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Results: 8

1.
FIG. 6

FIG. 6. From: Formation and function of hepatitis C virus replication complexes require residues in the carboxy-terminal domain of NS4B protein.

Effect of the CTD missense mutations on NS4B subcellular distribution. Wt and mutant NS4B-GFP constructs were transfected into Huh7.5 cells. At 48 h post-transfection, the cells were processed for fluorescence microscopy. Note that Wt NS4B-GFP as well as most of the mutant [D228A (ii), S239W (v), T241A (vi), L245D (vii), and H250E (viii)] proteins show the characteristic punctate foci and perinuclear distribution. Interestingly, V233R (iii) displays perinuclear, but elongated foci, whereas L237E (iv) exhibits a reticular distribution. Bars = 10 µm.

Jason Aligo, et al. Virology. ;393(1):68-83.
2.
FIG. 3

FIG. 3. From: Formation and function of hepatitis C virus replication complexes require residues in the carboxy-terminal domain of NS4B protein.

A. Subcellular distribution of GFP-fused NS4B CTD constructs. NS4B CTD constructs [(192–261)-GFP or GFP-(192–261)] were transfected into Huh7.5 cells. At 24 h post-transfection, the cells were processed for fluorescence microscopy. Notice that there are few foci and numerous hollow foci. Bars = 10 µm. B. Ultrastructural analysis of cells expressing NS4B-GFP (i) and NS4B (192–261)-GFP (ii) proteins. Cells were transfected as above and processed for TEM analysis. Square areas show vesicular structures. Higher magnification of the square areas is indicated by the arrows. Notice the presence of larger vesicles in (ii), in comparison to (i). Bars = 1 µm.

Jason Aligo, et al. Virology. ;393(1):68-83.
3.

FIG. 8. From: Formation and function of hepatitis C virus replication complexes require residues in the carboxy-terminal domain of NS4B protein.

Effect of the NS4B CTD mutations on the subcellular pattern of Rab5B protein. Huh7.5 cells were co-transfected with Wt or mutant constructs as described above except that IF was performed using anti Rab5B antibody. Notice that some Rab5B and NS5A foci merge in cells expressing Wt (i to iii) or mutant S239W (x to xii) polyprotein. Those Rab5B foci that merge with NS5A appear to be much larger than those with no NS5A overlap. In cells expressing mutant NS4B V233R and L237E proteins (iv to ix), NS5A displays a reticular distribution, which shows little overlap with Rab5B foci. Bars = 10 µm. Mock-transfected cells show typical Rab5B foci (xiii). Percent co-localization between NS5A and rab5B was calculated using Axiovision software (v. 4.5, Carl Zeiss). The data represent co-localization from at least five NS5A-expressing cells.

Jason Aligo, et al. Virology. ;393(1):68-83.
4.

FIG. 7. From: Formation and function of hepatitis C virus replication complexes require residues in the carboxy-terminal domain of NS4B protein.

Effect of the NS4B CTD mutations on the subcellular localization of NS5A and NS3. A. Huh7.5 cells were co-transfected with Wt or mutant DNA constructs expressing HCV polyprotein as described above. At 48 h post-transfection, the cells were processed for fluorescence (NS5AGFP) or immunofluorescence (IF) with NS4B-specific antibody. Notice that in cells with Wt polyprotein, NS4B and NS5A foci merge (i to iii). A mutation of valine to arginine (V233R; iv to vi) or leucine to glutamate (L237E; vii to ix) results in a reticular distribution of both NS4B and NS5A proteins. B. Huh7.5 cells were co-transfected with Wt or mutant constructs as described above except that IF was performed using anti NS3 antibody. Notice that NS3 and NS5A foci merge in the Wt construct (i to iii). In the mutant constructs (iv to ix), both NS5A and NS3 display a reticular distribution. Bars = 10 µm. Percent co-localization between NS3 and NS5A was calculated using Axiovision software (v. 4.5, Carl Zeiss). The data represent co-localization from five NS5A-expressing cells.

Jason Aligo, et al. Virology. ;393(1):68-83.
5.

FIG. 2. From: Formation and function of hepatitis C virus replication complexes require residues in the carboxy-terminal domain of NS4B protein.

A. Ultrastructural analysis of cells expressing Wt or truncated NS4B protein. Huh7.5 cells were transfected with GFP, NS4B-GFP or a CTD truncation [Δ (192–261)-GFP] construct. At 48 h posttransfection, the cells were processed for TEM analysis. The circled area shows the cluster of vesicles characteristic of the web (ii). Cells expressing NS4B Δ(192–261)-GFP show vesicles scattered in the cytosol (compare iii & iv to ii). No cluster of vesicles were found in control GFP-expressing cells (i). Bars = 1 µm. B. Membrane distribution of Wt and NS4B Δ(192–261)-GFP fusion proteins. Cells were transfected with constructs as described above. Three hundred micrograms of cell extract were subjected to membrane floatation, followed by western blot with GFP-, calnexin- or GAPDH-specific antibody. Lysate refers to crude lysate. Lanes 1 to 4: membrane fractions and lanes 5 to 8: soluble fractions.

