Plus-strand RNA viruses characteristically replicate their genome in association with altered cellular membranes. In the present study, the capacity of hepatitis C virus (HCV) proteins to elicit intracellular membrane alterations was investigated by expressing, in tetracycline-regulated cell lines, a comprehensive panel of HCV proteins individually as well as in the context of the entire HCV polyprotein. As visualized by electron microscopy (EM), expression of the combined structural proteins core-E1-E2-p7, the NS3-4A complex, and protein NS4B induced distinct membrane alterations. By immunogold EM (IEM), the membrane-altering proteins were always found to localize to the respective altered membranes. NS4B, a protein of hitherto unknown function, induced a tight structure, designated membranous web, consisting of vesicles in a membranous matrix. Expression of the entire HCV polyprotein gave rise to membrane budding into rough endoplasmic reticulum vacuoles, to the membranous web, and to tightly associated vesicles often surrounding the membranous web. By IEM, all HCV proteins were found to be associated with the NS4B-induced membranous web, forming a membrane-associated multiprotein complex. A similar web-like structure in livers of HCV-infected chimpanzees was previously described (Pfeifer et al., Virchows Arch. B., 33:233-243, 1980). In view of this finding and the observation that all HCV proteins accumulate on the membranous web, we propose that the membranous web forms the viral replication complex in HCV-infected cells.

RF ablation is superior to PEI with respect to local recurrence-free survival rates.

Formation of a membrane-associated replication complex, composed of viral proteins, replicating RNA, and altered cellular membranes, is a characteristic feature of plus-strand RNA viruses. Here, we demonstrate the presence of a specific membrane alteration, designated the membranous web, that contains hepatitis C virus (HCV) nonstructural proteins, as well as viral plus-strand RNA, in Huh-7 cells harboring autonomously replicating subgenomic HCV RNAs. Metabolic labeling with 5-bromouridine 5-triphosphate in the presence of actinomycin D revealed that the membranous web is the site of viral RNA synthesis and therefore represents the replication complex of HCV.Formation of a membrane-associated replication complex composed of viral proteins, replicating RNA, and altered cellular membranes is a hallmark of all of the plus-strand RNA viruses investigated thus far (4,11,23,27,29,32). This strategy may offer multiple potential advantages, including (i) compartmentalization and local concentration of viral products, (ii) physical support and organization of the RNA replication complex (20), (iii) tethering of the viral RNA during unwinding, (iv) provision of lipid constituents important for replication (1, 34), and (v) protection of the viral RNA from double-stranded RNA-mediated host defenses or RNA interference.Hepatitis C virus (HCV) contains a plus-strand RNA genome of approximately 9,600 nucleotides that encodes a polyprotein precursor of about 3,000 amino acid residues (17, 21). We have recently shown that expression of the HCV polyprotein in tetracycline-regulated human osteosarcoma cell lines induces distinct membrane alterations. A prominent alteration, designated the membranous web, was found to contain all of the viral structural and nonstructural proteins and therefore was proposed to represent the HCV replication complex (9). The membranous web could be induced by NS4B alone and was very similar to the "sponge-like inclusions" previously observed by electron microscopy (EM) in the livers of HCV-infected chimpanzees (24).The recent development of replicon systems for HCV has allowed, for the first time, the study of efficient and genuine HCV RNA replication in Huh-7 human hepatoma cells in vitro (6,13,19,25). The aims of the present study were to locate the site of HCV RNA replication and to identify the HCV replication complex in Huh-7 cells harboring a prototype subgenomic HCV replicon.First, Huh-7-derived clone 9-13, harboring a bicistronic subgenomic replicon (19), was investigated by immunofluorescence microscopy (IF) to determine the subcellular distribution of the viral nonstructural proteins. Fixation, permeabilization, and immunostaining of cells were performed as previously described (10). As shown in Fig. 1a, NS3 was found in the cytoplasm as brightly fluorescing dots and in a reticular staining pattern. Very similar staining patterns have consistently been observed with monoclonal antibodies (MAbs) directed against NS4A, NS4B, NS5A, and NS5B (data not shown). By double-labeling IF, HCV nonstruct...

