Mutagenic Analysis of the HIV Restriction Factor Shiftless

Figures

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  Figure 1. SFL inhibits HIV –1PRF in a cell-line-independent fashion. (A) Schematic illustration of the HIV-1 genome, including the site of –1PRF and the FSE. For quantification of –1PRF, the FSE sequence including the heptanucleotide slippery sequence, spacer, and stem-loop from the HIV-1 mRNA was inserted between Renilla luciferase (Rluc) (0 reading frame) and Firefly luciferase (Fluc) (–1 reading frame). (B) HEK293, A549, or TZM-bl cells were co-transfected with plasmids encoding the –1PRF reporter cassette and either SFL or SFLS. Co-transfection of a plasmid-encoding GFP served as a control. RLuc and FLuc signals were quantified at 48 h post-transfection. The FLuc signal was normalized against RLuc signal and signals measured upon co-transfection of the control plasmid were set as 100%. The average of three to ten biological replicates (HEK293, n = 3; A549, n = 10; TZM-bl, n = 8) carried out with technical triplicates is shown. Error bars indicate SEM. One-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test was performed to determine whether differences between cells expressing GFP (control) and SFL or SFLS were statistically significant (not significant [n.s.]; p ≥…

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  Figure 2. Deletions in the C-terminus of the RAA abrogate inhibition of –1PRF. (A) Sequences deleted from the RAA are shown. A C-terminal antigenic tag added for analysis of protein expression is shown in orange. (B) A549 cells were transfected with plasmids encoding SFL, SFLS, or SFL mutants together with –1PRF reporter vector. Luciferase signals in cell lysates were measured at 48 h post-transfection. A GFP expression plasmid served as a control. The average of five biological replicates conducted with technical triplicates is shown; error bars indicate SEM. One-way analysis of variance (ANOVA) with Dunnett‘s multiple comparison test was performed to determine whether differences between cells expressing GFP (control) and SFL, SFLS, or SFL mutants were statistically significant (not significant [n.s.]; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; n = 5). Unpaired Student’s t-test was performed to test whether differences between cells expressing SFL or SFLS and cells expressing SFLS and mutants Δ I, Δ II, or Δ III were statistically significant (not significant [n.s.]; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; n = 5). SFL, shiftless; SFLS, shiftless short.

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  Figure 3. Deletions in the RAA are not compatible with suppression of Gag-Pol expression. HEK293T cells were co-transfected with a vector encoding HIV-1 Gag-Pol and (A) different amounts of plasmids encoding SFL (1 µg, 0.75 µg, 0.5 µg, 0.1 µg, 0.05 µg) or SFLS (1 µg, 0.75 µg, 0.5 µg) or (B) constant amounts of plasmids encoding SFL, SFLS, and the indicated SFL mutants. Co-transfection of the Gag-Pol encoding plasmid with a GFP expression plasmid served as control. Cells were harvested 48 h post-transfection and protein expression was analyzed by immunoblot. Expression of SFL, SFLS, and SFL mutants was analyzed with an antibody against the C-terminal MYC antigenic tag. Gag-Pol (p160), Gag (p55), and capsid (p24) expression was detected using an anti-p24 antibody. Expression of β-actin (ACTB) served as a loading control. Representative blots are shown in the left panel; irrelevant lanes were excised. Three biological replicates were quantified using the software ImageJ and the ratio of Gag-Pol and Gag expression normalized to ACTB was calculated. Signals measured for the control (co-transfection of GFP expression plasmid) were set as 100%. The averages of three replicates are shown in the…

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  Figure 4. Deletions within the RAA abrogate SFL-mediated suppression of viral infectivity. HIV reporter particles containing a luciferase encoding vector genome and harboring the glycoprotein of vesicular stomatitis virus (VSV-G) were produced in the presence and absence of SFL, SFLS, or SFL mutants. Transfection of a GFP expression plasmid served as negative control. At 72 h post-transfection, supernatants were collected and equal volumes used for transduction of HEK293T cells. At 72 h post-transduction, the luciferase activity in cell lysates was measured. Transduction measured for control particles produced in the absence of SFL was set as 100%. The average of five biological replicates carried out with technical triplicates is shown; error bars indicate SEM. One-way analysis of variance (ANOVA) with Dunnett‘s multiple comparison test was performed to determine whether differences between cells expressing GFP (control) and SFL, SFLS, or SFL mutants were statistically significant (not significant [n.s.]; ***, p ≤ 0.001; n = 5). Unpaired Student’s t-test was performed to test whether differences between cells expressing SFL or SFLS were statistically significant (***, p ≤ 0.001; n = 5).…

