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      Hepatitis C Virus NS5A Protein Promotes the Lysosomal Degradation of Hepatocyte Nuclear Factor 1α via Chaperone-Mediated Autophagy

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          ABSTRACT

          Hepatitis C virus (HCV) infection is closely associated with type 2 diabetes. We reported that HCV infection induces the lysosomal degradation of hepatocyte nuclear factor 1 alpha (HNF-1α) via interaction with HCV nonstructural protein 5A (NS5A) protein, thereby suppressing GLUT2 gene expression. The molecular mechanisms of selective degradation of HNF-1α caused by NS5A are largely unknown. Chaperone-mediated autophagy (CMA) is a selective lysosomal degradation pathway. Here, we investigated whether CMA is involved in the selective degradation of HNF-1α in HCV-infected cells and observed that the pentapeptide spanning from amino acid (aa) 130 to aa 134 of HNF-1α matches the rule for the CMA-targeting motif, also known as KFERQ motif. A cytosolic chaperone protein, heat shock cognate protein of 70 kDa (HSC70), and a lysosomal membrane protein, lysosome-associated membrane protein type 2A (LAMP-2A), are key components of CMA. Immunoprecipitation analysis revealed that HNF-1α was coimmunoprecipitated with HSC70, whereas the Q130A mutation (mutation of Q to A at position 130) of HNF-1α disrupted the interaction with HSC70, indicating that the CMA-targeting motif of HNF-1α is important for the association with HSC70. Immunoprecipitation analysis revealed that increasing amounts of NS5A enhanced the association of HNF-1α with HSC70. To determine whether LAMP-2A plays a role in the degradation of HNF-1α protein, we knocked down LAMP-2A mRNA by RNA interference; this knockdown by small interfering RNA (siRNA) recovered the level of HNF-1α protein in HCV J6/JFH1-infected cells. This result suggests that LAMP-2A is required for the degradation of HNF-1α. Immunofluorescence study revealed colocalization of NS5A and HNF-1α in the lysosome. Based on our findings, we propose that HCV NS5A interacts with HSC70 and recruits HSC70 to HNF-1α, thereby promoting the lysosomal degradation of HNF-1α via CMA.

          IMPORTANCE Many viruses use a protein degradation system, such as the ubiquitin-proteasome pathway or the autophagy pathway, for facilitating viral propagation and viral pathogenesis. We investigated the mechanistic details of the selective lysosomal degradation of hepatocyte nuclear factor 1 alpha (HNF-1α) induced by hepatitis C virus (HCV) NS5A protein. Using site-directed mutagenesis, we demonstrated that HNF-1α contains a pentapeptide chaperone-mediated autophagy (CMA)-targeting motif within the POU-specific domain of HNF-1α. The CMA-targeting motif is important for the association with HSC70. LAMP-2A is required for degradation of HNF-1α caused by NS5A. We propose that HCV NS5A interacts with HSC70, a key component of the CMA machinery, and recruits HSC70 to HNF-1α to target HNF-1α for CMA-mediated lysosomal degradation, thereby facilitating HCV pathogenesis. We discovered a role of HCV NS5A in CMA-dependent degradation of HNF-1α. Our results may lead to a better understanding of the role of CMA in the pathogenesis of HCV.

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          Most cited references34

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          Chaperone-mediated autophagy: a unique way to enter the lysosome world.

          All cellular proteins undergo continuous synthesis and degradation. This permanent renewal is necessary to maintain a functional proteome and to allow rapid changes in levels of specific proteins with regulatory purposes. Although for a long time lysosomes were considered unable to contribute to the selective degradation of individual proteins, the discovery of chaperone-mediated autophagy (CMA) changed this notion. Here, we review the characteristics that set CMA apart from other types of lysosomal degradation and the subset of molecules that confer cells the capability to identify individual cytosolic proteins and direct them across the lysosomal membrane for degradation. Copyright © 2012 Elsevier Ltd. All rights reserved.
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            Efficient initiation of HCV RNA replication in cell culture.

