Autographa Californica Multiple Nucleopolyhedrovirus Ac142, a Core Gene That Is Essential for BV Production and ODV Envelopment ⁎ Christina B

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Virology 372 (2008) 325–339 www.elsevier.com/locate/yviro

Autographa californica multiple nucleopolyhedrovirus ac142, a core gene that is essential for BV production and ODV envelopment ⁎ Christina B. McCarthy a, Xiaojiang Dai a, Cam Donly b, David A. Theilmann a,

a Pacific Agri-Food Research Centre, Agriculture and Agri-Food Canada, Box 5000, Summerland, British Columbia, Canada V0H 1Z0 b Southern Crop Protection and Food Research Centre (London), Agriculture and Agri-Food Canada, 1391 Sandford Street, London, Ont., Canada N5V 4T3 Received 31 July 2007; returned to author for revision 27 August 2007; accepted 18 October 2007

Abstract

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac142 is a baculovirus core gene and encodes a protein previously shown to associate with occlusion-derived virus (ODV). To determine its role in the baculovirus life cycle, we used the AcMNPV bacmid system to generate an ac142 deletion virus (AcBACac142KO-PH-GFP). Fluorescence and light microscopy revealed that AcBACac142KO-PH-GFP exhibits a single-cell infection phenotype. Titration assays and Western blot confirmed that AcBACac142KO-PH-GFP is unable to produce budded virus (BV). However, viral DNA replication is unaffected and the development of occlusion bodies in AcBACac142KO-PH-GFP-transfected cells evidenced progression to very late phases of the viral infection. Western blot analysis showed that AC142 is expressed in the cytoplasm and nucleus throughout infection and that it is a structural component of BV and ODV which localizes to nucleocapsids. Electron microscopy indicates that ac142 is required for nucleocapsid envelopment to form ODV and their subsequent occlusion, a fundamental process to all baculoviruses. Crown Copyright © 2007 Published by Elsevier Inc. All rights reserved.

Keywords: ac142; Core gene; Nucleocapsid; BV; ODV; Envelopment; Occlusion body

Introduction epithelial cells to initiate secondary infections within the infected animal (Granados and Lawler, 1981; Keddie et al., The Baculoviridae are large, enveloped, double-stranded 1989). ODVs are highly infectious to the midgut epithelial cells DNA viruses (80- to 80-kbp genome) which are pathogens of and mediate animal-to-animal transmission but have very low insects, mostly from the order Lepidoptera but also from the infectivity when injected directly into the hemocoel of insects or orders Diptera and Hymenoptera. Their genomes are packaged when used to infect cultured insect cells. However, BVs are within rod-shaped capsids enclosed by a lipid envelope. During highly infectious for tissues of the hemocoel and for cultured the infection cycle, they typically produce two structurally and cells (Volkman and Summers, 1977; Volkman et al., 1976). functionally distinct virion phenotypes: occlusion-derived virus Even though the nucleocapsids of BV and ODV are (ODV) and budded virus (BV) (Theilmann et al., 2005). These genetically identical, their envelope origin and composition two virion forms have different and distinct functions in the viral differs and they are produced at different times during infection life cycle. ODVs are accumulated in a protein matrix that forms (Braunagel and Summers, 1994; Russell et al., 1997). Nucleo- polyhedra or occlusion bodies. ODVs are required for the oral capsids are initially shuttled out of the nucleus and are trans- infection of insects. ODVs are released from the occlusion body ported to the cytoplasmic membrane from which they bud by the alkaline environment within the midgut lumen of the larva forming BV (Monsma et al., 1996). During the very late phases and subsequently initiate primary infection of the mature co- of infection nucleocapsids are retained within the nucleus and lumnar epithelial cells of the midgut. BVs are produced when acquire a de novo envelope to form ODV, which are then em- nucleocapsids bud through the plasma membrane of the midgut bedded within the matrix of the occlusion body (Williams and Faulkner, 1997). ⁎ Corresponding author. Fax: +1 250 494 6415. Autographa californica multiple nucleopolyhedrovirus E-mail address: [email protected] (D.A. Theilmann). (AcMNPV) is the type species of the Baculoviridae and was

0042-6822/$ - see front matter. Crown Copyright © 2007 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.virol.2007.10.019 326 C.B. McCarthy et al. / Virology 372 (2008) 325–339 the first baculovirus to be completely sequenced (Ayres et al., vital structural function in the viral life cycle. Ac142 is predicted 1994). AcMNPV presents a genome of 134 kbp which contains to code for 55.4-kDa 477-aa protein that encodes a potential 154 predicted open reading frames (ORFs) (Ayres et al., 1994). transmembrane domain but its function in the viral life cycle has Comparative analysis of the over 30 completely sequenced remained unknown to date. baculovirus genomes reveals a set of 29 core genes that are To determine the role of ac142 in AcMNPV replication, this conserved in all baculovirus genomes and are therefore likely to study has used an AcMNPV bacmid to generate an ac142 deletion serve essential roles in baculovirus life cycles (Garcia-Maruniak mutant. We found that ac142 is essential for budded virus et al., 2004; Herniou et al., 2003). The core genes of known production and that its deletion produces a single cell infection function are required for RNA transcription, DNA replication phenotype. Viral DNA replication was unaffected, however. We and as structural and auxiliary proteins. Nine core genes 38K also generated an ac142-HA-tagged repair construct which (Ac98), p33 (Ac92), ac68, ac81, ac96, ac109, ac115, and enabled us to determine ac142 temporal subcellular expression. ac142, have no known function or sequence similarities to Furthermore, we show that ac142 is a structural component of BV, proteins of known functions (Herniou et al., 2003). Recently, which localizes mainly to its nucleocapsid fraction and confirmed proteomics analyses of proteins associated with ODV of three that it is a structural component of ODV. Electron microscopy baculoviruses, AcMNPV, Heliothis armigera NPV, and Culex analysis showed that ac142 is not required for nucleocapsid nigripalpus NPV, identified ac142 or its homologs (Hear9, formation but it is required for ODVenvelopment and subsequent Cuni30)(Braunagel et al., 2003; Perera et al., 2007). As it is a embedding of virions into polyhedra. These critical requirements core gene and is associated with ODV,AC142 plays a potentially provide a rationale for why ac142 is a core baculovirus gene.

