Interferon Resistance Research Articles

Interferon-alpha Induces High Expression of APOBEC3G and STAT-1 in Vitro and in Vivo

To investigate whether the JAK-STAT (Janus kinase-signal transducers and activators of transcription) pathway participates in the regulation of APOBEC3G (Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G) gene transcription and to study the molecular mechanisms of interferon resistance in patients with chronic hepatitis B (CHB), changes in APOBEC3G and STAT-1 expression levels in HepG2.2.15 cells after treatment with various concentrations of IFN-α, were detected using real-time RT-PCR and Western-blot. In addition, the differences in STAT-1 and APOBEC3G expression in liver tissues were also observed in patients with different anti-viral responses to IFN-α. It is found that IFN-α suppressed HBV replication and expression markedly in HepG2.2.15 cells, and simultaneously enhanced APOBEC3G expression in a dose- or time-dependent manner within a certain range. Moreover, a corresponding gradual increase in STAT-1 expression levels was also observed. The expression levels of STAT-1 and APOBEC3G in the liver of CHB patients with a complete response to IFN-α are significantly higher than that of the patients with non-response to IFN-α treatment. It is suggested that inducing intracellular APOBEC3G expression may be one of anti-HBV mechanisms of IFN-α, and IFN-α-induced APOBEC3G expression may be via the JAK-STAT signaling pathway. Moreover, interferon resistance may be related to the down-regulation of STAT-1 expression in the patients who had non-response to IFN-α treatment.

Modulation of interferon signaling by hepatitis C virus non-structural 5A protein: Implication of genotypic difference in interferon treatment

Interferon (IFN) response rate in hepatitis C virus (HCV) patients has been varied with genotypes. In this study, we investigated the effects of HCV NS5A protein on IFN resistance and compared the genotypic differences of NS5A. We showed that IFN-α-, poly I:C-, and Sendai virus-induced ISRE transcriptional activities were inhibited by both genotype 1b and 2a NS5A protein. We demonstrated that not only genotype 1b but also genotype 2a NS5A exerted the similar extent of IFN-α-induced antiviral activity. We showed that NS5A derived from both genotype 1b and 2a showed no significant differential IFN responses as seen in HCV patients. These data imply that some other host factor may be involved in genotypic differences of IFN antagonism in HCV patients.

The Cullin 3 substrate adaptor KLHL20 mediates DAPK ubiquitination to control interferon responses

Death-associated protein kinase (DAPK) was identified as a mediator of interferon (IFN)-induced cell death. How IFN controls DAPK activation remains largely unknown. Here, we identify the BTB-Kelch protein KLHL20 as a negative regulator of DAPK. KLHL20 binds DAPK and Cullin 3 (Cul3) via its Kelch-repeat domain and BTB domain, respectively. The KLHL20-Cul3-ROC1 E3 ligase complex promotes DAPK polyubiquitination, thereby inducing the proteasomal degradation of DAPK. Accordingly, depletion of KLHL20 diminishes DAPK ubiquitination and degradation. The KLHL20-mediated DAPK ubiquitination is suppressed in cells receiving IFN-alpha or IFN-gamma, which induces an enrichment/sequestration of KLHL20 in the PML nuclear bodies, thereby separating KLHL20 from DAPK. Consequently, IFN triggers the stabilization of DAPK. This mechanism of DAPK stabilization is crucial for determining IFN responsiveness of tumor cells and contributes to IFN-induced autophagy. This study identifies KLHL20-Cul3-ROC1 as an E3 ligase for DAPK ubiquitination and reveals a regulatory mechanism of DAPK, through blocking its accessibility to this E3 ligase, in IFN-induced apoptotic and autophagic death. Our findings may be relevant to the problem of IFN resistance in cancer therapy.

Ras V12 induces Survivin/AuroraB pathway conferring tumor cell apoptosis resistance

