Hepatitis C virus (HCV) proteins induce distinct membrane alterations, including a candidate viral replication complex. The study investigated the effects of HCV proteins on cellular membranes using tetracycline-regulated cell lines. Electron microscopy (EM) and immunogold EM (IEM) revealed that structural proteins (core-E1-E2-p7), NS3-4A complex, and NS4B induced distinct membrane alterations. NS4B formed a tight structure called the membranous web, consisting of vesicles in a membranous matrix. Expression of the entire HCV polyprotein led to membrane budding into rough endoplasmic reticulum (rER) vacuoles, the membranous web, and tightly associated vesicles. All HCV proteins were found to associate with the membranous web, forming a multiprotein complex. This structure was previously observed in HCV-infected chimpanzee livers, suggesting it may be the viral replication complex. HCV is a major cause of liver disease, including chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Despite the identification of HCV in 1989, its replication cycle and pathogenesis remain incompletely understood. The study used heterologous expression systems and subgenomic replicons to investigate the viral replication cycle. HCV has a positive-sense single-stranded RNA genome, with structural and nonstructural proteins involved in viral replication. NS4B, a protein of unknown function, induced the membranous web, while NS5A and NS5B did not induce distinct membrane alterations. The study found that HCV proteins interact with host cell membranes, either directly or via NS4A. The core protein is targeted to the rER, while NS2 and NS4B colocalize with other nonstructural proteins on rER-derived membranes. NS5A is an integral membrane protein, and NS5B functions as an RNA-dependent RNA polymerase (RdRp) in the viral replication complex. The formation of membrane-bound replication complexes is common in plus-strand RNA viruses, leading to structural changes in infected cells. The study suggests that the membranous web may be the HCV replication complex, as all HCV proteins associate with it. The findings indicate that HCV proteins contribute to cytopathology through membrane alterations, and the membranous web is a candidate for the viral replication complex. The study also highlights the importance of understanding HCV-induced membrane alterations in elucidating the pathogenesis of liver cell injury.Hepatitis C virus (HCV) proteins induce distinct membrane alterations, including a candidate viral replication complex. The study investigated the effects of HCV proteins on cellular membranes using tetracycline-regulated cell lines. Electron microscopy (EM) and immunogold EM (IEM) revealed that structural proteins (core-E1-E2-p7), NS3-4A complex, and NS4B induced distinct membrane alterations. NS4B formed a tight structure called the membranous web, consisting of vesicles in a membranous matrix. Expression of the entire HCV polyprotein led to membrane budding into rough endoplasmic reticulum (rER) vacuoles, the membranous web, and tightly associated vesicles. All HCV proteins were found to associate with the membranous web, forming a multiprotein complex. This structure was previously observed in HCV-infected chimpanzee livers, suggesting it may be the viral replication complex. HCV is a major cause of liver disease, including chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Despite the identification of HCV in 1989, its replication cycle and pathogenesis remain incompletely understood. The study used heterologous expression systems and subgenomic replicons to investigate the viral replication cycle. HCV has a positive-sense single-stranded RNA genome, with structural and nonstructural proteins involved in viral replication. NS4B, a protein of unknown function, induced the membranous web, while NS5A and NS5B did not induce distinct membrane alterations. The study found that HCV proteins interact with host cell membranes, either directly or via NS4A. The core protein is targeted to the rER, while NS2 and NS4B colocalize with other nonstructural proteins on rER-derived membranes. NS5A is an integral membrane protein, and NS5B functions as an RNA-dependent RNA polymerase (RdRp) in the viral replication complex. The formation of membrane-bound replication complexes is common in plus-strand RNA viruses, leading to structural changes in infected cells. The study suggests that the membranous web may be the HCV replication complex, as all HCV proteins associate with it. The findings indicate that HCV proteins contribute to cytopathology through membrane alterations, and the membranous web is a candidate for the viral replication complex. The study also highlights the importance of understanding HCV-induced membrane alterations in elucidating the pathogenesis of liver cell injury.