PMID- 29695419 OWN - NLM STAT- MEDLINE DCOM- 20180730 LR - 20220110 IS - 1098-5514 (Electronic) IS - 0022-538X (Print) IS - 0022-538X (Linking) VI - 92 IP - 13 DP - 2018 Jul 1 TI - Hepatitis C Virus NS5A Protein Promotes the Lysosomal Degradation of Hepatocyte Nuclear Factor 1alpha via Chaperone-Mediated Autophagy. LID - 10.1128/JVI.00639-18 [doi] LID - e00639-18 AB - 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-1alpha) via interaction with HCV nonstructural protein 5A (NS5A) protein, thereby suppressing GLUT2 gene expression. The molecular mechanisms of selective degradation of HNF-1alpha 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-1alpha in HCV-infected cells and observed that the pentapeptide spanning from amino acid (aa) 130 to aa 134 of HNF-1alpha 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-1alpha was coimmunoprecipitated with HSC70, whereas the Q130A mutation (mutation of Q to A at position 130) of HNF-1alpha disrupted the interaction with HSC70, indicating that the CMA-targeting motif of HNF-1alpha is important for the association with HSC70. Immunoprecipitation analysis revealed that increasing amounts of NS5A enhanced the association of HNF-1alpha with HSC70. To determine whether LAMP-2A plays a role in the degradation of HNF-1alpha protein, we knocked down LAMP-2A mRNA by RNA interference; this knockdown by small interfering RNA (siRNA) recovered the level of HNF-1alpha protein in HCV J6/JFH1-infected cells. This result suggests that LAMP-2A is required for the degradation of HNF-1alpha. Immunofluorescence study revealed colocalization of NS5A and HNF-1alpha in the lysosome. Based on our findings, we propose that HCV NS5A interacts with HSC70 and recruits HSC70 to HNF-1alpha, thereby promoting the lysosomal degradation of HNF-1alpha 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-1alpha) induced by hepatitis C virus (HCV) NS5A protein. Using site-directed mutagenesis, we demonstrated that HNF-1alpha contains a pentapeptide chaperone-mediated autophagy (CMA)-targeting motif within the POU-specific domain of HNF-1alpha. The CMA-targeting motif is important for the association with HSC70. LAMP-2A is required for degradation of HNF-1alpha caused by NS5A. We propose that HCV NS5A interacts with HSC70, a key component of the CMA machinery, and recruits HSC70 to HNF-1alpha to target HNF-1alpha for CMA-mediated lysosomal degradation, thereby facilitating HCV pathogenesis. We discovered a role of HCV NS5A in CMA-dependent degradation of HNF-1alpha. Our results may lead to a better understanding of the role of CMA in the pathogenesis of HCV. CI - Copyright (c) 2018 American Society for Microbiology. FAU - Matsui, Chieko AU - Matsui C AD - Division of Infectious Disease Control, Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe, Japan. FAU - Deng, Lin AU - Deng L AD - Division of Infectious Disease Control, Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe, Japan. FAU - Minami, Nanae AU - Minami N AD - Division of Infectious Disease Control, Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe, Japan. FAU - Abe, Takayuki AU - Abe T AD - Division of Infectious Disease Control, Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe, Japan. FAU - Koike, Kazuhiko AU - Koike K AD - Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan. FAU - Shoji, Ikuo AU - Shoji I AUID- ORCID: 0000-0002-0730-4379 AD - Division of Infectious Disease Control, Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe, Japan ishoji@med.kobe-u.ac.jp. LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't DEP - 20180613 PL - United States TA - J Virol JT - Journal of virology JID - 0113724 RN - 0 (HNF1A protein, human) RN - 0 (HSC70 Heat-Shock Proteins) RN - 0 (Hepatocyte Nuclear Factor 1-alpha) RN - 0 (Viral Nonstructural Proteins) RN - EC 2.7.7.48 (NS-5 protein, hepatitis C virus) SB - IM MH - *Autophagy MH - Carcinoma, Hepatocellular/metabolism/pathology/virology MH - HSC70 Heat-Shock Proteins/genetics/*metabolism MH - Hepacivirus/*pathogenicity MH - Hepatitis C/metabolism/*pathology/virology MH - Hepatocyte Nuclear Factor 1-alpha/genetics/*metabolism MH - Humans MH - Intracellular Membranes/metabolism MH - Liver Neoplasms/metabolism/pathology/virology MH - Lysosomes/*metabolism MH - Protein Binding MH - Proteolysis MH - Tumor Cells, Cultured MH - Viral Nonstructural Proteins/genetics/*metabolism PMC - PMC6002715 OTO - NOTNLM OT - HNF-1alpha OT - HSC70 OT - NS5A OT - chaperone-mediated autophagy OT - hepatitis C virus EDAT- 2018/04/27 06:00 MHDA- 2018/07/31 06:00 PMCR- 2018/12/13 CRDT- 2018/04/27 06:00 PHST- 2018/04/14 00:00 [received] PHST- 2018/04/17 00:00 [accepted] PHST- 2018/04/27 06:00 [pubmed] PHST- 2018/07/31 06:00 [medline] PHST- 2018/04/27 06:00 [entrez] PHST- 2018/12/13 00:00 [pmc-release] AID - JVI.00639-18 [pii] AID - 00639-18 [pii] AID - 10.1128/JVI.00639-18 [doi] PST - epublish SO - J Virol. 2018 Jun 13;92(13):e00639-18. doi: 10.1128/JVI.00639-18. Print 2018 Jul 1.