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To determine the effect of hepatitis C virus (HCV) proteins on cell growth, Huh-7 cells were transfected with a full-length HCV cDNA (pMO9.6-T7 Rz) clone and HCV proteins were expressed using a replication-defective adenovirus that encodes the gene for the T7 RNA polymerase. Expression of HCV proteins from this full-length clone resulted in reduction in viability of transfected cells as measured by trypan blue viability assay. For identification and separation of cells expressing hepatitis C virus proteins by fluorescence microscopy and flow cytometry, GFP was cloned in the HCV full-length clone. Cells transfected with the HCV-GFP chimera clone produced high levels of accurately processed structural and nonstructural proteins similar to those of the HCV full-length clone, which could be detected by Western blot analysis. Cells expressing all HCV proteins lost membrane permeability and underwent apoptotic cell death, indicated by the appearance of a sub-G0 peak in cell cycle analysis, DNA fragmentation in a TUNEL assay, and microscopic detection of nuclear condensation. Using double-channel flow analysis we confirmed that high-level expression of HCV proteins affected membrane permeability and cell survival. These results suggest that expression of all structural and nonstructural proteins from HCV cDNA in hepatic cells induces apoptotic cell death, which might be an important event in chronic hepatitis infection in humans.

The mechanisms of hepatocyte death and the events that lead to a high rate of chronic liver disease in patients infected with hepatitis C virus are not known. We established a HCV replication system in HepG2 cell culture and utilized this model to address the effect of HCV proteins on HepG2 cell growth and viability. After transfection of HepG2 cells with full-length RNA, a truncated RNA, or an antisense RNA, cell proliferation and cell viability were analyzed by thymidine uptake and the trypan blue exclusion method, respectively. Full-length RNA transfected HepG2 cells showed a decrease in cell proliferation and viability compared to cells transfected with HCV truncated RNA and antisense RNA control. A subset of cells expressing HCV proteins underwent apoptosis as documented by morphological studies, ultrastructural analysis, cell cycle analysis by flow cytometry, terminal transferase enzyme mediated end labeling of DNA, and DNA laddering. This study suggests that expression of HCV proteins can lead to cell death by apoptosis, which may be an important event in the pathogenesis of chronic hepatitis C virus infection in humans.

Acute infection of CD4+ lymphoid cells by human immunodeficiency virus type 1 (HIV-1) induces an increase in the intracellular concentration of potassium (K+). Media containing reduced or elevated concentrations of K+ were used to investigate the role of this ion in HIV-1 production and cytopathology. Incubation of CD4+ lymphoblastoid cells acutely infected by HIV-1 (strain LAI) in low K+ medium resulted in an approximately 50% decrease in HIV-1 production and markedly diminished HIV-1 induced cytopathic effects (CPE) relative to cells incubated in medium containing a normal K+ concentration (approximately 5 mM). Incubation of HIV-1 infected cells in media containing elevated concentrations of K+ (25 to 50 mM) increased HIV-1 production by two- to fivefold over the amount produced in cells incubated in normal K+ medium. Cells incubated in high K+ media also displayed enhanced HIV-1-induced cytopathology. The decrease in HIV-1 production by low K+ medium and increase by high K+ media could not be accounted for by effects on HIV-1 reverse transcription. However, low K+ medium inhibited HIV-1 protein synthesis and high K+ media increased HIV-1 protein synthesis. These results suggest that the HIV-1–induced increase in intracellular K+ concentration is required for efficient viral replication and to induce cytopathology.

The localization of ouabain-sensitive, K+-dependent p-nitrophenylphosphatase (K+-NPPase) activity of the Na+,K+-ATPase complex was studied ultracytochemically in the squirrel monkey inner ear. In the stria vascularis the reaction products showing K+-NPPase activity were limited to the cytoplasmic side of the plasmalemmal infoldings of the marginal cells. In the spiral prominence, a weak reaction was also found on the cytoplasmic process of the stromal cell, while no or little reaction was detected on the spiral prominence epithelium. In the dark cells of vestibular labyrinth the reaction products were observed on the basolateral interdigitation of the plasmalemma. In contrast, no reaction was observed on the apical cell surface. K+-NPPase activity was most intense in the strial marginal cell, followed by the dark cell of the ampulla and the utricle. The present results revealed that the dark cells in the vestibular labyrinth are involved in endolymph homeostasis.