Identification of Autographa californica ... - Journal of Virology

JOURNAL OF VIROLOGY, Nov. 2011, p. 11664–11674 0022-538X/11/$12.00 doi:10.1128/JVI.05275-11 Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Vol. 85, No. 22

Identification of Autographa californica Nucleopolyhedrovirus ac93 as a Core Gene and Its Requirement for Intranuclear Microvesicle Formation and Nuclear Egress of Nucleocapsids䌤 Meijin Yuan, Zhenqiu Huang, Denghui Wei, Zhaoyang Hu, Kai Yang,* and Yi Pang State Key Laboratory of Biocontrol, Sun Yat-sen University, Guangzhou 510275, China Received 1 June 2011/Accepted 24 August 2011

Autographa californica nucleopolyhedrovirus (AcMNPV) orf93 (ac93) is a highly conserved uncharacterized gene that is found in all of the sequenced baculovirus genomes except for Culex nigripalpus NPV. In this report, using bioinformatics analyses, ac93 and odv-e25 (ac94) were identified as baculovirus core genes and thus p33-ac93-odv-e25 represent a cluster of core genes. To investigate the role of ac93 in the baculovirus life cycle, an ac93 knockout AcMNPV bacmid was constructed via homologous recombination in Escherichia coli. Fluorescence and light microscopy showed that the AcMNPV ac93 knockout did not spread by infection, and titration assays confirmed a defect in budded virus (BV) production. However, deletion of ac93 did not affect viral DNA replication. Electron microscopy indicated that ac93 was required for the egress of nucleocapsids from the nucleus and the formation of intranuclear microvesicles, which are precursor structures of occlusionderived virus (ODV) envelopes. Immunofluorescence analyses showed that Ac93 was concentrated toward the cytoplasmic membrane in the cytoplasm and in the nuclear ring zone in the nucleus. Western blot analyses showed that Ac93 was associated with both nucleocapsid and envelope fractions of BV, but only the nucleocapsid fraction of ODV. Our results suggest that ac93, although not previously recognized as a core gene, is one that plays an essential role in the formation of the ODV envelope and the egress of nucleocapsids from the nucleus.

The Baculoviridae family encompasses a diverse group of insect-specific DNA viruses that have been reported worldwide from more than 600 host species and predominantly from insects of the orders Lepidoptera, Hymenoptera, and Diptera (13). A typical characteristic of baculoviruses is a biphasic infection process with the production of two virion phenotypes, the budded viruses (BVs) and the occlusion-derived viruses (ODVs). Although BVs and ODVs have a common nucleocapsid structure and carry identical genetic information, they differ in the composition of their envelopes, which parallel their different functional roles in the virus life cycle (5, 35, 38). During the early phase of infection, nucleocapsids egress through the nuclear membrane, migrate across the cytosol, and obtain their envelopes from plasma membranes modified by viral gene products to form BVs which are responsible for spreading infections between susceptible insect tissues and between cells in cell culture (41). Later in infection, the nucleocapsids are retained within the nucleus and acquire their envelopes from viral induced intranuclear membrane-derived microvesicles to form ODVs which can initiate primary infection in the midgut epithelium of infected insects and are required for the horizontal transmission of infection among insect hosts. The mature ODVs are then occluded within a proteinaceous crystal matrix to form occlusion bodies (OBs).

* Corresponding author. Mailing address: State Key Laboratory of Biocontrol, Sun Yat-sen University, Guangzhou 510275, China. Phone: 86(0)20 84036809. Fax: 86(0)20 84037472. E-mail: yangkai @mail.sysu.edu.cn. 䌤 Published ahead of print on 31 August 2011.

Baculovirus genomes are covalently closed, double-stranded DNAs that vary in size from approximately 80 to 180 kbp and encode 90 to 180 open reading frames (ORFs). More than 54 baculovirus genomes have been sequenced to date according to the National Center for Biotechnology Information (NCBI; (http://www.ncbi.nlm.nih.gov/genomes/GenomesGroup .cgi?taxid⫽10442)). Autographa californica nucleopolyhedrovirus (AcMNPV) was the first baculovirus genome to be completely sequenced and is now the archetypal species of the Baculoviridae. Based on phylogenetic evidence, genomic composition, and morphological characteristics, the family Baculoviridae is divided into four genera: Alphabaculovirus (lepidopteran-specific NPVs), Betabaculovirus (lepidopteranspecific Granuloviruses), Gammabaculovirus (hymenopteranspecific NPVs), and Deltabaculovirus (dipteran-specific NPVs) (16). Despite the diversity in the genetic content present in different baculovirus genomes, a set of 31 genes present in all of the sequenced baculovirus genomes has been identified and assigned as the baculovirus core genes; p33 (ac92) is one of these genes (13, 16, 27, 33). Ac92 is associated with both BV and ODV (4, 7, 30, 32, 44). The deletion of ac92 affects BV production (30, 44). Sequence-based and functional analyses showed that Ac92 has a flavin adenine dinucleotide-linked sulfhydryl oxidase activity (24). In addition to these core genes, there are 31 genes common to all of the sequenced lepidopteran-specific baculoviruses, and 12 of these genes are conserved in all of the sequenced baculoviruses except for the dipteranspecific NPVs (16). However, it is very likely that there are homologs of some of the lepidopteran virus core genes in the dipteran-specific and hymenopteran-specific NPVs but that the

