Warren N. Schmidt

Characterization of Hepatitis C Virus (HCV) and HCV E2 Interactions with CD81 and the Low-Density Lipoprotein Receptor

ABSTRACT Hepatitis C virus (HCV) or HCV–low-density lipoprotein (LDL) complexes interact with the LDL receptor (LDLr) and the HCV envelope glycoprotein E2 interacts with CD81 in vitro. However, E2 interactions with LDLr and HCV interactions with CD81 have not been clearly described. Using sucrose gradient-purified low-density particles (1.03 to 1.07 g/cm3), intermediate-density particles (1.12 to 1.18 g/cm3), recombinant E2 protein, or control proteins, we assessed binding to MOLT-4 cells, foreskin fibroblasts, or LDLr-deficient foreskin fibroblasts at 4°C by flow cytometry and confocal microscopy. Viral entry was determined by measuring the coentry of α-sarcin, a protein synthesis inhibitor. We found that low-density HCV particles, but not intermediate-density HCV or controls bound to MOLT-4 cells and fibroblasts expressing the LDLr. Binding correlated with the extent of cellular LDLr expression and was inhibited by LDL but not by soluble CD81. In contrast, E2 binding was independent of LDLr expression and was inhibited by human soluble CD81 but not mouse soluble CD81 or LDL. Based on confocal microscopy, we found that low-density HCV particles and LDL colocalized on the cell surface. The addition of low-density HCV but not intermediate-density HCV particles to MOLT-4 cells allowed coentry of α-sarcin, indicating viral entry. The amount of viral entry also correlated with LDLr expression and was independent of the CD81 expression. Using a solid-phase immunoassay, recombinant E2 protein did not interact with LDL. Our data indicate that E2 binds CD81; however, virus particles utilize LDLr for binding and entry. The specific mechanism by which HCV particles interact with LDL or the LDLr remains unclear.

Correlation between microRNA expression levels and clinical parameters associated with chronic hepatitis C viral infection in humans.

MicroRNAs (miRNAs) are small RNAs that regulate gene expression pathways. Previous studies have shown interactions between hepatitis C virus (HCV) and host miRNAs. We measured miR-122 and miR-21 levels in HCV-infected human liver biopsies relative to uninfected human livers and correlated these with clinical patient data. miR-122 is required for HCV replication in vitro, and miR-21 is involved in cellular proliferation and tumorigenesis. We found that miR-21 expression correlated with viral load, fibrosis and serum liver transaminase levels. miR-122 expression inversely correlated with fibrosis, liver transaminase levels and patient age. miR-21 was induced ∼twofold, and miR-122 was downregulated on infection of cultured cells with the HCV J6/JFH infectious clone, thus establishing a link to HCV. To further examine the relationship between fibrosis and the levels of miR-21 and miR-122, we measured their expression levels in a mouse carbon tetrachloride fibrosis model. As in the HCV-infected patient samples, fibrotic stage positively correlated with miR-21 and negatively correlated with miR-122 levels. Transforming growth factor β (TGF-β) is a critical mediator of fibrogenesis. We identified SMAD7 as a novel miR-21 target. SMAD7 is a negative regulator of TGF-β signaling, and its expression is induced by TGF-β. To confirm the relationship between miR-21 and the TGF-β signaling pathway, we measured the effect of miR-21 on a TGF-β-responsive reporter. We found that miR-21 enhanced TGF-β signaling, further supporting a relationship between miR-21 and fibrosis. We suggest a model in which miR-21 targeting of SMAD7 could increase TGF-β signaling, leading to increased fibrogenesis.

