Characterization of a late gene, ORF67 from Bombyx mori nucleopolyhedrovirus

FEBS Letters 581 (2007) 5836–5842

Characterization of a late gene, ORF67 from Bombyx mori nucleopolyhedrovirus Hui-qing Chena, Ke-ping Chena,*, Qin Yaoa, Zhong-jian Guoa, Ling-ling Wangb b

a Institute of Life Sciences, Jiangsu University, 301# Xuefu Road, Zhenjiang 212013, PR China College of Life Science, Chongqing Normal University, 12# Tianchen Road, Chongqing 212013, PR China

Received 7 August 2007; revised 1 November 2007; accepted 16 November 2007 Available online 29 November 2007 Edited by Hans-Dieter Klenk

Abstract Open reading frame 67 of Bombyx mori nucleopolyhedrovirus (BmORF67) is a homologue of Autographa californica multiple NPV ORF81. The gene is conserved among all baculoviruses and is thus considered a baculovirus core gene. The transcript of BmORF67 was detected at 18–72 h post-infection (p.i.). Polyclonal antiserum raised to a His-BmORF67 fusion protein recognized BmORF67 in infected cell lysates from 24 to 72 h p.i., suggesting that BmORF67 is a late gene. BmORF67 was not detected either in budded viruses or occlusion-derived virus. Immunofluoresence analysis showed that the protein located in the cytoplasm and interacted with host protein actin A3. In conclusion, BmORF67 is a late protein localized in the cytoplasm of infected cells that interacts with host protein.  2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: BmORF67; Transcription; Subcellular location; Pull-down; Co-immunoprecipitation; MALDI-TOF

1. Introduction Baculoviruses consist of a circular double-stranded DNA molecule of about 80–180 kbp. They have been reported worldwide from over 600 host species, mostly from insects of the order Lepidoptera but also from the orders Diptera and Hymenoptera [1]. Baculoviruses are currently subdivided into two genera, the nucleopolyhedroviruses (NPVs) and the granuloviruses (GVs) [2]. However, phylogenetic analyses based on gene and genome sequences suggested at least a third genus comprising the dipteran-specific Culex nigripalpus (Cuni) NPV (CuniNPV) [3]. Phylogenetic studies also indicate that the lepidopteran-specific NPVs can be further subdivided into two subgroups: groups I and II [4]. The infection of baculoviruses is characterized by a biphasic replication cycle during which two virion phenotypes are produced: (i) the budded virus (BV), which initiates secondary infections and spreads the infection throughout the host, and (ii) the occlusion-derived virus (ODV). The ODVs released from the diseased insects spread the infection in the host population by initiating primary infection of midgut epithelial cells [5]. China has a history of over 5000 years in raising silkworms (Bombyx mori L.). At present, over 30 million farmer house* Corresponding author. Fax: +86 511 8791923. E-mail address: [email protected] (K. Chen).

holds are involved in sericultural production in ChinaÕs over 10 provinces. Silkworm viral diseases are major diseases causing great loss in sericulture, among which the nucleopolyhedorosis caused by BmNPV infection is one of the most disastrous. BmNPV is also second in popularity only to Autographa californica nucleopolyhedrovirus (AcMNPV) as a baculovirus expression system. A BmNPV-based baculovirus expression system is a power tool for rapid and high-efficient expression of foreign genes, since the silkworm larvae, which are easy to rear at low cost of production, can be used instead of the cultured cell lines [6]. Since the first baculovirus to be completely sequenced [7], 29 other baculovirus genomes have been reported so far. BmNPV contains a covalently closed circular genome of 128 kbp, about 143 putative open reading frames based on the criterion that the ORF be a single, contiguous, nonoverlapping coding region. Recently, a number of BmNPV genes have been characterized, such as orf79 [8], orf8 [9], orf 68 [10]. But the functions of many other genes still remain unknown, including orf67. Sequence analysis of BmNPV genome revealed that a homologue of Bmorf67 which is one of the conserved or core genes among baculoviruses [11]. In this paper, we described the transcriptional analysis of Bmorf67 gene, demonstrated the expression pattern of BmORF67 protein and characterized the structural and subcellular localization.

