Fig 1.
Δ012 demonstrates heightened sensitivity to IFNα compared to LSDV. Growth curves were measured in various cell lines following infection with LSDV or LSDVΔ012 at a multiplicity of infection (MOI) of 0.01 pfu/cell. Viral titers were determined at 0, 24, 48, and 72 hours post-infection in the following cell types: MDBK cells (A), A549 cells (B), BHK-21 cells (C), and MDBK-012 cells(D). Effect of Interferon on LSDVΔ012 Replication. MDBK cells (E) and MDBK-012 cells (F) were pretreated with different concentrations of IFNα (2ng/ml) for 12 hours. Cells were then infected with either LSDV or LSDVΔ012 at 0.01 pfu/cell, and viral titers were measured at 0, 24, 48, and 72 hours post-infection. Additionally, MDBK cells were pre-stimulated with PBS or IFNα (2ng/ml) for 12 hours, followed by infection with LSDV-eGFP or LSDVΔ012 virus at 0.01 pfu/cell for 48 hours. Fluorescence images were captured (I), and viral titers (J), along with LSDV H3 protein levels (K and L), were evaluated from collected samples. Significance Levels: *p < 0.05, ** p < 0.01, *** p < 0.001, n.s: non-significant.
Fig 2.
IFIT1/5 identified as proteins binding to LSDV012.
Flowchart for LSDV012 co-immunoprecipitation and protein identification, sourced from SciDraw (https://scidraw.io/), a free repository of scientific illustrations (A). A total of 30 candidate proteins potentially interacting with LSDV012 were identified through mass spectrometry proteomics analysis (B). A schematic diagram displaying the candidate proteins involved in the interferon (IFN) signaling pathway was generated using the STRING database (C). To confirm the proteomics data, BHK-21 cells were co-transfected with IFIT1/5 or MX1 alongside LSDV012 for 24h. Co-immunoprecipitation was performed using myc magnetic beads (D). Impact of IFIT1/5 Overexpression on LSDV/LSDVΔ012 Replication. BHK-21 cells were transfected with IFIT1 or IFIT5, followed by infection with LSDV or LSDVΔ012 at an MOI of 0.01. After 48 hours, samples were collected to assess viral titers (E) and measure viral protein levels (F and G). Effect of IFIT1/5 Knockdown on LSDV/LSDVΔ012 Replication. A549 cells were transfected with siRNA targeting IFIT1 or IFIT5 for 12 hours, followed by treatment with IFNβ (5 ng/ml) for another 12 hours. The cells were then infected with LSDV or LSDVΔ012 at 0.1 pfu/cell for 48 hours, after which viral titers (H) and the levels of LSDV H3 protein (I-K) were measured. Significance Levels: *p < 0.05, ** p < 0.01, *** p < 0.001, n.s: non-significant.
Fig 3.
LSDV012 inhibits the degradation of IFIT1/2 by VACV C9L.
Effect of VACV and LSDV Infections on IFIT1 Expression in Cells. A549 cells were pretreated with IFNβ (5 ng/ml) for 24 hours. The expression levels of IFIT1 were then measured 12, 24, and 48 hours post-infection with either VACV WR (A) or LSDV (B) at a multiplicity of infection (MOI) of 0.1 pfu/ml. Impact of LSDV012 on VACV-Mediated IFIT1 Degradation. BHK-21 cells were transfected with IFIT1 and/or LSDV012 for 24 hours, followed by infection with PBS, VACV WR, or LSDV for an additional 24 hours. GFP-IFIT1 and myc-LSDV012 were detected using GFP and Myc antibodies, respectively (C). Interaction Between LSDV012 and IFIT1/2/5 from Different Species. BHK-21 cells were transfected with human IFIT1/2/3/5 (D) or bovine IFIT1/2/3/5 (E), alongside LSDV012 for 24 hours. Co-immunoprecipitation was then performed using myc magnetic beads to detect interactions. Interaction Between LSDV012 and Endogenous IFIT1/2/5. A549 cells were transfected with myc-LSDV012. Four hours later, the medium was replaced with an IFNβ-containing culture medium, and cells were incubated for an additional 24 hours. Co-immunoprecipitation was conducted using myc beads (G). A schematic diagram illustrates the protein domains of LSDV012, VACV C9, and MPXV D9 (F). Inhibition of VACV C9 and MPXV D9-Mediated Degradation of IFIT Proteins by LSDV012. BHK-21 cells were co-transfected with myc-LSDV012, flag-VACV C9 or flag-MPXV D9, and human GFP-IFIT1 (H and I), GFP-IFIT2 (J and K), GFP-IFIT3 (L and M), or GFP-IFIT5 (N and O) for 24 hours. Western blotting was performed using GFP, Myc, and Flag antibodies to assess the impact of LSDV012 on the stability of IFIT1/2/3/5 in the presence of VACV C9 and MPXV D9. Significance Levels: p < 0.05, ** p < 0.01, *** p < 0.001, n.s: non-significant.
