Journal: Viruses

Article Title: Hepatitis B Virus Protein X Induces Degradation of Talin-1

doi: 10.3390/v8100281

Figure Lengend Snippet: Hepatitis B virus (HBV) accessory protein X (HBx) transactivates transcription by inducing proteasomal degradation of a host protein. ( A ) HBV replication was initiated in HepG2 cells by transfection of a construct containing a 1.2-fold overlength HBV genome that initiates the full HBV replication cycle. The replication of a mutant lacking HBx was impaired but could be rescued by cotransfection of a construct that expresses HBx. ( B , C ) Activity of luciferase reporter constructs as a percentage of their basal activities 48 h after transfection in HEK 293 cells. Cells were cotransfected with plasmids expressing HBx or the HBx-R96E mutant, which does not interact with damaged DNA binding protein 1 (DDB1). The luciferase reporters were under control of the hepatitis B core (C), X (X), S1 (S1) and S2 (S2) promoters, or the HIV-1 LTR (LTR), the human elongation factor 1α (hEF1α), the phosphodiesterase 8A (PDE8A), or thyroglobulin (TG) promoters. ( D ) eCFP-HBx was expressed in HEK 293-T cells and lysates were immunoprecipitated with anti-green fluorescent protein (GFP). A Western blot of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) separated proteins was stained with anti-DDB1, showing that in these cells HBx interacts with DDB1. ( E ) Activity of the HBV core promoter 48 h after cotransfection with HBx in the absence and presence of proteasome inhibitor MG132, showing that transactivation by HBx critically depends on proteasomal degradation. ( F ) To assess the effect of HBx expression on integrated promoters, we analyzed the PDE8A mRNA production in HEK 293 cells after transfection with HBx. HBx expression did not affect the RNA transcription from the integrated PDE8A promoter, showing that transactivation by HBx is specific for extrachromosomal templates. * p < 0.05, ** p < 001, *** p < 0.001.

Article Snippet: The following antibodies were used to visualize proteins: mouse monoclonal anti-DDB1 (1:1000; BD transduction laboratories™); SC-1616 anti-β-actin antibody (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA); Mouse monoclonal anti-non-muscle Myosin IIA antibody (1:1000; Abcam, Cambridge, UK, ab55456); mouse monoclonal anti-talin-1 (1:200, Abcam [93E12] ab104913); and rabbit monoclonal anti-talin-2 antibody (1:10,000, Abcam ab108967).

Techniques: Transfection, Construct, Mutagenesis, Cotransfection, Activity Assay, Luciferase, Expressing, Binding Assay, Immunoprecipitation, Western Blot, Polyacrylamide Gel Electrophoresis, SDS Page, Staining