Prospective multicenter study of eligibility for antiviral therapy among 4,084 U.S. veterans with chronic hepatitis C virus infection

BACKGROUND:Many veterans may not be candidates for hepatitis C virus (HCV) treatment due to contraindications to therapy. The aims of this study were to determine the proportion of HCV-infected veterans who were eligible for interferon alfa and ribavirin therapy and to evaluate barriers to HCV treatment.METHODS:We prospectively enrolled 4,084 veterans who were referred for HCV treatment over a 1-yr period at 24 Veterans Affairs (VA) Medical Centers. Treatment candidacy was assessed using standardized criteria and the opinion of the treating clinician.RESULTS:Overall, 32.2% (95% CI, 30.8–33.7%) were candidates for HCV treatment according to standardized criteria, whereas 40.7% (95% CI, 39.2–42.3%) were candidates in the opinion of the treating clinician. Multivariable analysis identified ongoing substance abuse (OR = 17.68; 95% CI, 12.24–25.53), comorbid medical disease (OR = 9.62; 95% CI, 6.85–13.50), psychiatric disease (OR = 9.45; 95% CI, 6.70–13.32), and advanced liver disease (OR = 8.43; 95% CI, 4.42–16.06) as the strongest predictors of not being a treatment candidate. Among patients who were considered treatment candidates, 76.2% (95% CI, 74.0–78.3%) agreed to be treated and multivariable analysis showed that persons ≥50 yr of age (OR = 1.37; 95% CI, 1.07–1.76) and those with >50 lifetime sexual partners (OR = 1.44; 95% CI, 1.08–1.93) were more likely to decline treatment.CONCLUSIONS:The majority of veteran patients are not suitable candidates for HCV treatment because of substance abuse, psychiatric disease, and comorbid medical disease, and many who are candidates decline therapy. Multidisciplinary collaboration is needed to overcome barriers to HCV therapy in this population.

Full-Length GB Virus C (Hepatitis G Virus) RNA Transcripts Are Infectious in Primary CD4-Positive T Cells

ABSTRACT GB virus C (GBV-C or hepatitis G virus) is a recently described flavivirus which frequently leads to chronic viremia in humans. Although GBV-C is associated with acute posttransfusion hepatitis, it is not clear if the virus is pathogenic for humans. We constructed a full-length cDNA from the plasma of a person with chronic GBV-C viremia. Peripheral blood mononuclear cells (PBMCs) transfected with full-length RNA transcripts from this GBV-C clone resulted in viral replication. This was demonstrated by serial passage of virus from cell culture supernatants, detection of increasing concentrations of positive- and negative-sense GBV-C RNA over time, and the detection of the GBV-C E2 antigen by confocal microscopy. In addition, two types of GBV-C particles were identified in cell lysates; these particles had buoyant densities of 1.06 and 1.12 to 1.17 g/ml in sucrose gradients. PBMCs sorted for expression of CD4 contained 100-fold-more GBV-C RNA than CD4-negative cells. Taken together, these data demonstrate that RNA transcripts from GBV-C full-length cDNA are infectious in primary CD4-positive T cells. In contrast, RNA transcripts from an infectious hepatitis C virus clone did not replicate in the same cell culture system. Infectious RNA transcripts from GBV-C cDNA should prove useful for studying viral replication and may allow identification of differences between GBV-C and hepatitis C virus cultivation in vitro.

Hepatitis C Virus Infection and Hepatic Stellate Cell Activation Downregulate miR-29: miR-29 Overexpression Reduces Hepatitis C Viral Abundance in Culture

BACKGROUND Chronic hepatitis C virus (HCV)-induced liver fibrosis involves upregulation of transforming growth factor (TGF)-β and subsequent hepatic stellate cell (HSC) activation. MicroRNAs (miRNAs) regulate HCV infection and HSC activation. METHODS TaqMan miRNA profiling identified 12 miRNA families differentially expressed between chronically HCV-infected human livers and uninfected controls. To identify pathways affected by miRNAs, we developed a new algorithm (pathway analysis of conserved targets), based on the probability of conserved targeting. RESULTS This analysis suggested a role for miR-29 during HCV infection. Of interest, miR-29 was downregulated in most HCV-infected patients. miR-29 regulates expression of extracellular matrix proteins. In culture, HCV infection downregulated miR-29, and miR-29 overexpression reduced HCV RNA abundance. miR-29 also appears to play a role in HSCs. Hepatocytes and HSCs contribute similar amounts of miR-29 to whole liver. Both activation of primary HSCs and TGF-β treatment of immortalized HSCs downregulated miR-29. miR-29 overexpression in LX-2 cells decreased collagen expression and modestly decreased proliferation. miR-29 downregulation by HCV may derepress extracellular matrix synthesis during HSC activation. CONCLUSIONS HCV infection downregulates miR-29 in hepatocytes and may potentiate collagen synthesis by reducing miR-29 levels in activated HSCs. Treatment with miR-29 mimics in vivo might inhibit HCV while reducing fibrosis.