Table 1.
The count of ANK proteins across various poxviruses.
Fig 4.
Phylogenetic analysis of 636 ankyrin from poxvirus.
Table 2.
The binding capacity of LSDV012 orthologs to IFIT1 proteins from various species.
Fig 5.
The interaction of LSDV012 orthologs with IFIT1 shows host specificity.
Analysis of Binding Ability Between LSDV012 Orthologs and IFIT1 from Different Species. LSDV012 orthologs (myc-LSDV012, myc-DPV014, myc-TPXV08, myc-EPTV169) were co-transfected into 293T cells with bovine GFP-IFIT1 (A), human GFP-IFIT1 (D), and bat GFP-IFIT1 (G) for 24 hours. Following the incubation, co-immunoprecipitation was performed using Myc magnetic beads to assess the binding interactions. To evaluate the ability of LSDV012 orthologs to inhibit the degradation of IFIT1 by VACV C9 across different species, 293T cells were co-transfected with LSDV012 orthologs and Flag-VACV C9, along with bovine GFP-IFIT1 (B and C), human GFP-IFIT1 (E and F), and bat GFP-IFIT1 (H and I) for 24 hours. Western blot analysis was conducted using GFP, FLAG, and Myc antibodies. Significance Levels: p < 0.05, ** p < 0.01, *** p < 0.001, n.s: non-significant.
Fig 6.
LSDV012 binds to the C-terminus of IFIT1, impeding its mRNA-binding capability.
The structure of the human IFIT1 protein complex with RNA (PDB:5UDI) is shown in (A). The predicted structure of bovine IFIT1 by Alphafold3 is illustrated in (B), and the Alphafold3-predicted protein complex structure of IFIT1 with LSDV012 is depicted in (C). The interaction regions between LSDV012 and IFIT1 are segmented into section 1 (1–95 aa), section 2 (96–140 aa), section 3 (141–250 aa), section 4 (151–338 aa), section 5 (339–435 aa), and section 6 (436–478 aa) (D). Schematic representations of the N-terminal truncation (E) and C-terminal truncation (G) of Bovine IFIT1 are presented. LSDV012 validation of binding to different IFIT1 truncations. Myc-LSDV012 was co-transfected with the N-terminal truncation (F) and C-terminal truncation (H) of bovine IFIT1 into 293T cells for 24 hours, followed by co-immunoprecipitation using myc beads. A flow chart illustrating the use of Streptavidin Magnetic Beads to pull down Biotin-labeled RNA was sourced from SciDraw (J). Biotin-ppp-RNA and Biotin-Cap1-RNA (approximately 20 µg) were incubated with Streptavidin Magnetic Beads for 2 hours to prepare the RNA-bead complex. This mixture was then incubated with cell lysates from 293T cells, which were transfected with GFP-IFIT1 alone or in combination with LSDV012 for 24 hours. The incubation was conducted at 4°C for 4 hours. The protein content was analyzed using GFP and Myc antibodies. H5R ppp-RNA, Cap1-RNA, and A549/VACV RNA were incubated with 293T cell lysates at 4°C for 12 hours, which were transfected with GFP/GFP-IFIT1 alone or in combination with LSDV012 for 24 hours. GFP magnetic beads were then added for a 2-hour incubation. These GFP magnetic beads were used to pull down IFIT1, which may be bound to RNA. The protein content was detected using GFP and Myc antibodies (K). RNA was extracted using Trizol, and the concentration analysis was conducted using a spectrophotometer (L). Significant differences: * p < 0.05, ** p < 0.01, *** p < 0.001, n.s: non-significant.
Fig 7.
Evaluation of the pathogenicity of LSDV and LSDV
Δ012 in different mice models. BALB/c (A and B) or C57/BL6J (C and D) mice were infected with LSDV or LSDVΔ012 via intradermal injection at an infectious dose of 10^7 pfu/ml. Photos (B and D) were taken by the authors.The nodule size at the injection site was measured at 1, 5, 10, 15, and 20 days post-infection.