.) and pXMX (49.4 ary antibody at a 1:25,000 dilution. MultiMark colored to 49.8 m.u.). One strand of each plasmid was sequenced protein standards (Novex Exp. Tech., San Diego, CA) by the dideoxynucleotide chain termination method were used to approximate protein sizes. (Sanger et al., 1977) and the complementing strands BV, analyzed by Western analysis, was harvested from were sequenced using fluorescent DNA sequencing by the medium of wt-infected cells at 27Њ (Fig. 2B) or from the Molecular Genetics Instrumentation Facility (Univer- mock-, wt-, or tsB1074-infected cells at 33Њ (Fig. 3), first sity of Georgia, Athens, GA). The ts equivalents, pts74 by pelleting through a 25% sucrose cushion (w/w in 5 SMsc (49.0 to 49.4 m.u.) and pts74 Msma (49.4 to 49.8 mM NaCl, 10 mM EDTA), and then purified further by m.u.) were sequenced in the same manner. isopycnic centrifugation on a 25–60% sucrose step gradi- ent as previously described (O’Reilly et al., 1992). ODV Production of polyclonal serum were purified from wt-infected Sf-21 larvae by isopycnic centrifugation on a 40–65% sucrose step gradient as The TrpE/GP41 fusion protein plasmid construct was described (O’Reilly et al., 1992). BV and ODV were pel- grown in XL-1 Blue cells and induced to produce the leted and resuspended in 21 SDS buffer and analyzed fusion protein as previously described (Koerner et al., as described above. 1991). The fusion protein, in the insoluble fraction of cell lysates, was visualized by light staining with Coomassie Electron microscopy blue 250 (Sigma Chem. Co., St. Louis, MO) in water (Har- low and Lane, 1988) following separation by SDS–PAGE Sf-21 cells (5 1 106 cells per 100-mm-diameter tissue (Laemmli, 1970). The portion of the polyacrylamide gel culture dish) were mock-, wt-, or tsB1074-infected at a containing the fusion protein was excised and ground m.o.i. of 10 and propagated at 33Њ. At 16 hr, cells were using a dounce homogenizer. The homogenate was in- collected, washed with TC-100 medium lacking serum, jected into rabbits by the Animal Resources Facility (Uni- and pelleted at 1000 g for 5 min. Cell pellets were resus- versity of Georgia, Athens, GA) to produce polyclonal pended and fixed in a solution of 5% glutaraldehyde with antiserum to GP41. 0.1 M tetrabutylammonium fluoride in 0.025 M HEPES buffer, pH 7.2. Following a 1-hr incubation at room tem- Western analysis perature, cells were washed twice with HEPES buffer 6 and pelleted in a microcentrifuge for 30 sec. Pellets were Sf-21 cells, at a density of 2 1 10 cells per 60-mm- diameter dish, were mock-infected or infected with wt or resuspended in a solution of 1% osmium tetroxide in tsB1074 virus at a multiplicity of infection (m.o.i.) of 10 0.025 M phosphate buffer, pH 7.4, with 0.1 M potassium plaque forming units (PFU) per cell as previously de- fluoride (KF). Cells were incubated for 1 hr at 4Њ, pelleted, scribed (O’Reilly et al., 1992). At each time point, the cells and rinsed twice in phosphate buffer. Cells were resus- were washed off the dish with TC-100 medium without pended for 10 min in each of the following in a dehydra- serum, pelleted at 1000 g for 5 min, washed once with tion series: 50% acetone (with 0.1 M KF), 70% acetone (with 0.1 M KF), 80% acetone (with 0.05 M KF), 95% ace- 1 ml insect PBS, pH 6.2 (1 mM Na2HPO4r7H2O; 10.5 mM KH PO ; 140 mM NaCl; 40 mM KCl), and repelleted. The tone, 100% acetone (twice), and 100% propylene oxide. 2 4 Cells were embedded in Epon, sectioned, and negatively cells were resuspended in 0.6 ml 21 SDS buffer (0.125 M Tris, pH 6.8; 4.0% SDS; 20.0% glycerol; 10.0% 2-mercap- stained with uranyl acetate and lead citrate. Samples toethanol; 0.002% Bromophenol blue), supplemented were viewed using a JEOL 100CX II transmission electron with a protease inhibitor cocktail (Pharmingen, San microscope at an accelerating voltage of 80 kV. Diego, CA), and stored until use at 070Њ. Samples were boiled for 5 min and 1 ml(Ç3500 cells) of each was then RESULTS subjected to SDS–PAGE through a 10% polyacrylamide Marker rescue analysis running gel with a 5.2% stacking gel (Laemmli, 1970). Proteins were electrophoretically transferred to a PVDF Individual wt genomic clones that overlap, and collec- membrane (Immobilon, Millipore, Bedford, MA) and visu- tively span the entire AcMNPV genome (Passarelli and alized using a chemiluminescence detection kit (ECL kit, Miller, 1993), were cotransfected with tsB1074 viral