Site Of Hepatitis C Virus Replication Research Articles

Authentic Patient-Derived Hepatitis C Virus Infects and Productively Replicates in Primary CD4 + and CD8 + T Lymphocytes In Vitro

Accumulated evidence indicates that immune cells can support the replication of hepatitis C virus (HCV) in infected patients and in culture. However, there is a scarcity of data on the degree to which individual immune cell types support HCV propagation and on characteristics of virus assembly. We investigated the ability of authentic, patient-derived HCV to infect in vitro two closely related but functionally distinct immune cell types, CD4+ and CD8+ T lymphocytes, and assessed the properties of the virus produced by these cells. The HCV replication system in intermittently mitogen-stimulated T cells was adapted to infect primary human CD4+ or CD8+ T lymphocytes. HCV replicated in both cell types although at significantly higher levels in CD4+ than in CD8+ T cells. Thus, the HCV RNA replicative (negative) strand was detected in CD4+ and CD8+ cells at estimated mean levels ± standard errors of the means of 6.7 × 102 ± 3.8 × 102 and 1.2 × 102 ± 0.8 × 102 copies/μg RNA, respectively (P < 0.0001). Intracellular HCV NS5a and/or core proteins were identified in 0.9% of CD4+ and in 1.2% of CD8+ T cells. Double staining for NS5a and T cell type-specific markers confirmed that transcriptionally competent virus replicated in both cell types. Furthermore, an HCV-specific protease inhibitor, telaprevir, inhibited infection in both CD4+ and CD8+ cells. The emergence of unique HCV variants and the release of HCV RNA-reactive particles with biophysical properties different from those of virions in plasma inocula suggested that distinct viral particles were assembled, and therefore, they may contribute to the pool of circulating virus in infected patients.IMPORTANCE Although the liver is the main site of HCV replication, infection of the immune system is an intrinsic characteristic of this virus independent of whether infection is symptomatic or clinically silent. Many fundamental aspects of HCV lymphotropism remain uncertain, including the degree to which different immune cells support infection and contribute to virus diversity. We show that authentic, patient-derived HCV productively replicates in vitro in two closely related but functionally distinct types of T lymphocytes, CD4+ and CD8+ cells. The display of viral proteins and unique variants, the production of virions with biophysical properties distinct from those in plasma serving as inocula, and inhibition of replication by an antiviral agent led us to ascertain that both T cell subtypes supported virus propagation. Infection of CD4+ and CD8+ T cells, which are central to adaptive antiviral immune responses, can directly affect HCV clearance, favor virus persistence, and decisively influence the development and progression of hepatitis C.

High resolution sequencing of hepatitis C virus reveals limited intra-hepatic compartmentalization in end-stage liver disease

The high replication and mutation rate of hepatitis C virus (HCV) results in a heterogeneous population of viral sequences in vivo. HCV replicates in the liver and infected hepatocytes occur as foci surrounded by uninfected cells that may promote compartmentalization of viral variants. Given recent reports showing interferon stimulated gene (ISG) expression in chronic hepatitis C, we hypothesized that local interferon responses may limit HCV replication and evolution. To investigate the spatial influence of liver architecture on viral replication we measured HCV RNA and ISG mRNA from each of the 8 Couinaud segments of the liver from 21 patients undergoing liver transplant. HCV RNA and ISG mRNA levels were comparable across all sites from an individual liver but showed up to 500-fold difference between patients. Importantly, there was no association between ISG and HCV RNA expression across all sites in the liver or plasma. Deep sequencing of HCV RNA isolated from the 8 hepatic sites from two subjects showed a similar distribution of viral quasispecies across the liver and uniform sequence diversity. Single genome amplification of HCV E1E2-envelope clones from 6 selected patients at 2 hepatic sites supported these data and showed no evidence for HCV compartmentalization. We found no differences between the hepatic and plasma viral quasispecies in all patients sampled. We conclude that in end-stage liver disease HCV RNA levels and the genetic pool of HCV envelope sequences are indistinguishable between distant sites in the liver and plasma, arguing against viral compartmentalization. HCV is an RNA virus that exists as a quasispecies of closely related genomes that are under continuous selection by host innate and adaptive immune responses and antiviral drug therapy. The primary site of HCV replication is the liver and yet our understanding of the spatial distribution of viral variants within the liver is limited. High resolution sequencing of HCV and monitoring of innate immune responses at multiple sites across the liver identified a uniform pattern of diversity and argues against viral compartmentalization.