Fig. 1. Construction of ac142 knockout, repair and positive control AcMNPV bacmids. Schematic diagram describing the construction of the bacmids AcBACac142KO-PH-GFP, AcBACac142rep-PH-GFP and AcBACPH-GFP.Theac142 ORF was partially deleted by replacement with an EM7-zeocin gene resistance cassette (amplified with primers 684 and 1360) via homologous recombination in E. coli, to generate AcBAC-ac142KO-PH-. The deletion did not affect either the polyA signal for exon0 or the entire odv-e18 promoter, which includes three late transcriptional start sites. The deletion of ac142 was confirmed using the primer pairs 1013/1014 and 1239/1361; their relative positions are indicated. The lower part of the figure shows the genes inserted in the polyhedrin (polh) locus of AcBAC-ac142KO-PH− by Tn7-mediated transposition to generate AcBACac142KO-PH-GFP and AcBACac142rep-PH-GFP.Theac142 ORF inserted into AcBACac142rep-PH-GFP is driven by its own promoter and includes an in-frame C-terminal HA-epitope (indicated as a black triangle). The AcBACPH-GFP bacmid was generated by insertion of the polyhedrin and gfp genes into the polyhedrin locus of bMON14272 bacmid. C.B. McCarthy et al. / Virology 372 (2008) 325–339 327

Results the ac142 knockout virus we ensured the deletion did not affect either exon0 (ac141)orodv-e18 (ac143), as the ac142 Generation of ac142 knockout and repair bacmids coding region contains a potential polyA signal for exon0 and the entire odv-e18 promoter region (Fig. 1)(Braunagel et al., Using the bMON14272 bacmid, an ac142 knockout 1996; Dai et al., 2004; Harrison and Bonning, 2003). AcMNPV was generated through homologous recombination Therefore, in order to preserve both the polyA signal in Escherichia coli as previously described (Datsenko and sequence and the odv-e18 promoter region, the resulting Wanner, 2000; Lin and Blissard, 2002). When constructing deletion leaves 521 bp and 353 bp of the 5′ and 3′ ends of

Fig. 2. Viral replication analysis in Sf9 cells. (A) Fluorescence microscopy showing the progression of the infection in Sf9 cells transfected with AcBACac142KO-PH-GFP, AcBACac142rep-PH-GFP and AcBACPH-GFP. (B) Plaque assay analysis at 96 hpt showing that AcBACac142KO-PH-GFP infection does not progress beyond the initially transfected cells, indicating that no infectious budded virus is produced. The arrows indicate single-transfected cells as determined by fluorescence microscopy. (C) Light microscopy of Sf9 cells transfected with AcBACac142KO-PH-GFP, AcBACac142rep-PH-GFP and AcBACPH-GFP at 96 hpt. The arrows indicate single cells producing apparently normal occlusion bodies in AcBACac142KO-PH-GFP-transfected cells, compared to AcBACac142rep-PH-GFP and AcBACPH-GFP. 328 C.B. McCarthy et al. / Virology 372 (2008) 325–339 the ac142 ORF, respectively (Fig. 1). The deleted coding Light microscopy analysis showed that, at 96 hpt, normal sequences were replaced with the EM7-zeocin resistance gene appearing occlusion bodies formed in cells transfected with cassette (Fig. 1). all the constructs (Fig. 2C). Nevertheless, the number of The resulting ac142 knockout bacmid, AcBAC-ac142KO- AcBACac142KO-PH-GFP-transfected cells which contained PH−, was examined by PCR analysis (data not shown). To confirm the correct insertion of the zeocin gene cassette into the ac142 locus, two primer pairs, 1013/1014 and 1239/1361, were used to examine the recombination junctions of the upstream and downstream flanking regions (Fig. 1). The PCR results confirmed that the central region of ac142 had been deleted as expected (data not shown). To establish if the ac142 deletionhasanyeffectonocclusion body morphogenesis and to facilitate examination of virus infection, the polyhedrin and gfp genes were inserted into the polyhedrin locus of AcBAC-ac142KO-PH− by transposition (Fig. 1). This virus was named AcBACac142KO-PH-GFP.Torescueand confirm that the phenotype resulting from the ac142 knockout was not due to genomic effects, a repair bacmid, AcBACac142rep-PH-GFP, was generated. AcBACac142rep-PH-GFP contains ac142 encoding an in-frame C-terminal HA-epitope expressed by its own promoter, in addition to polyhedrin and gfp, inserted into the polyhedrin locus of AcBAC-ac142KO-PH− (Fig. 1). AcBACPH-GFP, which was used as a positive control, was constructed by transposing the polyhedrin and gfp genes into the polyhedrin locus of AcMNPV bacmid bMON14272 (Fig. 1). The resulting bacmids, AcBACac142KO-PH-GFP,AcBACac142rep-PH-GFP and AcBACPH-GFP, were confirmed by PCR analysis (data not shown).

Analysis of AcBACac142KO-PH-GFP, AcBACac142rep-PH-GFP and AcBACPH-GFP replication in transfected Sf9 cells