2. Materials and methods 2.1. Virus and cell lines BmNPV (Zhenjiang strain) virus was propagated in BmN cells, maintained at 27 C in TC-100 insect medium supplemented with 10% (v/v) fetal bovine serum (Gibco-BRL, Carlsbad). The titration of virus and other routine manipulations were performed according to the standard protocols. 2.2. Computer-assisted sequence analysis The protein sequence was analyzed using ExPASy server (www.expasy.ch) for the prediction of motifs, domains, transmembrane regions and signal peptides [12]. Homologues were explored by using BLASTP searching tool in the updated GenBank/EMBL and SWISS-PROT databases [13,14]. The sequence alignment was carried out with ClustalW (http://www.ebi.ac.uk/clustalw) and edited with Genedoc software (Ver.2.04) (Free Software Foundation, Inc., MA, USA). 2.3. Transcriptional analysis of BmORF67 For the transcriptional analysis, BmN cells were infected with BmNPV at a m.o.i. of 10. Total RNAs from transfected cells were isolated at mock, 0, 3, 6, 12, 18, 24, 48, 72 h post-infection with Trizol

0014-5793/$32.00  2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2007.11.059

H. Chen et al. / FEBS Letters 581 (2007) 5836–5842 reagent (Invitrogen, Carlsbad, USA) according to manufacturerÕs protocol. RNA was dissolved in DEPC-treated water and quantified by optical density measurement at 260 nm. For cDNA synthesis and PCR experiment, the total RNA was first treated with DNase to eliminate any potential genomic DNA contamination. Total RNA (2 lg) from each time point was transcribed by AMV reverse transcriptase and an oligo(dT) primer (Takara, Dalian, China) to synthesize the first strand cDNA. PCR was then carried out using orf67-specific primers of 5 0 -ATGACGACGACGACG-3 0 and 5 0 -TCATCTGTCATACTT-3 0 . 2.4. Prokaryotic expression of BmORF67 and preparation of antibody BmORF67 coding sequence was amplified with primers 5 0 -CGGGATCCATGACGACGACGACG-3 0 (containing the BamHI site) and 5 0 -CCGCTCGAGTCATCTGTCATACTT-3 0 (containing the XhoI site) from the BmNPV genomic DNA by PCR. The Bmorf67 was subcloned into the pET30a(+) expression vector (Novagen, USA) in frame with the N-terminal 6·His tag. The recombinant plasmid, pET-BmORF67, was verified by PCR, restriction analysis and DNA sequencing. The recombinant plasmid was transformed into E. coli BL21 cells and the fusion protein was expressed under induction conditions of 0.6 mM IPTG at 28 C for 10 h. The 6·His-tagged recombinant BmORF67 protein was purified on a Ni2+-NTA column (Novagen) and used to raise polyclonal antibodies in rabbits. The antibody was prepared using standard techniques [15]. Purified BmORF67 protein (about 2 mg) was injected subcutaneously to immunize New Zealand white rabbits in complete FreundÕs adjuvant, followed by two booster injections in incomplete FreundÕs adjuvant within a gap of 2 weeks before exsanguinations. The polyclonal rabbit antibody against BmORF67 was used for immunoassay. 2.5. Temporal expression of BmORF67 in infected BmN cells For the time course analysis, BmN cells were infected with BmNPV at a m.o.i. of 10. Cells were harvested at the designated times (mock, 0, 3, 6, 12, 18, 24, 72) and washed three times with 1· PBS. The protein concentrations of the cell extracts were determined by BradfordÕs method [16]. Cell lysates (20 lg) were analyzed by 10% SDS–PAGE and subsequently subjected to Western blot assay. Western blot was performed as described previously [17]. After SDS–PAGE electrophoresis, proteins were transferred onto a PVDF membrane in cold Towbin buffer (0.025 M Tris, 0.19 M Glycine, 20% methanol). The membranes were blocked in 3% skimmed milk powder in PBST for 1 h followed by incubation with the antiBmORF67 polyclonal antiserum diluted 1:5000 for 1 h at room temperature. Subsequently, the membrane was incubated with a goat anti-rabbit IgG conjugated to HRP diluted 1:5000 for 1 h at room temperature. The signal was detected with a DAB substrate solution. 2.6. Purification of BV and ODV Polyhedra produced in the infected larvae of Bombyx mori were analyzed by electron microscopy and purified as described by Summers and Smith [18]. The ODV was purified by ultracentrifugation on sucrose gradient [19]. Hemolymph-derived BVs were purified from BmNPV-infected larvae as described previously [20] with some modifications. Briefly, 3 days post-infection (p.i.) hemolymph was collected and clarified at 2000 · g for 10 min at 4 C. The supernatant was passed through a 0.45-lm-pore-size filter. BVs in the filtrate were pelleted through a 25–56% (wt/wt) continuous sucrose cushion made up in 0.1· TE by centrifugation at 100 000 · g for 60 min at 4C and resuspended in 0.1· TE. 2.7. Sub-cellular localization analysis of BmORF67 proteins in infected cells Monolayers of BmN cells infected with BmNPV (m.o.i. of 10) at 36 h p.i. were washed with cold PBS, and fixed with 2 ml of 4% paraformaldehyde for 20 min. Cells were then washed three times with PBS and permeabilized with 0.1% Triton X-100 in PBS for 15 min. After washing four times with cold PBS, cells were incubated with BmORF67 polyclonal antiserum (diluted with 3% BSA, 1:50) for 2 h at 37 C. Cells were washed three times with PBS and then incubated with the secondary antibody, FITC-conjugated goat anti-rabbit IgG