            Hepatitis C virus (HCV) infection is a global health problem affecting an estimated 170 million individuals worldwide. We report the identification of multiple independent adaptive mutations that cluster in the HCV nonstructural protein NS5A and confer increased replicative ability in vitro. Among these adaptive mutations were a single amino acid substitution that allowed HCV RNA replication in 10% of transfected hepatoma cells and a deletion of 47 amino acids encompassing the interferon (IFN) sensitivity determining region (ISDR). Independent of the ISDR, IFN-alpha rapidly inhibited HCV RNA replication in vitro. This work establishes a robust, cell-based system for genetic and functional analyses of HCV replication.
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              Acetylation targets the M2 isoform of pyruvate kinase for degradation through chaperone-mediated autophagy and promotes tumor growth.

              Most tumor cells take up more glucose than normal cells but metabolize glucose via glycolysis even in the presence of normal levels of oxygen, a phenomenon known as the Warburg effect. Tumor cells commonly express the embryonic M2 isoform of pyruvate kinase (PKM2) that may contribute to the metabolism shift from oxidative phosphorylation to aerobic glycolysis and tumorigenesis. Here we show that PKM2 is acetylated on lysine 305 and that this acetylation is stimulated by high glucose concentration. PKM2 K305 acetylation decreases PKM2 enzyme activity and promotes its lysosomal-dependent degradation via chaperone-mediated autophagy (CMA). Acetylation increases PKM2 interaction with HSC70, a chaperone for CMA, and association with lysosomes. Ectopic expression of an acetylation mimetic K305Q mutant accumulates glycolytic intermediates and promotes cell proliferation and tumor growth. These results reveal an acetylation regulation of pyruvate kinase and the link between lysine acetylation and CMA. Copyright © 2011 Elsevier Inc. All rights reserved.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                J Virol
                J. Virol
                jvi
                jvi
                JVI
                Journal of Virology
                American Society for Microbiology (1752 N St., N.W., Washington, DC )
                0022-538X
                1098-5514
                25 April 2018
                13 June 2018
                1 July 2018
                : 92
                : 13
                Affiliations
                [a ]Division of Infectious Disease Control, Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe, Japan
                [b ]Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
                University of Texas Southwestern Medical Center
                Author notes
                Address correspondence to Ikuo Shoji, ishoji@ 123456med.kobe-u.ac.jp .

                Citation Matsui C, Deng L, Minami N, Abe T, Koike K, Shoji I. 2018. Hepatitis C virus NS5A protein promotes the lysosomal degradation of hepatocyte nuclear factor 1α via chaperone-mediated autophagy. J Virol 92:e00639-18. https://doi.org/10.1128/JVI.00639-18.

                Article
                PMC6002715 PMC6002715 6002715 00639-18
                10.1128/JVI.00639-18
                6002715
                29695419
                c52b29cb-18a4-4560-903d-16735ad466a2
                Copyright © 2018 American Society for Microbiology.

                All Rights Reserved.

                Page count
                Figures: 8, Tables: 0, Equations: 0, References: 48, Pages: 14, Words: 7795
                Funding
                Funded by: Ministry of Health, Labour and Welfare (MHLW), https://doi.org/10.13039/501100003478;
                Award Recipient :
                Funded by: Japan Agency for Medical Research and Development (AMED), https://doi.org/10.13039/100009619;
                Award ID: JP17fk0210304
                Award ID: JP18fk0210040
                Award Recipient :
                Funded by: Ministry of Education, Culture, Sports, Science and Technology (MEXT), https://doi.org/10.13039/501100001700;
                Award Recipient :
                Categories
                Virus-Cell Interactions
                Custom metadata
                July 2018

                NS5A,HSC70,HNF-1α,hepatitis C virus,chaperone-mediated autophagy

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