Reconstitution of gene expression from a regulatory-protein-deficient hepatitis B virus genome by cell-permeable HBx protein.
Various functions are ascribed to the HBx regulatory protein of the hepatitis B virus (HBV). Due to the low expression level of HBx, it has been difficult to correlate spatial and temporal HBx expression levels with specific functions. Based on a novel cell-permeable peptide, known as the translocation motif (TLM), cell-permeable HBx fusion proteins were generated. The TLM-HBx fusion protein is rapidly internalized from the medium into almost all cells, whereas no significant internalization was seen with wild-type HBx. The major fraction of internalized HBx protein moves from the cytoplasm to the nucleus. The cytosolic fraction, however, activates c-RAF1/extracellular-signal-related kinase 2 signalling and causes activation of activator protein 1 (AP1) and nuclear factor-kappaB. The TLM-HBx protein rescues HBV gene expression from an activator-deficient HBV genome. These results indicate that cell-permeable regulatory proteins provide a novel, efficient tool for a clearly defined, dose-dependent analysis of regulatory protein function, without affecting the integrity of the cell, and can be used for the safe reconstitution of virus production from a regulatory-protein-deficient virus genome.
Hafner
Angela
A
Robert Koch-Institut, Nordufer 20, D-13353 Berlin, Germany.
Brandenburg
Boerries
B
Hildt
Eberhard
E
D045744:Cell Line, Tumor; D003600:Cytosol / Q000378:metabolism; D004279:DNA, Viral / Q000096:biosynthesis; D015870:Gene Expression; D006514:Hepatitis B Surface Antigens / Q000235:genetics / Q000378:metabolism; D006515:Hepatitis B virus / Q000235:genetics* / Q000502:physiology; D006801:Humans; D008928:Mitochondria / Q000502:physiology; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014162:Transfection; D054334:Viral Regulatory and Accessory Proteins; D014779:Virus Replication
D004279:DNA, Viral; D006514:Hepatitis B Surface Antigens; D016328:NF-kappa B; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D018808:Transcription Factor AP-1; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein; C050807:presurface protein 2, hepatitis B surface antigen; D019908:Proto-Oncogene Proteins c-raf; D019950:Mitogen-Activated Protein Kinase 1