Heme oxygenase‐1 suppresses hepatitis C virus replication and increases resistance of hepatocytes to oxidant injury

Oxidative injury to hepatocytes occurs as a result of hepatitis C virus (HCV) infection and replication. Modulation of host cell antioxidant enzymes such as heme oxygenase‐1 (HO‐1) may be useful therapeutically to minimize cellular injury, reduce viral replication, and attenuate liver disease. In this report, we evaluated the effects of HO‐1 overexpression on HCV replication and hepatocellular injury. Full‐length (FL) (Con1) or nonstructural (NS) replicons (I 389 NS3‐3′) were transfected with complete human HO‐1 sequences or empty vector for control. Cell lines overexpressing HO‐1 (twofold to sixfold above basal values) or empty vector were isolated, and their HCV RNA synthesis, pro‐oxidant levels, and resistance to oxidative injury were assessed. HO‐1 overexpression decreased HCV RNA replication in both FL and NS replicons without affecting cellular growth or DNA synthesis. The attenuation of HCV replication was significantly reversed in both replicon systems with HO‐1 small interfering RNA (siRNA) knockdown. Both FL and NS replicons that overexpress HO‐1 showed reduced prooxidant levels at baseline and increased resistance to oxidant‐induced cytotoxicity. HO‐1 induction with hemin also markedly decreased HCV replication in both parental FL and NS replicon cell lines. Conversely, knockdown of HO‐1 messenger RNA (mRNA) by siRNA in parental FL or NS replicons did not significantly affect HCV replication, suggesting that less than basal levels of HO‐1 had minimal effect on HCV replication. Conclusion: Overexpression or induction of HO‐1 results in decreased HCV replication as well as protection from oxidative damage. These findings suggest a potential role for HO‐1 in antiviral therapy and therapeutic protection against hepatocellular injury in HCV infection. (HEPATOLOGY 2008.)

Older donor livers show early severe histological activity, fibrosis, and graft failure after liver transplantation for hepatitis C.

Background. In hepatitis C virus (HCV)-positive liver transplant recipients, infection of the allograft and recurrent liver disease are important problems. Increased donor age has emerged as an important variable affecting patient and graft survival; however, specific age cutoffs and risk ratios for poor histologic outcomes and graft survival are not clear. Methods. A longitudinal database of all HCV-positive patients transplanted at our center during an 11-year period was used to identify 111 patients who received 124 liver transplants. Graft survival and histological endpoints (severe activity and fibrosis) of HCV infection in the allografts were compared as a function of donor age at transplantation. Results. By Kaplan-Meier analyses, older allografts showed earlier failure and decreased time to severe histological activity and fibrosis as compared with allografts from younger donors. By Cox proportional hazards analysis, older allografts were at greater risk for all severe histologic features and decreased graft survival as compared with younger allografts (P≤0.02 for all outcomes). Analysis of donor age as a dichotomous variable showed that donors greater than 60 yr were at high risk for deleterious histologic outcomes and graft failure. An age cutoff of 60 yr showed a sensitivity of 94% and specificity of 67% for worse graft survival by receiver operating characteristics curve. Conclusions. Advanced donor age is associated with more aggressive recurrent HCV and early allograft failure in HCV-positive liver transplant recipients. Consideration of donor age is important for decisions regarding patient selection, antiviral therapy, and organ allocation.

