Characterization of a Late Gene, ORF134, from Bombyx mori Nucleopolyhedrovirus

Objective: ORF134 of Bombyx mori nucleopolyhedrovirus (BmNPV) is a homolog of Autographa californica multiple NPV ORF5, but its function is unknown so far. The aim of this study is to characterize BmNPV ORF134 (Bm134). Methods: Transcription, protein expression and subcellular localization of Bm134 in BmN cells and silkworm midguts were analyzed using real-time polymerase chain reaction (RT-PCR), Western blot and immunofluorescence, respectively. Results: Both the transcription and protein expression of Bm134 were not detected until 24 h post-infection (p.i.) by RT-PCR and Western blot, indicating that Bm134 is a late gene. Western blot revealed that Bm134 encodes an expected 12.4-kDa structural protein that is associated with occlusion-derived virus (ODV), not with budded virus. Immunofluorescence analysis showed that the Bm134 was first detected in the cytoplasm 24 h p.i. and then transported to the nucleus during later infection. Transcripts of Bm134 and the corresponding protein were only detected 48–72 h p.i. in BmNPV-infected larvae of 306, a highly BmNPV-susceptible silkworm strain, but not in the NB strain that is resistant to BmNPV infection. Conclusion: Taken together, our data suggest that Bm134 is a late gene of BmNPV and may function as an ODV structural protein. Copyright © 2010 S. Karger AG, Basel Received: November 30, 2009 Accepted after revision: April 1, 2010 Published online: October 19, 2010 Keping Chen Institute of Life Sciences, Jiangsu University 301 Xuefu Road Zhenjiang 212013 (PR China) Tel./Fax +86 511 879 1923, E-Mail kpchen @ ujs.edu.cn © 2010 S. Karger AG, Basel 0300–5526/11/0543–0113$38.00/0 Accessible online at: www.karger.com/int D ow nl oa de d by : 54 .7 0. 40 .1 1 11 /4 /2 01 7 6: 10 :2 6 P M Lin/Chen/Xia/Yao/Gao/Chen Intervirology 2011;54:113–121 114 BmNPV (T3 strain) genome has been completely sequenced [11] . A number of BmNPV genes have been characterized, such as p95 [12] , DBP [13] , ie1 [14] , BRO [15] , Bm8 [16] , Bm68 [17] , Bm60 [1] , Bm51 [18] , Bm67 [19] , Bm122 [20] , and Bm79 [21] . Nevertheless, the functions of many other genes in the BmNPV genome still remain unknown, including orf134. In this study, we describe the transcription, expression and subcellular localization of the protein product of BmNPV ORF134 (Bm134) in BmN cells, a highly susceptible silkworm strain 306, and a resistant silkworm strain NB. Materials and Methods Viruses, Cells and Insects BmNPV (T3 strain) virus was propagated in BmN cells which were maintained at 27° in TC-100 media supplemented with 10% (v/v) fetal bovine serum (Gibco-BRL, Carlsbad, Calif., USA). The titration of virus and other routine manipulations were performed according to the standard protocol [22] . The silkworm B. mori was inbred in our laboratory. The highly BmNPV-susceptible silkworm strain 306 and the BmNPV-resistant silkworm strain NB were used for this study. Computer-Assisted Sequence Analysis The protein sequence was analyzed using software from ExPASy (www.expasy.ch) for the prediction of motifs, domains, transmembrane regions and signal peptides [23] . Homologs were explored using the BLASTP searching tool in the updated GenBank/EMBL and SWISS-PROT databases [24, 25] . The sequence alignment was carried out with ClustalW (http://www.ebi.ac.uk/ clustalw) and edited with Genedoc [26] . Transcription Analysis First, BmN cells were infected with BmNPV at a MOI of 10. Total RNAs from transfected cells were isolated at mock, 0, 6, 12, 24, 36, 48, 72 h post-infection (p.i.) with Trizo reagent (Invitrogen, Carlsbad, Calif., USA) according to manufacturer’s protocol. RNA was dissolved in diethylpyrocarbonate (DEPC)-treated water and quantified by optical density measurement at 260 nm. Second, larvae of 306 and NB were raised to the 5th instar. The larvae of newly metamorphosed 5th instars were orally fed with 5 l (TCD 50 /ml = 10 9 ) of BmNPV. Total RNA was extracted from the midgut 0, 6, 12, 24, 36, 48 and 72 h p.i. using the Trizol RNA extraction kit (Invitrogen). For cDNA synthesis and PCR, the total RNA was first treated with Dnase I (Takara) to eliminate any potential genomic DNA contamination. Total RNA (2 g) from each time point was transcribed by AMV reverse transcriptase (Takara) and an oligo (dT) primer (Takara) to synthesize the first-strand cDNA according to the manufacturer’s guidelines. Reactions without adding AMV reverse transcriptase were used as negative controls. The coding region of the Bm134 gene was amplified by PCR with two primers: Bm134-F, 5 -TCGCATCTCAACACGACTAT-3 ; Bm134-R, 5 TGTAGTCGGCAGTTCTTTTG-3 . The constantly expressed gene, ActinA3, was used as the internal control. Two primers ActinF (reverse 5 -ggatgtccacgtcgcacttca-3 ) and ActinR (forward 5 -gcgcggctactcgttcactacc-3 ) were designed on the basis of the sequence of the B. mori actin gene (accession No. BMU49854). Quantitative real-time polymerase chain reaction (qRT-PCR) information was obtained with SYBR Premix ExTaq (Takara) using Mx 3000P (Stratagene, San