Hepatitis C Viral Infection in Children: Updated Review.

Hepatitis C virus (HCV) infection is a major medical challenge affecting around 200 million people worldwide. The main site of HCV replication is the hepatocytes of the liver. HCV is a positive enveloped RNA virus from the flaviviridae family. Six major HCV genotypes are implicated in the human infection. In developed countries the children are infected mainly through vertical transmission during deliveries, while in developing countries it is still due to horizontal transmission from adults. Minimal nonspecific and brief symptoms are initially found in approximately 15% of children. Acute and chronic HCV infection is diagnosed through the recognition of HCV RNA. The main objective for treatment of chronic HCV is to convert detected HCV viremia to below the detection limit. Children with chronic HCV infection are usually asymptomatic and rarely develop severe liver damage. Therefore, the benefits from current therapies, pegylated-Interferon plus ribavirin, must be weighed against their adverse effects. This combined treatment offers a 50-90% chance of clearing HCV infection according to several studies and on different HCV genotype. Recent direct acting antiviral (DAA) drugs which are well established for adults have not yet been approved for children and young adults below 18 years. The most important field for the prevention of HCV infection in children would be the prevention of perinatal and parenteral transmission. There are areas of focus for new lines of research in pediatric HCV-related disease that can be addressed in the near future.

Soraphen A: A broad-spectrum antiviral natural product with potent anti-hepatitis C virus activity

Soraphen A (SorA) is a myxobacterial metabolite that inhibits the acetyl-CoA carboxylase, a key enzyme in lipid biosynthesis. We have previously identified SorA to efficiently inhibit the human immunodeficiency virus (HIV). The aim of the present study was to evaluate the capacity of SorA and analogues to inhibit hepatitis C virus (HCV) infection. SorA inhibition capacity was evaluated in vitro using cell culture derived HCV, HCV pseudoparticles and subgenomic replicons. Infection studies were performed in the hepatoma cell line HuH7/Scr and in primary human hepatocytes. The effects of SorA on membranous web formation were analysed by electron microscopy. SorA potently inhibits HCV infection at nanomolar concentrations. Obtained EC50 values were 0.70 nM with a HCV reporter genome, 2.30 nM with wild-type HCV and 2.52 nM with subgenomic HCV replicons. SorA neither inhibited HCV RNA translation nor HCV entry, as demonstrated with subgenomic HCV replicons and HCV pseudoparticles, suggesting an effect on HCV replication. Consistent with this, evidence was obtained that SorA interferes with formation of the membranous web, the site of HCV replication. Finally, a series of natural and synthetic SorA analogues helped to establish a first structure-activity relationship. SorA has a very potent anti-HCV activity. Since it also interferes with the membranous web formation, SorA is an excellent tool to unravel the mechanism of HCV replication.