Hepatitis C virus viremia in HIV-infected individuals with negative HCV antibody tests

Summary: Hepatitis C virus (HCV) viremia may occur in persons without detectable HCV antibodies and has been reported in as many as 5.5% of HIV‐positive persons. To better characterize serosilent HCV infection, the authors prospectively tested 131 HIV‐positive persons and 102 HIV‐negative control subjects with diabetes for the presence of HCV antibody (Ab) and HCV RNA. Thirty of 31 HCV Ab‐positive (AbP) HIV‐positive people tested positive for HCV RNA as did both HCV AbP, HIV‐negative control subjects. Similarly, none of the 100 HIV‐negative, HCV Ab‐negative (AbN) control subjects was HCV RNA positive (p < .001). In contrast, 19 of 100 HIV‐positive, HCV AbN persons met stringent criteria for HCV viremia, and 9 of these 19 people were HCV RNA positive when tested by a commercially available HCV RNA detection method. The mean duration of HCV viremia in HCV AbN people was 26.8 months (range, 1‐99 months). None of the subjects developed HCV antibody during the study. The HIV‐positive, HCV AbP, and RNA‐positive group was significantly more likely to have acquired HIV parenterally (p < .001), have higher initial CD4 counts (p = .029), and have higher ALT values than the HCV AbN group (p <.002). In summary, HCV infection appears to occur more frequently among HIV‐infected, HCV‐seronegative persons than appreciated, especially if HIV acquisition was through sexual as opposed to parenteral risk factors and was associated with a lower initial CD4 count and lower ALT values.

Surreptitious Hepatitis C Virus (HCV) Infection Detected in the Majority of Patients with Cryptogenic Chronic Hepatitis and Negative HCV Antibody Tests

Reverse transcription-polymerase chain reaction was used to identify hepatitis C virus (HCV) RNA in peripheral whole blood (WB) and plasma samples from 15 patients with chronic, unexplained hepatitis. These patients were serologically negative for hepatitis A, B, and C and were classified as having chronic non-A, non-B, non-C hepatitis (NANBNC). HCV RNA was repeatedly detected in WB samples from 10 (67%). In contrast, plasma samples from only 5 were intermittently positive. Statistically, HCV RNA detection in WB was significantly more sensitive than in plasma. Nucleic acid hybridization and HCV genotypic analysis confirmed the specificity of the HCV RNA assay. Liver biopsies from these patients suggested histopathologic differences between HCV RNA-positive and -negative groups. These data demonstrate that HCV infection is present in patients with unexplained chronic hepatitis more frequently than previously believed. Additionally, WB HCV RNA detection is more sensitive than plasma assays in identifying antibody-negative HCV infection.

Distribution of Hepatitis C Virus (HCV) RNA in Whole Blood and Blood Cell Fractions: Plasma HCV RNA Analysis Underestimates Circulating Virus Load

Previous experiments using a cationic surfactant to detect hepatitis C virus (HCV) RNA in whole blood (WB) suggested that WB was a more plentiful source of viral RNA than was plasma. The relative HCV RNA titers in WB, plasma, peripheral blood mononuclear cells (PBMC), neutrophils, and red blood cells (RBC)/platelets from 10 patients with chronic HCV infection were compared. WB contained significantly more HCV RNA than plasma, which contained more HCV RNA than PBMC, neutrophils, or RBC/platelets (P < .001). To determine if this increased sensitivity was clinically relevant, results of WB and plasma HCV RNA assays were compared with commercial quantitative and qualitative plasma HCV RNA assay results obtained for patients receiving interferon therapy. WB was significantly more sensitive than commercial plasma reverse transcription-polymerase chain reaction for detecting HCV RNA (P < .005). These data indicate that a significant proportion of HCV RNA in peripheral blood is not identified by standard plasma RNA detection methods.