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ROLE OF Ac93 IN INTRANUCLEAR MICROVESICLE FORMATION

genes are too highly divergent to be identified by BLAST searches (1, 13, 18, 33). Homologs of ac93 have been identified in all of the sequenced baculovirus genomes, with the exception of an NPV virus pathogenic for the dipteran Culex nigripalpus (CuniNPV) (16). Analyses with the InterProScan program revealed that Ac93 homologs constitute a functionally unknown DUF682 baculovirus protein family (IPR007773), and sequence-based queries showed that Ac93 does not have any significant sequence similarity to any other proteins or motifs in protein sequence databases. odv-e25 (ac94) is an integral ODV envelope protein (14, 34) and has been identified only in lepidopteran-specific baculovirus genomes and not in hymenopteran-specific NPVs or CuniNPV (16). In this research, we identified the ac93 homolog in CuniNPV and odv-e25 homologs in both hymenopteran-specific NPVs and CuniNPV through the inspection of the 54 sequenced baculoviruses. Moreover, p33, ac93, and odv-e25 group into a cluster and remain in the same relative position in all of the baculovirus genomes. These results indicate that ac93 and odv-e25, although previously not recognized as baculovirus core genes, are core genes and that p33, ac93, and odv-e25 constitute a core gene cluster. To investigate the functional role of ac93 in the baculovirus life cycle, an ac93 knockout virus was constructed via homologous recombination in the AcMNPV bacmid system. Our data show that ac93 was required for intranuclear microvesicle formation and the egress of nucleocapsids from the nucleus, thereby affecting BV production and ODV envelopment. Immunofluorescence microscopy analyses showed that, in late infection, Ac93 concentrates toward the cytoplasmic membrane in the cytoplasm and in the ring zone in the nucleus. Western blot analyses of purified virions and their nucleocapsid and envelope fractions showed that Ac93 was associated with the nucleocapsid fraction of both BV and ODV and the envelope fraction of BV. MATERIALS AND METHODS Cells and viruses. Sf9 insect cells were derived from the fall armyworm, Spodoptera frugiperda (39), and cultured at 27°C in TNM-FH medium (Invitrogen Life Technologies) supplemented with 10% fetal bovine serum, penicillin (100 ␮g/ml), and streptomycin (30 ␮g/ml). Bacmid bMON14272, which contains an AcMNPV genome, was propagated in Escherichia coli strain DH10B (25). Titers of BV were determined by a 50% tissue culture infective dose (TCID50) endpoint dilution assay in Sf9 cells (31). Total RNA preparation, RT-PCR, and 5ⴕRACE (5ⴕ rapid amplification of cDNA ends) analysis. Sf9 cells were infected with AcMNPV at a multiplicity of infection (MOI) of 10 TCID50. Total cellular RNA was isolated using an RNeasy minikit (Qiagen), according to the manufacturer’s instructions, at various time points postinfection (p.i.) and digested using an RNase-free DNase set (Qiagen). Reverse transcription-PCR (RT-PCR) was performed with an RNA PCR kit (v3.0; TaKaRa) using 2.0 ␮g of total RNA as the template for each time point. Synthesis of first-strand DNA complementary to the mRNA (cDNA) was carried out using avian myeloblastosis virus reverse transcriptase and oligo(dT) primers according to the manufacturer’s instructions. The ac93-specific primers Ac93P1 (5⬘-ATGGCGACTAGCAAAACGATCG-3⬘) and Ac93P2 (5⬘-TTAATTTACA ATTTCAATTCC-3⬘) were used for PCR amplification to detect the transcription of ac93. The 5⬘RACE procedure was performed using a second-generation 5⬘/3⬘RACE kit (Roche) with 1 ␮g of purified total RNA isolated from AcMNPV-infected Sf9 cells at 24 h p.i. An ac93-specific primer, Ac93SP1 (5⬘-GCTTGCTCCTGTTTG AGTTCAG-3⬘), and a nested primer, Ac93SP2 (5⬘-GGTCGTCCGTAAGACA TTCTGT-3⬘), were used for cDNA synthesis and PCR amplification, respectively. The obtained PCR products were gel purified and cloned into a pMD18-T vector (TaKaRa). Five clones were selected for sequencing.

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Generation of the ac93 knockout AcMNPV bacmid. An ac93 knockout AcMNPV bacmid was generated through ET homologous recombination, as previously described (47). First, a transfer vector in which the ac93 locus region was replaced with the chloramphenicol resistance gene (Cm) for antibiotic selection in E. coli was constructed. A 517-bp fragment homologous to the 5⬘ region of the ac93 ORF (AcMNPV nucleotides [nt] 79177 to 79693) was PCR amplified from bMON14272 using the primer pair of Ac93-US-1 (5⬘-GGTACC CTCTTGGGCTGGCACGAACG-3⬘, with the KpnI site underlined) and Ac93US-2 (5⬘-GGATCCGCACCAAGTGTTTGTATACT-3⬘, with the BamHI site underlined). A 588-bp fragment homologous to the 3⬘ region of the ac93 ORF (AcMNPV nt 79755 to 80342) was PCR amplified from bMON14272 using the primer pair Ac93-DS-1 (5⬘-CTGCAGGACATATGTGCATTTGGTCGA-3⬘, with the PstI site underlined) and Ac93-DS-2 (5⬘-AAGCTTGCGCGTCATGG TTACATTAC-3⬘, with the HindIII site underlined). The PCR products were digested with the corresponding enzymes and cloned into the pUC18-Cm plasmid (43), to generate the ac93 knockout transfer vector, pUC-US-Cm-DS. This transfer vector was digested with KpnI and HindIII, and the resulting linear ⬃2.1-kbp fragment containing the Cm cassette and ac93 flanking region was gel purified and resuspended in distilled water at a final concentration of 200 ng/␮l. Homologous recombination between the Cm gene and the linear 61-bp ac93 fragment was performed as previously described (43). The resulting ac93 knockout AcMNPV bacmid was designated bMON14272-ac93KO. To confirm the absence of the ac93 gene and its replacement with the Cm gene in bMON14272-ac93KO, the recombinant region was PCR amplified with the primer pair Ac93-US-1 and Ac94P2 (5⬘-GAGATAGGCGGTTTGTTCAG-3⬘) and confirmed by sequence analysis. Construction of the ac93 knockout, repair, and positive control AcMNPV bacmids containing polyhedrin and gfp. The ac93 knockout, the repair and the positive control AcMNPV bacmids containing the polyhedrin and egfp (enhanced green fluorescence protein gene, referred to as gfp in the present study) were constructed by site-specific transposition, as previously described (47). The pFB1-PH-GFP construct containing AcMNPV polyhedrin and gfp (43) was transformed into electrocompetent DH10B cells containing the pMON7124 helper plasmid and bMON14272-ac93KO or electrocompetent DH10B cells containing the pMON7124 helper plasmid and bMON14272 to generate the ac93 knockout bacmid (ac93KO) or the positive control bacmid (AcWT), respectively. To generate an ac93 repair bacmid tagged with hemagglutinin (HA), a donor plasmid (pFB1-Ac93HA-PH-GFP) was constructed. The gene fragment was PCR amplified from the AcMNPV bacmid using the Ac93RP1 (5⬘-GAATTCC TCTTGGGCTGGCACGAACG-3⬘, with the EcoRI site underlined) primer, which annealed 300 bp upstream of the ac93 ATG to include its native promoter region, and primer Ac93RP2 (5⬘-ACTAGTTTAAGCGTAATCTGGTACGTC GTATGGGTAATTTACAATTTCAATTCCA-3⬘, with the SpeI site underlined), which annealed just upstream of the ac93 native stop codon and also contained additional DNA encoding an in-frame HA epitope sequence. The fragment containing the AcMNPV ac93 native poly(A) signal was also PCR amplified from the AcMNPV bacmid using the primer pair Ac93RP3 (5⬘-ACT AGTAACAAATCATGTGGGGAATC-3⬘, with the SpeI site underlined) and Ac93RP4 (5⬘-CTGCAGCCTTGTTGGAGCCCTCTTTG-3⬘, with the PstI site underlined). The PCR products were digested with their corresponding enzymes and cloned into pFB1-PH-GFP to generate the donor plasmid, pFB1-Ac93HAPH-GFP. The donor plasmid was then transformed into electrocompetent DH10B cells harboring the pMON7124 helper plasmid and bMON14272ac93KO to generate the ac93 repair bacmid, ac93RepHA. The recombination products were confirmed by PCR, and the correct recombinant bacmids were electroporated into E. coli DH10B cells and screened for tetracycline sensitivity to ensure that the isolated bacmids were free of helper plasmids. Bacmid DNA was extracted and purified using a Qiagen large-construct kit and quantified by determining the optical density. Analysis of viral growth curve. Sf9 cells (106 cells/35-mm-diameter well of a six-well plate) were transfected in triplicate with 1.0 ␮g of the constructed bacmid (ac93KO, ac93RepHA, or AcWT) using the Cellfectin liposome reagent (Invitrogen Life Technologies) or infected in triplicate with BVs at an MOI of 5. At the indicated time points, the supernatants containing the BVs were harvested, and cell debris was removed by centrifugation (3,000 ⫻ g for 10 min). Titers of BV were determined by a TCID50 endpoint dilution assay in Sf9 cells (31). To further detect whether there was noninfectious progeny BVs produced in ac93KO, quantitative real-time PCR (qPCR) was performed to titrate the baculovirus stocks, as previously described (23, 26). The qPCR product corresponded to a 100-bp region of the AcMNPV essential gene, gp41 (36). A stock of wild-type (WT) AcMNPV, which was previously titrated using the TCID50 assay, was 10-fold diluted and used as a standard. Supernatants containing the BVs were harvested, and cellular debris was removed by centrifugation. An