HCV‐Host Cell Interactions: Role of Ebp‐1 in Hepatitis C virus Replication

Persistent hepatitis C virus ( HCV) infection leads to chronic hepatitis C (CHC), which often progresses to liver cirrhosis (LC) and hepatocellular carcinoma (HCC). The molecular mechanisms that establish chronic HCV infection are poorly understood. Also, the role of specific cellular factors in chronic HCV infection is not well established. We have recently identified a cellular factor ErbB3 binding protein‐1 (Ebp1) that specifically interacts with HCV RNA genome and is inhibitory to HCV replication. Ebp1 is a double stranded RNA binding protein that is a member of a family of proliferation‐regulating proteins. Ebp1 has two isoforms, p42 and p48, which are synthesized from two differently spliced mRNAs from the same gene. Our preliminary results show that overexpression of Ebp1 (the longer as well as the shorter isoform) has a strong negative impact on HCV replication. We also found that Ebp1 binds to the HCV 5′NTR. We established that Ebp1 is mainly localized in the cytosol, a site of HCV replication. Further experiments showed that Ebp1 interacts not only with HCV viral proteins NS5A and NS5B, but also with interferon‐inducible double stranded RNA activated protein kinase PKR, which is critical for cellular antiviral and anti proliferative responses induced by interferon. Taken together our results suggest the critical role of Ebp1 in negatively regulating Hepatitis C virus infection. We propose to further identify the role of PKR in Ebp1 mediated signaling in Hepatitis C virus infection. Elucidation of the role of Ebp1 in activating PKR or vice versa will provide valuable insight into the molecular mechanisms of these interactions and their implication on HCV‐replication and associated pathogenesis.

Chronic hepatitis C virus infection and neurological and psychiatric disorders: an overview.

Hepatitis C virus (HCV) infection is considered a systemic disease because of involvement of other organs and tissues concomitantly with liver disease. Among the extrahepatic manifestations, neuropsychiatric disorders have been reported in up to 50% of chronic HCV infected patients. Both the central and peripheral nervous system may be involved with a wide variety of clinical manifestations. Main HCV-associated neurological conditions include cerebrovascular events, encephalopathy, myelitis, encephalomyelitis, and cognitive impairment, whereas "brain fog", depression, anxiety, and fatigue are at the top of the list of psychiatric disorders. Moreover, HCV infection is known to cause both motor and sensory peripheral neuropathy in the context of mixed cryoglobulinemia, and has also been recently recognized as an independent risk factor for stroke. These extrahepatic manifestations are independent of severity of the underlying chronic liver disease and hepatic encephalopathy. The brain is a suitable site for HCV replication, where the virus may directly exert neurotoxicity; other mechanisms proposed to explain the pathogenesis of neuropsychiatric disorders in chronic HCV infection include derangement of metabolic pathways of infected cells, alterations in neurotransmitter circuits, autoimmune disorders, and cerebral or systemic inflammation. A pathogenic role for HCV is also suggested by improvement of neurological and psychiatric symptoms in patients achieving a sustained virologic response following interferon treatment; however, further ad hoc trials are needed to fully assess the impact of HCV infection and specific antiviral treatments on associated neuropsychiatric disorders.

New evidence of replication of hepatitis C virus in short-term peripheral blood mononuclear cell cultures

BackgroundEven though hepatocytes are the main site for hepatitis C virus (HCV) replication, peripheral blood mononuclear cells (PBMC) have also been proposed as a suitable site for HCV replication. However, this issue still remains under discussion. We have previously developed an innovative system where HCV-RNA can be recovered during PBMC culture from HCV infected patients. Thus, the aim of this work was to use this novel approach in order to observe the evolution and replication of HCV genotype 1b in the PBMC of an HIV-HCV coinfected patient. MethodsHCV-RNA was extracted from serum, uncultured PBMC and PBMC culture at day 6, 20 and 33. The evolutionary analysis was performed using the direct sequences of three viral regions: 5′UTR, E2 and NS5A. Additionally, E2 region was cloned in order to extend the evolutive analysis. ResultsIn the present work, the molecular characterization of HCV along the culture showed a clear dynamic evolving process with the appearance of several nucleotide or amino acid changes in the three regions analyzed. Furthermore, the population analysis of E2 clones showed emerging and loss of lineages which indicate the fast evolutive dynamics of this system. ConclusionsSince evolution can take place only if the virus is replicating in the culture, this finding constitutes an important evidence of viral replication in PBMC. Moreover, this extrahepatic compartment could be very important due to the presence of distinctive variants that could be responsible for resistance to treatment, viral pathogenesis and other clinical implications.