5837 (Sigma, USA), for 1 h at 37 C. Background staining was removed by washing with PBS three times. The cells were examined under a Leica confocal laser scanning microscope for fluorescence. The BmNPVinfected cells reacted only with FITC-conjugated goat anti-rabbit IgG as negative controls. 2.8. His pull-down and co-immunoprecipitation analysis For the pull-down assay, BmN cells were lysed by RIPA buffer. The His-BmORF67 fusion protein or the His protein was mixed with the control or BmNPV infected BmN cells for 5 h at 4 C in PBS containing 0.5% NP-40.The mixture was incubated with agarose beads. The beads were washed with wash buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0), and eluted with elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0). The eluted proteins were separated on a 10% SDS–polyacrylamide gel and stained with Commassie brilliant blue. Co-immunoprecipitation experiments were used to study BmORF67 interaction with host cell proteins. 6·106 BmN cells were infected with BmNPV (m.o.i. of 10). The cells were harvested via centrifuged and washed with cold PBS. The cells were lysated by RIPA buffer (50 mM Tris–HCl, pH 7.4, 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, 1 mM NaF) containing protease inhibitors (1 mM PMSF, 1 lg/ml aprotinin, 1 lg/ml leupeptin, 1 lg/ml pepstatin). After 1 h on ice, the lysates were centrifuged for 10 min at 9000 · g. For immunoprecipitation, the cleared extracts were incubated with anti-BmORF67 antibody for 1 h and then incubated with 30 ll of protein A agarose (Santa Cruz. Biotechnology, CA, USA) for 4 h at 4 C. The immunoprecipitates were washed three times in RIPA buffer, resuspended in 30 ll of 2· SDS–PAGE loading buffer, and the samples were heated for 5 min at 100 C, then the supernatant was collected and electrophoresed on a 10% SDS polyacrylamide gel, and stained with silver stain. The anti-BmORF67 antibody incubated only with protein A agarose was used as negative control. 2.9. In gel digestion and peptide analysis by MALDI-TOF The proteins located in the band visualized in the silver stain staining gel were digested with trypsin and analyzed by matrix-assisted laser desorption ionization-time-of flight mass spectrometry (MALDITOF) (Bruker Daltonics, Germany), which generated the peptide sequence in addition to peptide mass information. Masses of tryptic peptides obtained by MALDI-TOF were used as inputs to search corresponding proteins against the database NCBInr, via the program MASCOT.

