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. 1998 Dec;72(12):9722-8.
doi: 10.1128/JVI.72.12.9722-9728.1998.

Ectopic expression of hepatitis C virus core protein differentially regulates nuclear transcription factors

A Shrivastava  1 , S K Manna, R Ray, B B Aggarwal
Affiliations

Ectopic expression of hepatitis C virus core protein differentially regulates nuclear transcription factors

A Shrivastava et al. J Virol. 1998 Dec.

Abstract

The putative core protein of hepatitis C virus (HCV) regulates cellular growth and a number of cellular promoters. To further understand its effect, we investigated the role of the core protein in the endogenous regulation of two distinct transcription factors, nuclear factor-kappaB (NF-kappaB) and activating protein-1 (AP-1), and the related mitogen-activated protein kinase kinase (MAPKK) and c-Jun N-terminal kinase (JNK). Stable cell transfectants expressing the HCV core protein suppressed tumor necrosis factor (TNF)-induced NF-kappaB activation. Supershift analysis revealed that NF-kappaB consists of p50 and p65 subunits. This correlated with inhibition of the degradation of IkappaBalpha, the inhibitory subunit of NF-kappaB. The effect was not specific to TNF, as suppression in core protein-expressing cells was also observed in response to a number of other inflammatory agents known to activate NF-kappaB. In contrast to the effect on NF-kappaB, the HCV core protein constitutively activated AP-1, which correlated with the activation of JNK and MAPKK, which are known to regulate AP-1. These observations indicated that the core protein targets transcription factors known to be involved in the regulation of inflammatory responses and the immune system.

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Figures

FIG. 1
FIG. 1
Effect of HCV core protein expression on TNF-induced NF-κB activation. Empty-vector-transfected (A) and HCV core DNA-transfected (B) MCF-7 cells were incubated at 37°C for 0, 5, 10, 15, 30, and 60 min with TNF (100 pM). Nuclear extracts were prepared and analyzed for NF-κB activation. The units at the bottom show fold increases in TNF-induced NF-κB activation with time compared to that in untreated control cells.
FIG. 2
FIG. 2
Supershift analysis and specificity of TNF-induced NF-κB activation. The nuclear extracts from TNF-treated (100 pM, 30 min) control (A) and HCV core DNA-transfected (B) cells were incubated with different antibodies and a 50-fold excess of an unlabeled competitor for 30 min at 37°C. The reaction was monitored by EMSA with a labeled NF-κB probe.
FIG. 3
FIG. 3
Effect of HCV core protein on OA-H2O2-, and PMA-mediated activation of NF-κB. HCV core DNA-transfected and empty-vector-transfected MCF-7 cells were incubated with TNF (100 pM), PMA (100 ng/ml), H2O2 (250 μM), or OA (500 nM) for 30 min at 37°C and tested for NF-κB activation.
FIG. 4
FIG. 4
Time-dependent IκBα expression following TNF-α induction in control and HCV core-expressing cells. MCF-7 cells were incubated for different times with TNF (100 pM). Cytoplasmic extracts were prepared and analyzed for IκBα by Western blotting. The units at the bottom reflect densitometric scanning of the IκBα level. Similar results were obtained in three independent experiments.
FIG. 5
FIG. 5
TNF-induced AP-1 activation in control and HCV core-expressing cells. Empty-vector-transfected (A) and HCV core gene-transfected (B) MCF-7 cells were incubated at 37°C for 0, 30, 60, 90, 120, and 240 min with TNF (100 pM). Nuclear extracts were prepared and analyzed for AP-1 activation as described in the text. Results obtained with an unlabeled oligonucleotide as an unlabeled competitor are shown in panel A. The units at the bottom indicate fold increases in TNF-induced AP-1 activation compared to that in untreated control cells. FP, free probe.
FIG. 6
FIG. 6
Effect of HCV core protein expression on AP-1 activation in different cell lines. MCF-7, HeLa, and NIH 3T3 cells were stably transfected with the HCV core gene, and nuclear extracts from vector-transfected control and core DNA-transfected cells were analyzed for AP-1 activity by gel shift assay.
FIG. 7
FIG. 7
Autoradiograph of a representative CAT assay in which HepG2 cells were cotransfected with 0.5 μg of a reporter gene construct (phARE or pmut-hARE) or 2.5 μg of an empty vector (pPAC) and different concentrations of pHCV-core. After 48 h of transfection, the cytoplasmic extracts were analyzed for CAT activity. Fold CAT activation is indicated at the top of each lane, and the plasmids used in the transfection are shown below. mut, mutant.
FIG. 8
FIG. 8
Effect of HCV core protein on TNF-induced JNK activation. Empty-vector-transfected and HCV core DNA-transfected MCF-7 cells were treated with 1 nM TNF at 37°C for the indicated times. Cells were washed and lysed, and the JNK was immunoprecipitated from extracts by a specific antibody. JNK activity was measured by using the immune complex in a kinase assay with GST–C-Jun(1-79) as the substrate. Total JNK protein expression was determined by Western blot analysis. The units at the bottom indicate fold increases in JNK activity induced by TNF compared to that in untreated control cells.
FIG. 9
FIG. 9
TNF-induced MAPKK activity in control and HCV core-expressing cells. Empty-vector- and HCV core gene-transfected MCF-7 cells were stimulated with different concentrations of TNF for 30 min. Cell lysates were analyzed for MAPKK activity by Western blot analysis with a specific antibody to phosphorylated MAPK (phosphospecific anti-p44/42 MAPK).
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