Bombyx mori C-Type Lectin (BmIML-2) Inhibits the Proliferation of B. mori Nucleopolyhedrovirus (BmNPV) through Involvement in Apoptosis

Abstract

C-type lectins (CTLs) are widely distributed in mammals, insects, and plants, which act as pattern recognition receptors (PRRs) to recognize pathogens and initiate immune responses. In this study, we identified a C-type lectin gene called BmIML-2 from the silkworm Bombyx mori. Its open reading frame (ORF) encodes 314 amino acids, which contain dual tandem C-type lectin-like domain (CTLD). BmIML-2 is highly expressed in the fat body and is significantly induced at 24 h after BmNPV infection. Moreover, overexpression of BmIML-2 dramatically inhibited the proliferation of BmNPV, and knockdown assay via siRNA further validated the inhibition of BmIML-2 on viral proliferation. In addition, transcript level detection of apoptosis-related genes and observation of apoptosis bodies implied that overexpression of BmIML-2 promoted BmNPV-induced apoptosis. Immunofluorescence analysis indicated that BmIML-2 distributed throughout the cytoplasm and was slightly concentrated in the cell membrane. Taken together, our results suggest that BmIML-2 could inhibit in the proliferation of BmNPV by facilitating cell apoptosis.

Keywords: B. mori nucleopolyhedrovirus; Bombyx mori; C-type lectin; apoptosis.

Figure 1

Multiple sequence alignment of B. mori IML-2. The predicted signal peptides are shaded with a gray background. The conserved cysteine residues in CTLD are shown in red font. The asterisk below the sequences represents the consistent amino acid residues. The key residues that define sugar-binding specificity are indicated with a green background. The secondary structure elements of B. mori IML-2 are marked above or below the sequences. The GenBank accession numbers are as follows: BmIML-2, NP_001165396.1; MsIML-2, XP_030038244.1; HaIML-2, ACI32834; OfIML-2, AIR95998.1; SfCTL-4, XP_035441949.1.

Figure 2

The phylogenetic relationships of B. mori IML-2 with other homologous sequences. The branch containing B. mori IML-2 shown with a yellow background. The numerical values representing bootstrap values only over 70 are marked. The abbreviations are as follows: Aa: A. aegypti; Ag: A. gambiae; Bm: B. mori; Cb: Cerapachys biroi; Cq: Culex quinquefasciat; Dm: D. melanogaster; Dp: D. plexippus; Ha: H. armigera; Ms: M. sexta; Of: Ostrinia furnacalis; Ppo: Papilio polytes; Pxu: Papilio xuthus; Tc: T. castaneum.

Figure 3

The expression profiles analysis of B. mori IML-2. (A) The mRNA levels of BmIML-2 in various tissues. (B,C) Inducible expression profiles of BmIML-2 in fat body and malpighian tube upon BmNPV infection. The RNA samples of different tissues were extracted individually as described above. Then, the prepared RNA samples were converted into cDNA for qRT-PCR assay. The B. mori GAPDH gene was used as an internal reference. Error bars represent means ± S.D. of three independent values, and the asterisks indicate significant differences compared with the control group (unpaired t-test; *, p < 0.05; **, p < 0.01; ns, not significant).

Figure 4

Effects of BmNPV infection and replication by overexpression of BmIML-2 in BmN cells. (A) The infected cells after overexpression were observed under fluorescence microscope. White light, optical transmission; GFP, green; mCherry, red; scale bar = 200 µm. (B) Transcript level analysis of BmIML-2 at 48 h after transfection of the overexpression vector. (C) Transcript level analysis of VP 39 after overexpression of BmIML-2 at different times. Data were calibrated by using BmGAPDH and presented as means ± S.D. of three separate experiments. Asterisks represent significant differences compared the control (unpaired t-test; *, p < 0.05; ***, p < 0.001; ****, p < 0.0001; ns, not significant).

Figure 5

Effects of BmNPV infection and replication by knockdown of BmIML-2 in BmN cells. (A) The infected cells after knockdown of BmIML-2 were observed under a fluorescence microscope. White light, optical transmission; GFP, green; mCherry, red; scale bar = 200 µm. (B) Transcript level analysis of BmIML-2 at 48 h after transfection of siRNA. (C) Transcript level analysis of VP 39 after knockdown of BmIML-2 at different times. Data were calibrated by using BmGAPDH and presented as means ± S.D. of three separate experiments. Asterisks represent significant differences compared the control (unpaired t-test; *, p < 0.05; ****, p < 0.0001; ns, not significant).

Figure 6

Transcription level changes of selected apoptosis-related genes in virally infected BmIML-2 overexpressed cells (A–F). The mRNA levels of genes were detected by using qRT-PCR assay after transfection of BmIML-2 overexpression vector. The data were calibrated by using BmGAPDH. Error bars represent means ± S.D. of three separate experiments. Asterisks indicate significant differences compared with the control (unpaired t-test; *, p < 0.05; **, p < 0.01; ns, not significant).

Figure 7

Observation of apoptotic bodies induced by BmNPV after the overexpression of BmIML-2 in BmN cells. (A) The BmN cells overexpressing BmIML-2 were infected with BmNPV. After 48 h viral infection, cells were visualized under a fluorescence microscope. Nuclei stained with 4,6-diamidino-2-phenylindole (DAPI), blue, scale bar = 100 µm. Apoptotic bodies are marked with white arrows. (B) The percentage of cells in apoptotic morphology. Statistical analysis was carried out by using GraphPad Prism 6.0 software. Error bars represent means ± S.D. of three separate experiments. Asterisks indicate significant differences compared with the control (unpaired t-test; **, p < 0.01).

Figure 8

Subcellular localization of BmIML-2 in BmN cells. Cells were transfected with pIZ/V5-His-IML-2 and cultured for 48 h. Then, the cells were fixed, permeabilized, and stained. 4,6-Diamidino-2-phenylindole (DAPI) labels nuclei, shown in blue; Alexa-Flour-488-conjugated goat anti-mouse antibody labels BmIML-2, shown in green; scale bar represents 20 µm.