3. Results The coding region of Bmorf67 is 705 bp in length, which could encode a 234-aa peptide with a predicted molecular weight of 27 kDa. A putative late transcription motif, ATAAG, was found at 57 nts upstream of the start codon, ATG, suggesting that Bmorf67 might be a late transcriptional gene. A T-rich sequence was found 7 nt downstream of the stop codon, which might function as the mRNA transcription termination and polyadenylation signal [21]. Searches of protein databases, GenBank and SWISS-PROT, revealed that BmORF67 is conserved among baculoviruses and is shared by all baculoviruses whose complete genomes have been sequenced so far (Fig. 1). BmORF67 shared amino acid sequence identities ranging from 92% with AcMNPV ORF81 to 22% with CuniNPV ORF106 protein. To identify any similarities to potential biologically significant domains or motifs from the existing protein families, the deduced BmORF67 protein sequence was compared with proteins in the PROSITE database. Four Protein kinase C phosphorylation sites (aa 5–7, aa 16–18, aa 28–30 and aa 228–230), three cAMP- and cGMP-dependent protein kinase phosphorylation sites (aa 17–20, aa 29–32, aa 169–172), two

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Fig. 1. Amino acid sequence alignment of baculovirus BmORF67 homologues. The alignment was edited with GeneDoc software. Two shading levels are set: black for 100% similarity groups and grey for 80% similarity groups. The sources of sequences are: BmNPV (GenBank, NC_001962), AcMNPV (GenBank, NC_001623), RoMNPV (GenBank, NC_004323), CfMNPV (GenBank, NC_004778), OpMNPV (GenBank, NC_001875), EppoNPV (GenBank, NC_003083), CfdefNPV (GenBank, NC_005137), CpGV (GenBank, NC_002816), CrleGV (GenBank, NC_005068), PhopGV (GenBank, NC_004062), AdorGV (GenBank, NC_005038), PlxyGV (GenBank, NC_002593), XecnGV (GenBank, NC_002593), AgseGV (GenBank, NC_005839), SpltNPV (GenBank, NC_003102), SeMNPV (GenBank, NC_002169), AgseNPV (GenBank, NC_ 007921), MacoNPV-A (GenBank, NC_003529), MacoNPV-B (GenBank, NC_004117), HzSNPV (GenBank, NC_003349), HearNPV-G4 (GenBank, NC_002654), HearNPV (GenBank, NC_003094), TnSNPV (GenBank, NC_007383), ChChNPV (GenBank, NC 007151), AdhoNPV (GenBank, NC_004690), NeleNPV (GenBank, NC_005906) and NeseNPV (GenBank, NC_005905).

Casein kinase II phosphorylation site (aa 22–25, aa 32–35) and one N-glycosylation sites (aa 44–47) were found in the BmORF67 amino acid sequences. In addition, no other signif-

icant signal peptide sequence, transmembrane region, nuclear localization signal, or membrane retention signal was searched by protein searching tool.