DNA Amersham, Arlington Heights, IL). GP41 immune or pre- into Sf-21 cells at 33Њ. Transfection of tsB1074 DNA alone immune serum was diluted 1:10,000 in Tris-buffered sa- did not generate viruses which could form plaques at line, pH 7.6, with 0.1% Tween 20 (Sigma Chem. Co., St. 33Њ. However, transfection of tsB1074 viral DNA and wt Louis, MO) according to the manufacturer’s protocol. To AcMNPV fragments homologous to the mutant region detect the major capsid protein, VP39, rabbit polyclonal resulted in plaque formation at 33Њ. Rescue of the ‘‘single immune serum (Thiem and Miller, 1989) or preimmune cell infection’’ phenotype was scored by the presence of serum was used at a 1:8,000 dilution. In all cases, mouse plaques 4 to 5 days following cotransfection. anti-rabbit antibody conjugated with horseradish peroxi- Two overlapping AcMNPV genomic clones, PstH5 and dase (Promega, Madison, WI) was utilized as a second- PstH9, were able to rescue the ts phenotype. PstHI,

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FIG. 1. Marker rescue mapping and DNA sequencing of the mutation in gp41 from tsB1074. (A) TsB1074 viral DNA was cotransfected with wt genomic DNA clones into Sf-21 cells maintained at 33Њ. Rescue by homologous recombination was scored by the ability of the AcMNPV fragments to produce viruses which could form plaques. Boxes representing these fragments are shaded if they provided rescue. Relevant restriction sites are indicated along with their position in map units (m.u.) within the AcMNPV genome (1 m.u. Ç 1.34 kb). Black arrows indicate predicted coding sequences with the arrowhead showing directionality relative to the conventional orientation of the AcMNPV genome. The asterisk marks the position of the nucleotide change in gp41 from tsB1074. (B) Part of the nucleotide sequence (65,900 to 65,801) of the coding strand of gp41 with corresponding encoded amino acids (313 to 346) are indicated below each codon. The nucleotide substitution in gp41 from tsB1074 is indicated above the wt sequence at nucleotide 65,887. The corresponding amino acid change at position 317 is also indicated. which overlaps PstH5, and HC10, which overlaps the mid subclone from pXA7, pXABB 4.5, did not rescue the right end of HC9 (Fig. 1A), could not rescue the defect. ts phenotype. However, the BstEII to SmaI region of pXA7, All other AcMNPV genomic library clones were negative within plasmid pXBS 2.1 (48.2 to 49.8 m.u.), rescued well. for rescue and are not shown in Fig. 1A. A plasmid clone, Two plasmid subclones of pXBS 2.1, pXBS 1.0 and pXSS pXma17, which contains AcMNPV sequence from 39.5 1.1, were also tested. While pXBS 1.0 (48.2 to 49.0 m.u.) to 49.8 m.u. was able to rescue the ts phenotype. A was unable to rescue, pXSS 1.1 (49.0 to 49.8 m.u.) was subclone from the left end of pXma17, pSDP5, was not able to rescue and was the smallest clone to provide able to provide rescue, but a plasmid clone from the reproducible positive marker rescue results. The average right end of pXma17, pXA7, rescued. The SstItoBstEII number of plaques (from three independent experiments) AcMNPV sequence (45.1 to 48.2 m.u.) present in a plas- resulting from cotransfecting pXSS 1.1 and tsB1074 viral

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DNA was 27 { 6 per plate, while transfecting tsB1074 right panel). The 42-kDa polypeptide was first observed viral DNA alone resulted in less than one plaque per at 12 hr p.i. and was present through 48 hr p.i. (Fig. 2A, plate. The 1.1-kb AcMNPV sequence present in pXSS 1.1 left panel). The size and temporal expression pattern of encompasses the majority of the gp41 gene as well as this protein was consistent with late GP41 expression. the 5؅ end of the putative ORF 79 of AcMNPV (Ayres et Another polypeptide was detected at about 32 kDa with al., 1994) (Fig. 1A). both the immune and preimmune sera and is not specific for the GP41 immune serum (Fig. 2A). The size, abun- DNA sequencing of wt AcMNPV and tsB1074 from dance, and temporal expression of this protein suggests 49.0 to 49.8 m.u. that it corresponds to the baculovirus very late protein, polyhedrin. To identify the mutation in tsB1074 that can be rescued Sucrose gradient-purified wt BV and ODV were sub- by the wt AcMNPV sequence from 49.0 to 49.8 m.u., this jected to SDS–PAGE. Western blot analysis with the region was cloned from wt and tsB1074 and sequenced. GP41 polyclonal immune serum (Fig. 2B, GP41-I