Jason Aligo, et al. Virology. ;393(1):68-83.
6.
FIG. 4

FIG. 4. From: Formation and function of hepatitis C virus replication complexes require residues in the carboxy-terminal domain of NS4B protein.

Effect of NS4B CTD mutations on HCV genome replication. A. Schematic representation of the NS5AGFP subgenomic replicon construct (Moradpour et al., 2004) used in this study. B. Sequence of NS4B CTD from Con1. Shown are alpha helical structures 1 and 2 predicted by PSIPRED program. The stars indicate the mutated residues in helix 2 and the substitutions are shown underneath. C. Five micrograms of RNA transcripts, from Wt and NS4B CTD mutant replicons, were electroporated into Huh7.5 cells. After three weeks of selection, G418-resistant colonies were fixed and stained with crystal violet. Numbers below each dish indicate the colony forming units/µg (CFU/µg) of input in vitro replicon transcript. Notice that most of the NS4B CTD mutations resulted in a major decrease in the number of G418-resistant clones. Results represent values from at least two independent electroporations.

Jason Aligo, et al. Virology. ;393(1):68-83.
7.

FIG. 5. From: Formation and function of hepatitis C virus replication complexes require residues in the carboxy-terminal domain of NS4B protein.

Effect of NS4B CTD mutations on polyprotein processing. A. Huh7.5 cells were co-transfected with WT DNA construct expressing HCV polyprotein and T7 polymerase expression vector. At 48 h posttransfection, the cells were labeled with (35S)-methionine for 10 min and the lysates were immunoprecipitated (IP’d) with HCV-specific antibody to NS3, NS4B, NS5A or NS5B protein. Alternatively, the labeled cells were chased for 60 min and the lysates were IP’d with pooled HCV-specific antibodies to NS3, NS4B, NS5A or NS5B. IP’d proteins were separated on 10% SDS-PAGE and visualized by autoradiography using a phosphorImager. Notice that IP of NS4B protein results in the coIP of NS3 (indicated by asterisks in the αNS4B lane). Also notice the slowly migrating band (above NS4B) indicated by asterisks in the αNS3, αNS4B and αNS5A lanes. B. Processing of Wt and mutant HCV polyproteins. Huh7.5 cells were co-transfected with DNA constructs as described above. At 48 h post-transfection, the cells were pulse-labeled, followed by IP with pooled HCV-specific antibodies, SDS-PAGE and visualization as described above. Notice that the levels of the mature proteins are similar between Wt and mutant polyproteins. Precursor NS4A-B, NS4B-5A and NS5A-B appear to be processed by 60 min chase. C & D. Effect of NS4B CTD mutations on NS4B synthesis and stability. Huh7.5 cells were transfected as described above. At 48 h posttransfection, the cells were labeled with (35S)-methionine for 15 min, followed by a 30 min and 120 min chase. The cell lysates were IP’d with NS4B-specific antibody and processed as described above.

Jason Aligo, et al. Virology. ;393(1):68-83.
8.

FIG. 1. From: Formation and function of hepatitis C virus replication complexes require residues in the carboxy-terminal domain of NS4B protein.

A. The amino acid sequence of NS4B CTD from various HCV isolates. Highly conserved residues are highlighted, whereas stars indicate mutated residues. B. Schematic representation of Wt and NS4B truncation constructs. The predicted transmembrane domains (TMD1 to TMD4) (Lundin et al., 2003) are shown as dark boxes. Hatched boxes represent the N-terminus amphipathic alpha helix and the CTD. Note that all these constructs have a C-terminal GFP fusion. Also shown are summaries of the effect of NS4B truncations on its subcellular distribution when expressed alone. (+ +) Means foci resembling Wt NS4B distribution. (+) Refers to NS4B with smaller and numerous foci. (+/−) Means intermediate phenotype with mostly a reticular distribution and few foci, whereas (−) signifies reticular NS4B distribution. C. Huh7.5 cells were transfected with GFP, WT NS4BGFP or various NS4B deletion constructs. At 48 h posttransfection, 60 µg of cell lysates were separated on 10% SDS-PAGE and subjected to immunoblotting using anti GFP antibody. Lane 1: control GFP vector; lane 2: N4B-GFP; lane 3: Δ(192–261)-GFP; lane 4: Δ(227–261)-GFP; lane 5: Δ(227–250)-GFP; lane 6: Δ(1–191)-GFP. Arrowheads indicate the expected bands for GFP and NS4B fusion proteins. Notice that most of the constructs expressed similar levels of NS4B protein. D. Subcellular distribution of Wt and mutant NS4B constructs. Wt and various NS4B CTD truncations were transfected into Huh7.5 cells. At 48 h posttransfection, the cells were processed for fluorescence microscopy. Wt NS4B-GFP displays the characteristic NS4B foci (iv–vi), whereas truncations result in numerous and smaller foci (vii–ix and xvi–xviii), mostly reticular distribution and few foci (xiii–xv) or no visible foci (x–xii). Bars = 10 µm.

Jason Aligo, et al. Virology. ;393(1):68-83.

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