David R. Gretch

Control of PKR Protein Kinase by Hepatitis C Virus Nonstructural 5A Protein: Molecular Mechanisms of Kinase Regulation

ABSTRACT The PKR protein kinase is a critical component of the cellular antiviral and antiproliferative responses induced by interferons. Recent evidence indicates that the nonstructural 5A (NS5A) protein of hepatitis C virus (HCV) can repress PKR function in vivo, possibly allowing HCV to escape the antiviral effects of interferon. NS5A presents a unique tool by which to study the molecular mechanisms of PKR regulation in that mutations within a region of NS5A, termed the interferon sensitivity-determining region (ISDR), are associated with sensitivity of HCV to the antiviral effects of interferon. In this study, we investigated the mechanisms of NS5A-mediated PKR regulation and the effect of ISDR mutations on this regulatory process. We observed that the NS5A ISDR, though necessary, was not sufficient for PKR interactions; we found that an additional 26 amino acids (aa) carboxyl to the ISDR were required for NS5A-PKR complex formation. Conversely, we localized NS5A binding to within PKR aa 244 to 296, recently recognized as a PKR dimerization domain. Consistent with this observation, we found that NS5A from interferon-resistant HCV genotype 1b disrupted kinase dimerization in vivo. NS5A-mediated disruption of PKR dimerization resulted in repression of PKR function and inhibition of PKR-mediated eIF-2α phosphorylation. Introduction of multiple ISDR mutations abrogated the ability of NS5A to bind to PKR in mammalian cells and to inhibit PKR in a yeast functional assay. These results indicate that mutations within the PKR-binding region of NS5A, including those within the ISDR, can disrupt the NS5A-PKR interaction, possibly rendering HCV sensitive to the antiviral effects of interferon. We propose a model of PKR regulation by NS5A which may have implications for therapeutic strategies against HCV.

Negative Immune Regulator Tim-3 Is Overexpressed on T Cells in Hepatitis C Virus Infection and Its Blockade Rescues Dysfunctional CD4+ and CD8+ T Cells

ABSTRACT A number of emerging molecules and pathways have been implicated in mediating the T-cell exhaustion characteristic of chronic viral infection. Not all dysfunctional T cells express PD-1, nor are they all rescued by blockade of the PD-1/PD-1 ligand pathway. In this study, we characterize the expression of T-cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) in chronic hepatitis C infection. For the first time, we found that Tim-3 expression is increased on CD4+ and CD8+ T cells in chronic hepatitis C virus (HCV) infection. The proportion of dually PD-1/Tim-3-expressing cells is greatest in liver-resident T cells, significantly more so in HCV-specific than in cytomegalovirus-specific cytotoxic T lymphocytes. Tim-3 expression correlates with a dysfunctional and senescent phenotype (CD127low CD57high), a central rather than effector memory profile (CD45RAnegative CCR7high), and reduced Th1/Tc1 cytokine production. We also demonstrate the ability to enhance T-cell proliferation and gamma interferon production in response to HCV-specific antigens by blocking the Tim-3-Tim-3 ligand interaction. These findings have implications for the development of novel immunotherapeutic approaches to this common viral infection.

Hepatitis C Virus Nonstructural 5A Protein Induces Interleukin-8, Leading to Partial Inhibition of the Interferon-Induced Antiviral Response