Diego, Calif., USA) for thermal cycling, real-time fluorescence detection and subsequent analysis. The two-step amplification protocol is as following: 1 min at 94°, followed by 40 cycles at 94° for 20 s, 58° for 30 s and 72° for 15 s. The transcript levels of Bm134 were normalized against ActinA3 transcript levels in the same samples. Prokaryotic Expression of Bm134 and Preparation of Antibody The Bm134 coding region was amplified from the BmNPV genomic DNA by PCR using an upstream primer (5 -CG GGATCC ATGTATAGCACGTCAAAAATTAAC-3 ) with a Bam HI site (un derlined) and a downstream primer (5 -CCG CTCGAG TTATACGGTGCATCTGCCAT-3 ) with an Xho I site (underlined). The Bm134 was subcloned into the pET30a(+) expression vector (Novagen, USA) in frame with the N-terminal 6 ! His tag. The recombinant plasmid, pET-Bm134, was verified by PCR, restriction analysis and DNA sequencing. The recombinant plasmid was transformed into Escherichia coli BL21 (DE3) cells for protein expression under the induction conditions of 1 m M isopropylD -1thiogalactopyranoside (IPTG) at 37° for 6 h. The 6 ! His-tagged recombinant Bm134 protein was purified on a Ni 2+ -NTA column (Novagen) and used to raise polyclonal antibodies in rabbits. The antibody was prepared using the standard technique [27] , and was purified with protein A affinity column (Millipore). Mass Spectrometry Analysis The protein Bm134 was separated by 15% SDS-PAGE gel and then protein spots were manually excised from gels. Spots from Coomassie gels were washed with 100 ml of 50% acetonitrile/50 m M ammonium hydrocarbonate (pH 8) [28] . Gel pieces were then dehydrated with acetonitrile and vacuum dried. After rehydration in 10  l of 50 m M ammonium hydrocarbonate (pH 8), samples were incubated in the same buffer containing 0.5  g of porcine trypsin (Promega, France) overnight (16–18 h) at 37°. Peptide fragments from digested proteins were then crystallized with cyano-4-hydroxycinnamic acid as a matrix and subjected to MALDI-TOF (Bruker Daltonics, Germany) for peptide mass fingerprinting. The peak lists were the basis for peptide mass fingerprint analyzed by the Mascot software (Matrix Science; http:// www.matrixscience.com/search_form_select.html). Western Blot Analysis BmN cells were infected with BmNPV T3 strain BVs (MOI 10) at the designated times (0, 6, 12, 24, 36, 48 and 72 h p.i.), and then were washed three times with cold phosphate-buffered saline (PBS; 0.14 M NaCl, 2.7 m M KCl, 10.1 m M Na 2 HPO 4 , 1.8 m M KH 2 PO 4 , pH 7.4). The protein concentrations of the cell extracts were determined by the Bradford method [29] . Cell lysates (20  g) were analyzed by 15% SDS-PAGE and subsequently subjected to Western blot analysis. The 5th-instar larvae of 306 and NB were orally fed with 5  l (TCD 50 /ml = 10 9 ) of BmNPV and the midguts were harvested 12, 24, 36, 48, 72 and 96 h p.i. Protein samples of the midgut were separated on 15% SDS-PAGE and further analyzed by Western blot, as described previously [30] . D ow nl oa de d by : 54 .7 0. 40 .1 1 11 /4 /2 01 7 6: 10 :2 6 P M Characterization of ORF134 from Bombyx mori Nucleopolyhedrovirus Intervirology 2011;54:113–121 115 Preparation of BV and ODV Fifth-instar larvae of 306 and NB were orally fed with 5  l (TCD 50 /ml = 10 9 ) of BmNPV T3 BVs. The infected larvae were fed at 25° for 5 days, and the polyhedra were purified and verified by ordinary microscopy [31] . ODV was released from the occlusions by using alkaline treatment and purified from polyhedra as described by Braunagel and Summers [32] and Caballero et al. [33] . Hemolymph-derived BVs were purified from BmNPV-infected larvae as described previously [34] with minor modifications. Briefly, 3 days p.i. hemolymph was collected and clarified by centrifugation of 3,000 g for 5 min at 4°. The supernatant was further filtered (0.45 m filter, Millipore) and loaded onto a 25–56% (wt/ wt) continuous sucrose cushion in 0.1!TE and centrifuged at 100,000 g for 90 min at 4°. The BV band was collected and diluted in 0.1!TE, which was followed by centrifugation at 100,000 g for 90 min at 4° and resuspended in 0.1 ! TE. Subcellular Localization Analysis of Bm134 Proteins in Infected Cells Monolayers of BmN cells infected with BmNPV (MOI 10) were collected at designated time points (24, 48 and 72 h). The cells were washed three times in PBS and fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. The cells were washed three times and permeabilized with 0.2% Triton X-100 in PBS for 10 min at room temperature, and again washed three times with PBS. The cells were incubated with the antiBm134 antibody (dilution factor 1: 5,000) for 1 h at room temperature. The anti-Bm134 antibody was removed by washing three times with PBS. Then cells were incubated with the secondary antibody, FITC-conjugated goat anti-rabbit IgG (Sigma, USA), and then DAPI (Sigma, USA) for 1 h at 37°. Background staining was removed by washing with PBS three times. The cells were examined and photographed under a Zeiss LSM 5 Live (Carl Zeiss, Germany) confocal laser scanning microscope for f luorescence.