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aliquot of each of supernatant (100 ␮l) was processed using a Roche High-Pure viral nucleic acid kit. A 10-␮l aliquot of each purified DNA sample was mixed with 12.5 ␮l of Hot Start PCR Master Mix III (Chaoshi-Bio) and the qPCR primers in a 25-␮l reaction volume. The PCR was performed using an iQTM5.0 machine (Bio-Rad) under the following conditions: 95°C for 15 min and 45 cycles of 95°C for 30 s, 60°C for 20 s, and 72°C for 20 s. Quantitative analysis of viral DNA replication. To analyze viral DNA replication, qPCR was performed as previously described, with some modifications (36). Bacmid gp64KO was used as a noninfectious control as the deletion of gp64 results in a virus that is unable to propagate infection from cell to cell and may provide a more accurate comparison (29, 37). The qPCR product corresponded to a 100-bp region of the AcMNPV gp41 gene. The four DpnI restriction sites that allow discrimination between input, bacterially derived bacmid DNA and replicated DNA are located in this region (36). Sf9 cells were transfected in triplicate with ac93KO or gp64KO bacmid DNA, and cells were collected at various time points posttransfection (p.t.). Total DNA was purified from each sample using a Universal genomic DNA extraction kit (TaKaRa) and resuspended in 150 ␮l of double-distilled H2O. Prior to PCR, 5 ␮l of total DNA from each time point was digested with 20 U of DpnI restriction enzyme (NEB) overnight in a 50-␮l reaction volume. A 10-␮l aliquot of the digested DNA was mixed with 12.5 ␮l of Hot Start PCR Master Mix III (Chaoshi-Bio) and the qPCR primers in a 25-␮l reaction volume, and the PCR was performed as described above. The number of viral DNA genome copies within each sample was calculated by using a standard curve generated from a dilution series of AcMNPV bacmid DNA. Transmission electron microscopy (TEM). Sf9 cells (106 cells/35-mm-diameter dish) were transfected with 1.0 ␮g of AcWT, ac93KO, or ac93RepHA. At 48 and 72 h p.t., the cells were dislodged with a rubber policeman and centrifuged at 3,000 ⫻ g for 10 min. Cells were then fixed, dehydrated, embedded, sectioned, and stained as described previously (21). Samples were visualized with a JEOL JEM-1400 transmission electron microscope at an accelerating voltage of 120 kV. Time course analysis of Ac93 expression. Sf9 cells were infected with ac93RepHA at an MOI of 10. At the indicated time points p.i., the cells were washed twice with phosphate-buffered saline (PBS) and collected by centrifugation at 1,000 ⫻ g for 5 min. Equal numbers of pelleted cells were resuspended in PBS with an equal volumes of 2⫻ protein sample buffer (0.25 M Tris-Cl [pH 6.8], 4% sodium dodecyl sulfate [SDS], 20% glycerol, 10% 2-mercaptoethanol, and 0.02% bromophenol blue) and boiled at 100°C for 10 min. The samples were resolved by SDS–10% PAGE, electrophoretically transferred to nitrocellulose transfer membranes (Schleicher & Schuell), and then probed with a monoclonal HA antibody (1:1,000; Abcam) according to the manufacturer’s instructions. A goat anti-mouse horseradish peroxidase (HRP) antibody (1:5,000; Zymed Laboratories, Inc.) was used as the secondary antibody. Proteins were visualized using an enhanced chemiluminescence system (ECL; Amersham Biosciences) according to the manufacturer’s instructions. The apparent molecular masses of the bands were calculated by using Quantity One 1-D analysis software (Bio-Rad). Immunofluorescence. Cells were processed for immunofluorescence microscopy as previously described (8), with some modifications. Briefly, Sf9 cells on coverslips were infected with ac93RepHA at an MOI of 10. At 18, 24, 36, 48, and 72 h p.i., the supernatants were removed. After one wash with PBS, the cells were fixed with 3.0% paraformaldehyde and 0.1% glutaraldehyde for 30 min, washed three times in PBS for 10 min each, and permeabilized in 0.1% Triton X-100 in PBS for 15 min at room temperature. The cells were then blocked for 1 h in blocking buffer (PBS supplemented with 2% bovine serum albumin) and incubated with a mouse monoclonal anti-HA antibody (1:100; Abcam) for 1 h. After the incubation, the cells were washed three times in blocking buffer for 10 min each time, followed by 1 h of incubation with an Alexa 488-conjugated goat anti-mouse secondary antibody (1:500; Invitrogen/Molecular Probes). The cells were then washed three times in blocking buffer, stained with propidium iodide (PI; Invitrogen/Molecular Probes), and examined using a Leica TCS SP5 laserscanning confocal microscope with the same parameter settings. BV and ODV purification. BVs and ODVs were purified and fractionated into envelope and nucleocapsid fractions as previously described (5), with minor modifications. Briefly, Sf9 cells were infected with ac93RepHA at an MOI of 0.1. At 72 h p.i., 400-ml supernatants containing BVs were harvested from ten 175-cm2 flasks and centrifuged twice at 8,000 ⫻ g to remove the cell debris. Infected cells were supplemented with fresh culture medium and harvested at 6 days p.i. for the isolation of polyhedra. The BVs in the supernatants were pelleted by centrifugation at 100,000 ⫻ g (Beckman SW40 rotor) for 90 min at 4°C. The pellets were resuspended in 0.1⫻ TE (10 mM Tris [pH 7.4], 1.0 mM EDTA), overlaid onto a 25 to 56% (wt/vol)

