For many years our knowledge on hepatitis C virus (HCV) replication has been based on in vitro experiments or transfection studies. Recently, the first reliable system for studying viral replication in tissue culture cells was developed. Taking advantage of this system, we examined in detail the localization of viral nonstructural (NS) proteins in cells containing functional replication complexes. By fractionation experiments and immunomicroscopy, we observed that all NS proteins were associated with the endoplasmic reticulum (ER) membranes, confirming the hypothesis that the ER is the site of membrane-associated HCV RNA replication. Interestingly, NS3 and NS4A were preferentially localized in endoplasmic reticulum cisternae surrounding mitochondria, suggesting additional subcellular compartment-related functions for these viral proteins. Furthermore, the immunoelectron microscopy revealed the loss of the organization and other morphological alterations of the ER (convoluted cisternae and paracrystalline structures), resembling alterations observed in liver biopsies of HCV-infected individuals and in flavivirus-infected cells.

Melanogenesis and melanosome transfer from the melanocytes to the neighboring keratinocytes are induced by ultraviolet radiation and modulated by autocrine and paracrine factors. Keratinocyte growth factor (KGF/fibroblast growth factor (FGF)7) is a paracrine mediator of human keratinocyte growth and differentiation. We evaluated the influence of KGF on melanosome transfer in co-cultures of keratinocytes and melanocytes. Immunofluorescence analysis using anti-tyrosinase and anti-human cytokeratin antibodies, phagocytic assays using fluorescent latex beads, and ultrastructural analysis indicated that KGF is able to induce melanosome transfer acting only on the recipient keratinocytes and as a consequence of a general role of KGF in the promotion of the phagocytic process. Inhibition of proteinase-activated receptor-2, to block the Rho-dependent phagocytic pathway, or of the Src family tyrosine kinases, to inhibit the Rac-dependent pathway, showed that KGF promotes phagocytosis through both mechanisms. Increased expression of the KGF receptor (KGFR) on the keratinocytes by transfection led to increased phagocytosis of latex beads following KGF treatment, suggesting that the KGF effect is directly mediated by KGFR expression and activation. Moreover, confocal microscopic analysis revealed that KGFR localize in phagosomes during KGF-induced phagocytosis, suggesting a direct role of the receptor in regulating both the early steps of uptake and the intracellular traffic of the phagosomes.

Fibroblasts may be persistently activated by UV exposure to release melanogenic growth factors; this inducible cytokine network acts both directly and indirectly through keratinocytes and may contribute to the hyperpigmentation of SL.