ABSTRACT Hepatitis C virus (HCV), a major cause of liver disease worldwide, is frequently resistant to the antiviral alpha interferon (IFN). The HCV nonstructural 5A (NS5A) protein has been implicated in HCV antiviral resistance in many studies. NS5A antagonizes the IFN antiviral response in vitro, and one mechanism is via inhibition of a key IFN-induced enzyme, the double-stranded-RNA-activated protein kinase (PKR). In the present study we determined if NS5A uses other strategies to subvert the IFN system. Expression of full-length NS5A proteins from patients who exhibited a complete response (FL-NS5A-CR) or were nonresponsive (FL-NS5A-NR) to IFN therapy in HeLa cells had no effect on IFN induction of IFN-stimulated gene factor 3 (ISGF-3). Expression of mutant NS5A proteins lacking 110 (NS5A-ΔN110), 222 (NS5A-ΔN222), and 334 amino-terminal amino acids and mutants lacking 117 and 230 carboxy-terminal amino acids also had no effect on ISGF-3 induction by IFN. Expression of FL-NS5A-CR and FL-NS5A-NR did not affect IFN-induced STAT-1 tyrosine phosphorylation or upregulation of PKR and major histocompatibility complex class I antigens. However, NS5A expression in human cells induced interleukin 8 (IL-8) mRNA and protein, and this effect correlated with inhibition of the antiviral effects of IFN in an in vitro bioassay. NS5A induced transcription of a reporter gene driven by the IL-8 promoter, and the first 133 bp of the IL-8 promoter made up the minimal domain required for NS5A transactivation. NS5A-ΔN110 and NS5A-ΔN222 stimulated the IL-8 promoter to higher levels than did the full-length NS5A protein, and this correlated with increased nuclear localization of the proteins. Additional mutagenesis of the IL-8 promoter suggested that NF-κB and AP-1 were important in NS5A-ΔN222 transactivation in the presence of tumor necrosis factor alpha and that NF–IL-6 was inhibitory to this process. This study suggests that NS5A inhibits the antiviral actions of IFN by at least two mechanisms and provides the first evidence for a biological effect of the transcriptional activity of the NS5A protein. During HCV infection, viral proteins may induce chemokines that contribute to HCV antiviral resistance and pathogenesis.

Tim-3 expression on PD-1+ HCV-specific human CTLs is associated with viral persistence, and its blockade restores hepatocyte-directed in vitro cytotoxicity

Having successfully developed mechanisms to evade immune clearance, hepatitis C virus (HCV) establishes persistent infection in approximately 75%-80% of patients. In these individuals, the function of HCV-specific CD8+ T cells is impaired by ligation of inhibitory receptors, the repertoire of which has expanded considerably in the past few years. We hypothesized that the coexpression of the negative regulatory receptors T cell immunoglobulin and mucin domain-containing molecule 3 (Tim-3) and programmed death 1 (PD-1) in HCV infection would identify patients at risk of developing viral persistence during and after acute HCV infection. The frequency of PD-1-Tim-3- HCV-specific CTLs greatly outnumbered PD-1+Tim-3+ CTLs in patients with acute resolving infection. Moreover, the population of PD-1+Tim-3+ T cells was enriched for within the central memory T cell subset and within the liver. Blockade of either PD-1 or Tim-3 enhanced in vitro proliferation of HCV-specific CTLs to a similar extent, whereas cytotoxicity against a hepatocyte cell line that expressed cognate HCV epitopes was increased exclusively by Tim-3 blockade. These results indicate that the coexpression of these inhibitory molecules tracks with defective T cell responses and that anatomical differences might account for lack of immune control of persistent pathogens, which suggests their manipulation may represent a rational target for novel immunotherapeutic approaches.

Elevated Levels of Interleukin-8 in Serum Are Associated with Hepatitis C Virus Infection and Resistance to Interferon Therapy

ABSTRACT Hepatitis C virus (HCV), a major cause of liver disease worldwide, is frequently resistant to the antiviral alpha interferon (IFN). We have recently found that the HCV NS5A protein induces expression of the proinflammatory chemokine IL-8 to partially inhibit the antiviral actions of IFN in vitro. To extend these observations, in the present study we examined the relationship between levels of IL-8 in serum, HCV infection, and biochemical response to IFN therapy. Levels of IL-8 were significantly elevated in 132 HCV-infected patients compared to levels in 32 normal healthy subjects and were also significantly higher in patients who did not respond to IFN therapy than in patients who did respond to therapy. This study suggests that HCV-induced changes in levels of chemokine and cytokine expression may be involved in HCV antiviral resistance, persistence, and pathogenesis.