panel) There was only a single nucleotide difference between detected a protein from ODV that migrated at the same wt and tsB1074 sequence in this region, a T to C transi- size as GP41 from wt-infected cell lysates at 40 hr p.i. tion (for the bottom strand) at position 65,887 in the This protein is not detected with preimmune serum (Fig. AcMNPV genome (Fig. 1B). A T to C transition is consis- 2B, GP41-Pre). A GP41-specific protein was not, however, tent with mutagenesis of DNA with the base analog BrdU. found associated with BV (Fig. 2B, GP41-I). As a control This nucleotide substitution changes an ATA codon to for loading equivalent numbers of nucleocapsids from ACA and is predicted to alter the encoded amino acid both BV and ODV, immune serum to the major capsid at position 317 of GP41 from isoleucine to threonine. protein, VP39 (Fig. 2B, VP39-I) (Thiem and Miller, 1989), ORFs of ú50 codons are not predicted from the opposite or preimmune serum (Fig. 2B, VP39-Pre), were also used DNA strand or from alternate reading frames at this posi- to probe these same lysates. These results confirm the tion in the AcMNPV genome (Ayres et al., 1994), there- localization of GP41 exclusively to the ODV form. fore, gp41 is the only gene predicted to be affected by this change. The discovery that the gp41 sequence is mutated in Western analysis of BV production from wt- and tsB1074 was unexpected because GP41 was described tsB1074-infected cells at 33Њ as an ODV-specific structural protein localized to the Previous electron microscopic (EM) analysis of tegument and was not found associated with BV (Whit- tsB1074-infected Sf-21 cells revealed the presence of ford and Faulkner, 1992b). Thus, mutations in gp41 are normal-looking nucleocapsids within infected nuclei (Lee predicted to affect the infectivity of OV but not BV. Since and Miller, 1979). Therefore, we tested the possibility that tsB1074 is unable to infect neighboring cells at the non- BV is produced from tsB1074-infected cells at 33Њ, but permissive temperature, it was clear that BV production that these virions are noninfectious. Medium from wt- is somehow compromised by this mutation in gp41. Be- or tsB1074-infected Sf-21 cells propagated at 33Њ was cause localization of GP41 to the tegument of ODV was collected and BV was purified by isopycnic centrifugation performed with a monoclonal antibody (Whitford and through sucrose gradients. The fraction of the gradient Faulkner, 1992b), we considered the possibility that the containing wt BV and the equivalent fraction from the monoclonal antibody used might have recognized specif- tsB1074 sucrose gradient were collected and subjected ically a modified form of GP41 present in ODV but not to SDS–PAGE. Western analysis was performed with the BV. To test the possibility that BV contains an unmodified VP39 immune serum to detect the presence of nucleo- or differently modifed form of GP41 which could not be capsids from these fractions. Nucleocapsids from puri- detected by this monoclonal antibody, we raised a poly- fied BV were detected from the wt-infected medium at clonal rabbit antiserum to a TrpE/GP41 fusion protein 33Њ and are shown in a 1:5, 1:10, and 1:25 dilution series overexpressed in E. coli. The fusion protein consisted of (Fig. 3, lanes 1–3). The entire fraction collected from the N-terminal coding sequence of the bacterial TrpE tsB1074-infected cell medium at 33Њ was analyzed but protein fused in frame with amino acids 35 to 391 (of 409 the major capsid protein was not detected (Fig. 3, lane total amino acids) of GP41. 4). A mock-infected control lacked the major capsid pro- tein as well (Fig. 3, lane M). We also considered the Western blot analysis of wt-infected cell lysates, BV possibility that an abnormal BV form, which might migrate and ODV, with GP41 polyclonal antiserum differently through the sucrose gradient than wt BV, was Wt-infected Sf-21 cell lysates, subjected to SDS–PAGE produced from tsB1074-infected cells at 33Њ. Accordingly, and Western blot analysis with this rabbit polyclonal anti- we centrifuged the medium from tsB1074-infected cells serum, contained a polypeptide of 42 kDa which cross- to pellet all virions without prior purification through a reacted with the TrpE/GP41 immune serum (Fig. 2A, left sucrose gradient. No nucleocapsids were detected upon panel) but not the preimmune serum control (Fig. 2A, Western analysis of this pellet (Fig. 3, lane 5).