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3.1. Transcriptional analysis of BmORF67 To determine the time of Bmorf67 transcription, RT-PCR analysis was performed using total RNA isolated from BmNPV-infected host cells as template. A band with an expected size of 719 bp was amplified at 18 h p.i., which was remained detectable up to 72 h p.i. (Fig. 2). 3.2. Expression of BmORF67 and immunodetection of BmORF67 protein in infected cells Expression of a 6·His-BmORF67 fusion in E. coli resulted in the production of a 33-kDa protein. Western blot analysis using anti-His antiserum confirmed that the 33-kDa protein was the fusion protein (data not shown). The purified fusion protein was used to immunize rabbits to produce the specific antiserum against BmORF67. In order to study the expression of the BmORF67, a time course of BmNPV-infected BmN cells were analyzed by Western blot using anti-BmORF67antiserum. A specific immunoreactive band of approximately 27 kDa was first observed at 24 h p.i. and remained detectable up to 72 h p.i. (Fig. 3). These data are consistent with the analysis of transcripts and indicate that BmORF67 was synthesized at a late stage of infection. No immunoreactive band was detected in mock-infected control. The 27 kDa immunoreactive band was in agreement with predicted molecular weight. Fig. 4. Localization of BmORF67 protein in BmNPV BVs, ODVs and BmN cells. BV, ODV, cell lysates samples were separated by SDS– PAGE and analyzed by Western blotting. Lane 1, total proteins of infected cells; lane 2, purified BV; lane 3, purified ODV.

3.3. Localization of the BmORF67 protein in cell, BV and ODV To investigate whether the BmORF67 protein was a structural protein, Western blot analysis of purified BVs, ODVs and BmNPV-infected BmN cells were carried out for immunodetection (Fig. 4). A clear band was detected in the sample of BmNPV-infected cells. In contrast, no band was observed in the BV and ODV samples, indicating that the product of BmORF67 was a non-structurally functional protein. Fig. 2. RT-PCR analysis of the Bmorf67 transcription in mock- and BmNPV-infected BmN cells. Total RNA was isolated from mock- and BmNPV-infected cells at 0, 3, 6, 12, 24, 48 and 72 h p.i. (shown at the top) indicated above the lanes. Size of the transcript is indicated in bp on the left side of the panel.

Fig. 3. Western blot analysis of the BmORF67 protein in BmN cells. The cells were collected at mock, 0, 3, 6, 12, 18, 24, 48, 72 h p.i., and processed for Western blot using anti-BmORF67 antiserum followed by incubated with a goat antirabbit IgG conjugated to HRP. The signal was detected with DAB substrate. Molecular mass standards are shown on the right.

3.4. Cellular distribution of BmORF67 in BmN cells The intracellular localization of BmORF67 was determined by immunofluorescence using anti-BmORF67 antiserum and cells infected with BmNPV. Since BmORF67 protein was first detected at 24 h p.i., we chose time points of 24, 48 and 72 h p.i. for observation. The results revealed that the BmORF67 localized primarily in the cytoplasm and was not detectable in the nucleus (Fig. 5). As control experiment, no obvious fluorescence signal was observed in infected cells reacted with FITC-conjugated goat anti-rabbit IgG (data not shown). 3.5. Proteins associated with BmORF67 To identify BmORF67 associated factors, His pull-down experiments were carried out with the recombinant HisBmORF67 protein. One protein present both in mock-infected and infected cells, with molecular weight of 42.7, was found to associate with His-BmORF67 (Fig. 6). To confirm the result of pull-down experiments, co-immunoprecipitation was performed using anti-BmORF67. Western blot analysis with anti-BmORF67 showed that the 27 kDa protein was BmORF67 (data not shown). The other band was detected to be the protein that bound to BmORF67 (Fig. 7). The Mascot search was performed with carbamidomethyl as the fixed

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Fig. 5. Subcellular localization of BmORF67 in infected cells. BmNPV infected BmN cells were treated with anti-BmORF67 antibody, followed by treatment with FITC-conjugated goat anti-rabbit IgG, and examined in confocal microscope. From the left to the right: bright field, green fluorescence for BmORF67 and the merged images.