Daclatasvir-Like Inhibitors of NS5A Block Early Biogenesis of Hepatitis C Virus–Induced Membranous Replication Factories, Independent of RNA Replication

Direct-acting antivirals that target nonstructural protein 5A (NS5A), such as daclatasvir, have high potency against the hepatitis C virus (HCV). They are promising clinical candidates, yet little is known about their antiviral mechanisms. We investigated the mechanisms of daclatasvir derivatives. We used a combination of biochemical assays, in silico docking models, and high-resolution imaging to investigate inhibitor-induced changes in properties of NS5A, including its interaction with phosphatidylinositol-4 kinase IIIα and induction of the membranous web, which is the site of HCV replication. Analyses were conducted with replicons, infectious virus, and human hepatoma cells that express a HCV polyprotein. Studies included a set of daclatasvir derivatives and HCV variants with the NS5A inhibitor class-defining resistance mutation Y93H. NS5A inhibitors did not affect NS5A stability or dimerization. A daclatasvir derivative interacted with NS5A and molecular docking studies revealed a plausible mode by which the inhibitor bound to NS5A dimers. This interaction was impaired in mutant forms of NS5A that are resistant to daclatavir, providing a possible explanation for the reduced sensitivity of the HCV variants to this drug. Potent NS5A inhibitors were found to block HCV replication by preventing formation of the membranous web, which was not linked to an inhibition of phosphatidylinositol-4 kinase IIIα. Correlative light-electron microscopy revealed unequivocally that NS5A inhibitors had no overall effect on the subcellular distribution of NS5A, but completely prevented biogenesis of the membranous web. Highly potent inhibitors of NS5A, such as daclatasvir, block replication of HCV RNA at the stage of membranous web biogenesis-a new paradigm in antiviral therapy.

Review: Occult hepatitis C virus infection: still remains a controversy.

Occult hepatitis C virus (HCV) infection is characterized by the presence of HCV RNA in the liver cells or peripheral blood mononuclear cells of the patients whose serum samples test negative for HCV RNA, with or without presence of HCV antibodies. The present study reviews the existing literature on the persistence of occult hepatitis C virus infection, with description of the clinical characteristics and methods for identification of occult hepatitis C. Occult hepatitis C virus infection was detected in patients with abnormal results of liver function tests of unknown origin, with HCV antibodies and HCV RNA negativity in serum, and also in patients with spontaneous or treatment-induced recovery from hepatitis C. The viral replication in the liver cells and/or peripheral blood mononuclear cells was present in all clinical presentations of occult hepatitis C. The peripheral blood mononuclear cells represent an extra-hepatic site of HCV replication. The reason why HCV RNA was not detectable in the serum of patients with occult hepatitis C, could be the low number of circulating viral particles not detectable by the diagnostic tests with low sensitivity. It is uncertain whether occult hepatitis C is a different clinical entity or just a form of chronic hepatitis C virus infection. Data accumulated over the last decade demonstrated that an effective approach to the diagnosis of HCV infection would be the implementation of more sensitive HCV RNA diagnostic assays, and also, examination of the presence of viral particles in the cells of the immune system.

Hepatitis C Virus NS5A Hijacks ARFGAP1 To Maintain a Phosphatidylinositol 4-Phosphate-Enriched Microenvironment