Several cancers are treated by interferons α and γ in association with conventional chemotherapy due to the resistance observed with interferon treatment alone. The frequency of un-sensitive cancer depends on tumor origin and oncogenic genes. Preclinical studies have highlighted interferon resistance in many cancers such as colon carcinoma due to oncogenic Ras. However, the resistance mechanism remains elusive. Apoptosis and proliferation of Ras wt and mutated Ras V12 transformed colon carcinoma cells treated with several recombinant interferon combinations were analyzed by flow cytometer and immunoblot. Apoptotic pathways of resistant Ras V12 cells were investigated using siRNA strategy to determine key proteins involved in this process. We show that interferons α and γ synergized to induce human Ras wt colon carcinoma cell (HT29) apoptosis by caspases and PARP-1 cleavages in contrast to Ras V12 mutated colon carcinoma cells (SW480, HT29 clone). However, Ras V12 siRNA restored interferon sensitivity of Ras V12-HT29 clone to apoptosis. Survivin siRNA increased interferon apoptosis in Ras wt cells demonstrating the key role of this protein in cell survival. Ras V12 mutation in HT29 clone neutralized the interferon effect on Survivin suppression and maintained high level of phospho-Aurora-B/Histone H3, which protected cells from apoptosis. SiRNA strategy against both Aurora-B and Survivin in Ras V12 cells synergized to restore interferon -induced apoptosis. Ras V12 cells are less sensitive than Ras wt cells to interferon induced cell apoptosis due to a Survivin/Aurora-B survival alternative pathway. Taken together, these results may provide interest in siRNA-therapeutic strategy and diagnostic relevance for therapy.

Spatial and Temporal Genetic Homogeneity of Orf Viruses Infecting Japanese Serows (Capricornis crispus)

This study genetically characterized orf viruses (ORFVs) isolated from recent outbreaks in Japanese serows (Capricornis crispus) in 2007 and 2008, and from earlier outbreaks from 1985 to 2001. Nucleotide sequences of genes for the viral envelope, vascular endothelial growth factor (VEGF), and virus interferon resistance (VIR) were determined in two ORFVs isolated from recent outbreaks and in eight from earlier outbreaks. No deletions or insertions were observed in these genes. Surprisingly, the amino acid sequences of the envelope and VIR genes were identical among the 10 ORFVs, and only one amino acid substitution in the VEGF gene was found in one of the two recent ORFVs (2007). Genotyping by differential PCR for the A32L gene, which encodes an ATPase, classified all of the 10 ORFVs into the same genotype. In an ORFV isolated from a sheep in 1970, the three sequenced genes were almost the same as in the ORFVs isolated from Japanese serows, and the ORFV in 1970 was classified into the same genotype as the ORFVs from Japanese serows. These results suggest that recent outbreaks in Japanese serows are caused by ORFVs genetically closely related to the viruses in earlier outbreaks and that these genetically stable ORFVs have circulated among Japanese serows over a long period of time.

Phylogenetic analysis and characterization of Korean orf virus from dairy goats: case report

An outbreak of orf virus infection in dairy goats in Korea was investigated. Suspected samples of the skin and lip of affected goats were sent to the laboratory for more exact diagnosis. Orf virus was detected by electron microscopy and viral DNA was identified by PCR. To reveal the genetic characteristics of the Korean strain (ORF/09/Korea), the sequences of the major envelope protein (B2L) and orf virus interferon resistance (VIR) genes were determined and then compared with published reference sequences. Phylogenetic analysis revealed that the ORF/09/Korea strain was closest to the isolates (Taiping) from Taiwan. This is believed to be the first report on the molecular characterization of orf virus in Korea.

Absence of viral interference and different susceptibility to interferon between hepatitis B virus and hepatitis C virus in human hepatocyte chimeric mice

Both hepatitis B virus (HBV) and hepatitis C virus (HCV) replicate in the liver and show resistance against innate immunity and interferon (IFN) treatment. Whether there is interference between these two viruses is still controversial. We investigated the interference between these two viruses and the mode of resistance against IFN. We performed infection experiments with either or both of the two hepatitis viruses in human hepatocyte chimeric mice. Huh7 cell lines with stable production of HBV were also established and transfected with HCV JFH1 clone. Mice and cell lines were treated with IFN. The viral levels in mice sera and culture supernatants and messenger RNA levels of IFN-stimulated genes were measured. No apparent interference between the two viruses was seen in vivo. Only a small (0.3 log) reduction in serum HBV and a rapid reduction in HCV were observed after IFN treatment, regardless of infection with the other virus. In in vitro studies, no interference between the two viruses was observed. The effect of IFN on each virus was not affected by the presence of the other virus. IFN-induced reductions of viruses in culture supernatants were similar to those in in vivo study. No interference between the two hepatitis viruses exists in the liver in the absence of hepatitis. The mechanisms of IFN resistance of the two viruses target different areas of the IFN system.