Fig. 7. Co-immunoprecipitation to detect the interaction of BmORF67 with cell proteins. 1, negative control; 2, co-precipitation from the BmN cells. Immunoprecipitated protein is indicated with arrows. Fig. 6. His-BmORF67 fusion protein pull-down assay. The potential protein interacting with Ha128 was indicated with arrow. M, protein molecular weight marker; 1, eluted proteins from incubation of the His-BmORF67 protein with the lysate of BmN cells; 2, eluted proteins from incubation of the His-BmORF67 fusion protein incubated with the lysate of BmN cells infected with BmNPV; 3, eluted proteins from the His-BmORF67 protein without incubation with a host cell lysate (negative control).

modification of cysteine and variable N-terminal Gln-pyroGlu. These two proteins were confirmed to be actin A3 (Accession number: CAA28192) by a Mascot score of 100, with 7 peptides matched and 29% amino acid coverage (Fig. 8). The results indicated that BmORF67 interacted with actin in Bomby mori.

4. Discussion Among 29 fully sequenced genomes of baculoviruses in Genbank to date, all of them have a gene homologous to orf67 of

BmNPV. These include all type I and type II nucleopolyhedroviruses (NPV) and granuloviruses (GV). The phylogeny of BmORF67-like proteins (data not shown) represents that of baculovirus quite well [11], implicating the importance of the protein. Homology analysis indicated that BmORF67 has 58–92% and 50–57% identities with groups I and II NPVs BmORF67-like proteins, respectively. The very low identity (22%) of CuniNPV ORF106 with BmORF67 indicated that CuniNPV, which was isolated from a Diptera host, is distantly related to those isolated from the Lepidoptera. To elucidate BmORF67 function, we analyzed the transcription and translation of BmORF67 in BmNPV-infected BmN cells. Transcriptional analysis of RT-PCR showed that Bmorf67 transcription started at 18 h p.i. and remained detectable up to 72 h p.i., suggesting that Bmorf67 is a late gene whose transcription might be initiated in the baculovirus consensus late start site motif ATAAG. Western blot analysis further confirmed that Bmorf67 is a late gene

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Fig. 8. Identification of actin by MALDI-TOF analysis. (A) Peptide sequences identified by mass spectrometry. (B) Amino acid sequences of Bombyx mori actin A3. Matched peptide sequences are shown as bold characters.

detected from 24 to 72 h p.i. by BmORF67 polyclonal antiserum. Western blots showed that the translational product of BmORF67 was 27 kDa which corresponds with the size deduced from the sequence of Bmorf67. These results showed that no major post-translational modifications occurred in the BmORF67 protein, despite the presence of several potential post-modification motifs in BmORF67 amino acid sequence. The BmORF67 protein was not detected in the BV and ODV but was detected in the cell lysates, suggesting that the product of BmORF67 is a non-structurally functional protein. Fluorescent confocal laser scanning microscope examination further confirmed that BmORF67 product was localized in the cytoplasm from 24 to 72 h p.i. Motif analyses did not reveal any signal peptide sequence, transmembrane region, nuclear localization signal, or membrane retention signal, consistent with subcellular localization of BmORF67 in the cytoplasm of infected cells. The similar protein location was found in other baculovirus proteins, such as Ha128 [22]. Different from PP31, Bmorf67 seemed to be excluded from the virogenic stroma which is believed as the site of viral DNA replication [23]. Actin, an important protein expressed in all eukaryotic cells, composed of the microfilament (F-actin) systems of cell cytoskeleton, takes part in many vital activities. Baculovirus infection cycle is regulated by a succession of early and late genes [24,25]. The late phase referred to viral assembly, budding, and maturation of progeny virions [5]. All these important processes depend on or accompanied by the host F-actin cytoskeleton. Previous studies indicated that virus could utilize actin microfilaments of the host for transport to the plasma membrane of infected cells [26,27]. The vaccinia virus was found to move along microtubles and recruit actin tails to support membrane protrusion, and thus facilitate cell to cell spread [28,29]. The electron microscopy studies indicate that budding of the mouse mammary tumor virus occurs via surface protrusions containing actin filaments [30]. In the presence of the inhibitor of actin, normal AcMNPV was not observed to bud from the plasma membrane [31]. Recently, it has been found that IQGAP1 and cortactin, actin-binding proteins, play pivotal roles in virus assembly [32,33]. It has been suggested that the IQGAPs link the virus to the cytoskeleton for trafficking both into and out of the cell [34]. In this study, we found that BmORF67 was located in the cytoplasm and interacted with actin in the late stage of infection. Whether BmORF67 possess the ability to regulate the actin cytoskeleton or virus assembly like IQGAP1 or cortactin is yet to be determined. The further experiments shall be designed and conducted to elucidate them.

