Article Figures & Data
Figures
- FIG 1
Functional analysis of alanine scanning mutants of the N terminus of GBF1 in replication and secretion. (A) Schematic representation of the alanine substitutions in the GBF1 sequence. All GBF1 expression constructs are GFP tagged and contain the A795E BFA resistance mutation in the Sec7 domain. (B) Performance of the indicated mutants in the poliovirus replicon replication and cellular secretion assays. For the replication assay, cells were transfected with the plasmids expressing a corresponding GBF1 mutant, a full-length GBF1 A795E (positive control), or an empty vector (negative control). The next day, the cells were transfected with a poliovirus replicon RNA expressing Renilla luciferase and incubated in the presence or absence of 1 μg/ml BFA. For the secretion assay, the cells were cotransfected with plasmids coding for a corresponding GBF1 mutant, a full-length GBF1 A795E (positive control), or an empty vector (negative control) and a plasmid coding for a secreted Gaussia luciferase. The next day, they were washed and incubated in the medium with the indicated amount of BFA, and the amount of secreted luciferase was determined after 4 h. Secretion data are normalized to the signal obtained without BFA for each construct. Statistical significance of the difference between the signal in the positive control and in the sample expressing a mutant GBF1 for corresponding concentrations of BFA is indicated. RLU, relative light units.
- FIG 2
Functional analysis of GBF1/BIG2 N-terminal chimeras in replication and secretion. (A) Scheme of the BIG2-derived substitutions in the GBF1 sequence. All GBF1 expression constructs are GFP tagged and contain the A795E BFA resistance mutation in the Sec7 domain. (B) Performance of the indicated mutants in the poliovirus replicon replication and cellular secretion assays. For the replication assay, cells were transfected with the plasmids expressing a corresponding GBF1 mutant, a full-length GBF1 A795E (positive control), or an empty vector (negative control). The next day, the cells were transfected with a poliovirus replicon RNA expressing Renilla luciferase and incubated in the presence or absence of 1 μg/ml BFA. Expression of the 2-17/BIG2 and 47-62/BIG2 constructs, the most compromised in the replication assay, is additionally verified by Western blotting in the samples from the corresponding experiments. For the secretion assay, the cells were cotransfected with plasmids coding for a corresponding GBF1 mutant, a full-length GBF1 A795E (positive control), or an empty vector (negative control) and a plasmid coding for a secreted Gaussia luciferase. The next day, they were washed and incubated in the medium with the indicated amount of BFA, and the amount of secreted luciferase was determined after 4 h. Secretion data are normalized to the signal obtained without BFA for each construct. The statistical significance of the difference between the signal in the positive control and that in the sample expressing a mutant GBF1 for corresponding concentrations of BFA is indicated.
- FIG 3
Effects of mutations in the N terminus of GBF1 on interaction with the viral protein 3A. (A) Co-IP of 3A-FLAG with GFP fusions of alanine scanning mutants of GBF1. (B) Co-IP of 3A-FLAG with GFP fusions of GBF1/BIG2 chimeras. Cells were cotransfected with plasmids coding for corresponding GFP-tagged GBF1 mutants and a plasmid coding for 3A-FLAG-Y. IP was performed with anti-FLAG resin. GBF1s were detected with anti-GFP antibodies, and 3A was detected with anti-FLAG antibodies. Actin in the lysates is shown as a loading control. Relative recruitment is calculated by normalizing the GBF1-to-3A signal ratio in the pulldown material of the mutants to that of the positive-control (wt) sample. Each bar is an average of the results from at least three independent experiments. The performance of the corresponding GBF1 constructs in the replication assay is indicated.
- FIG 4
GBF1 mutants unable to activate Arf are defective in replication. (A) Schematic of GBF1 constructs containing the inactivating 7A mutation. All GBF1 expression constructs are GFP tagged. (B) GST-Arf1-GBF1 pulldown assay. GST or GST-Δ17 ARF1 was immobilized on glutathione beads and incubated with lysate from cells (designated SM [starting material]) expressing GFP-tagged GBF1/A795E, GBF1/A795E/A794K, or GBF1/A795E/7A. The bound material was analyzed by SDS-PAGE and the gel either stained with Coomassie blue (top) or transferred to nitrocellulose (NC) and immunoblotted with anti-GFP antibodies (bottom). GBF1/795/7A does not bind the ARF substrate, whereas GBF1/795 and GBF1/795/794 bind ARF. (C) Performance of the corresponding mutants in a poliovirus replication assay. Cells were transfected with the plasmids expressing a corresponding GBF1 mutant, a full-length GBF1 A795E (positive control), or an empty vector (negative control). The next day, the cells were transfected with a poliovirus replicon RNA expressing Renilla luciferase and incubated in the presence or absence of 1 μg/ml BFA. For the secretion assay, the cells were cotransfected with plasmids coding for a corresponding GBF1 mutant, a full-length GBF1 A795E (positive control), or an empty vector (negative control) and a plasmid coding for a secreted Gaussia luciferase. The next day, they were washed and incubated in the medium with the indicated amount of BFA, and the amount of secreted luciferase was determined after 4 h. Expression of the 7A constructs in the samples from the corresponding replication experiments is additionally verified by Western blotting. Representative positive-control (A795E) and negative-control (vector) samples are shown.
