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Results: 5

1.

Fig. 3. Reversal of BMS-790052-induced alterations on subcellular localization of NS proteins expressed from a replicon containing the BMS-790052-resistant mutation (Y93H) in NS5A. From: The hepatitis C virus NS5A inhibitor (BMS-790052) alters the subcellular localization of the NS5A non-structural viral protein.

(a) Immunofluorescence analysis of NS5A. Procedure was performed as described in Fig. 2a except that Bart79I plasmid, containing the BMS-790052-resistant mutation Y93H (NS5A) was used to express NS viral proteins.
(b) Immunofluorescence analysis of NS5A. Procedure was performed as described in Fig. 2a except that the plasmid used was pEF6-NS5A, which expresses NS5A by itself.

Choongho Lee, et al. Virology. ;414(1):10-18.
2.
Fig. 4

Fig. 4. BMS-790052 alters the subcellular fractionation patterns of NS proteins. From: The hepatitis C virus NS5A inhibitor (BMS-790052) alters the subcellular localization of the NS5A non-structural viral protein.

Huh7.5 cells were infected with a vaccinia virus expressing a T7 RNA polymerase and then transfected with a plasmid (Bart79I) containing the NS viral proteins downstream of a T7 promoter. Transfected cells were incubated with 0.1 % of DMSO (A) or 10 nM of BMS-790052 (B) for 9 hours. Membrane-enriched cell lysates were prepared and fractionated on idioxanol density gradients. Distribution patterns of NS5A, NS3, and E-cadherin proteins were examined by western blot analysis using anti-NS5A, anti-NS3, and anti-E-cadherin antibodies.

Choongho Lee, et al. Virology. ;414(1):10-18.
3.
Fig. 5

Fig. 5. BMS-790052 affects neither the in vitro replicase activity of pre-assembled RCs nor the self-dimerization of NS5A proteins. From: The hepatitis C virus NS5A inhibitor (BMS-790052) alters the subcellular localization of the NS5A non-structural viral protein.

(a) Effects of BMS-790052 on an in-vitro replicase activity from pre-assembled RCs from genotype 2a. 10 μM of 3′-dCTP, 0.1 % of DMSO, 1 or 10 μM of BMS-790052 was incubated with a crude membrane fraction isolated from Huh7.5 cells harboring replicating J6/JFH full-length replicons derived from genotype 2a. Newly replicated HCV RNAs were separated on an agarose gel and exposed to a phosphorimager.
(b) Effects of BMS-790052 on the dimerization of NS5A proteins. Equal amounts of pAct-NS5A, pBind-NS5A and pG5-Luc were transfected into Huh7.5 cells. Transfected cells were incubated with 0.1 % of DMSO or 10 nM of BMS-790052. Firefly luciferase assay was performed to measure levels of interaction between two NS5A fusion proteins. pAct and pBind are negative control vectors and pAct-Myo and pBind-ID are positive control vectors.

Choongho Lee, et al. Virology. ;414(1):10-18.
4.

Fig. 2. BMS-790052 alters the subcellular localization of NS viral proteins. From: The hepatitis C virus NS5A inhibitor (BMS-790052) alters the subcellular localization of the NS5A non-structural viral protein.

(a) Immunofluorescence analysis of NS5A. Huh7.5 cells were infected with a vaccinia virus expressing a T7 RNA polymerase and then transfected with a plasmid (Bart79I) expressing the HCV NS viral proteins downstream of a T7 promoter. Transfected cells were incubated with 0.1 % of DMSO (i), 100 pM (ii), or 1 nM of BMS-790052 (iii) for 9 hours. Cells were fixed, permeabilized, and stained with a monoclonal anti-NS5A mouse antibody. Anti-mouse Alexa 488-conjugated secondary antibody was used to visualize the NS5A protein in green. White scale bar represents 10 μm. The image on the right panel shows 4-fold enlarged view of the area marked with a white rectangle on the left panel, highlighting the differences in distribution patterns of NS5A.
(b) Immunofluorescence analysis of NS5B. Procedure was performed as described in Fig. 2a except that cells were stained with a polycolonal anti-NS5B rabbit antibody. Anti-rabbit Alexa 488-conjugated secondary antibody was used to visualize the NS5B protein in green.
(c) Immunofluorescence analysis of NS3. Procedure was performed as described in Fig. 2a except that cells were stained with a monoclonal anti-NS3 mouse antibody. Anti-mouse Alexa 488-conjugated secondary antibody was used to visualize the NS3 protein in green
(d) Immunofluorescence analysis of PDI. Procedure was performed as described in Fig. 2a except that cells were stained with a polyclonal anti-PDI rabbit antibody. Anti-rabbit Alexa 594-conjugated secondary antibody was used to visualize the PDI protein in red.

Choongho Lee, et al. Virology. ;414(1):10-18.
5.

Fig. 1. BMS-790052 blocks HCV genome replication. From: The hepatitis C virus NS5A inhibitor (BMS-790052) alters the subcellular localization of the NS5A non-structural viral protein.

(a) Structure of BMS-790052
(b) Effect of BMS-790052 on stable replication of a HCV replicon derived from genotype 1b. Huh7 cells harboring replicating subgenomic replicons derived from genotype 1b were treated with 0, 0.1, 1, 10, 100, or 1000 pM of BMS-790052 for 3 days. A total cell lysate from cells was prepared and an equal amount of each cell lysate was separated by SDS-PAGE. Levels of NS5A and β-actin protein expression were examined by western blot analysis using monoclonal anti-NS5A and anti-β-actin antibodies.
(c) Huh7.5 cells were infected with a vaccinia virus expressing a T7 RNA polymerase and then transfected with either Bart79I or Bartman (Con1) plasmid construct containing the NS viral proteins downstream of a T7 promoter. Transfected cells were incubated with 0.1 % of DMSO or 0.01, 0.1, 1, 10, or 100 nM of BMS-790052 for 9 hours. A total cell lysate was prepared and an equal amount of each cell lysate was separated by SDS-PAGE. Levels of NS5A protein expression were examined by western blot analysis using a monoclonal anti-NS5A antibody.
(d) All procedures were performed as in Fig. 1(c) except that levels of NS3 protein expression were examined by western blot analysis using a monoclonal anti-NS3 antibody.

Choongho Lee, et al. Virology. ;414(1):10-18.

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