Huh7 cells were transfected with these plasmids and, 40 hours later, we determined the subcellular distribution of the encoded proteins by fluorescence microscopy. The aa core protein fragment was found predominantly in the cytoplasm, whereas aa and aa were found solely in the nucleus. These experiments showed clearly that aa sequences and did not function as additional NES. Indeed, in the absence of the aa sequence identified in this study, these core protein fragments were not exported from the nucleus. We therefore conclude that the DAPT Gamma-secretase inhibitor cytoplasmic distribution of core requires a functional NES within aa. However, the presence of this sequence is not sufficient and an additional fragment aa is also required to maintain the protein in the cytoplasm. The hydrophobic C-terminal DII of HCV core, containing this sequence, is required for the binding of core protein to lipid droplets and membranes, and for core protein folding and stability. Indeed, residues of two amphipathic helices, aa and aa, in DII are critical for this association, as shown by extensive mutational analyses. Therefore, the core fragment aa is found mostly in the cytoplasm, because, in addition to the NES, it mediates interactions of core with the cytoplasmic lipid droplets and/or membranes via specific sites in the aa sequence. Quantitative analyses performed on HCV-infected and non infected cells after staining with anti-core antibodies confirmed the specificity of our observations and showed that a larger proportion of the gold-labeled HCV core was present in the cell nucleus than elsewhere in the cell at this stage of infection. No such staining was observed when unrelated monoclonal antibodies were used. These observations suggest that, at very early stages of HCV infection, at least some of the HCV core protein is directed to the nucleus. We provide here the first demonstration that a functional NES is present in HCV core protein. We show that amino acids 109 to 133 are responsible for the Nutlin-3 company active export of the HCV core protein out of the nucleus, via a CRM-1�Cmediated nuclear export pathway. This NES was functional in transfected cells and in an in vitro model of HCV replication. The trafficking of core protein into the nucleus early in infection may help to establish infection and facilitate the interaction of core with nuclear molecules, with potentially important pathological consequences. HCV core protein is thought to be a major viral factor promoting liver disease during HCV infection and the malignant transformation of hepatocytes, leading to the development of HCC through interactions with several host cell factors involved in a wide range of cellular processes. Indeed, in various experimental systems, HCV core has been reported to affect transcription mediated by various gene promoters and apoptosis, thereby contributing to cell transformation.