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FIG. 2. Western blot analysis with polyclonal antiserum to a GP41 fusion protein detects a polypeptide from infected cell lysates which is a component of ODV. (A) Mock- or wt-infected Sf-21 cell lysates from a time course infection were subjected to SDS–PAGE. Proteins were transferred to a PVDF membrane and probed with rabbit polyclonal sera. The right half of the membrane was probed with rabbit preimmune serum (GP41- Pre) while the left half was probed with the polyclonal immune serum raised against the TrpE/GP41 fusion protein (GP41-I). Protein size standards are indicated. (B) Sucrose gradient purified BV or ODV along with mock- or wt-infected cell lysates at 40 hr p.i. (40 h) were subjected to SDS– PAGE and Western blot analysis with the polyclonal preimmune or GP41 immune serum or with the major capsid protein immune (VP39-I) or preimmune serum (VP39-Pre).

EM analysis of mock-, wt-, and tsB1074-infected Sf-21 tsB1074-infected cells at 33Њ, however, nucleocapsids cells at 33Њ were not observed interacting with the nuclear mem- brane nor were they observed within the cytoplasm (e.g., Previous EM analysis of tsB1074-infected cells at 33Њ Fig. 4B). To study this in a quantitative manner, EM analy- did not specifically examine the cytoplasm and plasma sis of 25 cells each of wt- or tsB1074-infected cells at membrane of cells for budded virus precursors during 33Њ were randomly chosen and the presence of nucleo- the late phase of infection. We therefore examined mock-, capsids within the nucleus, exiting the nucleus, and/or wt-, and tsB1074-infected cells propagated at 33Њ at 16 within the cytoplasm were recorded (Table 1). Nucleo- hr p.i. As expected, nucleocapsids and virogenic stroma, capsids were always observed within the nucleus of in- the putative site of nucleocapsid assembly, were ob- fected cells for both the wt- and tsB1074-infections (Table served in the nuclei of virus-infected cells (e.g., Figs. 4B 1, nucleus). Nucleocapsids were in the process of exiting and 4C) but not in mock-infected cells (Fig. 4A). The through the nuclear membrane in 7/25 wt-infected cells number and appearance of nucleocapsids present in nu- but this was not observed for any of the tsB1074-infected clei from wt- and tsB1074-infected cells appeared to be cells examined (Table 1, nuclear membrane). While 22/25 similar (Figs. 4B–4D, arrowheads). In wt-infected cells wt-infected cells were observed to have nucleocapsids at 33Њ, nucleocapsids were frequently observed in the in the cytoplasm, none of the 25 tsB1074-infected cells process of exiting the nucleus (e.g., Fig. 4C, open arrow), examined showed nucleocapsids in the cytoplasm (Ta- or within the cytoplasm (Fig. 4D, black arrows). In ble 1, cytoplasm). Thus, nucleocapsids produced from

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al., 1994) and E2 (Kool et al., 1994) strains of AcMNPV. This is in contrast to the sequence of gp41 reported for the HR3 strain (Whitford and Faulkner, 1992a). All four strains have identical nucleotide sequence until nucleo- tide 65,806. The nucleotide sequence for gp41 in this region (Fig. 1B) for the L1, E2, and C6 strains of AcMNPV is GCCGCC (nucleotides 65,807 to 65,802) but is GCG- CCA for HR3 (Whitford and Faulkner, 1992a). The ab- sence of the second C nucleotide within this sequence shifts the reading frame for the protein from this point and predicts a smaller GP41 protein of 354 amino acids (instead of 409), with a different C-terminal sequence, encoded by the HR3 strain. Whether this represents a slightly different version of GP41 encoded by the HR3 strain or a sequencing error is not clear. GP41 encoded by SfNPV and HzNPV share a 52 and 59% identity, re- spectively, at the amino acid level with the L1, C6, and E2 strains of AcMNPV. Although GP41 from SfNPV and HzNPV have shorter C-termini than the L1, C6, and E2 strains