Vaccinia Virus K1L and C7L Inhibit Antiviral Activities Induced by Type I Interferons

Cellular tropism of vaccinia virus (VACV) is regulated by host range genes, including K1L, C7L, and E3L. While E3L is known to support viral replication by antagonizing interferon (IFN) effectors, including PKR, the exact functions of K1L and C7L are unclear. Here, we show that K1L and C7L can also inhibit antiviral effectors induced by type I IFN. In human Huh7 and MCF-7 cells, a VACV mutant lacking both K1L and C7L (vK1L-C7L-) replicated as efficiently as wild-type (WT) VACV, even in the presence of IFN. However, pretreating the cells with type I IFN, while having very little effect on WT VACV, blocked the replication of vK1L-C7L- at the step of intermediate viral gene translation. Restoring either K1L or C7L to vK1L(-)C7L(-) fully restored the IFN resistance phenotype. The deletion of K1L and C7L from VACV did not affect the ability of the virus to inhibit IFN signaling or its ability to inhibit the phosphorylation of PKR and the alpha subunit of eukaryotic initiation factor 2, indicating that K1L and C7L function by antagonizing an IFN effector(s) but with a mechanism that is different from those of IFN antagonists previously identified for VACV. Mutations of K1L that inactivate the host range function also rendered K1L unable to antagonize IFN, suggesting that K1L supports VACV replication in mammalian cells by antagonizing the same antiviral factor(s) that is induced by IFN in Huh7 cells.

Internal Initiation Stimulates Production of p8 Minicore, a Member of a Newly Discovered Family of Hepatitis C Virus Core Protein Isoforms

The hepatitis C virus (HCV) core gene is more conserved at the nucleic acid level than is necessary to preserve the sequence of the core protein, suggesting that it contains information for additional functions. We used a battery of anticore antibodies to test the hypothesis that the core gene directs the synthesis of core protein isoforms. Infectious viruses, replicons, and RNA transcripts expressed a p8 minicore containing the C-terminal portion of the p21 core protein and lacking the N-terminal portion. An interferon resistance mutation, U271A, which creates an AUG at codon 91, upregulated p8 expression in Con1 replicons, suggesting that p8 is produced by an internal initiation event and that 91-AUG is the preferred, but not the required, initiation codon. Synthesis of p8 was independent of p21, as shown by the abundant production of p8 from transcripts containing an UAG stop codon that blocked p21 production. Three infectious viruses, JFH-1 (2a core), J6/JFH (2a core), and H77/JFH (1a core), and a bicistronic construct, Bi-H77/JFH, all expressed both p8 and larger isoforms. The family of minicores ranges in size from 8 to 14 kDa. All lack the N-terminal portion of the p21 core. In conclusion, the core gene contains an internal signal that stimulates the initiation of protein synthesis at or near codon 91, leading to the production of p8. Infectious viruses of both genotype 1 and 2 HCV express a family of larger isoforms, in addition to p8. Minicores lack significant portions of the RNA binding domain of p21 core. Studies are under way to determine their functions.

STAT5 contributes to antiapoptosis in melanoma

Malignant melanoma is a cancer whose incidence is rising rapidly. Extensive studies of primary tumors and tumor-derived cell lines revealed that inappropriate activation of signal transducer and activator of transcription (STAT) proteins, particularly of STAT3 and 5, occurs with high frequency in various human cancers. We reported that in the Xiphophorus fish melanoma model, constitutive activation of STAT5 correlates with the aggressiveness of melanoma. Investigations in human melanoma mainly focussed on the function of STAT1, but we have shown recently that STAT5 is also activated in human melanoma. The objectives of this investigation were to get more information about the function of STAT5 in melanoma. Here we demonstrate that in murine melanocytes activation of STAT5 measured by its tyrosine phosphorylation and translocation to the nucleus parallels upregulation with its target gene bcl-XL. This indicates a role for STAT5 in antiapoptotic signaling in pigment cells. In human melanoma cell lines, we found that constitutive activation of STAT5 correlates with expression of bcl-XL. Expression of dominant negative STAT5 in the human melanoma cell line A375 leads to a reduced bcl-XL expression and a dramatic increase of apoptotic cells. In contrast to STAT1, which is known to transduce antiproliferative effects of interferons, our data support a significant role for STAT5 in melanoma cell proliferation and survival via the activation of the antiapoptotic protein bcl-XL. Keeping in mind that interferons activate both STAT proteins, STAT5 activation could be of importance in interferon resistance of melanoma.