To determine the effect of the ac142 deletion on virus replication, Sf9 cells were transfected with AcBACac142KO-PH-GFP, AcBACac142rep-PH-GFP and AcBACPH-GFP. As all the constructs express gfp under the control of the constitutive OpMNPV ie1 promoter, transfected cells were monitored by fluorescence. No difference was observed among the three viruses at 24 h post transfection (hpt), indicating comparable transfection efficiencies (Fig. 2A). By 48 hpt, AcBACac142KO-PH-GFP-transfected cells showed no increase in the number of infected cells (Fig. 2A), indicating that there was no spread of the virus beyond the initially transfected cells. Similar results for AcBACac142KO-PH-GFP were observed at 72 and 96 hpt. This result suggested that AcBACac142KO-PH-GFP was incapable of producing infectious Fig. 3. Replication kinetics of Sf9 cells transfected with AcBACac142KO-PH-GFP, BV. In contrast, fluorescence was observed in almost all ac142rep-PH-GFP PH-GFP PH-GFP ac142rep-PH-GFP AcBAC and AcBAC . (A) Virus growth curve determined by AcBAC and AcBAC -transfected cells by TCID50 end-point dilution and generated from Sf9 cells transfected with 48 hpt (Fig. 2A), indicating that both viruses were capable of AcBACac142KO-PH-GFP, AcBACac142rep-PH-GFP and AcBACPH-GFP. Each datum generating infectious BV from the initial transfection. point represents the average titer derived from two independent TCID50 To further compare the spread of virus between assays. Error bars represent standard deviation. (B) Virus titer independent of virion infectivity was determined by quantifying the number of viral AcBAC ac142KO-PH-GFP,AcBACac142rep-PH-GFP and PH-GFP genomes using qPCR analysis of supernatants of Sf9 cells transfected with AcBAC , plaque assays were performed to restrict AcBACac142KO-PH-GFP,AcBACac142rep-PH-GFP and AcBACPH-GFP at the movement of BV to adjoining cells. Under these conditions, designated time points. (C) Western blot analysis of purified BV particles. AcBACac142KO-PH-GFP produced only a single-cell pheno- BVs were purified from the supernatants of Sf9 cells transfected with ac142KO-PH-GFP ac142rep-PH-GFP PH-GFP type, confirming that no infectious BVs were being produced AcBAC ,AcBAC and AcBAC . Protein samples were separated by SDS-PAGE and analyzed by Western blot (Fig. 2B). In contrast, by 4 days post transfection, both ac142rep-PH-GFP PH-GFP using a monoclonal antibody specific for VP39 nucleocapsid protein. 1, AcBAC and AcBAC produced large AcBACac142KO-PH-GFP;2,AcBACac142rep-PH-GFP;3,AcBACPH-GFP;4, normal plaques. AcMNPV-E2; 5, Mock. C.B. McCarthy et al. / Virology 372 (2008) 325–339 329 occlusion bodies corresponded only to the single cells exhibiting AcBACac142KO-PH-GFP-infected cells. However, to determine fluorescence (Fig. 2C). In contrast, in AcBACac142rep-PH-GFP and if ac142 impacted viral DNA replication, a quantitative AcBACPH-GFP-transfected cells, almost every single cell analysis was performed to compare the initiation and levels contained occlusion bodies. of viral DNA replication in AcBACac142KO-PH-GFP and AcBACPH-GFP, over a 72-h time course. Equal amounts of Growth curve analysis bacmid-transfected Sf9 cells were collected at designated time points, cell lysates were prepared and total DNA was extracted Our results showed that deletion of ac142 leads to a defect and subjected to quantitative real-time PCR. Results showed in infectious BV production. To confirm these results and that, for both viruses, the onset of viral replication occurred further assess the effect of deleting ac142 on virus replication between 18 and 24 hpt (Fig. 4) and the levels of replication and determine the replication kinetics of the virus constructs, were the same up to 18 hpt. For AcBACPH-GFP, the levels of virus growth curve analysis was performed by 50% tissue DNA replication started increasing from 24 hpt, correlating culture infective dose (TCID50) and quantitative polymerase with the spread of the infection from the production of budded chain reaction (qPCR). Sf9 cells were transfected with the virus. For AcBACac142KO-PH-GFP, which does not produce different bacmid DNA constructs and, at selected time points, budded virus, DNA synthesis plateaus from 36 hpt at levels 4– PH-GFP the BV titers were determined by TCID50 end-point dilution. 5 times lower than AcBAC . This correlates with DNA No BV was detectable at any time point up to 96 hpt for replication occurring in the initially transfected cells. These AcBACac142KO-PH-GFP-transfected cells indicating no infec- results indicate that the onset and level of replication in tious virus was produced (Fig. 3A). As expected, Sf9 cells individual infected cells is unaffected by deletion of ac142. transfected with AcBACac142rep-PH-GFP and AcBACPH-GFP revealed a normal increase in virus production that reached ac142 transcriptional analysis equivalent titers (Fig. 3A). This confirmed that the ac142 repair virus was as proficient in virus production as the WT The ac142 promoter region contains a single late gene tran- virus and that the defect in BV production was not due to scriptional start site (ATAAG) 18 bp upstream from the trans- genomic effects at the site of the deletion. lational start site, but no putative early gene motifs (Fig. 5A). The TCID50 end-point dilution assays determine the pro- Previous studies have therefore predicted that it would be duction of infectious BV but cannot assay if any non-infectious expressed as a late gene (Ayres et al., 1994; Li et al., 2002). To BVs are produced. To further analyze this possibility, the BV examine ac142 temporal expression, we performed a Northern titers of our virus constructs were also determined by qPCR blot analysis using a strand-specific probe from within the ac142 analysis which detects viral genomes regardless of infectivity. coding region (Fig. 5A). The Northern blot analysis detected three Due to bacmid transfection there is detectable background of primaryRNAbandsfrom12to72hpost-infection(hpi)which viral genomes present at all time points analyzed. As expected were 2.3, 3.4 and 6.3 kb in size (Fig. 5B). The ac142 probe also for both AcBACac142rep-PH-GFP and AcBACPH-GFP, a steady in- detected transcripts originating from exon0 (ac141), which we crease in BV was detected up to 72 hpt. In contrast, for have previously shown to be 3.4, 4.2, and 6.3 kb in size (Daietal., AcBACac142KO-PH-GFP-transfected cells no increase in BV 2004). Since the 3.4-kb bands originating from the exon0 (ac141) production was detected above background at any time point and ac142 transcripts are indistinguishable in size, the only up to 72 hpt (Fig. 3B). The qPCR results therefore were unable transcript specific to ac142 would be the 2.3-kb band. The 2.3-kb to detect any BV production by the ac142 knockout, in RNA is detected after the onset of DNA replication from 12 hpi to agreement with the TCID50 results. Even though the transfection, end-point dilution assays and qPCR results had provided no evidence for BV production by AcBACac142KO-PH-GFP, an additional assay was performed to confirm these results. BVs were purified from bacmid- transfected cell supernatants, as described in Materials and methods, and analyzed by Western blot comparing the levels of nucleocapsid protein VP39 (Fig. 3C). VP39 was detected in the AcBACac142rep-PH-GFP and AcBACPH-GFP-transfected cells supernatants. In contrast, for AcBACac142KO-PH-GFP, no nucleocapsid protein VP39 was detected even with longer exposure times. This result confirmed what we observed with the other assays, indicating that the deletion of ac142 results in a viral phenotype incapable of producing BV.

Deletion of ac142 does not affect viral DNA replication in Sf9 cells Fig. 4. Quantitative real-time PCR analysis of viral DNA replication. Sf9 cells (1.0×106) were transfected with 1.0 μgofAcBACac142KO-PH-GFP and AcBACPH-GFP DNA. At various hpt, total DNA was extracted, DpnI digested to eliminate input The production of occlusion body-like structures (Fig. bacmid DNA and analyzed by qPCR. The graph shows the results of two 2C) suggested that DNA replication was occurring in independent replication assays. Error bars represent standard deviation. 330 C.B. McCarthy et al. / Virology 372 (2008) 325–339

Fig. 5. Northern blot analysis of orf142 and Western blot analysis of AC142 cellular localization and temporal expression. (A) Schematic map showing the relative sizes, positions and orientations of the exon0 (ac141), ac142, odv-e18 (ac143), odv-ec27 (ac144), ac145, ac146, ie-1 (ac147) and odv-e56 (ac148) ORFs. Potential transcripts detectable by the single-stranded probe (indicated as a black line) homologous to ac142 are indicated below the ORFs. Sizes are based on the location of late transcription start sites and polyadenylation signals. The solid arrow agrees with the unique ac142 2.3-kb band identified on the Northern blot; the dashed arrow indicates the 3.4-kb putative transcript of ac142 that, if expressed, is indistinguishable in size from one of the exon0 transcripts. Dotted arrows indicate transcripts originating from exon0 (Dai et al., 2004), which were also detected by the ac142 probe. (B) Northern blot analysis of ac142 transcription in Sf9 cells infected with AcMNPV from 3 to 72 hpi. The identified RNA bands and their sizes (kb) are indicated on the right. The 2.3-kb RNA band specific to ac142 is underlined. Size markers are shown on the left (kb). (C) Sf9 cells were infected with AcBACac142rep-PH-GFP (MOI 5), harvested at various times pi and separated into the nuclear and cytoplasmic fractions. The fractions were analyzed by SDS-PAGE and Western blot, probed with anti-HA, -IE1 and -GP64 monoclonal antibodies to detect HA tagged AC142, IE1 and GP64, respectively. C, cytoplasmic fraction; N, nuclear fraction.