Phosphatidylinositol 4-phosphate (PI4P) is well known to be upregulated during hepatitis C virus (HCV) replication. The role of PI4 kinases in HCV has been extensively investigated. Whether the PI4P phosphatase Sac1 is altered by HCV remains unclear. Here, we identified ARFGAP1 to be a novel host factor for HCV replication. We further show that Sac1 interacts with ARFGAP1 and inhibits HCV replication. The elevation of PI4P induced by HCV NS5A is abrogated when the coatomer protein I (COPI) pathway is inhibited. We also found an interaction between NS5A and ARFGAP1. Furthermore, we identified a conserved cluster of positively charged amino acids in NS5A critical for interaction between NS5A and ARFGAP1, induction of PI4P, and HCV replication. Our data demonstrate that ARFGAP1 is a host factor for HCV RNA replication. ARFGAP1 is hijacked by HCV NS5A to remove COPI cargo Sac1 from the site of HCV replication to maintain high levels of PI4P. Our findings provide an additional mechanism by which HCV enhances formation of a PI4P-rich environment. PI4P is enriched in the replication area of HCV; however, whether PI4P phosphatase Sac1 is subverted by HCV is not established. The detailed mechanism of how COPI contributes to viral replication remains unknown, though COPI components were hijacked by HCV. We demonstrate that ARFGAP1 is hijacked by HCV NS5A to remove COPI cargo Sac1 from the HCV replication area to maintain high-level PI4P generated by NS5A. Furthermore, we identify a conserved cluster of positively charged amino acids in NS5A, which are critical for interaction between NS5A and ARFGAP1, induction of PI4P, and HCV replication. This study will shed mechanistic insight on how other RNA viruses hijack COPI and Sac1.

Preclinical Evaluation of An Anti-HCV miRNA Cluster for Treatment of HCV Infection

We developed a strategy to treat hepatitis C virus (HCV) infection by replacing five endogenous microRNA (miRNA) sequences of a natural miRNA cluster (miR-17-92) with sequences that are complementary to the HCV genome. This miRNA cluster (HCV-miR-Cluster 5) is delivered to cells using adeno-associated virus (AAV) vectors and the miRNAs are expressed in the liver, the site of HCV replication and assembly. AAV-HCV-miR-Cluster 5 inhibited bona fide HCV replication in vitro by up to 95% within 2 days, and the spread of HCV to uninfected cells was prevented by continuous expression of the anti-HCV miRNAs. Furthermore, the number of cells harboring HCV RNA replicons decreased dramatically by sustained expression of the anti-HCV miRNAs, suggesting that the vector is capable of curing cells of HCV. Delivery of AAV-HCV-miR-Cluster 5 to mice resulted in efficient transfer of the miRNA gene cluster and expression of all five miRNAs in liver tissue, at levels up to 1,300 copies/cell. These levels achieved up to 98% gene silencing of cognate HCV sequences, and no liver toxicity was observed, supporting the safety of this approach. Therefore, AAV-HCV-miR-Cluster 5 represents a different paradigm for the treatment of HCV infection.

Hepatic enrichment and activation of myeloid dendritic cells during chronic hepatitis C virus infection

Chronic hepatitis C virus (HCV) infection is a serious disease that can result in numerous long-term complications leading to liver failure or death. Approximately 80% of people fail to clear their infection, largely as the result of weak, narrowly targeting or waning antiviral T-cell responses. Although professional antigen presenting cells (APCs) like dendritic cells (DCs) might serve as targets for modulation of T-cell immunity, the particular role of DCs in immunity to HCV is not known. Moreover the identity, phenotype, and functional characteristics of such populations in the liver, the site of HCV replication, have proven difficult to elucidate. Using a multicolor flow-based approach, we identified six distinct populations of professional APCs among liver interstitial leukocytes isolated from uninfected and HCV-infected patients. Although a generalized enrichment of DCs in the liver compared to blood was observed for all patients, HCV infection was characterized by a significant increase in the frequency of intrahepatic myeloid DCs (both CD1c+ and CD141+). Phenotypic analyses of liver plasmacytoid (pDC) and myeloid DCs (mDC) further revealed the HCV-induced expression of maturation molecules CD80, CD83, CD40, and programmed death ligand-1. Importantly, pDC and mDCs from HCV-infected liver were capable of secreting effector cytokines, interferon-alpha and interleukin-12, respectively, in response to Toll-like receptor stimulation in vitro. Chronic HCV infection facilitates the "customized" recruitment of liver DC subsets with established functional roles in antigen presentation. These DCs are characterized by a mature, activated phenotype and are functionally responsive to antigenic stimulation in vitro. Such findings highlight an important paradox surrounding liver DC recruitment during HCV infection, where despite their activation these cells do not provide adequate protection from the virus.