J. VIROL. continuous sucrose gradient, and centrifuged at 100,000 ⫻ g (Beckman SW40 rotor) for 90 min at 4°C. The virus band was collected, 1:4 diluted with 0.1⫻ TE, and centrifuged at 100,000 ⫻ g (Beckman SW40 rotor) for 90 min at 4°C. The BV pellets were resuspended in 0.1⫻ TE and stored at ⫺20°C. Polyhedra were isolated from infected cells collected above and purified as described by Braunagel and Summers (5). The concentration of the purified polyhedra was measured with a counting chamber. ODVs were purified as previously described (5, 42), with modifications. Briefly, 2 ⫻ 109 polyhedra were incubated in polyhedra lysis buffer (0.1 M Na2CO3, 0.166 M NaCl, and 0.01 M EDTA [pH 10.5]) at 37°C for 15 min, and undissolved polyhedra were removed by low-speed centrifugation for 5 min (500 ⫻ g). The supernatant was layered onto a 25 to 56% (wt/vol) continuous sucrose gradient and centrifuged at 100,000 ⫻ g (Beckman SW40 rotor) for 90 min at 4°C. The virus bands were collected, washed by dilution in 0.1⫻ TE, centrifuged at 50,000 ⫻ g (Beckman SW40 rotor) for 60 min at 4°C, resuspended in 0.1⫻ TE, and stored at ⫺20°C. BVs and ODVs were fractionated into envelope and nucleocapsid preparations as previously described (5). Immunoblotting was performed as described above with monoclonal HA antibody or one of the following primary antibodies according to the manufacturer’s instructions: (i) monoclonal GP64 AcC6 antibody (1:3,000; eBioscience), (ii) polyclonal antibody against AcMNPV ODVE25 (1:2,000) (40), or (iii) polyclonal antibody against AcMNPV VP39 (1:1,000) (20). A goat anti-rabbit HRP antibody (1:5,000; Amersham Biosciences) or goat anti-mouse HRP antibody were used as secondary antibody.

RESULTS ac93 is a core gene. To date, 54 baculovirus genomes have been sequenced and analyzed according to the NCBI database (http://www.ncbi.nlm.nih.gov/genomes/GenomesGroup.cgi?taxid ⫽10442). A total of 50 of these baculoviruses are pathogenic to lepidopteran hosts and three, including Neodiprion abietis NPV (NeabNPV), N. lecontei NPV (NeleNPV), and N. sertifer NPV (NeseNPV), are able to infect sawflies (Hymenoptera), whereas only CuniNPV has a mosquito (Diptera) host. According to previous analyses, there are 31 core genes, including p33 (ac92), that are conserved among all of the baculoviruses sequenced thus far (33). ac93 and odv-e25 (ac94) have not been identified as core genes. ac93 was found in all sequenced lepidopteran and hymenopteran baculovirus genomes but not in the dipteran baculovirus CuniNPV, and homologs of odve25 were found in all of the sequenced lepidopteran baculovirus genomes but not in those of the hymenopteran baculoviruses or CuniNPV (16). In lepidopteran baculoviruses, p33, ac93, and odv-e25 are grouped into a cluster and are found in the same position relative to each other and in the gene order p33, ac93, and odv-e25 (Fig. 1A). p33 is located upstream of ac93, and ac93 is found upstream of odv-e25. The transcription of ac93 and odv-e25 occurs in the same orientation but in the opposite direction of p33 (Fig. 1A). A careful analysis of the three sequenced hymenopteran-specific baculovirus genomes showed that neab10, nele18, and nese26, which are contiguous and cluster with the ac93 homologs neab09, nele17, and nese25, respectively, are in the same relative transcript orientation and similar in size to odv-e25 (Fig. 1A). Analysis of the CuniNPV genome showed that cuni13 and cuni15 are adjacent to the p33 homolog, cuni14 (the gene order for CuniNPV in GenBank [AF403738] is cuni14, cuni13, and cuni15) (1), and have the same transcript orientation as ac93 and odv-e25 (Fig. 1A). Moreover, the predicted molecular masses of their proteins are similar to Ac93 and ODV-E25, respectively. The gene family of cuni13, cuni15, and the ORFs adjacent to p33 in Neodiprion are unknown. Although the entire sequences of Neab10, Nele18, Nese26, and Cuni15 exhibit little homology to ODV-E25, a hydropho-

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FIG. 1. Evolutionary conservation of ac93. (A) Schematic map of the p33, ac93, and odv-e25 gene cluster. AcMNPV, NeleNPV, and CuniNPV are represented for lepidopteran-, hymenopteran-, and dipteran-specific baculoviruses, respectively. The relative positions and orientations of these three genes are conserved in all sequenced baculovirus genomes to date. The number of predicted amino acids is indicated above each ORF. (B and C) Amino acid sequence alignment of the ODV-E25 (Ac94) and Ac93 homologs. Ten representative sequences were selected and aligned using CLUSTAL X 1.83 and edited with GeneDoc software. Black shading denotes 100% conservation. Dark gray and light gray shading represents 80 and 60% conservation, respectively. Rules used to assign conservation are denoted as follows: A ⫽ G ⫽ S ⫽ T, V ⫽ L ⫽ I ⫽ M ⫽ F ⫽ Y ⫽ W, N ⫽ Q ⫽ D ⫽ E, and R ⫽ K ⫽ H (3). Transmembrane domain (TM) predictions were made using the TMpred program (http://www.ch .embnet.org/software/TMPRED_form.html). (B) Amino acid sequence alignment of the N terminus of the ODV-E25 homologs. The predicted TM found in all of the homologs is boxed. (C) Alignment of part of the amino acid sequences of the Ac93 homologs that show the predicted TM found in CuniNPV (solid box, TM1) and some other Ac93 homologs (dashed line box, TM2).

bic N-terminal sequence, which includes a predicted transmembrane (TM) domain, is highly conserved among them (Fig. 1B). The hydrophobic N-terminal sequence of ODV-E25 has been identified as an inner nuclear membrane-sorting motif (INM-SM), which is sufficient to traffic fusion proteins to intranuclear membranes and the ODV envelope (14). In addition, the predicted molecular masses of Neab10, Nele18, Nese26, and Cuni15 (24.967, 25.106, 25.239, and 25.117 kDa, respectively) are in the range of ODV-E25 (23.599 to 26.062 kDa). The presence of the INM-SM, the highly consistent genetic organization, and the molecular masses of the proteins are consistent with Neab10, Nele18, Nese26, and Cuni15 being ODV-E25 homologs and odv-e25 representing a baculovirus core gene. A comparison of the predicted amino acid sequences of ac93 homologs and cuni13 revealed that cuni13 was weakly homologous to ac93 homologs. When an amino acid sequence alignment was performed using CLUSTAL W at PBIL (Pole Bioinformatique Lyonnais; http://npsa-pbil.ibcp.fr/cgi-bin/npsa _automat.pl?page⫽npsa_clustalw.html), Cuni13 and Ac93 showed 16.05% identity, 24.07% strong similarity, 13.58% weak similarity, and 46.3% difference. However, these scores were very close to those of the Nele17/Ac93 comparison, which were 14.53, 25.58, 11.63, and 48.26%, respectively. In addition, the level of amino acid conservation between CuniNPV and the lepidopteran baculovirus ORFs only ranged from 18 to 54% (1). Meanwhile, Cuni13 retains the distinctive hydropathic profile of Ac93 homologs, including two possible TM domains. One possible TM domain is quite conserved between Ac93 homologs and Cuni13 (Fig. 1C, solid box, TM1). Although the TMpred scores of other Ac93 homologs were low in this region, this domain might be a TM region because the

