Fig 1.
A schematic representation of the FMDV proteins and a multiple alignment of the 2B protein among FMDV serotypes.
One isolate of each serotype of the 2B protein sequences was listed in the alignment. The sequences labeled in red represent the two transmembrane domains predicted by the online software.
Fig 2.
The subcellular localization of the FMDV 2B protein in BHK-21 cells.
BHK-21 cells were transfected with pEGFPN1 (A) and pEGFPN1-2B (B) and evaluated using a LSCM. The blue fluorescence represents the nucleus, the green fluorescence represents the GFP protein or GFP-2B protein, and the red fluorescence represents the ER.
Fig 3.
The topological structure of the FMDV 2B protein.
Three groups of cells were transfected with pXJ-FLAG-2B, pXJ-2B-HA, or pXJ-2BĪ(110ā111). (A) Cells cultured without any treatment. (B) Cells treated with Triton X-100. (C) Cells treated with digitonin. (D) A schematic representation of the predicted topology of the FMDV 2B protein in the ER membrane. The blue fluorescence represents the nucleus, and the green fluorescence represents the FLAG-2B protein or the 2B-HA protein.
Fig 4.
The cytotoxicity of the 2B protein in Escherichia coli.
(A) The proliferation curves of the recombinant bacteria carrying different plasmids are presented. The pSUMO and pSUMO-2B plasmids were transfected into the Escherichia coli BL21(DE3)pLysS or C43(DE3)pLysS strain. The OD600 was determined at 0h, 1h, 2h, 3h, 4h, and 5h post-induction. (B) Expression of the 2B protein in the bacteria. The pSUMO and pSUMO-2B plasmids were transfected into the Escherichia coli BL21(DE3)pLysS or C43(DE3)pLysS strain. The products were analyzed by Western blot analysis with an anti-His antibody.
Fig 5.
The effect of amantadine on the release of FMDV virions.
BHK-21 cell cultures were infected with FMDV (MOI/0.1) and then treated with amantadine. The virus in the supernatant was collected at 4 hours post-infection. The virus titer was determined by TCID50. Asterisks indicate significant differences between the indicated samples (*P<0.05,** P<0.01).
Fig 6.
The pore-forming activity of the 2B protein.
(A) Purified Sumo-2B protein was incubated with a glutaraldehyde cross-linker at the indicated concentrations (0, 0.1, 0.5, 1.0, 1.5, 2.0, and 3.0 mM). The monomers and oligomers of the 2B protein were detected by immunoblot analysis with an anti-His monoclonal antibody. (B) BHK-21 cells were transfected with pXJ-FLAG-2B or pXJ-2B-HA. The cell lysates were subjected to immunoprecipitation using anti-HA antibodies.
Fig 7.
The effects of the 2B protein on the Ca2+ content and membrane integrity in host cells.
Untreated BHK-21 cells (A) and cells transfected with pXJ-2B-HA (B) were stained with Fluo-3 AM and propidium iodide (PI) at 12 hours post-transfection. The increase in intracellular Ca2+ (Fluo-3 AM) is shown in Q4. This increase was associated with a change in the PI in BHK-21 cells. A histogram was constructed to reveal the changes in the intracellular Ca2+ concentration (C). Asterisks indicate significant differences between the indicated samples (*P<0.05,** P<0.01).
Fig 8.
Autophagy induced by the 2B protein in BHK-21 cells and H1299 cells.
Four groups of BHK-21 cells were cultured with different treatments, and the LC3-I and LC3-II levels were determined by Western blot analysis with an anti-LC3 antibody (A). Untreated cells (B, F), cells transfected with pCS2 (C, G) or pCS2-2B (D, H), or treated with rapamycin (E, I) were cultured and observed under a LSCM to evaluate LC3 aggregation using IFA in BHK-21 cells (B-E) or based on the green and red fluorescence in H1299 cells (F-I). The blue fluorescence represents the nucleus, the green fluorescence represents the LC3 protein, and the red fluorescence represents the RFP-2B protein.