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Interferon-α inducible protein 6 impairs EGFR activation by CD81 and inhibits hepatitis C virus infection.

Meyer K, Kwon YC, Liu S, Hagedorn CH, Ray RB, Ray R - Sci Rep (2015)

Bottom Line: HCV RNA level or infectious foci were inhibited significantly by ectopic expression of IFI6.Taken together, the results from our study support a model where IFI6 inhibits HCV entry by impairing EGFR mediated CD81/CLDN1 interactions.This may be relevant to other virus entry processes employing EGFR.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, Saint Louis University.

ABSTRACT
Viral entry requires co-operative interactions of several host cell factors. Interferon (IFN) and the IFN-stimulated genes (ISGs) play a central role in antiviral responses against hepatitis C virus (HCV) infection. We examined the effect of interferon-α inducible protein 6 (IFI6) against HCV infection in human hepatoma cells. HCV RNA level or infectious foci were inhibited significantly by ectopic expression of IFI6. IFI6 impaired CD81 co-localization with claudin-1 (CLDN1) upon HCV infection or CD81 cross-linking by specific antibody. Activation of epidermal growth factor receptor (EGFR), a co-factor involved in CD81/CLDN1 interactions, was reduced in IFI6 expressing cells in response to HCV infection or CD81 cross linking by antibody, but not by treatment with EGF. Taken together, the results from our study support a model where IFI6 inhibits HCV entry by impairing EGFR mediated CD81/CLDN1 interactions. This may be relevant to other virus entry processes employing EGFR.

No MeSH data available.


Related in: MedlinePlus

IFI6 localizes on both the cell surface and intracellularly, and does not associate with CD81.FLAG tagged IFI6 expression was detected in lysates of Huh7.5 stable transfectants by Western blotting with a FLAG specific antibody. Note that cropped gel images are used in this figure and the gels were run under the same experimental conditions (panel A). Expression of IFI6 is shown during transient, stable, and IFN-α stimulated conditions; and standard deviations are shown as error bars (panel B). Detection of IFI6 on the cell surface of unfixed (panel C, section a) and in the cytoplasm of fixed cells (panel C, section b) by FLAG antibody. Mitochondria were stained with a mitotracker dye (panel C, section c) and merged fluorescence of cytoplasmic IFI6 and stained mitochondria (panel C, section d) are shown. Basal expression of CD81 was determined in Huh7.5 cells (panel D, Section a) and IFI6 transfected Huh7.5 cells (panel D, section b). Huh7.5 and IFI6 expressing cells were cross-linked with a CD81 specific antibody, followed by fixation. CD81 translocation after antibody mediated cross-linking was observed in control (panel D, section c) and was not inhibited in IFI6 expressing cells (panel D, section d). The presence of IFI6 (red) was also observed (panel C, section D) and did not co-localize with CD81 (green). Cell nuclei were stained with DAPI (blue). Each panel is representative of three independent experiments.
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f2: IFI6 localizes on both the cell surface and intracellularly, and does not associate with CD81.FLAG tagged IFI6 expression was detected in lysates of Huh7.5 stable transfectants by Western blotting with a FLAG specific antibody. Note that cropped gel images are used in this figure and the gels were run under the same experimental conditions (panel A). Expression of IFI6 is shown during transient, stable, and IFN-α stimulated conditions; and standard deviations are shown as error bars (panel B). Detection of IFI6 on the cell surface of unfixed (panel C, section a) and in the cytoplasm of fixed cells (panel C, section b) by FLAG antibody. Mitochondria were stained with a mitotracker dye (panel C, section c) and merged fluorescence of cytoplasmic IFI6 and stained mitochondria (panel C, section d) are shown. Basal expression of CD81 was determined in Huh7.5 cells (panel D, Section a) and IFI6 transfected Huh7.5 cells (panel D, section b). Huh7.5 and IFI6 expressing cells were cross-linked with a CD81 specific antibody, followed by fixation. CD81 translocation after antibody mediated cross-linking was observed in control (panel D, section c) and was not inhibited in IFI6 expressing cells (panel D, section d). The presence of IFI6 (red) was also observed (panel C, section D) and did not co-localize with CD81 (green). Cell nuclei were stained with DAPI (blue). Each panel is representative of three independent experiments.

Mentions: Huh7.5 cells were transiently or stably transfected with IFI6 plasmid DNA (Huh7.5/IFI6). Rep2a cells were also transiently transfected to express IFI6. Transient and stable expression of IFI6 in Huh7.5 cells was documented by Western blot analysis using antibody to FLAG, as compared to controls (Fig. 2, panel A). Relative levels of IFI6 mRNA were measured by real time PCR following stable and transient expression. IFI6 expression in the presence of IFN-α was examined as a positive control. Expression of IFI6 was detected in unstimulated transiently transfected Huh7.5 cells. Expression of IFI6 at the RNA level in stable transfectants was modest and was approximately six fold less than that seen during transient transfection, and three fold less than that seen in an IFN-α treated cell line (Figure 2, panel B). As noted previously (Fig. 1, panels A and C) there is a decreased ability of IFI6 to reduce HCVcc infection in transiently transfected cells, and this difference is likely due to the reduced number of cells expressing IFI6 in the total cell population. Immunofluorescence staining of IFI6-FLAG expressing cells exhibited protein localization as punctate dots on the surface of the unfixed cells (Fig. 2, panel C, section a), as well as a more localized expression upon intracellular staining (Fig. 2, panel C, section b). Mitotracker Red was used to stain mitochondria (Fig. 2, panel C, section c). Intracellular staining of IFI6 was done in fixed cells and determined that there was significant co-localization of IFI6 with mitochondria (28%) using a mitotracker stain (Fig. 2, panel C, section d).