TMpred scores of Cuni13, Neab09, and Nele17 were 506, 444, and 444, which were similar to the threshold limit value of 500. Another possible TM region predicted in most Ac93 homologs is relatively conserved and hydrophobic in Cuni13, even though the TMpred score of Cuni13 was 125 (Fig. 1C, dashed line box, TM2). In addition, the predicted molecular mass of Cuni13 (16.413 kDa) is quite similar to that of the Ac93 homologs (16.532 to 21.430 kDa). The similarity in the hydropathic profiles and the molecular masses of the protein and the presence of a highly conserved p33-ac93-odv-e25 gene cluster indicated that Cuni13 is an Ac93 homolog and that ac93 is another previously unidentified baculovirus core gene. ac93 transcripts in Sf9 cells after AcMNPV infection. As an initial characterization of ac93, the temporal expression was examined using total RNA extracted from AcMNPV-infected Sf9 cells at different time points by RT-PCR analysis and 5⬘RACE analysis. The RT-PCR analyses showed that the 486-bp ac93-specific transcripts were detectable at 9 h p.i., increased in abundance by 12 h p.i., and remained detectable up to 72 h p.i. (Fig. 2A). No signals were obtained when reverse transcriptase was not added prior to the PCR step (data not shown), which indicated no possible contamination of the AcMNPV DNA. A search of the 200 nt 5⬘ to the predicted start codon of the ac93 ORF (ATG) showed that there are two contiguous late promoter TAAG elements within ATAAGTAAG located at positions ⫺129 and ⫺133 upstream of the ATG. 5⬘RACE analysis indicated that the ac93 mRNA initiated at the fourth A of this sequence, indicating that the downstream promoter element (⫺129) was used for transcription of Ac93 (Fig. 2B). Construction of the ac93 knockout and repair AcMNPV bacmids. Using the commercially available WT AcMNPV

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FIG. 2. Transcriptional analysis of ac93 in AcMNPV-infected Sf9 cells. (A) RT-PCR analysis of ac93 transcripts. Total RNAs were extracted from AcMNPV-infected Sf9 cells at the designated time points. The size of the PCR products is indicated on the right. (B) 5⬘RACE analysis of the ac93 transcriptional start site. The sequence derived from 5⬘RACE analysis is shown below the AcMNPV genome. The late promoter, GTAAG (boxed), and the transcriptional start site (arrow) are shown. The translational start codon (ATG) is indicated as ⫹1. Primer Ac93SP2 is underlined.

bacmid, bMON14272, an ac93-null mutant, bMON14272ac93KO, was constructed via the ␭ Red recombination system, as previously described (2, 22). To avoid affecting the transcription of adjacent genes, 217 bp of the 5⬘ end and 208 bp of the 3⬘ end of the ac93 coding region were retained. The remaining 61-bp coding sequences were deleted and replaced with a Cm gene for antibiotic selection in E. coli (Fig. 3). PCR analysis of the recombinant region with primer pair Ac93-US1/Ac94P2 (Fig. 3) and further sequence analysis confirmed the correct deletion of ac93 and the correct insertion of the Cm gene in bMON14272-ac93KO (data not shown). To examine the effect of the ac93 deletion on OB morphogenesis and to facilitate the examination of virus infection, the polyhedrin and gfp genes were transposed into the polyhedrin locus of bMON14272-ac93KO to generate ac93KO (Fig. 3). As a positive control, AcWT was also generated by inserting polyhedrin and gfp into the polyhedrin locus of bMON14272 (Fig. 3). To confirm that the phenotype resulting from the ac93 knockout was not due to genomic effects, we constructed a repair bacmid, ac93RepHA, which contained the ac93 ORF driven by its native promoter with an HA epitope-encoding sequence at the 3⬘ terminus, in addition to the polyhedrin and gfp sequences (Fig. 3). ac93 is critical for BV production. To determine the effect of ac93 deletion on viral replication, Sf9 cells were transfected with ac93KO, ac93RepHA, or AcWT. Transfected cells were monitored with fluorescence microscopy. No differences in the numbers of GFP-positive cells were observed among these three viruses at 24 h p.t., which indicated relatively equal levels and efficiencies of transfection (Fig. 4A). By 72 h p.t., widespread fluorescence was observed in ac93RepHA- and AcWTtransfected cells. However, there was no detectable increase in the number of fluorescent cells in the ac93KO-transfected cells (Fig. 4A), which suggested that the ac93 knockout bacmid was unable to produce infectious BVs to initiate secondary infection. Light microscopy analyses revealed OBs in infected cells when all of the three constructs were used, and no differences in the numbers of cells containing OBs were observed at 48 h p.t. (data not shown). However, by 96 h p.t., most of the ac93RepHA- and AcWT-transfected cells contained OBs, whereas the number of the ac93KO-transfected cells containing OBs showed no increase (Fig. 4A). The bacmid transfec-

tion experiments showed that deletion of ac93 led to a defect in the production of infectious BVs but did not affect OB formation. To further assess the effect of ac93 deletion on viral replication, Sf9 cells were transfected with each bacmid DNA, and

FIG. 3. Construction of the ac93 knockout and repair AcMNPV bacmids. A 61-bp fragment of the ac93 ORF was replaced by a chloramphenicol resistance gene (Cm) via ET homologous recombination in E. coli to generate bMON14272-ac93KO. Correct deletion and insertion were confirmed by PCR with the primer pair Ac93-US-1/ Ac94P2 and sequencing. The relative positions of these primers are shown on the diagram. The upper part of the figure is a schematic diagram of AcWT, which was generated by inserting polyhedrin (polh) and gfp gene into the polh locus of bMON14272. The lower part of the figure shows the genes inserted into the polyhedrin locus of bMON14272-ac93KO by Tn7-mediated transposition to generate ac93KO and ac93RepHA. In ac93RepHA, the ac93 ORF is under the control of its native promoter and tagged with an in-frame HA epitope sequence (indicated as a black triangle) at the 3⬘ terminus.

