Alpha-Glucosidase of Manduca sexta Is an Entry Factor for Daphnis nerii Cypovirus-23

Figure 1. VP3 mediates DnCPV-23 binding to HBBMVs. (A) Binding of DnCPV-23 virions to HBBMVs. Attachment of virions to BSA (control) or HBBMVs was determined according to OD450 values by ELISA. Data are presented as the mean ± SEM of triplicates; (B) Binding of GST-tagged viral structural proteins to HBBMVs. Binding levels were determined according to OD450 values. Each bar represents the mean ± SEM of triplicates; (C) Inhibition of viral attachment via antibodies targeting viral structural proteins. HBBMVs were incubated with pre-immune serum antibodies (control), antibodies against viral structural proteins, or the corresponding antibody mixture prior to treatment with DnCPV-23 virions. The antibody mixture was prepared by mixing equal proportions of antibodies against viral structural proteins and then diluted to the indicated folds. The binding rate of DnCPV-23 virions treated with pre-immune rabbit serum was defined as 100%. *: p < 0.05; **: p < 0.01; ***: p < 0.001; ns: not significant, p > 0.05.

Figure 2. Structural homology analysis of DnCPV-23 VP3 with BmCPV VP2. (A) Sequence alignment showing identity and similarity percentages; (B) Predicted secondary structure of VP3, modeled on BmCPV VP2, showing the N-terminal domain (NTD, orange), body 1 domain (green), claw domain (red), body 2 domain (cyan), and C-terminal domain (CTD, blue); (C) Predicted domain organization of VP3, inferred from the VP2 template, illustrating the same domain arrangement: NTD (orange), body 1 (green), claw (red), body 2 (cyan), and CTD (blue).

Figure 3. Identification of HBBMV proteins that interact with VP3 on the basis of far-Western blotting. HBBMV proteins were separated in a 10% SDS-PAGE gel and probed with purified GST-VP3 or GST alone. Lane M: protein molecular mass marker. The red arrow in lane 3 indicates HBBMV proteins that interact with VP3.

Figure 4. Interaction between MsAGL and VP3. (A) Co-immunoprecipitation analysis. Immunoprecipitated complexes (lanes 1–3) along with GST (lane 4) and MsAGL (lane 5) protein controls were separated by SDS-PAGE, transferred to a PVDF membrane, and probed using anti-GST or anti-Flag pAbs; (B) GST pull-down assay. Input lysates containing GST-VP3 and MsAGL-3Flag (lane 1) and proteins pulled down by GST-VP3 (lane 2) or GST alone (lane 3) were analyzed by immunoblotting with anti-GST or anti-Flag pAbs.

Figure 5. Role of MsAGL in DnCPV-23 attachment. The effects of MsAGL knockdown and overexpression on viral attachment were assessed by measuring the viral gene S10 (encoding the capsid protein PH) levels and corresponding protein levels at 1 h post-infection (h p.i.). The time point designated as h p.i. corresponds to the duration of virus–cell interaction. (A,E) Relative S10 gene levels following MsAGL knockdown (A) or overexpression (E) in QB-MS 2-2 cells, indicating the number of virions that attached to the cells. (B,F) Corresponding relative PH protein levels after MsAGL knockdown (B) or overexpression (F), reflecting the viral attachment level. (C,G) Validation of MsAGL expression after knockdown (C) or overexpression (G). (D,H) Western blot analysis confirming protein levels of MsAGL, PH, and GAPDH under knockdown (D) or overexpression (H) conditions. Data are shown as mean ± SEM from three independent experiments, each performed in triplicate. ***: p < 0.001.

Figure 6. Role of MsAGL in DnCPV-23 internalization. The contribution of MsAGL to viral internalization was evaluated through changes in S10/PH expression after virus internalization in host cells at 2 h p.i. The time point designated as h p.i. corresponds to the duration of virus–cell interaction. (A,E) Relative S10 (encoding PH) mRNA levels after MsAGL knockdown (A) or overexpression (E) in QB-Ms2-2 cells, representing the number of virions that internalized into host cells. (B,F) Relative PH protein levels following MsAGL knockdown (B) or overexpression (F). (C,G) MsAGL expression levels upon knockdown (C) or overexpression (G). (D,H) Western blot analysis of MsAGL, PH, and GAPDH after knockdown (D) or overexpression (H). Data are shown as mean ± SEM from three independent experiments, each performed in triplicate. ***: p < 0.001.

Figure 7. Role of MsAGL in DnCPV-23 entry. The effects of MsAGL knockdown and overexpression on viral attachment were assessed by measuring the expression of viral gene S10 (encoding the capsid protein PH) and corresponding protein levels at 24 h p.i. and 48 h p.i. The time point designated h p.i. corresponds to the duration of virus–cell interaction. (A,E) Relative S10 mRNA levels following MsAGL knockdown (A) or overexpression (E) in QB-Ms2-2 cells, indicating transcriptional activity during viral entry. (B,F) Corresponding relative PH protein levels after MsAGL knockdown (B) or overexpression (F), reflecting translational output. (C,G) Validation of MsAGL expression after knockdown (C) or overexpression (G). (D,H) Western blot analysis confirming protein levels of MsAGL, PH, and GAPDH under knockdown (D) or overexpression (H) conditions. Data are shown as mean ± SEM from three independent experiments, each performed in triplicate. ***: p < 0.001.

Figure 8. MsAGL neutralizes the infection of host cells by DnCPV-23. (A,B) Relative S10 (encoding PH) mRNA level (A) and PH protein level (B) in Sf9 cells following a pre-incubation with MsAGL-EXR at 48 h p.i.; (C) Western blot analysis of the PH protein in Sf9 cells after the virus was pre-incubated with increasing concentrations of GST-MsAGL-EXR or GST alone at 48 h p.i. Data are shown as mean ± SEM from three independent experiments, each performed in triplicate.**: p < 0.01; ***: p < 0.001; ****: p < 0.0001; ns: not significant, p > 0.05.

Figure 9. MsAGL neutralizes the infection of D. nerii larvae by DnCPV-23. (A,B) Survival rates of D. nerii larvae (n = 24 per group) in two independent bioassays. Larvae were fed DnCPV-23 virions (positive control, DnCPV), MsAGL alone (MsAGL), DnCPV-23 virions pre-treated with BSA (BSA + DnCPV-23),DnCPV-23 virions pre-treated with MsAGL (MsAGL + DnCPV-23) or BSA alone (CK).