Enhancement of antiproliferative activity of interferons by RNA interference‐mediated silencing of SOCS gene expression in tumor cells

The suppressor of cytokine signaling (SOCS) proteins, negative regulators of interferon (IFN)-induced signaling pathways, is involved in IFN resistance of tumor cells. To improve the growth inhibitory effect of IFN-beta and IFN-gamma on a murine melanoma cell line, B16-BL6, and a murine colon carcinoma cell line, Colon26 cells, SOCS-1 and SOCS-3 gene expression in tumor cells was downregulated by transfection of plasmid DNA expressing short hairpin RNA targeting one of these genes (pshSOCS-1 and pshSOCS-3, respectively). Transfection of pshSOCS-1 significantly increased the antiproliferative effect of IFN-gamma on B16-BL6 cells. However, any other combinations of plasmids and IFN had little effect on the growth of B16-BL6 cells. In addition, transfection of pshSOCS-1 and pshSOCS-3 produced little improvement in the effect of IFN on Colon26 cells. To understand the mechanism underlining these findings, the level of SOCS gene expression was measured by real time polymerase chain reaction. Addition of IFN-gamma greatly increased the SOCS-1 mRNA expression in B16-BL6 cells. Taking into account the synergistic effect of pshSOCS-1 and IFN-gamma on the growth of B16-BL6 cells, these findings suggest that IFN-gamma-induced high SOCS-1 gene expression in B16-BL6 cells significantly interferes with the antiproliferative effect of IFN-gamma. These results indicate that silencing SOCS gene expression can be an effective strategy to enhance the antitumor effect of IFN under conditions in which the SOCS gene expression is upregulated by IFN.

NS5A Sequences of Hepatitis C Virus Genotype 1 and Interferon Resistance: Where Are We?

NS5A Sequences of Hepatitis C Virus Genotype 1 and Interferon Resistance: Where Are We?

Interaction of Hepatitis C Virus Nonstructural Protein 5A with Core Protein Is Critical for the Production of Infectious Virus Particles

Nonstructural protein 5A (NS5A) of the hepatitis C virus (HCV) possesses multiple and diverse functions in RNA replication, interferon resistance, and viral pathogenesis. Recent studies suggest that NS5A is involved in the assembly and maturation of infectious viral particles; however, precisely how NS5A participates in virus production has not been fully elucidated. In the present study, we demonstrate that NS5A is a prerequisite for HCV particle production as a result of its interaction with the viral capsid protein (core protein). The efficiency of virus production correlated well with the levels of interaction between NS5A and the core protein. Alanine substitutions for the C-terminal serine cluster in domain III of NS5A (amino acids 2428, 2430, and 2433) impaired NS5A basal phosphorylation, leading to a marked decrease in NS5A-core interaction, disturbance of the subcellular localization of NS5A, and disruption of virion production. Replacing the same serine cluster with glutamic acid, which mimics the presence of phosphoserines, partially preserved the NS5A-core interaction and virion production, suggesting that phosphorylation of these serine residues is important for virion production. In addition, we found that the alanine substitutions in the serine cluster suppressed the association of the core protein with viral genome RNA, possibly resulting in the inhibition of nucleocapsid assembly. These results suggest that NS5A plays a key role in regulating the early phase of HCV particle formation by interacting with core protein and that its C-terminal serine cluster is a determinant of the NS5A-core interaction.

Modelling and analysis of dynamics of viral infection of cells and of interferon resistance

Interferons are active biomolecules, which help fight viral infections by spreading from infected to uninfected cells and activate effector molecules, which confer resistance from the virus on cells. We propose a new model of dynamics of viral infection, including endocytosis, cell death, production of interferon and development of resistance. The novel element is a specific biologically justified mechanism of interferon action, which results in dynamics different from other infection models. The model reflects conditions prevailing in liquid cultures (ideal mixing), and the absence of cells or virus influx from outside. The basic model is a nonlinear system of five ordinary differential equations. For this variant, it is possible to characterise global behaviour, using a conservation law. Analytic results are supplemented by computational studies. The second variant of the model includes age-of-infection structure of infected cells, which is described by a transport-type partial differential equation for infected cells. The conclusions are: (i) If virus mortality is included, the virus becomes eventually extinct and subpopulations of uninfected and resistant cells are established. (ii) If virus mortality is not included, the dynamics may lead to extinction of uninfected cells. (iii) Switching off the interferon defense results in a decrease of the sum total of uninfected and resistant cells. (iv) Infection-age structure of infected cells may result in stabilisation or destabilisation of the system, depending on detailed assumptions. Our work seems to constitute the first comprehensive mathematical analysis of the cell-virus-interferon system based on biologically plausible hypotheses.