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FIG. 4. Viral replication analysis in Sf9 cells. (A) Microscopy analyses. Fluorescence microscopy shows the progression of viral infection in Sf9 cells transfected with AcWT, ac93RepHA, or ac93KO from 24 to 72 h p.i. Light microscopy shows the formation of occlusion bodies in AcWT-, ac93RepHA-, or ac93KO-transfected cells at 96 h p.i. (B) Virus growth curves as determined by TCID50 endpoint dilution assays. For the transfection growth curves, Sf9 cells were transfected with each bacmid DNA. The supernatants were then harvested at the indicated time points p.t., and the titers were determined using TCID50 assays. For the infection growth curves, cells were infected at an MOI of 5 for each virus, and the supernatants were harvested at the selected time points for the titer assay. Each data point was determined from the average of three independent transfections or infections, and error bars represent the standard deviations. (C) BV production independent of virion infectivity was determined by quantifying the number of viral genomes via real-time PCR analysis of the supernatants harvested from ac93KO- or AcWTtransfected Sf9 cells at the designated time points. Each value represents the average of three independent transfections, and the error bars indicate the standard deviations.

the BV titers were determined by the TCID50 endpoint dilution assay at selected time points. As expected, the supernatants from Sf9 cells transfected with ac93RepHA or AcWT revealed a steady increase in virus production, and the ac93 repair virus showed similar growth kinetics in titer to the WT virus (Fig. 4B). No virus was detected in the supernatants from ac93KO-transfected cells at any time points up to 120 h p.t. (Fig. 4B). The BV titers of the supernatants prepared from cells infected with BVs obtained from ac93RepHA- or AcWTtransfected cells were also determined using the TCID50 assay. The resulting growth curves derived from the supernatants of

ac93RepHA- or AcWT-infected cells also revealed a steady increase in virus production, and their growth kinetics were similar (Fig. 4B). Thus, these data confirmed that the deletion of ac93 led to a defect in infectious BV production. Because this defect was rescued by insertion of the ac93 gene into the polyhedrin locus of the ac93-null bacmid, this defect was not due to genomic effects at the site of the deletion. Because the TCID50 assay determines the production of only infectious BVs, the BV titers were also tested by qPCR analysis for detection of copies of viral genomes regardless of infectivity. The present study determines whether ac93KO pro-

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FIG. 5. Real-time PCR analysis of viral DNA replication. Sf9 cells were transfected with each bacmid DNA (ac93KO or gp64KO). At the designated time points, total intracellular DNA was extracted, digested with the restriction enzyme DpnI to eliminate input bacmid DNA, and analyzed by real-time PCR. The y axis value indicates the number of viral DNA genome copies within each sample. The graph shows the results of three independent replication assays, with error bars indicating the standard deviations.

duced any noninfectious, viral DNA containing BVs. Sf9 cells were transfected with ac93KO and AcWT, and BV production was analyzed at various time points p.t. (Fig. 4C). Due to the bacmid transfection, there was a detectable background of viral genomes present at all of the time points analyzed. The growth curves showed that there was no increase in BV production detected above the background in ac93KO-transfected cell supernatants at any time points up to 96 h p.i. As expected, AcWT generated a pronounced increase in BV production (Fig. 4C). In combination (Fig. 4), these findings indicated that ac93 is required for BV production. ac93 is not required for viral DNA replication. To determine whether the lack of BV production in ac93KO-transfected cells was due to a defect in AcMNPV DNA replication, the levels of viral DNA synthesis in bacmid-transfected cells were monitored by qPCR analyses (Fig. 5). A gp64 knockout bacmid, gp64KO, a mutation that has been reported to prevent the propagation of infections from cell to cell but that does not affect viral DNA replication, served as a noninfectious positive control (29, 37, 47). Total intracellular DNA was harvested from the same number of bacmid-transfected cells at designated time points and treated with DpnI to eliminate all of the input bacmid DNA prior to PCR amplification. When DNA replication levels were analyzed over a 96-h time course, cells transfected with the ac93 knockout bacmid and the gp64 knockout bacmid exhibited a comparable level of viral DNA replication throughout the time course, with DNA synthesis reaching a plateau at 72 h p.t. for both viruses. This result indicated that the rate and level of viral DNA synthesis in cells transfected with these two bacmids were parallel and the deletion of ac93 did not affect viral DNA replication in Sf9 cells. Therefore, the defect in BV production in ac93 knockout virus must be due to some other reason(s). ac93 is critical for nuclear egress of nucleocapsids and intranuclear microvesicle formation. To further investigate the barrier to BV production in the ac93 knockout virus and to analyze whether the deletion of ac93 has any effect on virus

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morphogenesis, electron microscopy analyses were performed on ac93KO-, ac93RepHA-, or AcWT-transfected cells. As expected, cells transfected with the control bacmid, ac93RepHA, showed the typical characteristics of AcMNPV infection at 48 and 72 h p.t., such as a net-shaped virogenic stroma (VS) inundated with electron-dense rod-shaped nucleocapsids (Fig. 6Aa), nucleocapsids budding through the nuclear membrane and the cytoplasmic membrane (Fig. 6 Ab), numerous virus-induced intranuclear microvesicles emerging and nucleocapsids aligning with membranous profiles end-on and acquiring their envelopes (Fig. 6Ac), and polyhedra containing mature virions in the ring zone (Fig. 6 Ad). Cells transfected with AcWT showed characteristics similar to those transfected with ac93RepHA (data not shown). In ac93KO bacmid-transfected cells, a well-defined VS and abundant electron-dense rod-shaped nucleocapsids (Fig. 6 Ba) were also observed in the center of nuclei, and the nucleocapsids were morphologically indistinguishable from those observed in AcWT-transfected (data not shown) or ac93RepHAtransfected (Fig. 6 Aa) cells. However, all of the nucleocapsids were observed in nuclei, and no nucleocapsids were found budding from the nuclear membrane to the cytoplasm or budding through the cytoplasmic membrane (Fig. 6 Bb). Although nucleocapsids accumulated at the ring zone, no virus-induced intranuclear microvesicles were observed, and though bundles of nucleocapsids were observed these were not enveloped to form ODVs (Fig. 6 Bc). Consequently, even though the shape and size of the polyhedra were comparable to those in WT bacmid-transfected (data not shown) or repair bacmid-transfected (Fig. 6 Ad) cells, the polyhedra observed in the ac93KO bacmid-transfected cells did not appear to contain ODVs (Fig. 6 Bd). Taken together, these observations indicated that the deletion of ac93 did not affect the formation of VS, nucleocapsids, or polyhedra. However, it did affect the egress of nucleocapsid from the nucleus, intranuclear microvesicle formation, and subsequent BV formation, ODV envelopment, and embedding of ODVs into polyhedra. ac93 is a late gene. To determine whether the HA tag fused to the C terminus of Ac93 was detectable and to analyze the temporal expression of Ac93 in virus-infected cells, ac93RepHA-infected cells were collected at designated time points and analyzed by immunoblotting with a monoclonal anti-HA antibody. An immunoreactive band of ⬃18 kDa, the predicted molecular mass of ac93, was first detected at low levels at 12 h p.i., increased in intensity by 18 h p.i., and persisted at 96 h p.i. (Fig. 7). The expression time course of ac93 was the same as for vp39, a late gene (data not shown). This result confirmed that ac93 is a late gene, which is consistent with the results of the transcription analyses. Ac93 is localized in both the cytoplasm and nuclei of AcMNPV-infected cells. To further characterize the function of Ac93 in the baculovirus life cycle, the subcellular localization of Ac93 was analyzed by immunofluorescence microscopy. Cells infected with AcWT or ac93RepHA were fixed and analyzed by immunofluorescence using an HA antibody (Fig. 8). As a negative control, AcWT-infected cells showed only background level fluorescence, which demonstrated the specificity of the HA antibody and no interference from the GFP signal (Fig. 8). In ac93RepHA-infected cells, at 18 h p.i., Ac93 was