72 hpi. The steady-state levels increase up to 24 hpi and then required to confirm that ac142 initiates from a late promoter decline up to 72 hpi. The mRNA size agrees with transcriptional motif. initiation at the ac142 late promoter motif ATAAG and ter- mination at the first polyA signal sequence (AATAAA) down- Subcellular localization of AC142 during infection stream of its coding region (Fig. 1A). These results support the conclusion that AcMNPV ac142 is transcribed as a late gene but Deletion of ac142 resulted in the elimination of BV production further studies using 5′RACE or aphidicolin inhibition will be indicating that ac142 is an essential gene. To further understand C.B. McCarthy et al. / Virology 372 (2008) 325–339 331 the function of ac142 in the viral life cycle it was necessary to specific envelope protein) which were found only in the establish its temporal expression and cellular localization. A time nucleocapsid and envelope fractions, respectively, even with course analysis of ac142 expression was performed using longer exposures. These results show that AC142 is AcBACac142rep-PH-GFP, which expresses an HA-tagged version of associated with both BV and ODV virions and localizes AC142. Sf9 cells infected with AcBACac142rep-PH-GFP were primarily to the nucleocapsid fraction of BV. collected at various times hpi and the nuclear and cytoplasmic fractions were analyzed by SDS-PAGE and Western blot Electron microscopic analysis of AcBACac142KO-PH-GFP-, (Fig. 5C). This analysis detected a band of approximately AcBACac142rep-PH-GFP- and AcBACPH-GFP-transfected cells 50 kDa for AC142, in agreement with its predicted size, expressed from 24 hpi to 96 hpi and distributed in both the Our results showed that the lack of ac142 did not stall the nucleus and the cytoplasm. Levels in the nucleus and progression of the viral infection to very late phases in Sf9 cytoplasm were approximately the same except at 24 hpi cells, as evidenced by DNA replication which was during BV peak production, when AC142 localized to a unaffected and by the presence of occlusion bodies in the greater extent in the cytoplasm. The presence of the nuclear nuclei of transfected cells. TCID50 and qPCR results protein IE1 and the cytosolic protein GP64 exclusively in the indicated that AC142 is essential for BV production and nuclear and cytoplasmic fractions, respectively, confirmed the Western blots showed that it is associated with nucleocap- efficiency of the cell fractionation. sids. Nevertheless, we did not know if the loss of ac142 affected nucleocapsid morphogenesis of BV as well as Western blot analysis of purified BV and ODV in association ODV and, in addition, if the occlusion of ODV to form with AC142 functional occlusion bodies was affected. To further analyze these aspects, electron microscopy was performed Since ac142 affects BV production, it was possible that it with thin sections generated from AcBACac142KO-PH-GFP-, could be required for either the regulation of BV production AcBACac142rep-PH-GFP-andAcBACPH-GFP-transfected cells or it could be a structural component that was essential for (Figs.7,8and9). BV processing. Previous proteomic studies have suggested At 60 hpt cells infected with AcBACac142KO-PH-GFP, that AC142 is a structural component of ODV (Braunagel AcBACac142rep-PH-GFP or AcBACPH-GFP all exhibited the et al., 2003). Therefore, to determine if AC142 was also a development of virogenic stroma and occlusion bodies of structural component of BV, virions were produced from similar size. The virogenic stroma is the active site for viral AcBACac142rep-PH-GFP-infected cells, purified and analyzed by DNA replication and nucleocapsid assembly in its periph- Western blot for the presence of AC142. As shown in Fig. 6, ery, termed the ring zone (Figs. 7A, B, C). The most AC142 co-purified with both BV and ODV. BV particles were obvious difference between AcBACac142KO-PH-GFP-and biochemically separated into their nucleocapsid and envelope AcBACac142rep-PH-GFP-orAcBACPH-GFP-transfected cells, fractions and analyzed by Western blot and AC142 localized was that the ac142 knockout virus occlusion bodies did not principally to the nucleocapsid fraction (Fig. 6). With longer contain any ODV (Figs. 7D–F). exposure times a faint band that corresponded to AC142 was The electron-dense rod-shaped nucleocapsids present in repeatedly detected in the envelope fraction (data not shown). AcBACac142KO-PH-GFP-transfected cells were morphologically The efficacy of the fractionation was demonstrated by the indistinguishable from those observed in the AcBACPH-GFP- and control proteins VP39 (nucleocapsid protein) and GP64 (BV- AcBACac142rep-PH-GFP-transfected cells (Figs. 7G–I). Cross- sections and longitudinal sections of these nucleocapsids showed an electron-dense core, indicative of a nucleoprotein core (Fig. 7G), suggesting that the viral DNA genomes were being condensed and packaged into the tubular nucleocapsid structures. Electron-dense nucleocapsids associating with edges of the virogenic stroma, forming bundles, aligning with de novo devel- oped nuclear envelopes, acquiring envelopes and being embedded into the developing occlusion bodies, were easily observed in both AcBACPH-GFP-andAcBACac142rep-PH-GFP-infected cells (Figs. 8A, B). In AcBACac142KO-PH-GFP-transfected cells bundles of nucleocapsids were also clearly observed. On occasion, these Fig. 6. Western blot analysis of AC142 in purified BVand ODVand fractionated bundles of nucleocapsids were seen in close proximity to BV. Sf9 cells were infected with AcBACac142rep-PH-GFP and at 96 hpi BV and envelopes or vesicle-like structures and the developing occlusion ODV were purified by sucrose gradient. Triplicate blots were probed bodies. However, for AcBACac142KO-PH-GFP, these bundles of individually with anti-HA to detect HA-tagged AC142, anti-VP39 to detect nucleocapsids never aligned with the envelopes or vesicles or the nucleocapsid protein VP39 and MAb AcV5 to detect the BV-specific became embedded into occlusion bodies (Figs. 8C, D). envelope protein GP64. BV, budded virus; ODV, occlusion-derived virus; NC, PH-GFP ac142rep-PH-GFP nucleocapsid; E, envelope. The locations of the AC142-, VP39- and GP64- In AcBAC - and AcBAC -transfected specific bands are indicated on the left. cells, most nucleocapsids were mainly concentrated in the 332 C.B. McCarthy et al. / Virology 372 (2008) 325–339

Fig. 7. TEM analysis of nucleocapsid and occlusion body morphogenesis. Sf9 cells transfected with AcBACac142KO-PH-GFP (A, D, G), AcBACac142rep-PH-GFP (B, E, H) and AcBACPH-GFP (C, F, I) were analyzed by TEM. Panels A–C show whole cell low magnification micrographs displaying enlarged nucleus (Nu), virogenic stroma (vs), and polyhedra (P). Panels D, E, and F show higher magnification micrographs of polyhedra and the production of ODV (arrows) in AcBACac142rep-PH-GFP and AcBACPH-GFP but not in AcBACac142KO-PH-GFP. Panels G, H, and I compare nucleocapsids (arrows) and the association of nucleocapsids with de novo envelopes (triangles). C, cytoplasm; nm, nuclear membrane. nucleus, but a significant number was observed in the cytoplasm Discussion and budding at the cytoplasmic membrane (Figs. 9B, C). In contrast, in all the AcBACac142KO-PH-GFP-transfected cells that All baculoviruses sequenced to date contain homologs of 29 were observed up to 60 hpt, the nucleocapsids were consistently core genes, suggesting that they perform key functions in the found only in the nucleus (Fig. 9A). baculovirus life cycle. In this study we have examined the role of Taken together, these observations indicated that deletion of one of these core genes, AcMNPV ac142, and have determined ac142 did not abrogate either nucleocapsid or occlusion body it is an essential gene in the viral life cycle. Using the AcMNPV morphogenesis, but it did preclude virion envelopment and bacmid system (Luckow et al., 1993), an ac142 knockout virus, occlusion. In addition, nucleocapsids were incapable of exiting AcBACac142KO-PH-GFP, was generated and our results show that the nucleus to form BV. ac142 is essential for BV and ODV production. C.B. McCarthy et al. / Virology 372 (2008) 325–339 333

