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In the eukaryotic nucleus, the genomic DNA and histones are packaged and organized into a nucleoprotein complex called "chromatin". The fundamental packaging unit of chromatin is the nucleosome, an octamer of histones around which 147 base pairs (bp) of DNA is wrapped twice[21]. The linker histone H1 interacts with both the nucleosome core and the linker DNA, and promotes higher-order folding and compaction of chromatin (reviewed in[24]). Besides assembling DNA into chromatins to form higher-order structures in the eukaryotic nucleus, the histones also play an active role in the regulation of gene transcription through establishing a dynamic molecular interface for transcription factors and RNA polymerases to bind to DNA sequences[27].
Protamines are a group of relatively small (4.0-12.0 kDa) and structurally heterogeneous proteins. A chemical definition of the protamine can be deduced from its sequence composition of ≥40% arginine with a few or no lysine[18]. Protamines reportedly serve as functional counterparts of histones, and the remodeling from a histone-to a protamine-based chromatin will usually result in higher condensation of genomic DNA and gene transcription down-regulation (reviewed in[3]).
Besides cellular chromatin, viral chromatin also exists and plays an important role in the life cycle of many viruses (reviewed in[19]). Viruses such as simian virus 40 (SV40) and polyomavirus which use host enzymes to replicate their DNA tend to use host histones to package viral genomic DNA into virions[4]. Alternatively, for viruses such as adenoviruses using virus-encoded replication machinery, they tend to form viral chromatin via virus-encoded histone-like proteins[28].
Baculoviruses are large double-stranded DNA viruses and among them, Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) is one of the most extensively studied prototypes. The AcMNPV basic DNA-binding protein P6.9 exhibits a protamine-like amino acid composition (44% arginine and no lysine)[29]. By 10 hours post infection (hpi), P6.9 becomes associated with the viral DNA[34]. By 24 hpi, the nucleosome-like structures are completely substituted by subnucleosome-sized DNA fragments of 120 and 90 bp chromatin structure containing exclusively viral DNA[33]. As a protamine-like chromosomal protein, P6.9 was supposed to form a higher condensed chromatin and down-regulate AcMNPV gene transcription. However, Wilson et al provided evidence that the subnucleosome-sized AcMNPV chromatin is sensitive to micrococcal nuclease digestion, which is correlated to transcriptional activity[33]. This unexpected phenotype implies that P6.9's role in regulation of viral gene transcription is probably distinct from the protamines or other protamine-like proteins. However, the detailed role of P6.9 in regulation of AcMNPV gene transcription remains unknown.
Previous research demonstrates that P6.9 is essential for viral nucleocapsid assembly, but it has no influence on viral genome replication[31]. In the present study, the epigenetic role of P6.9 in regulation of AcMNPV gene transcription was characterized. We found that P6.9 as a protamine-like chromosomal protein up-regulates viral gene transcription at 12-24 hpi, which is opposite to the protamines or other protamine-like proteins that usually down-regulate gene transcription.
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Sf9 cells were cultured at 27℃ in Grace's media containing 10% fetal bovine serum (FBS) (Gibco). AcMNPV recombinant bacmids were derived from bMON14272 (Invitrogen)[20], and propagated in Escherichia coli strain DH10B. The AcMNPV p6.9-nulled bacmid AcBacΔp6.9 was provided by Prof. Just Vlak, Wageningen University, the Netherlands[31]. And the bacmid gp64-KO which lacks of the viral envelope protein encoding gene gp64 was provided by Prof. George Rohrmann, Oregon State University, USA[30].
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The open reading frame (ORF) of p6.9 was amplified by polymerase chain reaction (PCR) from the AcMNPV genome (strain E2) with the primers 6.9F and 6.9R (Table 1). The PCR product was then cloned into the BamH I-EcoR I sites of pFB-mCMV-eGFP[25] to generate pFB-polh-p6.9-eGFP, which was subsequently transformed to DH10B harboring bMON14272 or AcBacΔp6.9 to generate recombinant viruses Ac-p6.9eGFP and Ac-p6.9eGFPrp by the Bac-to-Bac system according to the manufacturer's protocols (Fig. 1A) (Invitrogen).
Table 1. PCR primers used in this paper
Figure 1. A: Schematic of recombinant baculovirus constructs. Three bacmid constructs were generated by using the Bac-to-Bac system. The Ac-p6.9eGFP and Ac-eGFP were produced by transposing either Ppolh-p6.9-egfp or p10-egfp expression cassettes into bMON14272, respectively. The Ac-p6.9eGFPrp was generated by transposing Ppolh-p6.9-egfp expression cassette into AcBac∆p6.9.B: Infectivity assay of the recombinant baculovirus constructs. Sf9 cells were infected with either Ac-p6.9eGFP, Ac-eGFP at 5 MOI, or supernatant from Ac-p6.9eGFPrp transfected cells at 144 hpt. The viral titers were determined by monitoring EGFP expression at the indicated time points. The data represents the averages of infections with virus from triplicate transfections, and the error bars indicate standard deviations. Note that the supernatant of Ac-p6.9eGFPrp transfected cells failed to generate any EGFP (+) cells at any indicated time points.