Formation and function of hepatitis C virus replication complexes require residues in the carboxy-terminal domain of NS4B protein

During replication, hepatitis C virus (HCV) NS4B protein rearranges intracellular membranes to form foci, or the web, the putative site for HCV replication. To understand the role of the C-terminal domain (CTD) in NS4B function, mutations were introduced into NS4B alone or in the context of HCV polyprotein. First, we show that the CTD is required for NS4B-induced web structure, but it is not sufficient to form the web nor is it required for NS4B membrane association. Interestingly, all the mutations introduced into the CTD impeded HCV genome replication, but only two resulted in a disruption of NS4B foci. Further, we found that NS4B interacts with NS3 and NS5A, and that mutations causing NS4B mislocalization have a similar effect on these proteins. Finally, we show that the redistribution of Rab5 to NS4B foci requires an intact CTD, suggesting that Rab5 facilitates NS4B foci formation through interaction with the CTD.

Roles for endocytic trafficking and phosphatidylinositol 4-kinase III alpha in hepatitis C virus replication

Hepatitis C virus (HCV) reorganizes cellular membranes to establish sites of replication. The required host pathways and the mechanism of cellular membrane reorganization are poorly characterized. Therefore, we interrogated a customized small interfering RNA (siRNA) library that targets 140 host membrane-trafficking genes to identify genes required for both HCV subgenomic replication and infectious virus production. We identified 7 host cofactors of viral replication, including Cdc42 and Rock2 (actin polymerization), EEA1 and Rab5A (early endosomes), Rab7L1, and PI3-kinase C2gamma and PI4-kinase IIIalpha (phospholipid metabolism). Studies of drug inhibitors indicate actin polymerization and phospholipid kinase activity are required for HCV replication. We found extensive co-localization of the HCV replicase markers NS5A and double-stranded RNA with Rab5A and partial co-localization with Rab7L1. PI4K-IIIalpha co-localized with NS5A and double-stranded RNA in addition to being present in detergent-resistant membranes containing NS5A. In a comparison of type II and type III PI4-kinases, PI4Ks were not required for HCV entry, and only PI4K-IIIalpha was required for HCV replication. Although PI4K-IIIalpha siRNAs decreased HCV replication and virus production by almost 100%, they had no effect on initial HCV RNA translation, suggesting that PI4K-IIIalpha functions at a posttranslational stage. Electron microscopy identified the presence of membranous webs, which are thought to be the site of HCV replication, in HCV-infected cells. Pretreatment with PI4K-IIIalpha siRNAs greatly reduced the accumulation of these membranous web structures in HCV-infected cells. We propose that PI4K-IIIalpha plays an essential role in membrane alterations leading to the formation of HCV replication complexes.

Reply:

Our article1 reported that viral RNA could not be detected in the peripheral blood mononuclear cells (PBMCs) of aviremic seropositive blood donors, a conclusion supported by another recent article which also concluded that there was no evidence for persistent hepatitis C virus (HCV) infection in PBMCs after clearance of viremia in plasma.2 Decreasing or disappearing anti-HCV antibody titers in aviremic subjects also argue against continued HCV protein expression.3 Whether other reservoirs of HCV RNA, such as hepatocytes, exist in aviremic subjects remains unclear with some4-6 although not all7, 8 studies finding no HCV RNA in the liver of treated sustained viral responders. We clearly acknowledged1 that in chronically viremic subjects, PBMCs could be a site of HCV replication and that in vitro mitogen plus cytokine treatment might increase PBMC-associated HCV levels.8-10 With respect to the relative sensitivities of the HCV RNA assays used, we performed two different tests for HCV RNA in PBMCs (cell-associated transcription-mediated amplification [CA-TMA] and reverse transcription nested polymerase chain reaction [RT-nPCR]). We demonstrated a sensitivity of 2-50 HCV RNAs in 5 million PBMCs for the CA-TMA assay and 15-150 HCV RNAs in 5 million cells using RT-nPCR (adjusted from a sensitivity of 1-10 HCV RNAs for RT-nPCR, because only a fraction of total PBMC RNA and derived complementary DNA is sampled).1 Pham et al. report a sensitivity for their RT-nPCR-NAH (nucleic acid hybridization) assay of <10 viral genome equivalent (vge)/mL for serum (and in their letter, <5 vge/million PBMCs). We quantified the sensitivities of our assays using serial dilutions of plasma samples of known viral loads (prequantified using a commercial viral load assay) in the presence of total cellular RNA, as well as of total RNA from the PBMC of viremic subjects and showed the actual replicate assay results.1 Pham et al. used dilutions of a plasmid without interfering cellular nucleic acids for “semiquantitative estimation” in a “preliminary experiment” whose results where not shown.10 The fraction of total complementary DNA derived from PBMCs or 250 μL serum they used as input for their nPCR was not specified.10 With regard to the total number of cell equivalents analyzed in respective assays, we indeed analyzed only 0.16 million PBMCs by RT-nPCR but in parallel, and with consistent results, also used CA-TMA on 2.5 million PBMCs,1 slightly more than the 1 million to 2 million PBMCs Pham and Michalak reported in their letter. We agree with Pham and Michalak that difference in assay sensitivity is a likely explanation for the reported discrepancy but feel that the more relevant assay is that used for detecting HCV RNA in plasma. In our case, we used an extensively validated plasma HCV RNA assay used for blood-donation screening (Transcription mediated amplification, Procleix human immunodeficieny virus-1/HCV assay, Gen-probe, with a 95% detection sensitivity of 30 HCV RNA/mL).11-13 This TMA assay was used in duplicate assays (consuming a total of 1 mL plasma) to identify a total of 69 plasma aviremic blood donors, none of whom possessed detectable PBMC-associated HCV RNA.1 Pham et al. analyzed and found HCV RNA in the PBMCs of two aviremic subjects (so classified using their RT-nPCR-NAH).10 We suspect that further testing of the plasma from these two subjects using TMA assays might have detected a low-level viremia. Also, mostly consistent with our results, Pham et al. reported in 2008 detecting HCV RNA in only one of 10 PBMCs from aviremic subjects (although stimulation with interleukin-2 plus mitogen and testing of purified immune cell subsets lead to the detection of HCV RNA in all 10 PBMCs).14 Our selection criterion for the absence of plasma viremia (based on a highly sensitive, extensively validated assay used in duplicate tests to increase sensitivity) may therefore have been more stringent than that used by Pham et al. and account for the discrepant results seen in PBMCs. Ultimately, we agree with Pham and Michalak that the question of a residual HCV reservoir in the PBMCs of truly aviremic subjects will best be resolved using the demonstrably most sensitive detection assays for HCV RNA in both plasma and PBMC. The use of the same HCV RNA quantitation standards15 by different groups will help compare assay sensitivities. Eric Delwart Ph.D.*, * Blood Systems Research Institute, Department of Laboratory Medicine, University of California, San Francisco, San Francisco CA.

Quantitative Analysis of the Hepatitis C Virus Replication Complex

The hepatitis C virus (HCV) encodes a large polyprotein; therefore, all viral proteins are produced in equimolar amounts regardless of their function. The aim of our study was to determine the ratio of nonstructural proteins to RNA that is required for HCV RNA replication. We analyzed Huh-7 cells harboring full-length HCV genomes or subgenomic replicons and found in all cases a >1,000-fold excess of HCV proteins over positive- and negative-strand RNA. To examine whether all nonstructural protein copies are involved in RNA synthesis, we isolated active HCV replication complexes from replicon cells and examined them for their content of viral RNA and proteins before and after treatment with protease and/or nuclease. In vitro replicase activity, as well as almost the entire negative- and positive-strand RNA, was resistant to nuclease treatment, whereas <5% of the nonstructural proteins were protected from protease digest but accounted for the full in vitro replicase activity. In consequence, only a minor fraction of the HCV nonstructural proteins was actively involved in RNA synthesis at a given time point but, due to the high amounts present in replicon cells, still representing a huge excess compared to the viral RNA. Based on the comparison of nuclease-resistant viral RNA to protease-resistant viral proteins, we estimate that an active HCV replicase complex consists of one negative-strand RNA, two to ten positive-strand RNAs, and several hundred nonstructural protein copies, which might be required as structural components of the vesicular compartments that are the site of HCV replication.