Acknowledgements: This work was supported by National Basic Research Program of China (No. 2005CB121000), and National Natural Science Foundation of China (No. 30370773).

References [1] Moscardi, F. (1999) Assessment of the application of baculoviruses for control of Lepidoptera. Annu. Rev. Entomol. 44, 257– 289. [2] Blissard, G., Black, B., Crook, N., Keddie, B.A., Possee, R., Rohrmann, G.F., Theilmann, D.A. and Volkman, L. (2000) Family Baculoviridae, Academic Press, San Diego. [3] Afonso, C.L., Tulman, E.R., Lu, Z., Balinsky, C.A., Moser, B.A., Becnel, J.J., Rock, D.L. and Kutish, G.F. (2001) Genome sequence of a baculovirus pathogenic for Culex nigripalpus. J. Virol. 75, 11157–11165. [4] Herniou, E.A., Luque, T., Chen, X., Vlak, J.M., Winstanley, D., Cory, J.S. and OÕReilly, D.R. (2001) Use of whole genome sequence data to infer baculovirus phylogeny. J. Virol. 75, 8117– 8126. [5] Keddie, B.A., Aponte, G.W. and Volkman, L.E. (1989) The pathway of infection of Autographa californica nuclear polyhedrosis virus in an insect host. Science 243, 1728– 1730. [6] Sehgal, D. and Gopinathan, K.P. (1998) Recombinant Bombyx mori nucleopolyhedrovirus harboring green fluorescent protein. Biotechniques 25, 997–1000, 1002, 1004 passim. [7] Ayres, M.D., Howard, S.C., Kuzio, J., Lopez-Ferber, M. and Possee, R.D. (1994) The complete DNA sequence of Autographa californica nuclear polyhedrosis virus. Virology 202, 586–605. [8] Xu, H.J., Yang, Z.N., Wang, F. and Zhang, C.X. (2006) Bombyx mori nucleopolyhedrovirus ORF79 encodes a 28-kDa structural protein of the ODV envelope. Arch. Virol. 151, 681–695. [9] Kang, W., Imai, N., Kawasaki, Y., Nagamine, T. and Matsumoto, S. (2005) IE1 and hr facilitate the localization of Bombyx mori nucleopolyhedrovirus ORF8 to specific nuclear sites. J. Gen. Virol. 86, 3031–3038. [10] Iwanaga, M., Kurihara, M., Kobayashi, M. and Kang, W. (2002) Characterization of Bombyx mori nucleopolyhedrovirus orf68 gene that encodes a novel structural protein of budded virus. Virology 297, 39–47. [11] Jehle, J.A. et al. (2006) On the classification and nomenclature of baculoviruses: a proposal for revision. Arch. Virol. 151, 1257– 1266. [12] Appel, R.D., Bairoch, A. and Hochstrasser, D.F. (1994) A new generation of information retrieval tools for biologists: the example of the ExPASy WWW server. Trends Biochem. Sci. 19, 258–260. [13] Pearson, W.R. (1990) Rapid and sensitive sequence comparison with FASTP and FASTA. Methods Enzymol. 183, 63–98. [14] Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W. and Lipman, D.J. (1997) Gapped BLAST and PSIBLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402. [15] Harlow, E. and Lane, D. (1988) Antibodies: A Laboratory Manual, 1st edn, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NewYork.

