Oliver Bauhofer

Classical Swine Fever Virus Npro Interacts with Interferon Regulatory Factor 3 and Induces Its Proteasomal Degradation

ABSTRACT Viruses have evolved a multitude of strategies to subvert the innate immune system by interfering with components of the alpha/beta interferon (IFN-α/β) induction and signaling pathway. It is well established that the pestiviruses prevent IFN-α/β induction in their primary target cells, such as epitheloidal and endothelial cells, macrophages, and conventional dendritic cells, a phenotype mediated by the viral protein Npro. Central players in the IFN-α/β induction cascade are interferon regulatory factor 3 (IRF3) and IRF7. Recently, it was proposed that classical swine fever virus (CSFV), the porcine pestivirus, induced the loss of IRF3 by inhibiting the transcription of IRF3 mRNA. In the present study, we show that endogenous IRF3 and IRF3 expressed from a cytomegalovirus (CMV) promoter are depleted in the presence of CSFV by means of Npro, while CSFV does not inhibit CMV promoter-driven protein expression. We also demonstrate that CSFV does not reduce the transcriptional activity of the IRF3 promoter and does not affect the stability of IRF3 mRNA. In fact, CSFV Npro induces proteasomal degradation of IRF3, as demonstrated by proteasome inhibition studies. Furthermore, Npro coprecipitates with IRF3, suggesting that the proteasomal degradation of IRF3 is induced by a direct or indirect interaction with Npro. Finally, we show that Npro does not downregulate IRF7 expression.

Classical Swine Fever Virus Can Remain Virulent after Specific Elimination of the Interferon Regulatory Factor 3-Degrading Function of Npro

ABSTRACT Pestiviruses prevent alpha/beta interferon (IFN-α/β) production by promoting proteasomal degradation of interferon regulatory factor 3 (IRF3) by means of the viral Npro nonstructural protein. Npro is also an autoprotease, and its amino-terminal coding sequence is involved in translation initiation. We previously showed with classical swine fever virus (CSFV) that deletion of the entire Npro gene resulted in attenuation in pigs. In order to elaborate on the role of the Npro-mediated IRF3 degradation in classical swine fever pathogenesis, we searched for minimal amino acid substitutions in Npro that would specifically abrogate this function. Our mutational analyses showed that degradation of IRF3 and autoprotease activity are two independent but structurally overlapping functions of Npro. We describe two mutations in Npro that eliminate Npro-mediated IRF3 degradation without affecting the autoprotease activity. We also show that the conserved standard sequence at these particular positions is essential for Npro to interact with IRF3. Surprisingly, when these two mutations are introduced independently in the backbones of highly and moderately virulent CSFV, the resulting viruses are not attenuated, or are only partially attenuated, in 8- to 10-week-old pigs. This contrasts with the fact that these mutant viruses have lost the capacity to degrade IRF3 and to prevent IFN-α/β induction in porcine cell lines and monocyte-derived dendritic cells. Taken together, these results demonstrate that contrary to previous assumptions and to the case for other viral systems, impairment of IRF3-dependent IFN-α/β induction is not a prerequisite for CSFV virulence.

Interleukin-32: a new proinflammatory cytokine involved in hepatitis C virus-related liver inflammation and fibrosis.

Interleukin 32 (IL‐32) is a recently described proinflammatory cytokine that activates p38 mitogen‐activated protein kinase (MAPK) and nuclear factor kappa B (NF‐κB), thereby inducing proinflammatory cytokines such as IL‐1β and tumor necrosis factor alpha (TNF‐α). We investigated the role of IL‐32 in patients with chronic hepatitis C virus (HCV) infection. Steady‐state hepatic messenger RNA (mRNA) levels of IL‐32 were determined in a cohort of 90 subjects; anti‐IL‐32 staining was used in a second cohort of 132 consecutive untreated chronic HCV patients. Correlations with histological features of steatosis, inflammation, and fibrosis were made. In vitro, endogenous IL‐32 in monocytes and in the human hepatoma cell line Huh‐7.5 were examined. The effects of IL‐32‐overexpression and IL‐32‐silencing on HCV replication were studied using HCV luciferase reporter viruses. There were highly significant positive associations between hepatic IL‐32 mRNA expression and liver steatosis, inflammation, fibrosis, smooth muscle actin (SMA) area, and serum alanine aminotransferase (ALT) levels. IL‐32 protein expression was positively associated with portal inflammation, SMA area, and ALT. In vitro, IL‐1β and TNF‐α significantly induced IL‐32 expression in human Huh‐7.5 cells. Alone, stimulation with interferon alpha (IFN‐α) did not induce IL‐32 expression in Huh‐7.5. However, IFN‐α exerted a significant additive effect on TNF‐α‐induced but not IL‐1β‐induced IL‐32 expression, particularly in CD14+ monocytes. This effect was dependent both on NF‐κB and Jak/STAT signaling. Viral infection of Huh‐7.5 cells resulted in a significant (11‐fold) induction of IL‐32 mRNA expression. However, modulation of IL‐32 in Huh‐7.5 cells by overexpression or silencing did not influence HCV virus replication as determined by luciferase assays. Conclusion: IL‐32 is a novel proinflammatory cytokine involved in HCV‐associated liver inflammation/fibrosis. IL‐32 is expressed by human hepatocytes and hepatoma cells and its expression is regulated by proinflammatory stimuli. (HEPATOLOGY 2011;)

Persistence of HCV in Quiescent Hepatic Cells Under Conditions of an Interferon-Induced Antiviral Response