of AcMNPV, the sequence at the C-termini resem- FIG. 3. Budded virions are not produced from tsB1074-infected Sf- bles these strains of AcMNPV more than it does the 21 cells at 33Њ. BV from mock (M), wt- (1–3), or tsB1074-infected cells predicted C-terminus for the HR3 strain of AcMNPV. (4) at 33Њ was purified by isopycnic centrifugation through sucrose gradients, subjected to SDS–PAGE and Western blot analysis with Mapping the ts defect to GP41 was unexpected be- polyclonal immune serum to the major capsid protein, VP39. Lanes 1– cause of its prior localization to ODV but not BV (Whitford 3 are 1/5, 1/10, and 1/25 dilutions, respectively, of the BV fraction and Faulkner, 1992b). Western blot analysis with a poly- collected from the gradient containing wt-infected medium. Lane 4 clonal antiserum to a TrpE/GP41 fusion protein confirmed represents the equivalent fraction, undiluted, from the gradient con- the presence of GP41 exclusively in purified ODV. Since taining tsB1074-infected cell medium. Lane 5 contains all virions col- lected from tsB1074-infected cell medium at 33Њ by direct pelleting nucleocapsids could not be recovered from tsB1074-in- without purification through a gradient. fected cell medium at the nonpermissive temperature, it was clear that BV production was compromised in this mutant. GP41, therefore, has a role in BV production and tsB1074-infected cells at 33Њ appeared to be restricted is not simply an ODV structural protein. to the nucleus. This inability of nucleocapsids to exit the EM analysis of wt- and tsB1074-infected Sf-21 cells nucleus is consistent with the lack of BV in infected cell at 33Њ showed the presence of nucleocapsids, which medium as determined by Western analysis, and the in- appeared normal, within the nuclei of infected cells from ability of tsB1074 to form plaques on an Sf-21 cell mono- both infections. Nucleocapsids from wt-infected cells layer at the restrictive temperature. were observed interacting with the nuclear membrane, apparently in the process of leaving the nucleus, as well DISCUSSION as within the cytoplasm in transit to the plasma mem- brane. This was not observed in the tsB1074-infected Marker rescue mapping of the defect in tsB1074 and cells at the nonpermissive temperature. It appears that DNA sequencing of this region led to the identification nucleocapsids fail to egress from the nucleus so that of a single nucleotide substitution within gp41. The tran- BV is not produced. Initial immunolocalization studies sition is predicted to alter coding of the amino acid iso- indicate that GP41 localizes to the nucleus (data not leucine to threonine, a substitution that could potentially shown). GP41 may mediate the exit of nucleocapsids affect the three-dimensional folded structure of a poly- from the nucleus directly, or it may be required to facili- peptide and is consistent with a ts version of GP41. Addi- tate or direct the nucleocapsids to the nuclear mem- tional evidence that this isoleucine residue is important brane. to the function of GP41 is the observation that it is con- The mechanism by which nucleocapsids enter the cy- served in gp41 homologous sequences from related nu- toplasm from the nucleus is not known. They may pass clear polyhedrosis viruses, Bombyx mori (BmNPV) (Na- through nuclear pores, bud directly through the nuclear gamine et al., 1991), Spodoptera frugiperda (SfNPV) (Liu membrane, or both. Evidence to support the theory that and Maruniak, 1995), and Helicoverpa zea (HzNPV) (Ma nucleocapsids bud through the nuclear membrane stems et al., 1993). from the observation that enveloped nucleocapsids are Our wt (L1 strain) sequence of gp41 exactly matched seen within the cytoplasm by electron microscopy, but the nucleotide sequences reported for the C6 (Ayres et this transient envelope is lost before reaching the plasma