Fig. 8. Nucleocapsid envelopment in AcBACac142KO-PH-GFP-, AcBACac142rep-PH-GFP- and AcBACPH-GFP-transfected cells. Transmission electron micrographs showing the aligning of bundles of nucleocapsids (NC) with envelopes and their subsequent envelopment to form ODV in AcBACac142rep-PH-GFP- and AcBACPH-GFP- infected cells (A, B). Panels C and D show ac142KO-transfected cells and nucleocapsids forming bundles in proximity to envelopes or vesicle-like structures which never become enveloped. Arrows indicate either bundles of nucleocapsids, ODV in the process of becoming enveloped or fully enveloped ODV; triangles show the de novo nuclear envelopes. Nu, nucleus; c, cytoplasm; nm, nuclear membrane; P, polyhedra.

The role of ac142 in the context of an AcMNPV infection unable to propagate after transfection and the infection was in Sf9 cells was analyzed with the ac142 knockout bacmid restricted to the initially transfected Sf9 cells. TCID50 end- that expressed both GFP and polyhedrin. This virus was point dilution, qPCR and Western blot assays confirmed that

Fig. 9. Nuclear and cytoplasmic localization of nucleocapsids in AcBACac142KO-PH-GFP-, AcBACac142rep-PH-GFP- and AcBACPH-GFP-transfected cells. Transmission electron micrographs of AcBACac142rep-PH-GFP- and AcBACPH-GFP (B, C)-transfected cells showing nucleocapsids in the cytoplasm (c) and budding from the plasma membrane (pm) (arrows). In AcBACac142KO-PH-GFP-transfected cells (A) nucleocapsids are observed in the nucleus but none are seen in the cytoplasm. Nu, nucleus; nm, nuclear membrane; pm, plasma membrane. 334 C.B. McCarthy et al. / Virology 372 (2008) 325–339 the deletion of ac142 prevents BV production. Comparison of therefore showing that AC142 is required for both ODVand BV the initiation and levels of viral DNA replication via qPCR in formation. In the AcBACac142KO-PH-GFP-infected cells nucleo- AcBACac142KO-PH-GFPand AcBACPH-GFP-transfected cells, capsids were consistently found only in the nucleus in similar showed that the onset and level of replication in individual numbers to those found in the nuclei of AcBACac142rep-PH-GFP- infected cells are unaffected by deletion of ac142. and AcBACPH-GFP-infected cells, but they were never observed HA-tagged AC142 was expressed from 24 hpi to 96 hpi as a in the cytoplasm or budding from the plasma membrane. 50-kDa protein, in agreement with the predicted size for AC142, AC142 therefore functions at a juncture in the viral life cycle and distributed in both the cytoplasm and nucleus at similar levels. that is required for both BV and ODV. Several other AcMNPV Western blot analysis also determined that AC142 is a structural genes have been shown to affect the production of BVand ODV. component of nucleocapsids of both BV and ODV. This is in Core genes vp1054 (ac54) and vlf-1 (ac77) express nucleocap- agreement with previous proteomic analyses which identified sid proteins which are structural components of both BV and AC142 or its homologs as a component of ODV of AcMNPV, ODV and have been shown to be required for the assembly of HearNPV, and CuniNPV (Braunagel et al., 2003; Deng et al., in nucleocapsids in the nucleus (Olszewski and Miller, 1997a; press; Perera et al., 2007). Thus, AC142 is the tenth nucleocapsid Vanarsdall et al., 2006; Yang and Miller, 1998). gp41 (ac80)is protein found to associate with both AcMNPV BV and ODV, also a core gene that expresses an O-glycosylated protein which along with P78/83 (Russell et al., 1997), VP1054 (Olszewski and affects both BV and ODV but is only a structural component of Miller, 1997a), FP25 (Braunagel et al., 1999), VLF-1 (Yang and ODV (Lee and Miller, 1979; Olszewski and Miller, 1997b; Miller, 1998), VP39 (Thiem and Miller, 1989), BV/ODV-C42 Whitford and Faulkner, 1992). The nucleocapsid protein P78/ (Braunagel et al., 2001), P87 (Lu and Carstens, 1992), P24 83, associated with both BVand ODV, is essential for AcMNPV (Wolgamot et al., 1993) and AC141 (EXON0) (Fang et al., in viability (Possee et al., 1991; Russell et al., 1997; Vialard and press). The fractionation of BV particles indicated that AC142 Richardson, 1993). The baculovirus core gene 38K (ac98) has localized predominantly to the nucleocapsid fraction of BV but been shown to be essential for the formation of normal with a very minor amount consistently detected in the BV nucleocapsids, affecting both BV and ODV production, but it envelope fraction. It is not yet known if there is any functional has not been shown to be a structural component of significance to this minor component. nucleocapsids (Wu et al., 2006). Electron microscopy showed that the nucleocapsids pro- During the preparation of this manuscript, (Vanarsdall et al., duced by AcBACac142KO-PH-GFP were morphologically indis- 2007) also described the deletion of ac142. Similar to the tinguishable from those observed in either AcBACPH-GFP-or results of this study, the ac142 deletion exhibited a single cell AcBACac142rep-PH-GFP-transfected cells. However, only phenotype and did not produce any BV. However, unlike our AcBACPH-GFP and AcBACac142rep-PH-GFP nucleocapsid ends results, they did not detect mature nucleocapsids but instead could be observed aligning with envelope-like structures and found aberrant-looking empty capsid structures and concluded forming enveloped ODV bundles. The deletion of ac142 also that ac142 is required for normal nucleocapsid production. This results in the dramatic effect of producing occlusion bodies that major difference with our findings could be attributed to the fact were completely devoid of ODV. The AcBACac142KO-PH-GFP that the ac142 deletion mutants used in each study are different. nucleocapsids formed non-enveloped bundles that were never The ac142 coding region contains a potential polyA signal for incorporated into the growing occlusion bodies, despite being exon0 and our AcBACac142KO-PH-GFP construct maintains its found in close proximity, indicating that envelopment of the integrity (Fig. 1). In contrast, the ac142 KO construct used by nucleocapsid bundles is a pre-requisite for occlusion. These (Vanarsdall et al., 2007) eliminates this signal for exon0. Since results therefore suggest that AC142 is required for association exon0 codes for another nucleocapsid protein (Fang et al., in of nucleocapsids with envelopes and the subsequent formation press), the phenotype they observe could be due to the fact that of ODV. In agreement with previous studies (Lee and Miller, both exon0 and ac142 expression were affected. Alternatively, 1979, Wu et al., 2006), our results show that the formation of the nucleotides left intact in the 5′ region of our AcBACac142KO- occlusion bodies does not require ODV assembly. PH-GFP construct could putatively express a 174-aa AC142 Homologs of AC142 are found in all baculoviruses suggesting which contains a potential transmembrane domain, whereas the that it is required for a function utilized by all members of the nucleotides left intact in the 5′ region of the construct