HCV NS4B targets Scribble for proteasome-mediated degradation to facilitate cell transformation

Abstract

Hepatitis C virus (HCV) nonstructural protein 4B (NS4B) is a multi-transmembrane protein, but little is known about how NS4B contributes to HCV replication and tumorigenesis. Its C-terminal domain (CTD) has been shown to associate with intracellular membrane, and we have previously shown that NS4B CTD contains a class I PDZ-binding motif (PBM). Here, we demonstrated that NS4B PBM interacts with the PDZ-containing tumor suppressor protein, Scribble, using immunofluorescence and co-immunoprecipitation assays, and this interaction requires at least three contiguous PDZ domains of Scribble. In addition, NS4B PBM specifically induced Scribble degradation by activating the proteasome-ubiquitin pathway. Similar Scribble degradation was also observed in HCV-infected cells, suggesting NS4B could work in the context of HCV. Finally, NS4B PBM mutants showed reduced colony formation capacity compared with its wild-type counterpart, indicating that NS4B PBM plays important roles in NS4B-mediated cell transformation. Altogether, we provide a mechanism by which NS4B induces cell transformation through its PBM, which specifically interacts with the PDZ domains of Scribble and targets Scribble for degradation.

Keywords: HCV; NS4B; PBM; Scribble; Tumorigenesis; Ubiquitin.

Fig. 1

HCV NS4B co-localized with Scribble protein. Cells were fixed for immunofluorescence analysis at 24 h post-transfection. Cell nuclei were stained by DAPI (in blue). The co-localization between NS4B and Scribble was shown in the merged images (fourth columns)

Fig. 2

HCV NS4B PBM is required to bind Scribble. Cell extract was incubated with anti-FLAG resin, and the co-immunoprecipitated Scribble was analyzed by Western blot with anti-Scribble antibody

Fig. 3

Three adjacent PDZ domains of Scribble are required to bind HCV NS4B. a Diagram showing the structure of Scribble protein. The Myc-tagged PDZ domains of Scribble used in the Co-IP assay were indicated, and the binding activities of Scribble PDZ domains for HCV NS4B were summarized. b–j 293T cells were transfected with pFLAG-CMV2 or pFLAG-CMV2-NS4B and Myc-tagged Scribble PDZ (b), PDZ1 (c), PDZ2 (d), PDZ3 (e), PDZ4 (f), PDZ1-2 (g), PDZ2-3 (h), PDZ3-4 (i), and PDZ2-4 (j). Cell lysates were mixed with anti-FLAG resin, and the precipitates were analyzed by Western blot with indicated antibodies. k 293T cells were co-transfected with pFLAG-CMV2-NS4B, pCDNA4b, or Myc-tagged Scribble PDZ1-3. Cell lysates were mixed with anti-Myc resin, and the precipitates were analyzed by Western blot with indicated antibodies

Fig. 4

HCV NS4B induced Scribble degradation. a, b 293T (a) and HepG2 (b) cells were transfected with 1, 3, and 5 μg pCDNA3.1-NS4B expression plasmid. Twenty-four hours post-transfection, cell lysates were prepared and analyzed by Western blot with indicated antibodies. pCDNA3.1(−) plasmid was used as a negative control. c, d 293T (c) and HepG2 (d) cells were transfected with 1 μg of pCDNA3.1-NS4B expression plasmid. Twenty-four and 48 h post-transfection, the protein levels of NS4B, Scribble, and LGL2 were analyzed by Western blot. e, f Relative protein levels of Scribble (e) and LGL2 (f) were quantitated with standard deviation (SD). GAPDH levels were used to normalize these two proteins. *P < 0.05; **P < 0.01

Fig. 5

HCV NS4B PBM induced Scribble degradation via the proteasome. a 293T and HepG2 cells were transfected with 1 μg pCDNA3.1(−) or pCDNA3.1-NS4B. Twenty-four and 48 h post-transfection, the protein levels of NS4B, USP14, and GAPDH were analyzed by Western blot. b 293T cells and HepG2 cells were transfected with pFLAG-CMV2 or pFLAG-NS4B, and proteasome inhibitor was added to the cells 5 h before harvesting cells. The ubiquitination levels of Scribble were analyzed by Western blot using anti-ubiquitin antibody. c 293T and HepG2 cells were transfected with full-length NS4B, NS4BD, NS4BM, pFLAG-CMV2, and pEFGPC1. Twenty-four hours post-transfection, cell lysates were prepared and analyzed by Western blot with indicated antibodies

Fig. 6

HCV infection induced Scribble protein degradation. Huh7.5.1 cells were infected by HCV JFH1 and harvested at day 3 and day 5. a Total mRNA was extracted, and RT-PCR was performed using primers for Scribble and β-actin. Data were presented as mean ± SE (n = 3, P>0.05). b Cells lysates were subjected to Western blot analysis using antibodies against HCV Core protein, Scribble, and β-actin

Fig. 7

NS4B PBM facilitated HepG2 cells transformation. Soft-agar colony formation assay for HepG2 cells transfected with pEGFP-C1 or pEGFP-C1-NS4B or pEGFP-C1-NS4B or pEGFP-C1-NS4BM. Quantification of foci was counted. Values are means ± SD (n = 3). *P < 0.01,**P < 0.05