Blunted Cytopenias and Weight Loss: New Correlates of Virologic Null Response to Re-treatment of Chronic Hepatitis C

Peginterferon with ribavirin therapy for chronic hepatitis C leads to sustained loss of serum hepatitis C virus (HCV) RNA in half of treated patients. Although viral kinetic profiles in treatment responders shed light on the mechanism of action of interferon and ribavirin, minimal information is available to explain why some patients experience little or no virologic suppression. We evaluated factors that might be associated with the lack of a week-20 virologic response in previous nonresponder patients undergoing peginterferon and ribavirin retreatment. Among 1145 patients enrolled in the Hepatitis C Antiviral Long-term Treatment against Cirrhosis Trial's lead-in treatment phase, 588 who received more than 80% of prescribed therapy for 20 weeks were analyzed. By week 20, 245 patients (41.7%) had undetectable HCV RNA (full response), 186 (31.6%) had a 1 log(10) or greater decrease in HCV RNA (partial response), and 157 (26.7%) had less than a 1 log(10) decrease (null response) in HCV RNA. Null response was associated independently with African American race, genotype-1 infection, and less reduction in body weight, platelets, and white blood cells during treatment. On-treatment variables associated with null response suggest a blunted systemic response to interferon. These observations have important implications for the design and analysis of trial results in which novel agents are evaluated. Further studies are needed to discern whether host interferon resistance, viral drug resistance, or both are playing a role in patients who have no virologic response to interferon treatment.

Advances in hepatitis C virus core protein

Hepatitis C virus (HCV), a blood-borne virus, is one of the leading causes responsible for chronic hepatitis, liver cirrhosis and even hepatocellular carcinoma. At present precise mechanism of HCV infection has not been known yet, and no effective treatment and vaccination against HCV are available. However, recent findings show that apart from the function of viral partial package as the nucleocapsid protein, HCV core protein also participates in modulating cell apoptosis, lipid metabolism, transcription and antigen presentation, and has a close relationship with interferon resistance. HCV core protein has intense trans-activation effect and its interaction with the host protein accounts for persistent infection and hepatocellular tumorigenesis. Further cognition and analysis of molecular features of HCV core protein is of significance to elucidate persist HBV infection, mechanism underlying tumorigenesis induced by HCV, as well as impact of HCV on hepatic steatosis and interferon resistance.

Loss of Protein Kinase PKR Expression in Human HeLa Cells Complements the Vaccinia Virus E3L Deletion Mutant Phenotype by Restoration of Viral Protein Synthesis

The E3L proteins encoded by vaccinia virus bind double-stranded RNA and mediate interferon resistance, promote virus growth, and impair virus-mediated apoptosis. Among the cellular proteins implicated as targets of E3L is the protein kinase regulated by RNA (PKR). To test in human cells the role of PKR in conferring the E3L mutant phenotype, HeLa cells stably deficient in PKR generated by an RNA interference-silencing strategy were compared to parental and control knockdown cells following infection with either an E3L deletion mutant (DeltaE3L) or wild-type (WT) virus. The growth yields of WT virus were comparable in PKR-sufficient and -deficient cells. By contrast, the single-cycle yield of DeltaE3L virus was increased by nearly 2 log(10) in PKR-deficient cells over the impaired growth in PKR-sufficient cells. Furthermore, virus-induced apoptosis characteristic of the DeltaE3L mutant in PKR-sufficient cells was effectively abolished in PKR-deficient HeLa cells. The viral protein synthesis pattern was altered in DeltaE3L-infected PKR-sufficient cells, characterized by an inhibition of late viral protein expression, whereas in PKR-deficient cells, late protein accumulation was restored. Phosphorylation of both PKR and the alpha subunit of protein synthesis initiation factor 2 (eIF-2alpha) was elevated severalfold in DeltaE3L-infected PKR-sufficient, but not PKR-deficient, cells. WT virus did not significantly increase PKR or eIF-2alpha phosphorylation in either PKR-sufficient or -deficient cells, both of which supported efficient WT viral protein production. Finally, apoptosis induced by infection of PKR-sufficient HeLa cells with DeltaE3L virus was blocked by a caspase antagonist, but mutant virus growth was not rescued, suggesting that translation inhibition rather than apoptosis activation is a principal factor limiting virus growth.