In contrast to a detailed understanding of antiviral cellular immune responses, the impact of neutralizing antibodies for the resolution of acute hepatitis C is poorly defined. The analysis of neutralizing responses has been hampered by the fact that patient cohorts as well as hepatitis C virus (HCV) strains are usually heterogeneous, and that clinical data from acute-phase and long-term follow-up after infection are not readily available. Using an infectious retroviral HCV pseudoparticle model system, we studied a cohort of women accidentally exposed to the same HCV strain of known sequence. In this single-source outbreak of hepatitis C, viral clearance was associated with a rapid induction of neutralizing antibodies in the early phase of infection. Neutralizing antibodies decreased or disappeared after recovery from HCV infection. In contrast, chronic HCV infection was characterized by absent or low-titer neutralizing antibodies in the early phase of infection and the persistence of infection despite the induction of cross-neutralizing antibodies in the late phase of infection. These data suggest that rapid induction of neutralizing antibodies during the early phase of infection may contribute to control of HCV infection. This finding may have important implications for understanding the pathogenesis of HCV infection and for the development of novel preventive and therapeutic antiviral strategies.vaccines ͉ pathogenesis ͉ host reponses

CD8 + T lymphocytes play a key role in host defense, in particular against important persistent viruses, although the critical functional properties of such cells in tissue are not fully defined. We have previously observed that CD8 + T cells specific for tissue-localized viruses such as hepatitis C virus express high levels of the C-type lectin CD161. To explore the significance of this, we examined CD8 + CD161 + T cells in healthy donors and those with hepatitis C virus and defined a population of CD8 + T cells with distinct homing and functional properties. These cells express high levels of CD161 and a pattern of molecules consistent with type 17 differentiation, including cytokines (e.g., IL-17, IL-22), transcription factors (e.g., retinoic acid-related orphan receptor γ-t, P = 6 × 10 −9 ; RUNX2, P = 0.004), cytokine receptors (e.g., IL-23R, P = 2 × 10 −7 ; IL-18 receptor, P = 4 × 10 −6 ), and chemokine receptors (e.g., CCR6, P = 3 × 10 −8 ; CXCR6, P = 3 × 10 −7 ; CCR2, P = 4 × 10 −7 ). CD161 + CD8 + T cells were markedly enriched in tissue samples and coexpressed IL-17 with high levels of IFN-γ and/or IL-22. The levels of polyfunctional cells in tissue was most marked in those with mild disease ( P = 0.0006). These data define a T cell lineage that is present already in cord blood and represents as many as one in six circulating CD8 + T cells in normal humans and a substantial fraction of tissue-infiltrating CD8 + T cells in chronic inflammation. Such cells play a role in the pathogenesis of chronic hepatitis and arthritis and potentially in other infectious and inflammatory diseases of man.

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Chronic hepatitis C virus (HCV) infection is associated with impaired proliferative, cytokine, and cytotoxic effector functions of HCV-specific CD8؉ T cells that probably contribute significantly to viral persistence. Here, we investigated the potential role of T cells with a CD4؉ CD25 ؉ regulatory phenotype in suppressing virusspecific CD8؉ T-cell proliferation during chronic HCV infection. In vitro depletion studies and coculture experiments revealed that peptide specific proliferation as well as gamma interferon production of HCVspecific CD8؉ T cells were inhibited by CD4 ؉ CD25 ؉ T cells. This inhibition was dose dependent, required direct cell-cell contact, and was independent of interleukin-10 and transforming growth factor beta. Interestingly, the T-cell-mediated suppression in chronically HCV-infected patients was not restricted to HCV-specific CD8 ؉ T cells but also to influenza virus-specific CD8 ؉ T cells. Importantly, CD4 ؉ CD25 ؉ T cells from persons recovered from HCV infection and from healthy blood donors exhibited significantly less suppressor activity. Thus, the inhibition of virus-specific CD8 ؉ T-cell proliferation was enhanced in chronically HCV-infected patients. This was associated with a higher frequency of circulating CD4 ؉ CD25؉ cells observed in this patient group. Taken together, our results suggest that chronic HCV infection leads to the expansion of CD4 ؉ CD25 ؉

Exhausted CD8+ T cell responses during chronic viral infections are defined by a complex expression pattern of inhibitory receptors. However, very little information is currently available about the coexpression patterns of these receptors on human virus-specific CD8+ T cells and their correlation with antiviral functions, T cell differentiation and antigen recognition. We addressed these important aspects in a cohort of 38 chronically HCV infected patients and found a coexpression of inhibitory receptors such as 2B4, CD160 and KLRG1 in association with PD-1 in about half of the HCV-specific CD8+ T cell responses. Importantly, this exhaustive phenotype was associated with low and intermediate levels of CD127 expression, an impaired proliferative capacity, an intermediate T cell differentiation stage and absence of sequence variations within the corresponding epitopes, indicating ongoing antigen triggering. In contrast, a low expression of inhibitory receptors by the remaining HCV-specific CD8+ T cells occurred in concert with a CD127hi phenotype, an early T cell differentiation stage and presence of viral sequence variations within the corresponding epitopes. In sum, these results suggest that T cell exhaustion contributes to the failure of about half of HCV-specific CD8+ T cell responses and that it is determined by a complex interplay of immunological (e.g. T cell differentiation) and virological (e.g. ongoing antigen triggering) factors.