Interferon-α inducible protein 6 impairs EGFR activation by CD81 and inhibits hepatitis C virus infection.

Meyer K, Kwon YC, Liu S, Hagedorn CH, Ray RB, Ray R - Sci Rep (2015)

IFI6 localizes on both the cell surface and intracellularly, and does not associate with CD81.FLAG tagged IFI6 expression was detected in lysates of Huh7.5 stable transfectants by Western blotting with a FLAG specific antibody. Note that cropped gel images are used in this figure and the gels were run under the same experimental conditions (panel A). Expression of IFI6 is shown during transient, stable, and IFN-α stimulated conditions; and standard deviations are shown as error bars (panel B). Detection of IFI6 on the cell surface of unfixed (panel C, section a) and in the cytoplasm of fixed cells (panel C, section b) by FLAG antibody. Mitochondria were stained with a mitotracker dye (panel C, section c) and merged fluorescence of cytoplasmic IFI6 and stained mitochondria (panel C, section d) are shown. Basal expression of CD81 was determined in Huh7.5 cells (panel D, Section a) and IFI6 transfected Huh7.5 cells (panel D, section b). Huh7.5 and IFI6 expressing cells were cross-linked with a CD81 specific antibody, followed by fixation. CD81 translocation after antibody mediated cross-linking was observed in control (panel D, section c) and was not inhibited in IFI6 expressing cells (panel D, section d). The presence of IFI6 (red) was also observed (panel C, section D) and did not co-localize with CD81 (green). Cell nuclei were stained with DAPI (blue). Each panel is representative of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4355636&req=5

f2: IFI6 localizes on both the cell surface and intracellularly, and does not associate with CD81.FLAG tagged IFI6 expression was detected in lysates of Huh7.5 stable transfectants by Western blotting with a FLAG specific antibody. Note that cropped gel images are used in this figure and the gels were run under the same experimental conditions (panel A). Expression of IFI6 is shown during transient, stable, and IFN-α stimulated conditions; and standard deviations are shown as error bars (panel B). Detection of IFI6 on the cell surface of unfixed (panel C, section a) and in the cytoplasm of fixed cells (panel C, section b) by FLAG antibody. Mitochondria were stained with a mitotracker dye (panel C, section c) and merged fluorescence of cytoplasmic IFI6 and stained mitochondria (panel C, section d) are shown. Basal expression of CD81 was determined in Huh7.5 cells (panel D, Section a) and IFI6 transfected Huh7.5 cells (panel D, section b). Huh7.5 and IFI6 expressing cells were cross-linked with a CD81 specific antibody, followed by fixation. CD81 translocation after antibody mediated cross-linking was observed in control (panel D, section c) and was not inhibited in IFI6 expressing cells (panel D, section d). The presence of IFI6 (red) was also observed (panel C, section D) and did not co-localize with CD81 (green). Cell nuclei were stained with DAPI (blue). Each panel is representative of three independent experiments.
Mentions: Huh7.5 cells were transiently or stably transfected with IFI6 plasmid DNA (Huh7.5/IFI6). Rep2a cells were also transiently transfected to express IFI6. Transient and stable expression of IFI6 in Huh7.5 cells was documented by Western blot analysis using antibody to FLAG, as compared to controls (Fig. 2, panel A). Relative levels of IFI6 mRNA were measured by real time PCR following stable and transient expression. IFI6 expression in the presence of IFN-α was examined as a positive control. Expression of IFI6 was detected in unstimulated transiently transfected Huh7.5 cells. Expression of IFI6 at the RNA level in stable transfectants was modest and was approximately six fold less than that seen during transient transfection, and three fold less than that seen in an IFN-α treated cell line (Figure 2, panel B). As noted previously (Fig. 1, panels A and C) there is a decreased ability of IFI6 to reduce HCVcc infection in transiently transfected cells, and this difference is likely due to the reduced number of cells expressing IFI6 in the total cell population. Immunofluorescence staining of IFI6-FLAG expressing cells exhibited protein localization as punctate dots on the surface of the unfixed cells (Fig. 2, panel C, section a), as well as a more localized expression upon intracellular staining (Fig. 2, panel C, section b). Mitotracker Red was used to stain mitochondria (Fig. 2, panel C, section c). Intracellular staining of IFI6 was done in fixed cells and determined that there was significant co-localization of IFI6 with mitochondria (28%) using a mitotracker stain (Fig. 2, panel C, section d).

Bottom Line: HCV RNA level or infectious foci were inhibited significantly by ectopic expression of IFI6.Taken together, the results from our study support a model where IFI6 inhibits HCV entry by impairing EGFR mediated CD81/CLDN1 interactions.This may be relevant to other virus entry processes employing EGFR.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, Saint Louis University.

ABSTRACT
Viral entry requires co-operative interactions of several host cell factors. Interferon (IFN) and the IFN-stimulated genes (ISGs) play a central role in antiviral responses against hepatitis C virus (HCV) infection. We examined the effect of interferon-α inducible protein 6 (IFI6) against HCV infection in human hepatoma cells. HCV RNA level or infectious foci were inhibited significantly by ectopic expression of IFI6. IFI6 impaired CD81 co-localization with claudin-1 (CLDN1) upon HCV infection or CD81 cross-linking by specific antibody. Activation of epidermal growth factor receptor (EGFR), a co-factor involved in CD81/CLDN1 interactions, was reduced in IFI6 expressing cells in response to HCV infection or CD81 cross linking by antibody, but not by treatment with EGF. Taken together, the results from our study support a model where IFI6 inhibits HCV entry by impairing EGFR mediated CD81/CLDN1 interactions. This may be relevant to other virus entry processes employing EGFR.

No MeSH data available.


Related in: MedlinePlus