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FIG. 6. TEM analyses of Sf9 cells transfected with ac93KO or ac93RepHA DNA. (A) Cells transfected with ac93RepHA. (a) Electron-dense nucleocapsids in the virogenic stroma (VS). (b) Nucleocapsids budding from the nuclear membrane (nm) or cytoplasmic membrane (cm) (white arrows). (c) Intranuclear microvesicles (triangles) and nucleocapsids aligning with the membranous profiles end-on (black arrow) in the ring zone. (d) Polyhedra with embedded virions. (B) Cells transfected with ac93KO. (a) Normal-looking nucleocapsids in the VS. (b) A portion of a cell showing a lack of nucleocapsids residing in the cytoplasm or budding at the cytoplasmic membrane. (c) Bundles of nucleocapsids in the ring zone with no evidence for the presence of intranuclear microvesicles or mature virions. (d) Polyhedra devoid of embedded virions. Nu, nucleus. Scale bar, 500 nm.

predominantly localized within the nucleus, with low levels of Ac93 detected within the cytoplasm. By 24 h p.i., Ac93 became condensed at the periphery of the virogenic stroma (stained red by PI, which stains DNA), which corresponded to the intranuclear ring zone. By 36 h p.i., the Ac93 in the cytoplasm was more concentrated toward the cytoplasmic membrane. In

FIG. 7. Time course analysis of Ac93 expression. Sf9 cells were infected or mock infected with ac93RepHA at an MOI of 10. At the indicated time points, the cells were collected, resolved by SDS–10% PAGE, and analyzed by immunoblotting with a monoclonal anti-HA antibody. Mi, mock-infected cells. The numbers to the left indicate the molecular masses (in kilodaltons) of protein standards.

the nucleus, Ac93 was mainly localized to the ring zone (Fig. 8). This localization pattern remained at 48 h p.i., but with higher levels of expression. By 72 h p.i., the Ac93 signal in both the cytoplasm and the nucleus was lower (Fig. 8). These findings indicated that Ac93 was localized to both the cytoplasm and the nucleus and concentrated near the cytoplasmic membrane and ring zone. Ac93 is localized to both BV and ODV. Since Ac93 affects both BV and ODV morphogenesis, it is necessary to gain insight into whether Ac93 is associated with virions. Sf9 cells were infected with ac93RepHA, and virions were purified and fractionated into envelope and nucleocapsid fractions. HAtagged Ac93 was detected by Western blotting with monoclonal anti-HA antibody. Ac93 was detected in both BV and ODV, as shown in Fig. 9. Meanwhile, Ac93 was associated with both nucleocapsid and envelope fractions of BV, but only the nucleocapsid fraction of ODV. As a control, the nucleocapsid

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FIG. 8. Subcellular localization of Ac93 as demonstrated by immunofluorescence. Sf9 cells were infected with ac93RepHA at an MOI of 10. At the designated time points, cells were fixed, immunostained with a mouse monoclonal anti-HA antibody to detect Ac93HA, and visualized using an Alexa 488-conjugated goat anti-mouse antibody (green). PI was used to identify the nucleus and DNA-rich regions (red). Cells infected with AcWT were used as a control.

protein VP39, the BV envelope specific protein GP64, and the BV/ODV envelope-associated protein ODV-E25 were probed in the identical samples, and all of these three proteins were only detected in the expected fractions (Fig. 9). These results indicated that Ac93 was associated with both BV and ODV and sublocalized to both nucleocapsid and envelope fractions of BV, but only the nucleocapsid fraction of ODV. DISCUSSION Previous studies have shown that the three contiguous genes p33, ac93, and odv-e25 are highly conserved in baculoviruses, but only p33 had been identified as a core gene, and both ac93 and odv-e25 had not been identified as core genes by BLAST analysis (13, 16). We demonstrated here that ac93 and odv-e25 were conserved among all baculoviruses and were baculovirus core genes, thus increasing the total number of core genes from 31 to 33 and designating p33-ac93-odv-e25 as the third core gene cluster (13, 27, 33). The failure to identify ac93 and

FIG. 9. Western blot analysis of Ac93 in purified and fractionated virions. BVs and ODVs were purified from ac93RepHA-infected cell supernatants and pellets, respectively, and fractionated into envelope and nucleocapsid fractions. Proteins were detected by immunoblotting with anti-HA antibody to detect HA-tagged Ac93, anti-VP39 antibody to detect the major capsid protein VP39, anti-GP64 antibody to detect the BV envelope specific protein GP64, and anti-ODV-E25 to detect the BV/ODV envelope-associated protein ODV-E25. NC, nucleocapsid fraction; E, envelope fraction.

odv-e25 homologs in hymenopteran and dipteran baculoviruses by BLAST searches might simply be because they are too highly divergent to be identified by this technique (1, 13, 18, 33). The amino acid conservation between CuniNPV and lepidopteran baculovirus ORFs or hymenopteran and lepidopteran baculoviruses ORFs is low: 18 to 54% between CuniNPV and lepidopteran baculoviruses and 24.9% between NeleNPV and SeMNPV (1, 18). To investigate the functional role of ac93 in the baculovirus life cycle, a recombinant bacmid lacking ac93 was successfully constructed via homologous recombination in E. coli. A repair bacmid containing an HA epitope-coding sequence in the 3⬘ terminus of ac93 was also constructed to rescue the phenotype of the ac93 knockout and to analyze the localization of the ac93 gene product further. Our results showed that ac93 was essential for both BV and ODV formation. Importantly, no intranuclear microvesicles and no egress of nucleocapsids from nuclei were observed in ac93 knockout bacmid-transfected cells. To the best of our knowledge, ac93 is the first baculovirus gene identified that affects both ODV envelope morphogenesis and egress of nucleocapsids from nuclei. Western blot analysis showed that Ac93 is a structural component of both BV and ODV. To our surprise, though Ac93 affected the intranuclear microvesicle formation, it was not detected in the envelope fraction of ODV obtained from three separate purifications and fractionations. It cannot be ruled out that the amount of Ac93 associated with ODV envelope was too low to be detected by immunoblotting. Also, it is possible that Ac93 does not function as a structural component in ODV envelope formation. Braunagel et al. (4) performed an extensive proteomic analysis of AcMNPV ODV and produced a list of 44 ODV-associated viral proteins, but Ac93 was not included in the list. It may be because Ac93 is produced in low abundance and may not be easily detected in AcMNPV ODV preparations, as in ODV nucleocapsid, the concentration of Ac93 is quite low compared to VP39, as shown in Fig. 9. There are some other ODV-associated proteins not identified by