Risk of death among chronic dialysis patients infected with hepatitis C virus.

Hepatitis C virus (HCV) infection is highly prevalent among chronic dialysis patients (10% to 40%) and is the most common cause of chronic liver disease. However, there are no studies estimating the risk for death among dialysis patients infected with HCV compared with those not infected. We conducted a prospective cohort study to estimate the risk for death among chronic dialysis patients infected with HCV compared with those not infected. In 1992, 200 patients (91%) who had been undergoing dialysis therapy for at least 6 months consented to be screened for HCV infection by enzyme immunoblot assay and polymerase chain reaction (PCR). Information about potential confounders and potential risk factors for death and HCV infection was obtained from the dialysis center database. Patient outcomes collected included death, transplantation, and loss to follow-up. The Cox proportional hazards model was used to estimate the odds of death among dialysis patients who were positive for the HCV antibody and HCV RNA compared with negative patients. Forty-four patients (22%) were HCV antibody positive. Thirty-four patients (17%) were HCV RNA positive. Patients in the HCV RNA-positive group were more likely to be younger (51.8+/-12.6 v 57.2+/-17.3 years of age), men (77% v 54%), and black (65% v 37%). None of the home hemodialysis or peritoneal dialysis patients were HCV RNA positive, whereas one of the home hemodialysis and one of the peritoneal dialysis patients were HCV antibody positive. Two patients became infected with HCV during the follow-up period. Patients who were HCV RNA positive and those who were HCV antibody positive were at increased risk for death compared with patients who were negative (adjusted relative risk [aRR]=1.78; 95% confidence interval [CI], 1.01 to 3.14; P=0.045; and aRR=1.97; 95% CI, 1.16 to 3.33; P=0.012, respectively), after adjusting for time on dialysis, race, transplantation, and age. We conclude that HCV infection increased the risk for death during the study period compared with those not infected. Further studies should assess the measures used to prevent and treat HCV infection.

Decreased NK cell frequency in chronic hepatitis C does not affect ex vivo cytolytic killing

Prior studies have suggested that natural killer (NK) cell function might be impaired in chronic hepatitis C virus (HCV) infection. Circulating NK cell frequency and cytolytic activity were examined freshly ex vivo in HCV‐infected and uninfected subjects. Surprisingly, the intrinsic cytolytic activity of peripheral blood NK‐enriched cells was similar between HCV‐infected and uninfected groups (P = .91). Although the percentage of circulating CD3−CD16/56+NK cells was 30% lower in HCV‐infected compared with uninfected subjects (P = .02) paralleled by a decrease of CD56dim cytolytic NK cells (P = .02), overall K562 cytolysis by unfractionated peripheral blood mononuclear cells was not affected (P = .29). Analysis of the relationships between NK cytolytic activity and other clinical information revealed an inverse association with liver fibrosis stage (P = .035). In conclusion, NK cell cytolytic function does not appear to be impaired in chronic hepatitis C, but higher levels of NK cell cytolysis are associated with less liver fibrosis. (HEPATOLOGY 2006;43:573–580.)

Association of multispecific CD4+ response to hepatitis C and severity of recurrence after liver transplantation*