used by Baculoviridae (Herniou et al., 2003). Our results support this, as (Vanarsdall et al., 2007) could potentially express only a 24-aa the nuclear envelopment and occlusion of nucleocapsids are a AC142. Consequently, if the N-terminus of AC142 is required fundamental process exhibited by all known baculoviruses (Funk for capsid formation, this could account for the different et al., 1997). There are a number of additional core baculovirus phenotypes. The (Vanarsdall et al., 2007) construct does not genes that have been identified in ODV envelopes which include express polyhedrin, so it is unknown whether that deletion also odv-ec27 (ac144), odv-e56 (ac148),andp74 (ac138)(Braunagel affects occlusion bodies. Further studies will be required to et al., 1996; Faulkner et al., 1997; Kuzio et al., 1989). It is possible determine the cause for the intriguing differences in phenotype that interaction between AC142, a nucleocapsid protein, and the between these two ac142 deletions. core viral ODV envelope proteins is required for ODV In conclusion, this study has shown that ac142, one of the 29 envelopment and occlusion. baculovirus core genes, is essential for BV and ODV The deletion of ac142 also completely eliminated BV production. For ODV, AC142 appears to be required for the production and limited the virus to a single cell infection, acquisition of the nuclear de novo synthesized viral envelope by C.B. McCarthy et al. / Virology 372 (2008) 325–339 335 bundles of nucleocapsids to form ODV, which enables their generate bacmids containing the polyhedrin and gfp genes to subsequent occlusion. This is a fundamental process common to enable the analysis of occlusion body morphogenesis and facili- all baculoviruses and additional studies dissecting the structure tate the examination of virus infection, the transfer vector pFAcT- and function of AC142 should provide valuable insight into this GFP was used as previously described (Daietal.,2004). Briefly, key function of the baculovirus life cycle in insect cells. pFAcT-GFP contains polyhedrin, driven by its own promoter, a multiple cloning site and gfp, driven by the OpMNPV ie1 Materials and methods promoter, between two Tn7 transposition excision sites. The genes cloned between these transposition sites are transposed into Viruses and cells the mini ATT region located in the AcMNPV bacmid. pFAcT- GFP was used to generate AcBAC-ac142KO-GFP+PH+ (called The AcMNPV bacmid bMON14272 (Invitrogen Life AcBACac142KO-PH-GFP) and AcMNPV bacmid bMON14272- Technologies) was derived from the AcMNPV strain E2 and GFP+PH+ (called AcBACPH-GFP)(Fig. 1). maintained in DH10B cells as described previously (Luckow To generate an ac142 knockout bacmid repaired with an HA- et al., 1993). Sf9 (Spodoptera frugiperda IPLB-Sf21-AE clonal tagged version of ac142, the repair transfer vector pFAcT-GFP- isolate 9) insect cells were cultured in suspension at 27 °C in OpIE2pA-ac142 HA was constructed as follows. Primers 1017 TC100 medium supplemented with 10% fetal bovine serum. (5′-GGCCTGCAGCTACCCGGGATCTTAGTTTGTATTGT- CATGTT-3′) and 1018 (5′-GGCTCTAGATCGGGTGCG- Construction of ac142 knockout AcMNPV bacmid CACGCGCTTGA-3′), which contain PstIandXbaIsites (underlined sequences), respectively, were used to amplify the An AcMNPV bacmid (bMON14272) was used to generate an OpMNPV IE2 polyA using p2ZOp2F as template. The PCR ac142 knockout virus by recombination in E. coli,aspreviously fragment, digested with the corresponding enzymes, was ligated described (Hou et al., 2002; Lin and Blissard, 2002; Lung et al., to the pFAcT-GFP backbone digested with the same enzymes. 2003; Stewart et al., 2005). A zeocin resistance cassette with The resulting vector was called pFAcT-GFP-OpIE2pA. Primers ac142 flanking regions was amplified using primers 684 (5′- 1019 (5′-GCCCTGCAGACGGCCACCACGCACGCAAA-3′) GGTTTCTTGTTCGACGATGCGTACGTGGATTG- and1021(5′-TCACCCGGGTTAGGCGTAGTCGGG- GAATGGTGTGCGAATGTGTTTCGGATCTCTGCAGC-3′) CACGTCGTAGGGGTATTGTACCGAGTCGGGGAT-3′), and1360(5′-TGCTTGGGCGCGTAGAACGCGTTCA- which contain PstIandXmaI sites (underlined sequences), TAGTGCCTTTCAATTGCAAAAAGTCTCGAGGTC- respectively, were used to amplify ac142 ORF and its promoter GACCCCCCTG-3′) using p2ZeoKS as template. These primers region. An in frame HA-epitope sequence was included in the 3′ contain 51-bp and 50-bp homologous to upstream and down- end of the ac142 ORF (sequence highlighted in italics in primer stream coding regions of ac142 (underlined sequences), respec- 1021). The digested PCR fragment was sub-cloned into pBS+. tively (Fig. 1). The zeocin cassette PCR fragment was gel purified Subsequently ac142 was cloned by digestion with PstIand and electroporated into E. coli BW25113-pKD46 cells, which XmaI, gel purification and ligation into pFAcT-GFP-OpIE2pA contained the AcMNPV bacmid bMON14272. The electropo- digested with the same enzymes. The resulting repair transfer rated cells were incubated at 37 °C for 2 h in 1 ml of LB medium vector was called pFAcT-GFP-OpIE2pA-ac142 HA and was and plated onto agar medium containing 30 μg/ml of zeocin and used to generate AcBAC-ac142KO-GFP+PH+ac142 HA repair 50 μg/ml of kanamycin. Plates were incubated at 37 °C overnight (named AcBACac142rep-PH-GFP). and colonies resistant to both zeocin and kanamycin were selected The polyhedrin and gfp plus AcMNPV bacmids were and further confirmed by PCR. generated by Tn7-mediated transposition as previously described Two different primer pairs were used to confirm that ac142 by Luckow et al. (1993). Briefly, electrocompetent DH10B cells had been deleted from the ac142 locus of the AcMNPV bacmid containing helper plasmid pMON7124 and bacmid DNA were genome. Primers 1013 (5′-GATGATGGCTTTCCTGTACGCT- transformed with the transfer vectors described above. Cells were GAA-3′) and 1014 (5′-CCGATATACTATGCCGATGATT-3′) plated onto agar medium containing 50 μg/ml kanamycin, 7 μg/ml were used to detect the correct insertion of the zeocin gene gentamicin, 10 μg/ml tetracycline, 100 μg/ml X-Gal and 40 μg/ml cassette in the upstream region of the ac142 locus (Fig. 1). IPTG. After 5-h incubation at 37 °C in 1 ml LB, cells were plated Primers 1239 (5′-CTGACCGACGCCGACCAA-3′) and 1361 onto agar medium containing 50 μg/ml kanamycin, 7 μg/ml (5′-AACTCTGCCGGTACACGA-3′) were used to detect the gentamicin, 10 μg/ml tetracycline, 30 μg/ml zeocin, 100 μg/ml X- correct insertion of the zeocin gene cassette in the downstream Gal and 40 μg/ml IPTG. Plates were incubated at 37 °C for 24 h; region of the ac142 locus (Fig. 1). One recombinant bacmid with white colonies were selected and streaked onto fresh plates to the correct PCR confirmations was selected and named AcBAC- verify the phenotype which was then confirmed by PCR. ac142KO-PH−. Transposition events were confirmed by GFP expression and occlusion body formation in bacmid DNA-transfected Sf9 cells. Construction of knockout, repair and positive control AcMNPV bacmids containing polyhedrin and gfp Time course analysis of BV production

