Biochemical characterization of Periplaneta fuliginosa densovirus non-structural protein NS1

Yang, Bo; Zhang, Jiamin; Cai, Dawei; Li, Doulin; Chen, Wuguo; Jiang, Hong; Hu, Yuanyang

doi:10.1016/j.bbrc.2006.02.053

Cited by 24 publications

(25 citation statements)

References 44 publications

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“…Far-Western blot assays were conducted according to our standard protocol (53). Bacterial expressions of His-B2 and His-B2⌬N20 were separated by SDS-PAGE, electrophoretically transferred to PVDF membranes, and gradually renatured at 4°C in HEPES buffer [20 mM HEPES-KOH, 50 mM NaCl, 5 mM MgCl 2 , 1 mM EDTA, 1 mM DTT, and 10% glycerol (pH 7.4)] containing 5% nonfat milk.…”

Section: Vol 85 2011mentioning

confidence: 99%

RNA Binding by a Novel Helical Fold of B2 Protein from Wuhan Nodavirus Mediates the Suppression of RNA Interference and Promotes B2 Dimerization

Qiu

Xie

et al. 2011

J Virol

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Wuhan nodavirus (WhNV) is a newly identified member of the Nodaviridae family with a bipartite genome of positive-sense RNAs. A nonstructural protein encoded by subgenomic RNA3 of nodaviruses, B2, serves as a potent RNA silencing suppressor (RSS) by sequestering RNA duplexes. We have previously demonstrated that WhNV B2 blocks RNA silencing in cultured Drosophila cells. However, the molecular mechanism by which WhNV B2 functions remains unknown. Here, we successfully established an RNA silencing system in cells derived from Pieris rapae, a natural host of WhNV, by introducing into these cells double-stranded RNA (dsRNA)-expressing plasmids or chemically synthesized small interfering RNAs (siRNAs). Using this system, we revealed that the WhNV B2 protein inhibited Dicer-mediated dsRNA cleavage and the incorporation of siRNA into the RNA-induced silencing complex (RISC) by sequestering dsRNA and siRNA. Based on the modeled B2 3-dimensional structure, serial single alanine replacement mutations and N-terminal deletion analyses showed that the RNA-binding domain of B2 is formed by its helices ␣2 and ␣3, while helix ␣1 mediates B2 dimerization. Furthermore, positive feedback between RNA binding and B2 dimerization was uncovered by gel shift assay and far-Western blotting, revealing that B2 dimerization is required for its binding to RNA, whereas RNA binding to B2 in turn promotes its dimerization. All together, our findings uncovered a novel RNA-binding mode of WhNV B2 and provided evidence that the promotion effect of RNA binding on dimerization exists in a viral RSS protein.

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Section: Vol 85 2011mentioning

confidence: 99%

RNA Binding by a Novel Helical Fold of B2 Protein from Wuhan Nodavirus Mediates the Suppression of RNA Interference and Promotes B2 Dimerization

Qiu

Xie

et al. 2011

J Virol

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“…However, expression levels declined sharply when the NS1 gene was deleted from the recombinant genome, presumably because of the multiple activities that this densovirus replication initiator protein possesses. These activities include sequence-and strand-specific nicking activity, 19 ATP-dependent helicase activity, 20 and especially promoter transregulation 21,22 that influences the expression of target genes. If NS1 is preserved in its entirety, the size of the inserted sequence is limited to approximately 1,000 bases; this limit excludes most genes for insecticidal proteins.…”

Section: Discussionmentioning

confidence: 99%

“…However, according to previous reports, the functions of the NS1 fusion protein should be preserved and will be manifested by its nuclear localization. 21,28,29 Under the control of the p61 promoter, the N-terminal fused NTS will target the protein into nucleus and its function was confirmed by its nuclear localization. Our results showed a similar pattern of intracellular GFP distribution between p61NTS-BIT-GFP and p7NS1-BIT-GFP compared with p7NS1-GFP, which confirmed that the NS1-BmK IT1-GFP and NTS-BmK IT1-GFP fusion proteins, NS1 and NTS, retained their functions well.…”

Section: Gu Dong and Othersmentioning

confidence: 99%

A Recombinant AeDNA Containing the Insect-Specific Toxin, BmK IT1, Displayed an Increasing Pathogenicity on Aedes albopictus