BACKGROUND & AIMS After liver transplantation for hepatitis C virus (HCV), reinfection of the allograft invariably occurs. Indirect evidence suggests that the cellular immune response may play a central role. The purpose of this analysis was to determine the correlation between HCV-specific peripheral CD4(+) T-cell responses and the severity of recurrence after liver transplantation. METHODS Fifty-eight HCV-seropositive patients, including 43 liver transplant recipients with at least 1 year of histological follow-up, were studied. Peripheral blood mononuclear cells (PBMCs) were isolated from fresh heparinized blood and stimulated with either recombinant HCV antigens (core, E2, NS3, NS4, and NS5) or control antigens. RESULTS Fourteen (40%) of 35 patients with mild or no evidence of histological recurrence within their allografts responded to at least 1 of the HCV antigens. Eleven responded to NS3, 5 to all the nonstructural antigens, and 3 to the HCV core polypeptide alone. In contrast, in the 8 patients with severe HCV recurrence, no proliferation in response to any of the HCV antigens was seen (P = 0. 03) despite responses to the control antigens. CONCLUSIONS Despite immunosuppression, HCV-specific, major histocompatibility complex class II- restricted CD4(+) T-cell responses are detectable in patients with minimal histological recurrence after liver transplantation. In contrast, PBMCs from patients with severe HCV recurrence, despite being able to proliferate in response to non-HCV antigens, fail to respond to the HCV antigens. These findings suggest that the inability to generate virus-specific T-cell responses plays a contributory role in the pathogenesis of HCV-related graft injury after liver transplantation. It is hoped that further characterization of the immunoregulatory mechanisms related to recurrent HCV will provide the rationale for novel therapeutic strategies and diminish the incidence of inevitable graft loss.

Cytomegalovirus viremia : Risk factor for allograft cirrhosis after liver transplantation for hepatitis C

BACKGROUND Despite recent advances in diagnosis and treatment, cytomegalovirus (CMV) infection continues to be a common cause of morbidity in liver transplant (LT) recipients. Because CMV infection suppresses cell-mediated immunity, which seems to be important in neutralizing hepatitis C virus (HCV) infection, we assessed the impact of CMV infection on histopathological HCV recurrence after LT. METHODS The study group was comprised of 43 consecutive LT recipients with at least 6 months of histologic follow-up. Group 1 consisted of the 8 patients who developed CMV viremia after LT; group 2 comprised the 35 patients without CMV viremia. There was no significant difference with regard to age, initial immunosuppression, incidence of rejection, distribution of HCV genotypes, or mean follow-up between the groups. Semiquantitative histopathologic assessment of allograft hepatitis was performed using the Knodells score. RESULTS The mean total Knodell score of the final allograft biopsy was significantly greater in group 1 patients (P=0.016), with most of the difference due to periportal/bridging necrosis (P=0.009) and lobular activity subitem (P=0.01) scores. Half of the CMV viremic patients eventually developed allograft cirrhosis as compared with 11% of the CMV-negative patients (P=0.027). Accordingly, the cirrhosis-free actuarial survival by Kaplan-Meier estimates was significantly diminished in the CMV viremic patients. Glycoprotein B genotype analysis of CMV isolates revealed no significant differences between patients who did and those who did not develop allograft cirrhosis. CONCLUSIONS After LT for chronic HCV, patients who develop CMV viremia incur a significantly greater risk of severe HCV recurrence.

Assessment of Hepatitis C Viremia Using Molecular Amplification Technologies: Correlations and Clinical Implications