The AcMNPV bacmid genome (bMON14272) has an Sf9 cells (1.0×106 cells/35-mm-diameter well of a 6-well occlusion-minus phenotype (Luckow et al., 1993). In order to plate) were transfected with 1.0 μg of each bacmid 336 C.B. McCarthy et al. / Virology 372 (2008) 325–339 construct (AcBACac142KO-PH-GFP, AcBACac142rep-PH-GFP or nated hpt, cells were washed once with 1× phosphate-buffered AcBACPH-GFP). At various hpt, the supernatant containing saline (1× PBS, 137 mM NaCl, 10 mM phosphate, 2.7 mM the BV was harvested and cell debris was removed by KCl, pH 7.4), scraped with a rubber policeman and pelleted centrifugation (8000×g for 5 min). BV production was (8000×g for 5 min). Each pellet was resuspended in 1 ml of determined in duplicate by end-point dilution in Sf9 cells 0.4 M NaOH–125 mM EDTA and incubated at 100 °C for with 96-well microtiter plates. 10 min to disrupt the cells. An aliquot of the lysed cells (50 μl) was treated with 250 μl Tris–Cl pH 7.5 and 20 μg/ml RNAase Plaque assay A at 37 °C for 30 min before adding 80 μg/ml proteinase K and incubating overnight at 50 °C. Total DNA was extracted with Sf9 cells (1.0×106 cells/35-mm-diameter well of a 6-well 300 μl phenol–chloroform and 300 μl chloroform; 50 μl were plate) were transfected with 1.0 μg of each bacmid construct carefully removed from the aqueous layer. Prior to the PCR, 2 μl (AcBACac142KO-PH-GFP,AcBACac142rep-PH-GFP or AcBACPH-GFP). of total DNA from each time point were digested with 40 U of After a 3-h incubation period, the transfection supernatant was DpnI restriction enzyme (New England Biolabs) overnight in removed and the cells were washed with 1 ml of Grace's medium. 20 μl total reaction volume. An aliquot of the digested DNA Cells were overlaid with 2 ml of TC100 medium containing 1.5% (2 μl) was combined with the New England Biolabs SYBR low-melting-point agarose (SeaPlaque) pre-equilibrated to 37 °C, Master Mix (DyNAmo HS SYBR Green qPCR Kit) and the followed by incubation at 27 °C in a humid box for 4 days. The qPCR primers 1483 (5′-CGTAGTGGTAGTAATCGCCGC-3′) cells were observed by fluorescence and light microscopy as from and 1484 (5′-AGTCGAGTCGCGTCGCTTT-3′)ina20μl 48 hpt. reaction. The samples were analyzed in a Stratagene MX4000 qPCR cycler under the following conditions: 1 cycle of 95 °C BV titration using quantitative real time PCR for 15 min; 40 cycles of 95 °C for 30 s, 52 °C for 24 s, 72 °C for 30 s; 1 cycle of 95 °C for 1 min; 41 cycles of 55 °C for 30 s. The The use of qPCR to titrate baculovirus stocks has been results were analyzed by the MX4000 software. previously described (Lo and Chao, 2004). This method is based on the amplification of approximately 150-bp fragments Transcriptional analysis of ac142 located in the coding regions of selected genes. Primers 850 (5′- TTTGGCAAGGGAACTTTGTC-3′) and 851 (5′- For the synthesis of a single-stranded RNA Northern blot ACAAACCTGGCAGGAGAGAG-3′) were designed which probe homologous to ac142, plasmid pBS+-ac142 was amplify a 100-bp genomic fragment of ac126 (chitinase). A constructed. A 606-nt EcoRI-BamHI fragment from within stock of wild-type AcMNPV (2.5×108 pfu/ml), previously the AcMNPV 142 ORF was PCR amplified with upper primer titered by end-point dilution and qPCR, was used as standard 671 (5′-ATAGAATTCTACCGCCGCACCCTTC-3′) and lower and 10-fold diluted. Viral DNA was purified separately from primer 672 (5′-ACACGAGCAGGATCCGATGAAAGTC-3′) these 10-fold diluted solutions as described below. using AcMNPV-E2 DNA as template. This fragment was To prepare DNA for analysis, supernatant containing BV was cloned into EcoRI/BamHI-digested pBS+ to give pBS+-ac142. harvested and cell debris was removed by centrifugation (8000×g The probe was generated by digesting pBS+-ac142 with EcoRI, for 5 min). An aliquot of each of these supernatants (100 μl) was followed by RNA synthesis with T3 RNA polymerase by processed using the Roche High Pure Viral Nucleic Acid Kit. standard methods (Harlow, 1999). An aliquot of each purified DNA sample (8 μl) was RNA was extracted from Sf9 cells (2.0×106/35-mm- combined with the New England Biolabs SYBR Master Mix diameter 6-well plate) infected with AcMNPV at a multiplicity (DyNAmo HS SYBR Green qPCR Kit) and the qPCR primers of infection (MOI) of 10 at the designated times post-infection in a 20 μl reaction. The samples were analyzed in a Stratagene (pi). RNA from each time point was isolated using RNeasy MX4000 qPCR cycler under the following conditions: 1 cycle mini-kit (QIAGEN). Total RNA (5 μg) was separated by of 95 °C for 15 min; 40 cycles of 95 °C for 30 s, 52 °C for 24 s, electrophoresis in a 1% formaldehyde gel, blotted, and 72 °C for 30 s; 1 cycle of 95 °C for 1 min; 41 cycles of 55 °C for hybridized to a α-32P-radiolabed ac142 probe. The blot was 30 s. The results were analyzed by the MX4000 software. visualized by exposure to high performance chemiluminescence film (Amersham Hyperfilm™ ECL). DNA replication using quantitative real time PCR BV partial purification and concentration Viral DNA replication was assayed by real-time PCR, as previously described by (Vanarsdall et al., 2004)with Sf9 cells (1.0×106 cells/35-mm-diameter well of a 6-well modifications. Briefly, this method is based upon the amplifi- plate) were transfected in duplicate with 1.0 μg of each bacmid cation of a 100-bp region of the gp41 gene which contains four construct (AcBACac142KO-PH-GFP,AcBACac142rep-PH-GFP or DpnI restriction sites that allow discrimination between input AcBACPH-GFP). At 120 hpt, medium was harvested and BVs and replicated DNA. To prepare total DNA for analysis, Sf9 were purified as previously described (Oomens and Blissard, cells (1.0×106 cells/35-mm-diameter well of a 6-well plate) 1999). Briefly, supernatants were cleared of cell debris (2000×g were transfected with 1.0 μg of bacmid DNA (AcBACac142KO- for 20 min) at room temperature in a GS-6R centrifuge. PH-GFP, AcBACac142rep-PH-GFP or AcBACPH-GFP) and at desig- Supernatant (3 ml) was loaded onto a 25% sucrose cushion and C.B. McCarthy et al. / Virology 372 (2008) 325–339 337 centrifuged at 80,000×g for 90 min at 4 °C in an SW41 Ti rotor. concentrated by centrifugation (10,000×g for 30 min at 4 °C) BV pellets were resuspended in 39.6 μlof250mMTris–Cl (pH and the pellet was resuspended in 10 mM Tris–HCl pH 7.4. The 7.8) and 0.4 μl of 100× protease inhibitor cocktail. An appropriate pelleted nucleocapsids were dissolved in 10 mM Tris–HCl pH amount of 4× protein sample buffer (PSB) (400 mM Tris–Cl, pH 7.4. 