Reconstitution of gene expression from a regulatory‐protein‐deficient hepatitis B virus genome by cell‐permeable HBx protein

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Abstract
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‐κB. 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.