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  Figure 5. Effect of RAA deletions for SFL self-interactions. (A) Analytical gel filtration profiles for (i) EF-G and EF-Tu, (ii) SFL and (iii) SFL treated with 6 M urea. The indicated proteins were loaded on a Superdex 200 Increase 10/300 GL column and elution profiles were recorded. EF-G and EF-Tu served as controls for protein size. (B) HEK293T cells were co-transfected with plasmids encoding SFL or SFLS equipped with a C-terminal HA- or MYC-antigenic tag. At 48 h post-transfection, proteins carrying an HA-tag were immunoprecipitated (IP) and detected with an antibody against the HA antigenic tag. Co-precipitated proteins were detected with an antibody against the MYC antigenic tag. Transfection of empty vector or single transfection of plasmids expressing either cMYC-tagged SFLS or HA-tagged SFL served as negative controls. Expression of the proteins in whole cell lysates (WCL) served as control of input proteins. Expression of β-actin (ACTB) was determined as loading control. A representative immunoblot is shown from which irrelevant lanes were excised. Similar results were obtained in two separate experiments. (C) HEK293T cells were co-transfected with plasmids encoding SFL, SFLS, or…

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  Figure 6. Effect of RAA deletions on ribosome binding. HEK293T cells were transfected with plasmids encoding SFL, SFLS, and the indicated SFL variants equipped with a C-terminal MYC antigenic tag. At 48 h post-transfection, cells were lysed and WCL was loaded on a sucrose cushion and centrifuged for 60 min at 100,000 rpm at 4 °C. The ribosomal pellet (RP) and WCL were analyzed by immunoblot using anti-MYC antibody. The large ribosomal subunit protein uL3 served as a loading control. A representative blot is shown in the (left panel); irrelevant lanes were excised. Up to five replicates were quantified by the software ImageJ, and the cMYC signals in RP and WCL were normalized against the corresponding uL3 signals and the ratio of cMYC signals in RP versus WCL was calculated (right panel). The ratio obtained for SFL was set as 100%. The average of four (mutants ΔI, ΔII, ΔIII, ΔIV, ΔV, ΔVI) or five (SFL, SFLS, Δ I–III, Δ IV–VI) independent experiments is shown; error bars indicate standard error of the mean (SEM). One-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test was performed to determine whether differences between cells expressing SFL, SFLS, SFL mutants, or empty…

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  Figure 7. Deletions in the N-terminus of the RAA abrogate SFL binding to the HIV FSE. (A) HEK293T cells were transfected with plasmids encoding SFL, SFLS, or the indicated SFL mutants equipped with a C-terminal MYC-tag. A plasmid encoding GFP served as negative control. At 48 h post-transfection, the cells were lysed, fluorescence labeled HIV-1 FSE mRNA was added to cell lysates, and proteins were precipitated with anti-MYC antibody. Co-precipitated HIV-1 FSE mRNA was loaded on a urea gel and the subsequent fluorescence signal was quantified. Expression of SFL, SFLS, and SFLS mutants in WCL was confirmed via immunoblot using anti-MYC antibody. β-Actin (ACTB) expression in WCL served as a loading control. A single representative blot is shown in the left panel; irrelevant lanes were excised. Three biological replicates were quantified by the software ImageJ (right panel). SFL, SFLS, or SFL mutants were normalized to ACTB and in turn used for normalization of HIV-1 FSE mRNA signals. RNA signal measured upon precipitation of SFL was set as 100%. The average of three independent experiments is shown; error bars indicate the standard error of the mean (SEM). One-way analysis of variance (ANOVA)…