Replication of hepatitis C virus subgenomes in nonhepatic epithelial and mouse hepatoma cells.

The hepatitis C virus (HCV) pandemic affects the health of more than 170 million people and is the major indication for orthotopic liver transplantations. Although the human liver is the primary site for HCV replication, it is not known whether extrahepatic tissues are also infected by the virus and whether nonprimate cells are permissive for RNA replication. Because HCV exists as a quasispecies, it is conceivable that a viral population may include variants that can replicate in different cell types and in other species. We have tested this hypothesis and found that subgenomic HCV RNAs can replicate in mouse hepatoma and nonhepatic human epithelial cells. Replicons isolated from these cell lines carry new mutations that could be involved in the control of tropism of the virus. Our results demonstrated that translation and RNA-directed RNA replication of HCV do not depend on hepatocyte or primate-specific factors. Moreover, our results could open the path for the development of animal models for HCV infection.

Possible mechanisms of action and reasons for failure of antiviral therapy in chronic hepatitis C

The known mechanisms of hepatitis C virus (HCV) clearance and their failure in persistent infection are discussed. Interferon-α is the main treatment in chronic HCV but has shown poor sustained virological response rates when used as a monotherapy. The effects of interferon-α may include inhibition of HCV virion production by an effect on viral RNA and protein synthesis, enhancement of immune lysis of HCV infected cells, inhibition of hepatic fibrosis by an effect on TGFβ, and an effect on HCV induced carcinogenesis. Mathematical modelling studies have provided insights into the mechanisms of action of interferon-α in chronic HCV. The two-phase plasma HCV RNA disappearance curve may reflect the presence of an interferon-resistant second site of HCV replication either within or outside the liver. Clinical observations and cerebral magnetic resonance scans provide evidence of functional cerebral impairment in HCV infected patients, raising the issue of the central nervous system (CNS) as a site for HCV replication. Recent studies using ribavirin in combination with interferon suggest that this approach doubles the sustained response rates obtained without having a major effect on the initial rate of HCV clearance (see Zeuzem paper). The potential mechanisms of action of ribavirin, although not yet fully understood, include inhibition of synthesis of GTP by an effect on inosine monophosphate dehydrogenase thereby limiting viral RNA synthesis, and enhancement of TH1 responses, which may assist viral clearance. There is no significant effect on HCV RNA polymerase activity. It is possible that ribavirin may have activity at extrahepatic sites of HCV infection, thus explaining the marked reduction in relapse rates with combination therapy, without an appreciable effect on initial antiviral response.

Hepatitis C virus RNA in different blood lymphocyte subsets

Objective: Lymphocytes play an important role in the pathogenesis of chronic viral hepatitis. Apart from intrahepatic replication of hepatitis C Virus (HCV), there is also strong evidence for an extrahepatic site in mononuclear blood cells. To further characterize the site of HCV replication, we tested resting peripheral blood lymphocytes (PBL) and stimulated PBL for the presence of positive- and negative-stranded HCV RNA. Method: PBL from 10 patients with histologically proven chronic active hepatitis C were sorted into CD4+ (T helper) and CD8+ (T suppressor) T cells, CD20+ B cells and CD16+ natural killer (NK) cells by a fluorescence-activated cell sorter. Results: HCV RNA was detected by a nested polymerase chain reaction