Marie-Sophie Hiet1, Oliver Bauhofer1, Margarita Zayas1, Hanna Roth1, Yasuhito Tanaka2, Peter Schirmacher3, Joschka Willemsen4, Oliver Grünvogel1, Silke Bender1, Marco Binder4, Volker Lohmann1, Vincent Lotteau5, Alessia Ruggieri6, Ralf Bartenschlager7. 1. Department for Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany. 2. Department of Virology and Liver Unit, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan. 3. Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany. 4. Department for Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany; Division of Virus-Associated Carcinogenesis, German Cancer Research Center (DKFZ), Heidelberg, Germany. 5. CIRI U1111/UMR5308, Tour Inserm-CERVI, Lyon, France. 6. Department for Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany. Electronic address: Alessia_Ruggieri@med.uni-heidelberg.de. 7. Department for Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany. Electronic address: Ralf_Bartenschlager@med.uni-heidelberg.de.
Abstract
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.
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.
Authors: Katharina Esser-Nobis; Lauren D Aarreberg; Justin A Roby; Marian R Fairgrieve; Richard Green; Michael Gale Journal: J Virol Date: 2019-06-14 Impact factor: 5.103
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