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The virus infection and infectivity assay were performed as described previously[32]. Briefly, the virus stocks of Ac-p6.9eGFP and Ac-eGFP at a multiplicity of infection (MOI) of 5 or supernatant from Ac-p6.9eGFPrp transfected cells were used to infect fresh Sf9 cells. After 1 hour of infection, the viral supernatant was removed and the cells were replenished with fresh medium. Viral supernatants were collected at 0, 24, 48, 72, 96 hpi and titre was determined by endpoint dilution assay with EGFP expression as an indicator of virus infection.
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The transcriptional patterns of viral genes were analyzed by quantitative reverse transcription-PCR (qRT-PCR). Sf9 cells plated at 5×105 per well on 6-well plates were transfected with 2 µg of AcBacΔp6.9, Ac-p6.9eGFPrp, or gp64-KO, respectively. Total RNAs were isolated by TRIzol reagent (Invitrogen) at 12 and 24 hours post transfection (hpt), and dissolved in 20 µL of RNase-free water, respectively. Total RNA (1000 ng) of each sample was digested with 1 unit of RQ1 RNase-Free DNase (Promega) for 30 min. The resultant DNA-free total RNAs in each sample (250 ng) were collected and submitted to qRT-PCR assay using a QuantiTect SYBR Green RT-PCR Kit (Qiagen) and a StepOne realtime PCR machine (Applied Biosystems). Two genes, pe38 and p10, (primer sequences are listed in Table 1) were chosen to investigate the transcriptional level of representative genes transcribed by either host RNA polymerase Ⅱ (pe38) or virus-encoded RNA polymerase (p10) and host 18s rRNA was selected as the endogenous reference. The experiment was performed in triplicate and each with three replicates.
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Sf9 cells (5×105) were plated on slides and infected with Ac-p6.9eGFP inoculum (MOI=5). For visualization of DNA, cells at indicated time points were incubated with 100 μg/mL RNase A for 30 min at 37℃ and subsequently fixed with 4% paraformaldehyde in 0.1 mol/L PIPES, pH 6.9, 0.010 mol/L EGTA and 0.010 mol/L MgCl2 (PEM) buffer for 10 min before being permeabilized by 0.01% Triton X-100 in PBS with 2% BSA. A final incubation with 3 μg/mL propidium iodide (PI) for 2 min was then performed. For immunostaining, host RNA polymerase Ⅱ was detected with 1:500 diluted anti-ARNA-3 monoclonal antibody (Millipore) followed by addition of secondary Cy3-conjugated rabbit anti-mouse IgG (Millipore). Virus-encoded RNA polymerase was incubated with 1:500 diluted anti-LEF-8 antiserum (provided by Prof. Lorena Passarelli, Kansas State University, USA) and secondary Cy3-conjugated goat anti-rabbit IgG (Millipore). Cells were captured with a FluoView FV1000 confocal laser scanning microscope (Olympus).
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Isolation of S1, S2, and P fractions from Sf9 nuclear chromatin was performed according to the method described previously with minor modifications[17, 26]. Nuclei of Ac-p6.9eGFP infected cells were isolated using a Nuclear Extract Kit (Active Motif) at 24 hpi, and suspended in 200 µL of nuclear buffer (20 mmol/L Tris-HCl, pH 7.5, 70 mmol/L NaCl, 20 mmol/L KCl, 5 mmol/L MgCl2, and 3 mmol/L CaCl2 supplemented with protease inhibitors). The nuclear suspension was incubated with 30 U of micrococcal nuclease (TaKaRa) at room temperature. The digestion was terminated by the addition of EDTA and EGTA 5 mmol/L of each, and the mixture was then centrifuged at 5000r/min for 3 min. This supernatant was designated the S1 fraction. The nuclear pellet was further lysed in 2 mmol/L EDTA for 15 min at 4℃, followed by centrifugation, and the supernatant and the pellet were designated as the S2 and P fractions, respectively. For Western blotting, 1:1000 diluted anti-GFP, anti-ARNA-3 or anti-LEF-8 antiserum and a 1:10, 000 diluted HRP-labeled secondary goat anti-rabbit IgG (Beyotime) were used.
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Sf9 cells were infected with Ac-p6.9eGFP (MOI=5). At indicated time points, the cells were submitted to Chromatin immunoprecipitation (ChIP) assay using a ChIP kit (Upstate Biotechnology) and monoclonal anti-GFP antibody (Sigma) according to the manufacturer's recommendations. PCR of input virus dilutions and the bound ChIP fraction were performed simultaneously with ExTaq DNA polymerase (TakaRa). The primers used for PCR are listed in Table 1. Thirty cycles were performed and each cycle was carried out at 94 ℃ for 30 s, 55 ℃ for 30 s, and 72 ℃ for 60 s. The PCR products were separated by 2% agarose gels, visualized by staining with ethidium bromide (EB) and the bands of each PCR product were densitometrically assayed using a Gel-pro32 imager (Media Cybernetics).
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Representative results from two to three independent experiments repeated in triplicate were shown in each figure. All experimental data values shown were calculated from triplicate samples. Data were analyzed using independent sample t-tests and were expressed as means ± standard deviation (SD), except for qRT-PCR. P-values less than 0.05 were considered significant.