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FIG. 4. Electron microscopic analysis of mock-, wt-, and tsB1074-infected cells at 33Њ. Sf-21 cells at 33Њ were infected with (A) Mock-, (B) tsB1074-, or (C and D) wt-virus, harvested at 16 hr p.i., fixed, sectioned, and stained for EM analysis. The nuclear (N) and cytoplasmic (C) regions are indicated as well as the virogenic stroma (VS). Arrowheads point to representative nucleocapsids (B, C, and D), an open arrow points toward a nucleocapsid leaving the nucleus (C), and black arrows indicate nucleocapsids within the cytoplasm (D) of a wt-infected cell. Scale bars are 1 mm. membrane (Fraser, 1986). GP41 may be required for nu- tsB1074-infected cells at 33Њ appear normal within the cleocapsids to move from the virogenic stroma, a virus- resolution of EM analysis, we cannot rule out the possi- induced structure thought to be the site of nucleocapsid bility that GP41 is directly involved in nucleocapsid as- assembly, to the nuclear membrane. Alternatively, GP41 sembly and the apparent transport defect is a secondary may be directly involved in facilitating the movement of result of aberrant nucleocapsid formation. However, it nucleocapsids through the nuclear membrane or its is clear that GP41 is not simply a structural protein of pores. Although nucleocapsids produced from the nucleocapsids. It is interesting that other proteins with the O-linked N-acetylglucosamine (O-GlcNAc) modification like that TABLE 1 present on GP41 are typically phosphoproteins which Quantitative Analysis of Nucleocapsid Location in wt- and tsB1074- form protein multimers (Hart et al., 1996). Some proteins infected Sf-21 Cells at the Nonpermissive Temperature bearing this modification are found within the cytoplasm, such as neurofilaments which seem to require O-GlcNA- Number of cells with nucleocapsids associated cylation for their function in cytoskeletal assembly (Hart Nuclear et al., 1996). Studies using highly purified rat liver subcel- Virus Nucleus membrane Cytoplasm lular fractions demonstrated that the majority of O- GlcNAc-modified proteins are located in the nucleus, wt 25/25 7/25 22/25 most of which are within the nucleoplasm. The highest tsB1074 25/25 0/25 0/25 density of protein O-GlcNAcylations are associated with

AID VY 8612 / 6a39$$$103 06-04-97 14:08:20 vira AP: Virology 300 OLSZEWSKI AND MILLER the nuclear membrane (e.g., nuclear pore proteins) (Hart Braunagel, S., Elton, D., Ma, H., and Summers, M. D. (1996a). Identifica- et al., 1996). tion and analysis of an Autographa californica nuclear polyhedrosis virus structural protein of the occlusion-derived virus envelope: ODV- To reconcile the data that GP41 is contained within E56. Virology 117, 97–110. ODV yet is required for BV production, GP41 may be Braunagel, S., He, H., Ramamurthy, P., and Summers, M. D. (1996b). localized to the nucleoplasm or associated loosely with Transcription, translation, and cellular localization of three Auto- the inner leaflet of the nuclear membrane and to viral grapha californica nuclear polyhedrosis virus structural proteins: membranes elaborated within the nucleus very late in ODV-E18, ODV-E35, and ODV-EC27. Virology 222, 100–114. Braunagel, S. C., and Summers, M. D. (1994). Autographa californica infection. ODV are formed at very late times when bun- nuclear polyhedrosis virus, PDV, and ECV viral envelopes and nu- dles of nucleocapsids are enveloped within the nucleus. cleocapsids: Structural proteins, antigens, lipid and fatty acid profiles. After these enveloped bundles are embedded within the Virology 202, 315–328. polyhedrin paracrystalline matrix, there is an apparent Fraser, M. J. (1986). Ultrastructural observations of virion maturation in compression of the space between the nucleocapsids Autographa californica nuclear polyhedrosis virus infected Spodopt- era frugiperda cell cultures. J. Ultrastruct. Mol. Struct. Res. 95, 189– and their envelope (Knudson and Harrap, 1976) to form 195. the tegument. The tegument is thus likely to be com- Granados, R. R., and Lawler, K. A. (1981). In vivo pathway of Autographa posed primarily of nucleoplasmic proteins and the pres- californica baculovirus invasion and infection. Virology 108, 297– ence of GP41 in the tegument of ODV may be an adventi- 308. tious association based on its presence in the nucleo- Harlow, E., and Lane, D. (1988). ‘‘Antibodies: A Laboratory Manual.’’ Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. plasm. Since nucleocapsids presumably lose any Harrap, K. (1972). The structure of nuclear polyhedrosis viruses. III. associated nucleoplasm during transit through the cyto- Virus assembly. Virology 50, 133–139. plasm, BV would not be predicted to contain any GP41. Hart, G. W., Kreppel, L. K., Comer, F. I., Arnold, C. S., Snow, D. M., Ye, Previous examination of envelope fractions from ODV Z., Cheng, X., DellaManna, D., Caine, D. S., Earles, B. J., Akimoto, Y., did not detect GP41, as is consistent with a polypeptide Cole, R. N., and Hayes, B. K. (1996). O-GlcNAcylation of key nuclear and cytoplasmic proteins: reciprocity with O-phosphorylation and with a hydrophilic profile (Whitford and Faulkner, 1992b). putative roles in protein multimerization. Glycobiology 6, 711–716. The possibility still exists that GP41 is a peripheral pro- Hong, T., Braunagel, S. C., and Summers, M. D. (1994). Transcription, tein which is loosely associated with the envelope and is translation, and cellular localization of PDV-E66: A structural protein solubilized upon biochemical fractionation in vitro. GP41 of the PDV envelope of Autographa californica nuclear polyhedrosis would not fractionate with the envelope nor the nucleo- virus. Virology 204, 210–222. Knudson, D. L., and Harrap, K. A. (1976). Replication of a nuclear polyhe- capsids but would appear to reside within the tegument drosis virus in a continuous cell culture of Spodoptera frugiperda: by default. Microscopy study of the sequence of events of the virus infection. While GP41 is clearly required for BV production, its J. Virol. 17, 254–268. function in ODV, if any, has yet to be determined. The Koerner, T. J., Hill, J. E., Myers, A. M., and Tzagoloff, A. (1991). High- occlusion process at 33Њ is defective for wt virus, produc- expression vectors with multiple cloning sites for construction of trpE fusion genes: pATH vectors. Methods Enzymol. 194, 477–490. ing occlusion bodies that are primarily composed of the Kool, M., Broer, R., Zuidema, D., Goldbach, R. W., and Vlak, J. M. (1994). polyhedrin paracrystalline matrix without virions embed- Nucleotide sequence and genetic organization of a 7.3 kb region ded within them (Lee and Miller, 1979). Therefore, we (map unit 47 to 52.5) of Autographa californica nuclear polyhedrosis could not address whether OV produced by an infection virus fragment EcoRI-C. J. Gen. Virol. 75, 487–494. with tsB1074 at 33Њ exhibit normal infectivity in vivo. Laemmli, U. K. (1970). Cleavage of structural proteins during the assem- bly of the head of bacteriophage T4. Nature 227, 680–685. Lee, H. H., and Miller, L. K. (1978). Isolation of genotypic variants of ACKNOWLEDGMENTS Autographa californica nuclear polyhedrosis virus. J. Virol. 27, 754– 767. We thank Jeanne McLachlin for providing the pXA7 series of plasmid Lee, H. H., and Miller, L. K. (1979). Isolation, complementation, and constructs used in this study and Cathy Kelloes at the Electron Micros- initial characterization of temperature sensitive mutants of the bacu- copy Laboratory, the University of Georgia, for assistance with the EM lovirus Autographa californica nuclear polyhedrosis virus. J. Virol. 31, work. This work was supported in part by Public Health Service Grant 240–252. AI23719, from the National Institute of Allergy and Infectious Diseases, Liu, J. C., and Maruniak, J. E. (1995). 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