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proteomic analysis of ODV, including 38K, PIF-1, PIF-3, Ac150, EXON0, and Ac96 (8, 9, 35, 42). With the identification of Ac93, there are up to 51 proteins associated with ODV, and among them over 20 proteins are identified to be associated with both AcMNPV BV and ODV (8, 9, 35, 42). The deletion of ac93 affected both BV and ODV morphogenesis, as demonstrated by electron microscopy. Baculoviruses have a biphasic replication cycle. During the early stages of infection, viral DNA replication occurs within a virus-induced specific nuclear region, the VS. The newly replicated viral genomes are prepackaged with the highly basic DNAbinding protein, P6.9, within the stromal matte (46), and the nucleoprotein complexes are subsequently packaged into a lucent capsid shell within the intrastromal spaces to form nucleocapsids (10). These nucleocapsids egress from the nucleus, bud through the plasma membrane, and acquire envelopes to form BVs. Later during infections, the nucleocapsids retained within the nuclear ring zone bundle together and acquire envelopes from intranuclear microvesicles to form ODVs. The resulting ODVs are embedded within a paracrystalline matrix to form OBs (41). In the present study, the qPCR analyses showed that the rate and level of viral DNA synthesis in the ac93KO-transfected cells was similar to those of the gp64KO control, an observation which suggested that ac93 was not involved in viral DNA synthesis. Electron microscopy showed that electron-dense rod-shaped nucleocapsids were present in the intrastromal space of the virogenic stroma of ac93KOtransfected cells and bundled in the ring zone. These nucleocapsids were morphologically indistinguishable from those in the WT or repair bacmid-transfected cells, which indicated that the deletion of ac93 did not affect the condensation and packaging of the newly replicated viral genomes into the empty capsids or the trafficking of the nucleocapsids out of the virogenic stroma. The remaining question is what hinders the nucleocapsids from forming BVs and ODVs in ac93KO-transfected cells? Notably, our data indicated that the deletion of ac93 led to a defect in the intranuclear microvesicle formation, as well as the egress of nucleocapsids from the nucleus. The formation of the intranuclear membrane is thought to be the result of the budding of discrete regions of the INM into the nucleoplasm (6). With regard to the egress of progeny nucleocapsids from the nucleus for exit at the plasma membrane, several routes have been suggested and include exit via nuclear pores, migration into or through the endoplasmic reticulum, or passage through discontinuities in the nuclear membrane (41). However, the most common method of egress observed in the electron microscopy studies of NPV involves a budding process in which nucleocapsids acquire a double membrane vesicle derived from the nuclear membrane (41). Both the formation of the intranuclear membrane and the egress of nucleocapsids through the nuclear membrane require a more fluid nuclear membrane (6, 41). Since no intranuclear microvesicles and no budding process for nucleocapsids through the nuclear membrane were observed, it appears that the barrier for the morphogenesis of BV and ODV in ac93KO-transfected cells was the nuclear membrane. The nuclear envelope of eukaryotic cells is composed of double lipid bilayer membranes, the membrane-connected nuclear pore, and an underlying nuclear lamina network. The nuclear lamina, which is composed of lamin proteins, provides an

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intact meshwork for the structural integrity of the nuclei and also presents a natural barrier against most DNA viruses (11, 19). How then can assembled progeny viral nucleocapsids break this rigid nuclear lamina wall, traverse the INM and outer nuclear membrane, and gain access to the cytoplasm? For herpesviruses, which are large DNA viruses, most data support an envelopment/de-envelopment/re-envelopment model that entails primary envelopment by the budding of capsids at the INM, resulting in the formation of primary virions in the perinuclear space, the envelopes of which then fuse with the outer nuclear membrane to release the capsids into the cytoplasm (17, 28). This hypothetical model claims that the nuclear lamina needs to be dissolved through a kinase-mediated phosphorylation mechanism before viral nucleocapsids can contact the inner leaflet of the INM. Two conserved herpesvirus proteins, which are designated pUL31 and pUL34 for alphaherpesviruses, are required for the nuclear egress of herpesviruses (17). These pUL31/pUL34 homolog pairs are codependent for subcellular localization to the nuclear rim. Furthermore, the interaction between these two proteins and the formation of a complex is required for the local dissolution of the nuclear lamina, the modification of the host cell chromatin, and the efficient release of nucleocapsids from the INM (12, 45). Coexpression of pUL31 and pUL34 from pseudorabies virus results in the formation of vesicles in the perinuclear space that resemble primary envelopes without a nucleocapsid, suggesting that coexpression of only the pUL34 and pUL31 homologs is sufficient to form vesicles and to promote the budding of nucleocapsids from the INM (17, 19). The nucleocapsids of baculoviruses are proposed to egress from the nucleus by budding at the nuclear membrane. Thus, viral proteins similar to pUL31 and pUL34 could also exist in baculoviruses and mediate the egress of nucleocapsids from the nuclei of host cells. The similarity between the budding of vesicles from the nuclear envelope during the egress of herpesvirus nucleocapsids from the nucleus (17) and the budding of INM to form intranuclear microvesicles to serve as envelopes for baculovirus ODVs (6) has prompted a hypothesis that these two processes may share a mechanism. Because Ac93 is essential for both the egress of nucleocapsids from the nuclear membrane and the formation of intranuclear microvesicles, Ac93 might also interact with other viral or cellular proteins to facilitate the egress of nucleocapsids and the budding of INM to form intranuclear microvesicles in a manner similar to pUL31 and pUL34 of herpesviruses. The elucidation of the protein that interact with Ac93, especially Ac76, which is also required for intranuclear microvesicle formation (15), may help to shed light on the mechanism of BV egress from nuclear membrane and morphogenesis of the ODV envelope from the INM. ACKNOWLEDGMENTS We thank Zhihong Hu (Wuhan Institute of Virology) for her generous gift of ODV-E25 polyclonal antiserum. This research was supported by the National Basic Research Program of China (973 Program; no. 2009CB118903), the National Nature Science Foundation of China (no. 30900941), the Hi-Tech Research and Development Program of China (863 Program; no. 2011AA10A204), and the Fundamental Research Funds for the Central Universities (no. 09lgpy39). REFERENCES 1. Afonso, C. L., et al. 2001. Genome sequence of a baculovirus pathogenic for Culex nigripalpus. J. Virol. 75:11157–11165.

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