- FIG 5
Functional analysis of GBF1 mutants targeting conserved elements in the C-terminal noncatalytic domains in replication and secretion. (A) Scheme of the mutations in the GBF1 sequence. All GBF1 expression constructs are GFP tagged and contain the A795E BFA resistance mutation in the Sec7 domain. (B) Performance of the corresponding mutants in a poliovirus replicon replication assay. Cells were transfected with the plasmids expressing a corresponding GBF1 mutant, a full-length GBF1 A795E (positive control), or an empty vector (negative control). The next day, the cells were transfected with a poliovirus replicon RNA expressing Renilla luciferase and incubated in the presence or absence of 1 μg/ml BFA.
- FIG 6
GBF1 constructs containing a Sec7d from another GEF support poliovirus replication. (A) Scheme of the GBF1/ARNO chimeras. All GBF1 expression constructs are GFP tagged. (B) Analysis of the corresponding constructs in a poliovirus replicon replication assay. Cells were transfected with the plasmids expressing a corresponding GBF1 mutant, a full-length GBF1 A795E (positive control), or an empty vector (negative control). The next day, the cells were transfected with a poliovirus replicon RNA expressing Renilla luciferase and incubated in the presence or absence of 1 μg/ml BFA.
- FIG 7
C-terminal GBF1 mutants show differential capacity to support replication of a wild-type versus the 3A-2 mutant replicon. (A) Top, scheme of the full-length and 1060 truncated GBF1 constructs; bottom, amino acid sequence of 3As from the wt poliovirus and 3A-2 mutant. All GBF1 expression constructs are GFP tagged and contain the A795E BFA resistance mutation in the Sec7 domain. (B) Performance of the full-length and 1060 truncated GBF1 constructs in a replication assay with a wt replicon and a replicon bearing the 3A-2 mutation, severely inhibiting 3A-GBF1 interaction. Cells were transfected with the plasmids expressing a full-length GBF1 A795E, a 1060 GBF1 A795E truncated construct, or an empty vector (negative control). The next day, the cells were transfected with a wt or 3A-2 poliovirus replicon RNAs expressing Renilla luciferase and incubated in the presence or absence of 1 μg/ml BFA. (C) Performance of the GBF1 mutants in the HDS1, HDS2, and HDS3 domains in a replication assay with a replicon containing 3A-2 mutation. Scheme of the mutation positions in the GBF1 domains is shown. All GBF1 expression constructs are GFP tagged and contain the A795E BFA resistance mutation in the Sec7 domain. Cells transfected with the full-length GBF1 A795E serve as a positive control, and those transfected with the 1060 truncated GBF1 A795 serve as a negative control. Expression of constructs L1246R, LF1266AA, and FPL1594AAA, severely impaired in rescuing the replication of the 3A-2 poliovirus replicon, is additionally verified by Western blotting in the samples from the corresponding experiments.
- FIG 8
C-terminal part of GBF1 can compensate for the defects in 3A-GBF1 interaction. (A) The C-terminal part of GBF1 facilitates its recruitment by 3A. Top, scheme of the full-length and 1060 truncated GBF1 constructs and amino acid sequence of 3As from the wt poliovirus and 3A-2 mutant. HeLa cells were cotransfected with plasmids expressing wt 3A or 3A-2 mutant proteins together with plasmids expressing either full-length or 1060 truncated GBF1 constructs. The next day, the cells were fixed and stained for 3A upon mild permeabilization, and the distribution of GBF1 signal in 3A-expressing cells was evaluated. Graph shows quantification of three independent experiments, with at least 200 cells counted for each sample. Statistical significance is indicated. (B) The C-terminal part of GBF1 can functionally compensate for the defect of GBF1-3A interaction in poliovirus replication. Top, scheme of a full-length GBF1 construct with substitution of amino acids 32 to 46 for those derived from a corresponding segment of BIG2; 1060 truncated GBF1 construct with wt N-terminal sequence; and 1060 truncated GBF1 construct with BIG2-derived substitution of amino acids 32 to 46. HeLa cells were transfected with the plasmids expressing indicated GBF1 constructs. The next day, the cells were transfected with a wt poliovirus replicon RNA coding for the Renilla luciferase gene, and incubated in the presence or absence of 1 μg/ml of BFA. Expression of the GBF1 constructs was verified by Western blot.