Domain 2 of Nonstructural Protein 5A (NS5A) of Hepatitis C Virus Is Natively Unfolded

Nonstructural protein 5A protein (NS5A) of hepatitis C virus (HCV) plays an important role in the regulation of viral replication, interferon resistance, and apoptosis. HCV NS5A comprises three domains. Recently the structure of domain 1 has been determined, revealing a structural scaffold with a novel zinc-binding motif and a disulfide bond. At present, the structures of domains 2 and 3 remain undefined. Domain 2 of HCV NS5A (NS5A-D2) is important for functions of NS5A and involved in molecular interactions with its own NS5B and PKR, a cellular interferon-inducible serine/threonine specific protein kinase. In this study we performed structural analysis of domain 2 by multinuclear nuclear magnetic resonance (NMR) spectroscopy. The analysis of the backbone 1H, 13C, and 15N resonances, 3JHNalpha coupling constants ,and 3D NOE data indicates that NS5A-D2 lacks secondary structural elements and reveals characteristics of unfolded proteins. NMR relaxation parameters confirmed the lack of rigid structure in the domain. The absence of an ordered conformation and the observation of a highly dynamic behavior of NS5A-D2 may provide an underlying molecular basis on its physiological function to allow NS5A-D2 to interact with a variety of biological partners.

Reduced expression of Jak-1 and Tyk-2 proteins leads to interferon resistance in Hepatitis C virus replicon

BackgroundAlpha interferon in combination with ribavirin is the standard therapy for hepatitis C virus infection. Unfortunately, a significant number of patients fail to eradicate their infection with this regimen. The mechanisms of IFN-resistance are unclear. The aim of this study was to determine the contribution of host cell factors to the mechanisms of interferon resistance using replicon cell lines.ResultsHCV replicons with high and low activation of the IFN-promoter were cultured for a prolonged period of time in the presence of interferon-alpha (IFN-alpha2b). Stable replicon cell lines with resistant phenotype were isolated and characterized by their ability to continue viral replication in the presence of IFN-alpha. Interferon resistant cell colonies developed only in replicons having lower activation of the IFN promoter and no resistant colonies arose from replicons that exhibit higher activation of the IFN promoter. Individual cell clones were isolated and nine IFN resistant cell lines were established. HCV RNA and protein levels in these cells were not altered by IFN- alpha2b. Reduced signaling and IFN-resistant phenotype was found in all Huh-7 cell lines even after eliminating HCV, suggesting that cellular factors are involved. Resistant phenotype in the replicons is not due to lack of interferon receptor expression. All the cell lines show defect in the JAK-STAT signaling and phosphorylation of STAT 1 and STAT 2 proteins were strongly inhibited due to reduced expression of Tyk2 and Jak-1 protein.ConclusionThis in vitro study provides evidence that altered expression of the Jak-Stat signaling proteins can cause IFN resistance using HCV replicon cell clones.

Glycyrrhizin injection therapy prevents hepatocellular carcinogenesis in patients with interferon‐resistant active chronic hepatitis C

There is no useful and effective treatment for patients with non-sustained response to interferon, from the viewpoint of cancer prevention. Our aim was to elucidate the influence of a glycyrrhizin therapy on hepatocarcinogenesis rate in interferon-resistant hepatitis C Methods: We retrospectively analyzed 1249 patients with chronic hepatitis with or without cirrhosis. Among 346 patients with high alanine transaminase values of twice or more of the upper limit of normal, 244 patients received i.v. glycyrrhizin injection and 102 patients did not, after judgment of interferon resistance. Crude carcinogenesis rates in the treated and untreated group were 13.3%, 26.0% at the fifth year, and 21.5% and 35.5% at the 10th year, respectively (P = 0.021). Proportional hazard analysis using time-dependent covariates disclosed that fibrotic stage, gender and glycyrrhizin treatmentwere significantly associated with future carcinogenesis. A long-term glycyrrhizin injection therapy decreased the hepatocarcinogenesis rate (hazard ratio, 0.49; 95% confidence interval, 0.27-0.86, P = 0.014) after adjusting the background features with significant covariates. Cancer preventive activity was also found in a subgroup of older patients of 60 years or more. Glycyrrhizin injection therapy significantly decreased the incidence of hepatocellular carcinoma in patients with interferon-resistant active chronic hepatitis C, whose average aminotransferase value was twice or more of the upper limit of normal after interferon.