6.8; 8% w/v sodium dodecyl sulphate (SDS); 400 mM Polyhedra and ODV were purified from cells infected with dithiothreitol; 20% v/v glycerol; 0.1% bromophenol blue) was AcBACac142rep-PH-GFP, harvested at 4 days pi, as described added and the samples were placed at 100 °C for 10 min. One previously (Braunagel and Summers, 1994). quarter of the AcBACPH-GFP and AcBACac142rep-PH-GFP samples and the totality of the AcBACac142KO-PH-GFP sample were Western blot analysis analyzed by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Protein samples from the nuclear and cytoplasmic fractionation, purified BV and ODV and the BV nucleocapsid and Cellular localization of AC142 by nuclear and cytoplasmic envelope fractions, were mixed with appropriate volumes of fractionation 4× PSB and incubated at 100 °C for 10 min. Protein samples were electrophoresed in 10% SDS-PAGE gels according to Sf9 cells (1.0×106 cells/35-mm-diameter well of a 6-well Laemmli (1970) with a Bio-Rad Mini-Protean II apparatus plate) were infected with AcBACac142rep-PH-GFP at an MOI of 5. and transferred to Millipore Immobilon-P membrane with a At 6, 12, 24, 48, 72 and 96 h post-infection (hpi), cells were Bio-Rad liquid transfer apparatus, according to the manufac- washed with 1× PBS, scraped with a rubber policeman and turers' recommended protocols. Western blot hybridizations pelleted (8000×g for 5 min). The cell pellets were resuspended were performed by standard methods (Harlow and Lane, in 0.1 ml NP-40 lysis buffer (10 mM Tris–HCl pH 7.9, 10 mM 1988). The blots were probed with one of the following NaCl, 5 mM MgCl2, 1 mM dithiothreitol, 0.5% v/v NP40), antibodies: (i) mouse monoclonal HA antibody (Covance, gently mixed and kept on ice for 5 min. Nuclei were pelleted by HA11), 1:1000; (ii) mouse monoclonal OpMNPV VP39, centrifugation at 1000×g for 3 min. The supernatant containing 1:1000 (Pearson et al., 1988); (iii) mouse monoclonal GP64 the cytosolic fraction was transferred to a new tube and mixed AcV5 antibody, 1:500 (Hohmann and Faulkner, 1983); or (iv) with an equal volume of 2× protein sample buffer (PSB) mouse monoclonal IE1 antibody, 1:5000 (Ross and Guarino, (200 mM Tris–HCl pH 6.8; 4% SDS; 20% v/v glycerol; 1997). After incubation with a 1:10,000 dilution of peroxi- 200 mM dithiothreitol; 0.05% bromophenol blue). The nuclei dase-conjugated secondary antibody, signals were detected were resuspended in the original volume of lysis buffer and using the Enhanced Chemiluminescence System (ECL; mixed with an equal volume of 2× PSB. The nuclear and Amersham). cytoplasmic fractions were incubated at 100 °C for 10 min and analyzed by 10% SDS-PAGE electrophoresis and Western Transmission electron microscopy (TEM) blotting. Sf9 cells (1.0×106 cells/35-mm-diameter well of a 6-well Purification of BV and ODV plate) were transfected with 1.0 μg of each bacmid construct (AcBACac142KO-PH-GFP, AcBACac142rep-PH-GFP or BVs were purified from Sf9 cells infected with AcBACPH-GFP). At 20, 42 and 60 hpt, the supernatant was AcBACac142rep-PH-GFP (MOI 0.1) as described previously removed, cells were washed once with 1× cacodylate buffer (Braunagel and Summers, 1994; IJkel et al., 2001; Wang (0.1 M NaCl; 0.05 M cacodylate pH 7.5) and then fixed in et al., 2002). Four days pi, 150 ml of media were collected 2.5% glutaraldehyde in 0.1 M Na cacodylate for 30 min. The and centrifuged at 8000×g in a Beckman JA-25.50 rotor. cells were then washed twice with 1× cacodylate buffer and The supernatant was then centrifuged at 100,000×g at 4 °C fixed with 1:1 osmium tetroxide:cacodylate buffer for 30 min, (Beckman SW 28 rotor; 28,000 rpm) to pellet the BV. The washed once with 1× cacodylate buffer and stained with 2% BV pellet was resuspended in 0.4 ml of 0.1× TE, loaded on uranyl acetate for 30 min. After dehydration through a series of a25–60% sucrose gradient and centrifuged at 100,000×g 30–100% ethanol washes, cells were embedded in Spur resin. (Beckman SW41 Ti rotor; 28,000 rpm) for 90 min. The BV Ultra thin sections were obtained and subsequently stained band was collected, washed twice by dilution in 1× PBS with 1% uranyl acetate and Sato's lead (Takagi et al., 1990). and centrifuged at 100,000×g for 30 min at 4 °C (Beckman Images were obtained using a Hitachi transmission electron SW41 Ti rotor; 28,000 rpm). The virus pellet was microscope. resuspended overnight in 200 μl 0.1× TE at 4 °C. In a 250 μl reaction, 250 μg of BV were incubated in 1.0% Acknowledgments NP-40 and 10 mM Tris–HCl pH 8.5 at room temperature for 30 min with gentle agitation. The solution was then layered onto We give special thanks to Les Willis for excellent a 4 ml 30% (w/v) glycerol/10 mM Tris–HCl pH 8.5 cushion and technical support and Michael Weis for his expertise and centrifuged at 150,000×g for 60 min at 4 °C (Beckman SW60 help with TEM. This study was supported by the Canadian rotor; 34,000 rpm). The envelope proteins were recovered from Crop Genomics Initiative from Agriculture and Agri-Food the top of the cushion, trichloroacetic acid (TCA) precipitated, Canada. 338 C.B. McCarthy et al. / Virology 372 (2008) 325–339

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