Dong²,

Peng³

et al. 2010

The American Journal of Tropical Medicine and Hygiene

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Abstract. The Aedes aegypti densovirus (AeDNV) has previously shown potential in mosquito control. To improve its efficacy as a biopesticide, the gene for an excitatory insect-specific toxin from Buthus martensii Karsch (BmK IT1) was inserted into the AeDNV genome and cloned into pUCA plasmid. The coding sequence for green fluorescent protein was ligated to the C-terminus of the BmK IT1 gene as a screening marker. Recombinant and helper plasmids were cotransfected into C6/36 cells; wild-type viruses were the controls. The recombinant viruses were identified and quantified by real-time polymerase chain reaction and exposed to Ae. albopictus larvae for the evaluation of its bioinsecticidal activity. LT 50 and LD 50 bioassays showed that the recombinant AeDNV had stronger and faster pathogenic effects on Ae. albopictus than the wild-type virus. This is the first report on the recombinant AeDNA containing the insect-specific toxin, BmK IT1, which may be used to develop a novel type of insecticide.* Address correspondence to Xiao-Guang Chen, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou 510515, China. E-mail: xgchen@fimmu.com † These authors contributed equally to this work. THE PATHOGENICITY OF RECOMBINANT AEDNA ON AEDES ALBOPICTUS1 protein (NS1), and structural protein (VP) genes were transcribed from the P7 and P61 promoters in the genome, respectively, which has been described in detail previously. 15The p7NS1-GFP expresses an NS1-GFP fusion protein from the p7 promoter. The construction of p7NS1-GFP is described in detail elsewhere. 15The p7NS1-BIT-GFP was derived from p7NS1-GFP, and BmK IT1 coding sequences were inserted at the AgeI site of p7NS1-GFP. As a result, the BmK IT1-GFP construct was fused to the C-terminus of NS1. An additional flexible linker (GGGGS) was used to link these two fragments. 16 The NS1-linker-BmK IT1-GFP fusion protein was expressed from the p7 promoter.The p61NTS-BIT-GFP was derived from pUCA. The BmK IT1-GFP coding sequence was cloned into the structural protein, ORF (2,791-3,635 nt). As a result, the 77 aa of the N-terminal of the structural protein, VP1, which harbors a putative nuclear targeting signal (NTS) sequence, 17 were fused to the N-terminus of the BmK IT1-GFP protein, and the fusion protein was expressed from the p61 promoter.The p7NS1-BIT was derived from p7NS1-BIT-GFP by deleting GFP from C-terminus of BmK IT1. As a result, the NS1-linker-BmK IT1 fusion protein was expressed from the p7 promoter.All of the constructions were confirmed by sequencing (data not shown). The plasmids that were used in this study are depicted in Figure 1 .Mosquito cell maintenance and transfection. Aedes albopictus C6/36 cells (ATCC CRL-1660) were grown at 28°C in RPMI 1640 medium (Gibco BRL, USA ) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco BRL) and 1% antibiotics (Penicillin-Streptomycin; Gibco BRL). The C6/36 cells were grown to a 50-70% confluent monolayer in 24-well cell culture plates (Corning costar, USA ) and washed onc...

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“…It displays various biochemical activities which are required for viral genome amplification, such as intrinsic ATPase and ATPdependent helicase activities [15]. On the other hand, the NS1 protein has been shown to control viral gene expression.…”

Section: Introductionmentioning

confidence: 99%

Non-structural proteins of Periplaneta fuliginosa densovirus inhibit cellular gene expression and induce necrosis in Sf9 cell cultures

Yang

Dong

et al. 2009

Virus Genes

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The non-structural protein NS1 of Periplaneta fuliginosa densovirus (PfDNV) is a multifunctional protein that has previously been shown to possess ATP-binding, ATPase, site-specific DNA-binding, helicase, and transcription activation activities. We report here an investigation of the cytopathogenicity of this viral non-structural (NS) protein, as well as other two NSs, NS2, and NS3, in cultured insect cells. The expression of NS1 alone potently inhibited cellular gene expression, whereas NS2 and NS3 did not produce a similar effect. The inhibition of gene expression by NS1 was confirmed to be specific and not a simple manifestation of toxicity. For example, NS1 inhibited expression of several reporter genes under the control of different RNA polymerase II promoters, whereas it did not inhibit expression from a T7 RNA polymerase promoter construct. Mapping analysis identified the carboxy-terminal peptide of this protein as the region important for the inhibition of cellular gene expression, suggesting that this inhibition is independent of its DNA-binding activity. Next, the mutagenesis assay showed that ATP-binding was essential for the unique function of this protein. Furthermore, we found that NS2 and NS3 cooperatively enhanced the NS1-induced transcription inhibition. Co-expression of all the three NS proteins in Sf9 cells also led to necrotic cell death by ATP depletion.

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Biochemical characterization of Periplaneta fuliginosa densovirus non-structural protein NS1

Cited by 24 publications

References 44 publications

RNA Binding by a Novel Helical Fold of B2 Protein from Wuhan Nodavirus Mediates the Suppression of RNA Interference and Promotes B2 Dimerization

RNA Binding by a Novel Helical Fold of B2 Protein from Wuhan Nodavirus Mediates the Suppression of RNA Interference and Promotes B2 Dimerization

A Recombinant AeDNA Containing the Insect-Specific Toxin, BmK IT1, Displayed an Increasing Pathogenicity on Aedes albopictus

Non-structural proteins of Periplaneta fuliginosa densovirus inhibit cellular gene expression and induce necrosis in Sf9 cell cultures

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