BACKGROUND & AIMS Hepatitis C virus (HCV) is a common cause of chronic liver disease. Many patients do not clear the viral infection; little is known about the mechanisms of HCV persistence or the frequent failure of interferon (IFN) to eliminate it. Better culture systems are needed to study viral replication in quiescent liver cells. METHODS We used human hepatoma (Huh7.5) cells and those that had undergone proliferation arrest and differentiation (Huh7.5(dif)) to study the persistence of HCV infection following exposure of the cells to IFN-α and to compare the antiviral effects of IFN-α and IFN-λ. We validated these results with primary human hepatocytes and Huh7 cells that expressed an IFN-inducible fluorophore. RESULTS Following infection of Huh7.5(dif) cells, HCV replicated persistently and released infectious particles. Long-term exposure of the cells to IFN-α reduced HCV replication ∼1000-fold but did not eliminate the virus; viral replication rebounded after withdrawal of IFN, as it does in patients with chronic HCV infection. HCV replicated at higher levels, but not exclusively, in cells that had a low level of response to IFN-α. Following incubation of cells with equipotent concentrations of IFN-α or IFN-λ, Huh7.5(dif) cells expressed a wider pattern of IFN-stimulated genes than undifferentiated Huh7.5 cells or primary human hepatocytes, indicating that the antiviral response depends on the differentiation status of the cells. CONCLUSIONS We developed a cell culture system using hepatoma cells to study persistent HCV infection during the type I or type III IFN-induced antiviral response. The level and range of the antiviral responses were associated with the differentiation status of the cells. We propose that HCV exploits the stochastic nature of the response of hepatocytes to IFN to sustain persistence.

Low Oxygen Tension Enhances Hepatitis C Virus Replication

ABSTRACT Low oxygen tension exerts a significant effect on the replication of several DNA and RNA viruses in cultured cells. In vitro propagation of hepatitis C virus (HCV) has thus far been studied under atmospheric oxygen levels despite the fact that the liver tissue microenvironment is hypoxic. In this study, we investigated the efficiency of HCV production in actively dividing or differentiating human hepatoma cells cultured under low or atmospheric oxygen tensions. By using both HCV replicons and infection-based assays, low oxygen was found to enhance HCV RNA replication whereas virus entry and RNA translation were not affected. Hypoxia signaling pathway-focused DNA microarray and real-time quantitative reverse transcription-PCR (qRT-PCR) analyses revealed an upregulation of genes related to hypoxic stress, glycolytic metabolism, cell growth, and proliferation when cells were kept under low (3% [vol/vol]) oxygen tension, likely reflecting cell adaptation to anaerobic conditions. Interestingly, hypoxia-mediated enhancement of HCV replication correlated directly with the increase in anaerobic glycolysis and creatine kinase B (CKB) activity that leads to elevated ATP production. Surprisingly, activation of hypoxia-inducible factor alpha (HIF-α) was not involved in the elevation of HCV replication. Instead, a number of oncogenes known to be associated with glycolysis were upregulated and evidence that these oncogenes contribute to hypoxia-mediated enhancement of HCV replication was obtained. Finally, in liver biopsy specimens of HCV-infected patients, the levels of hypoxia and anaerobic metabolism markers correlated with HCV RNA levels. These results provide new insights into the impact of oxygen tension on the intricate HCV-host cell interaction.

Control of temporal activation of hepatitis C virus-induced interferon response by domain 2 of nonstructural protein 5A

BACKGROUND & AIMS Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a multifunctional protein playing a crucial role in diverse steps of the viral replication cycle and perturbing multiple host cell pathways. We showed previously that removal of a region in domain 2 (D2) of NS5A (mutant NS5A(D2Δ)) is dispensable for viral replication in hepatoma cell lines. By using a mouse model and immune-competent cell systems, we studied the role of D2 in controlling the innate immune response. METHODS In vivo replication competence of NS5A(D2Δ) was studied in transgenic mice with human liver xenografts. Results were validated using primary human hepatocytes (PHHs) and mechanistic analyses were conducted in engineered Huh7 hepatoma cells with reconstituted innate signaling pathways. RESULTS Although the deletion in NS5A removed most of the interferon (IFN) sensitivity determining-region, mutant NS5A(D2Δ) was as sensitive as the wild type to IFN-α and IFN-λ in vitro, but severely attenuated in vivo. This attenuation could be recapitulated in PHHs and was linked to higher activation of the IFN response, concomitant with reduced viral replication and virus production. Importantly, immune-reconstituted Huh7-derived cell lines revealed a sequential activation of the IFN-response via RIG-I (retinoic acid-inducible gene I) and MDA5 (Myeloma differentiation associated factor 5), respectively, that was significantly higher in the case of the mutant lacking most of NS5A D2. CONCLUSIONS Our study reveals an important role of NS5A D2 for suppression of the IFN response that is activated by HCV via RIG-I and MDA5 in a sequential manner.

785 INNATE SIGNALING BY HEPATITIS C VIRUS IS RIG-I AND MDA5 DEPENDENT AND MODULATED BY NS5A DOMAIN II

785 INNATE SIGNALING BY HEPATITIS C VIRUS IS RIG-I AND MDA5 DEPENDENT AND MODULATED BY NS5A DOMAIN II M.-S. Hiet, O. Bauhofer, M. Zayas-Lopes, A. Ruggieri, F. Eberle, Y. Tanaka, P. Schirmacher, V. Lohmann, M. Binder, R. Bartenschlager. Department of Infectious Diseases, University of Heidelberg, Heidelberg, Germany; Department of Virology & Liver Unit, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan; Institute of Pathology, University Hospital, Heidelberg, Germany E-mail: marie-sophie_hiet@med.uni-heidelberg.de