Hepatitis C virus, a positive-strand RNA virus classified within the Flaviviridae family, is an important cause of chronic hepatitis. Hepatic complications associated with chronic HCV infection include chronic active hepatitis in approximately 60% of cases, bridging fibrosis and cirrhosis in at least 20%, end-stage liver disease, and possibly hepatocellular carcinoma [1-5]. Several extrahepatic syndromes are also associated with HCV infection, including essential mixed cryoglobulinemia and membranoproliferative glomerulonephritis [6-9]. Serologic tests that detect antibodies to HCV structural and nonstructural antigens are useful screening assays, and the prevalence of HCV infection in the United States is currently estimated to be between 1% and 2% of the general population. Sensitive qualitative tests for detection of viral RNA in serum by reverse transcription polymerase chain reaction (PCR) have been developed and shown to be useful for diagnosing active hepatitis C [10]. In one cross-sectional study of seropositive blood donors and clinic patients, more than 90% of persons with antibodies to HCV also had evidence of HCV viremia, as evidenced by the detection of the viral RNA genome in serum by reverse transcription PCR testing [11]. Accurate quantitative methods for measuring viral nucleic acid levels in patient serum have recently been developed. Several recent studies have indicated that the level of HCV viremia correlates with the clinical stage of disease; patients with advanced stages of liver disease such as severe chronic active hepatitis, cirrhosis, and endstage liver disease had higher serum levels of HCV RNA than patients with mild HCV infections. Moreover, the HCV viremia titer may predict a subsequent response to antiviral therapy [11-15]. Two different technologies have been used to assess HCV viremia: the branched-DNA (bDNA) assay [13, 16] and quantitative PCR [11, 12]. The bDNA assay uses a novel approach to viral detection called signal amplification technology. Briefly, viral nucleic acids in a clinical specimen (for example, plasma or serum) are first solubilized by the addition of denaturing reagents and are then hybridized to microtiter plates through the use of virus-specific capture probes. The bound viral nucleic acid is then reacted with virus-specific extender probes followed by bDNA polymers. The bDNA polymers contain multiple repetitive binding sites for oligonucleotide-linked enzymes that catalyze the activation of chemiluminescent substrates; thus, signal amplification is achieved without amplification of viral nucleic acid. Quantitative PCR relies on amplification of viral nucleic acids in the presence of a specific competitor molecule. In this assay, the ratio of amplification products derived from the native viral RNA and the competitive template are compared. In the quantitative competitive reverse transcription PCR assay, known amounts of synthetic internal control HCV RNA are titrated directly into clinical specimens and are then coextracted with the wild-type HCV RNA before analysis by competitive reverse transcription PCR [11]. Adding the competitor RNA directly into the clinical specimen allows the assay to be internally controlled for variations in efficiency of RNA extraction, complementary DNA synthesis, and PCR because the competitor is present during all steps. Quantitative PCR assays that use synthetic competitor templates as internal controls have been shown to be highly accurate for measuring various viral nucleic acids in clinical specimens. The current study compared the bDNA assay with a quantitative competitive reverse transcription PCR assay (quantitative PCR) for detecting and measuring HCV RNA levels in serum specimens in various patient populations. Methods Clinical Specimens We selected for analysis 400 specimens from four different sources: prospective blood donors at the Puget Sound Blood Center (Seattle, Washington) or Irwin Memorial Blood Centers (San Francisco, California), patients attending hepatology clinics at either the University of Washington Medical Center or Harborview Medical Center (Seattle), patients receiving long-term renal dialysis at the Northwest Kidney Center (Seattle), and HCV-infected liver transplant recipients followed at the University of Washington. All serum samples were collected and processed within 4 hours of venipuncture to optimize detection of viral RNA [17] and were analyzed by laboratory personnel blinded to the clinical data. All specimens were tested for HCV antibodies by second-generation enzyme immunoassay (EIA2; Abbott Laboratories, North Chicago, Illinois) and by second-generation recombinant immunoblot assay (RIBA-II; Ortho Diagnostics, Raritan, New Jersey) when results of the enzyme immunoassay were positive. All 400 specimens were initially screened for HCV RNA by reverse transcription PCR combined with a specific radioactive probe hybridization, which is a highly sensitive and specific qualitative (nonquantitative) screening assay for HCV RNA [18, 19]. The analytical sensitivity of our reverse transcription PCR assay is less than 10 molecules of purified synthetic HCV RNA [19]; a clinical specificity of greater than 99% has been shown in previous studies and confirmed by ongoing proficiency testing approved by the College of American Pathology. We selected 101 of the 400 specimens as negative controls because they were negative for antibody to HCV by enzyme immunoassay and for HCV RNA by reverse transcription PCR; 299 specimens were selected as viremic specimens because they were positive for both antibody to HCV and HCV RNA by the screening reverse transcription PCR assay. To evaluate the bDNA and quantitative PCR assays for detecting and measuring HCV RNA levels in patients followed prospectively, serum specimens were obtained at monthly intervals from 19 consecutive HCV-infected patients; 18 of these were treated for 6 months with recombinant interferon-, 3 million U three times per week, and 1 was treated with an escalating dose regimen of interferon for 48 months. Patients were classified as having a complete virologic response to interferon if their serum tested negative for HCV RNA by reverse transcription PCR at the end of therapy; we chose this virologic end point because reverse transcription PCR is the most sensitive test available for detection of HCV viremia. Branched-DNA Assay The bDNA assay was done as recommended by the manufacturer (Chiron Corp.; Emeryville, California); the design and principles of the assay are discussed above and in a previous report [16]. Quantitation of HCV RNA (expressed as equivalents per mL) was calculated by comparing relative luminescence with that obtained from an HCV RNA standard curve. All samples were assayed in duplicate, and the mean value of the duplicate tests was used for data analysis. Quantitative Polymerase Chain Reaction Known quantities of synthetic internal control HCV RNA were directly titrated into clinical specimens before nucleic acid extraction. Viral and internal control RNAs were coextracted from serum through the guanidinium thiocyanate method and were reverse transcribed; complementary DNA was amplified by PCR using primers derived from the highly conserved 5-noncoding region of the HCV genome. Polymerase chain reaction amplification products were detected by either agarose gel electrophoresis plus ethidium bromide staining or Southern blot analysis in which a radiolabeled internal probe was used. The RNA copy number was deduced by comparing the PCR amplification product band intensity with the intensity of the internal control bands. The linear range of quantitative PCR ranged from 103 to 1010 RNA molecules per mL (3 to 10 logs of HCV RNA per mL) by comparison with end-point dilution analysis of HCV RNA [11]. Results Linearity of Hepatitis C Virus RNA Quantitation in Vitro We assessed the linearity of HCV RNA quantitation in vitro using a serum sample obtained from an immunosuppressed patient with previously determined high-titer HCV viremia: The undiluted serum HCV RNA titer was 9.5 logs per mL by quantitative PCR. Eighteen 0.5-log dilutions of the high-titer serum samples into noninfected human sera were prepared, and HCV RNA was tested in duplicate by bDNA assay, quantitative PCR, and the qualitative screening assay, reverse transcription PCR (Figure 1). Dilutions 1 through 15 all tested positive for HCV RNA by reverse transcription PCR, whereas dilutions 16 through 18 tested negative. In contrast, only dilutions 1 through 6 were positive by bDNA assay; dilution 7 was indeterminate; and dilutions 8 to 18 were negative. The bDNA assay showed a nearly perfect slope within the linear range of the assay, from 6.0 logs to 8.5 logs when expressed as molecules per mL, or 5.5 logs to 7.9 logs when expressed as equivalents per mL as defined by the manufacturer (Figure 1). The bDNA assay went off scale at HCV RNA levels greater than 7.9 log-equivalents per mL. Figure 1. Analysis of hepatitis C virus (HCV) RNA in serial dilutions of high-titer patient serum by two different quantitative assays, branched-DNA (bDNA) and quantitative polymerase chain reaction (Q-PCR), and a highly sensitive qualitative assay for HCV RNA, reverse transcription PCR (RT-PCR). open circles closed circles The quantitative PCR assay gave linear results on the serially diluted serum specimen, from 4-log molecules per mL to 9.5-log molecules per mL. The reverse transcription PCR end point was estimated to be 100 molecules per mL or approximately 35 log-equivalents per mL; thus, reverse transcription PCR was approximately 4 orders of magnitude more sensitive than bDNA assay for detecting HCV RNA in vitro. The linear range of HCV RNA quantitation by quantitative PCR exceeded that of the bDNA assay by at least 3 orders of magnitude, although quantitative PCR varied slightly more than the bDNA assay. Within the linear range of the bDNA assay, the in vitro correlation between