5842 [16] Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem. 72, 248–254. [17] Towbin, H., Staehelin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamidae gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350–4354. [18] Summers, M.D. and Smith, G.E. (1978) Baculovirus structural polypeptides. Virology 84, 390–402. [19] Braunagel, S.C. and Summers, M.D. (1994) Autographa californica nuclear polyhedrosis virus, PDV, and ECV viral envelopes and nucleocapsids: structural proteins, antigens, lipid and fatty acid profiles. Virology 202, 315–328. [20] Palhan, V.B. and Gopinathan, K.P. (1996) Characterization of a local isolate of Bombyx mori nuclear polyhedrosis virus. Curr. Sci. 70, 147–153. [21] Jin, J. and Guarino, L.A. (2000) 3Õ-end formation of baculovirus late RNAs. J. Virol. 74, 8930–8937. [22] An, S.H., Wang, D., Zhang-Nv, Y., Guo, Z.J., Xu, H.J., Sun, J.X. and Zhang, C.X. (2005) Characterization of a late expression gene, Open reading frame 128 of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus. Arch. Virol. 150, 2453– 2466. [23] Guarino, L.A., Dong, W., Xu, B., Broussard, D.R., Davis, R.W. and Jarvis, D.L. (1992) Baculovirus phosphoprotein pp31 is associated with virogenic stroma. J. Virol. 66, 7113–7120. [24] Friesen, P.D. (1997) Regulation of baculovirus early gene expression in: The Baculoviruses (Miller, L.K., Ed.), pp. 141– 166, Plenum, New York, NY.

H. Chen et al. / FEBS Letters 581 (2007) 5836–5842 [25] Lu, A. and Miller, L.K. (1997) Regulation of baculovirus late and very late gene expression in: The Baculoviruses (Miller, L.K., Ed.), pp. 193–216, Plenum, New York, NY. [26] Rey, O., Canon, J. and Krogstad, P. (1996) HIV-1 Gag protein associates with F-actin present in microfilaments. Virology 220, 530–534. [27] Liu, B., Dai, R., Tian, C.J., Dawson, L., Gorelick, R. and Yu, X.F. (1999) Interaction of the human immunodeficiency virus type 1 nucleocapsid with actin. J. Virol. 73, 2901–2908. [28] Cudmore, S., Cossart, P., Griffiths, G. and Way, M. (1995) Actinbased motility of vaccinia virus. Nature 378, 636–638. [29] Smith, G.L., Murphy, B.J. and Law, M. (2003) Vaccinia virus motility. Annu. Rev. Microbiol. 57, 323–342. [30] Damsky, C.H., Sheffield, J.B., Tuszynski, G.P. and Warren, L. (1977) Is there a role for actin in virus budding? J. Cell Biol. 75, 593–605. [31] Hess, R.T., Goldsmith, P.A. and Volkman, L.E. (1989) Effect of cytochalasin D on cell morphology and AcMNPV replication in a Spodoptera frugiperda cell line. J. Invertebr. Pathol. 53, 169–182. [32] Bensenor, L.B., Kan, H.M., Wang, N., Wallrabe, H., Davidson, L.A., Cai, Y., Schafer, D.A. and Bloom, G.S. (2007) IQGAP1 regulates cell motility by linking growth factor signaling to actin assembly. J. Cell Sci. 120, 658–669. [33] Tehrani, S., Tomasevic, N., Weed, S., Sakowicz, R. and Cooper, J.A. (2007) Src phosphorylation of cortactin enhances actin assembly. Proc. Natl. Acad. Sci. USA 104, 11933–11938. [34] Leung, J., Yueh, A., Appah Jr., F.S., Yuan, B., de los Santos, K. and Goff, S.P. (2006) Interaction of Moloney murine leukemia virus matrix protein with IQGAP. Embo. J. 25, 2155–2166.