XB-ART-49756PLoS One January 1, 2014; 9 (6): e99200.
Identification of a new Sprouty protein responsible for the inhibition of the Bombyx mori nucleopolyhedrovirus reproduction.
The rat sarcoma-extracellular signal regulated kinase mitogen-activated protein kinases pathway, one of the most ancient signaling pathways, is crucial for the defense against Bombyx mori nucleopolyhedrovirus (BmNPV) infection. Sprouty (Spry) proteins can inhibit the activity of this pathway by receptor tyrosine kinases. We cloned and identified a new B. mori gene with a Spry domain similar to the Spry proteins of other organisms, such as fruitfly, mouse, human, chicken, Xenopus and zebrafish, and named it BmSpry. The gene expression analysis showed that BmSpry was transcribed in all of the examined tissues and in all developmental stages from embryo to adult. BmSpry also induced expression of BmNPV in the cells. Our results indicated: (1) the knock-down of BmSpry led to increased BmNPV replication and silkworm larvae mortality; (2) over-expression of BmSpry led to reduced BmNPV replication; and (3) BmSpry regulated the activation of ERK and inhibited BmNPV replication. These results showed that BmSpry plays a crucial role in the antiviral defense of the silkworm both in vitro and in vivo.
PubMed ID: 24915434
PMC ID: PMC4051654
Article link: PLoS One
Species referenced: Xenopus
Genes referenced: mapk1 spry1
Article Images: [+] show captions
|Figure 2. Expression profile of BmSpry in different tissues, developmental stages and inducible expression by BmNPV.(A) Expression of BmSpry in multiple tissues of 3rd-day 5th instar larvae. Lanes: 1, head; 2, integument; 3, hemocyte; 4, malpighian tubule; 5, midgut; 6, fat body; 7, silk gland; 8, testis; and 9, ovary. (B) Expression of BmSpry at developmental stages from embryo to adult. Lanes: 1, 2 days after egg laying (AEL); 2, 4 days AEL; 3, 6 days AEL; 4, 8 days AEL; 5, newly hatched larvae; 6, molting larvae (ML) of the 1st instar; 7, newly exuviated larvae (NEL) of the 2nd instar; 8, ML of the 2nd instar; 9, NEL of the 3rd instar; 10, ML of the 3rd instar;11, NEL of the 4th instar; 12, ML of the 4th instar; 13, NEL of the 5th instar; 14, pupation in (PI) 2 days; 15, PI 4 days; 16, PI 6 days; 17, PI 8 days; and 18, adult moth. (C) Analysis of BmSpry gene expression in BmNPV-infected BmE cells at MOI of 10. Total RNA was extracted from the BmE cells at the indicated time points post-infection. Non-infected cells were used as the control and qPCR of BmSpry was done. A representative of triplicate experiments is shown. Data are given as mean ±SD (n = 3). Statistically significant differences: ** P<0.01.|
|Figure 3. BmSpry inhibited BmNPV replication in cultured cells.(A) BmE cells pretreated with dsRNA as indicated, the dsRNA of dsRed was used as a negative control. At 3 days post transfection, total RNA was extracted and qPCR was used to analyze the BmSpry expression level. (B) BmE cells treated with dsRNA against the indicated genes were infected with BmNPV-GFP at MOI of 1 for 3 days and processed for immunofluorescence. (C) BmE cells treated with the indicated dsRNA were infected with BmNPV-GFP at MOI of 1 and infection total genomes were extracted for qPCR at 3 days post. (D) BmE cells were used for transient transfection and the empty vector 1180 was used as a negative control. BmSpry-oe was an over-expression vector of BmSpry. At 3 days post-transfection, total RNA was extracted for qPCR. (E) BmE cells were subjected to transient transfection with BmSpry expression vectors and the empty 1180 vector, as indicated. At 2 days post-transfection, the cells were infected with BmNPV-GFP at MOI of 1 for 3 days and then processed for immunofluorescence. (F) BmE cells transfected with the indicated vectors were infected with BmNPV at MOI of 1 and total genomes were extracted for qPCR at 3 days post-infection. A representative of triplicate experiments is shown. Data are given as mean ±SD (n = 3). Statistically significant differences: ** P<0.01.|
|Figure 4. BmSpry regulation of ERK activation inhibited BmNPV replication.(A) Firstly, BmE cells were transfected. The empty vector 1180 was used as a negative control and the BmSpry-oe vector was used as the experimental group with over-expression of BmSpry. At 2 days post-transfection, BmE cells were infected with BmNPV at MOI of 10. Post infection for 24 h, total proteins were extracted for western blotting using anti-phospho-ERK antibody. Total protein levels were measured with anti-GAPDH antibody. (B) BmN4-SID1 cells were used. After RNAi of 5 days, the BmN4-SID1 cells were infected with BmNPV at MOI of 10. Post infection for 24 h, total proteins were extracted for western blotting. A representative of triplicate experiments is shown.|
|Figure 5. BmSpry was essential for antiviral defense in vivo.(A) The silkworm Nm DZ line was used for the RNAi experiment. Injection of dsRNA is indicated, the dsRNA of dsRed was used as a negative control with dsRNA of BmSpry as the experimental group. At 3 days post-injection of dsRNA, total RNA was extracted for qPCR to measure the expression level of BmSpry. Data are given as mean ±SD (n = 3). (B) Nm DZ silkworms treated with dsRNA against the indicated genes were infected with BmNPV (106 pfu/mL) by stab inoculation for 3 days and processed for qPCR. Data are given as mean ±SD (n = 3). (C) Analysis of the expression level of BmSpry in Nm DZ and the mutant DZ SN. Total RNA extracted from newly exuviated Nm DZ and DZ SN 5th instar larvae was used for qPCR. Data are given as mean ±SD (n = 3). (D) Analysis of mortality of DZ SN and Nm DZ after oral inoculation with BmNPV. Nm DZ and DZ SN newly exuviated 4th instar larvae were used for this experiment. DZ SN and Nm DZ silkworms were infected with BmNPV per os using a dose of 104 or 106 OB/larva and mortality was monitored until the adult stage (22 days). (E) Newly exuviated 2nd-instar larvae were used to investigate mortality after inoculation per os with 5×103 OB/larva. Mortality was monitored until the pupa stage (16 days). A representative of triplicate experiments is shown. Statistically significant differences: ** P<0.01.|
|Figure 1. Characteristics of the BmSpry sequence.The nucleotide sequence is 1398-hand margin begins with the A of the presumptive initiating AUG codon. The 213 amino acid sequence is presented, terminated by an asterisk (*). The N terminus has a short conserved motif (underlined) and a conserved residue Y (indicated by a circle). A Cys-rich region (21 residues) is marked C rich at the right.|
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