Glucosamine promotes hepatitis B virus replication through its dual effects in suppressing autophagic degradation and inhibiting MTORC1 signaling

Yong Lin; Chunchen Wu; Xueyu Wang; Shi Liu; Kaitao Zhao; Thekla Kemper; Haisheng Yu; Mengqi Li; Jiming Zhang; Mingzhou Chen; Ying Zhu; Xinwen Chen; Mengji Lu

doi:10.1080/15548627.2019.1632104

Glucosamine promotes hepatitis B virus replication through its dual effects in suppressing autophagic degradation and inhibiting MTORC1 signaling

Autophagy. 2020 Mar;16(3):548-561. doi: 10.1080/15548627.2019.1632104. Epub 2019 Jun 23.

Authors

Yong Lin^{1

2}, Chunchen Wu³, Xueyu Wang¹, Shi Liu⁴, Kaitao Zhao³, Thekla Kemper¹, Haisheng Yu⁴, Mengqi Li⁴, Jiming Zhang⁵, Mingzhou Chen⁴, Ying Zhu⁴, Xinwen Chen³, Mengji Lu¹

Affiliations

¹ Institute of Virology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany.
² The Key Laboratory of Molecular Biology of Infectious Diseases designated by the Chinese Ministry of Education, Institute for Viral Hepatitis, Department of Infectious Diseases, The Second Affiliated Hospital, Chongqing Medical University, Chongqing, China.
³ State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Science, Wuhan, China.
⁴ State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China.
⁵ Department of Infectious Diseases, Huashan Hospital, Fudan University, Shanghai, China.

Abstract

Glucosamine (GlcN), a dietary supplement widely utilized to promote joint health and effective in the treatment of osteoarthritis, is an effective macroautophagy/autophagy activator in vitro and in vivo. Previous studies have shown that autophagy is required for hepatitis B virus (HBV) replication and envelopment. The objective of this study was to determine whether and how GlcN affects HBV replication, using in vitro and in vivo experiments. Our data demonstrated that HBsAg production and HBV replication were significantly increased by GlcN treatment. Confocal microscopy and western blot analysis showed that the amount of autophagosomes and the levels of autophagic markers MAP1LC3/LC3-II and SQSTM1 were clearly elevated by GlcN treatment. GlcN strongly blocked autophagic degradation of HBV virions and proteins by inhibiting lysosomal acidification through its amino group. Moreover, GlcN further promoted HBV replication by inducing autophagosome formation via feedback inhibition of mechanistic target of rapamycin kinase complex 1 (MTORC1) signaling in an RRAGA (Ras related GTP binding A) GTPase-dependent manner. In vivo, GlcN application promoted HBV replication and blocked autophagic degradation in an HBV hydrodynamic injection mouse model. In addition, GlcN promoted influenza A virus, enterovirus 71, and vesicular stomatitis virus replication in vitro. In conclusion, GlcN efficiently promotes virus replication by inducing autophagic stress through its dual effects in suppressing autophagic degradation and inhibiting MTORC1 signaling. Thus, there is a potential risk of enhanced viral replication by oral GlcN intake in chronically virally infected patients.Abbreviations: ACTB: actin beta; ATG: autophagy-related; CMIA: chemiluminescence immunoassay; ConA: concanavalin A; CQ: chloroquine; CTSD: cathepsin D; DAPI: 4',6-diamidino-2-phenylindole; EV71: enterovirus 71; GalN: galactosamine; GFP: green fluorescence protein; GlcN: glucosamine; GNPNAT1: glucosamine-phosphate N-acetyltransferase 1; HBP: hexosamine biosynthesis pathway; HBV: hepatitis B virus; HBcAg: hepatitis B core antigen; HBsAg: hepatitis B surface antigen; HBeAg: hepatitis B e antigen; HBV RI: hepatitis B replicative intermediate; IAV: influenza A virus; LAMP1: lysosomal associated membrane protein 1; LAMTOR: late endosomal/lysosomal adaptor, MAPK and MTOR activator; ManN: mannosamine; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; PHH: primary human hepatocyte; RAB7: RAB7A, member RAS oncogene family; RPS6KB1: ribosomal protein S6 kinase B1; RRAGA: Ras related GTP binding A; RT-PCR: reverse transcriptase polymerase chain reaction; SEM: standard error of the mean; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; UAP1: UDP-N-acetylglucosamine pyrophosphorylase 1; VSV: vesicular stomatitis virus.

Keywords: Autophagy; HBV; MTORC1 signaling; glucosamine; lysosomal acidification.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Autophagy / drug effects*
Disease Models, Animal
Enterovirus / drug effects
Gene Expression Regulation / drug effects
Glucosamine / pharmacology*
Hep G2 Cells
Hepatitis B Surface Antigens / metabolism
Hepatitis B virus / drug effects
Hepatitis B virus / physiology*
Humans
Hydrodynamics
Influenza A virus / drug effects
Liver / drug effects
Liver / metabolism
Liver / pathology
Liver / virology
Lysosomes / drug effects
Lysosomes / metabolism
Male
Mechanistic Target of Rapamycin Complex 1 / metabolism*
Mice, Inbred C57BL
Signal Transduction* / drug effects
Vesiculovirus / drug effects
Virus Replication / drug effects*

Substances

Hepatitis B Surface Antigens
Mechanistic Target of Rapamycin Complex 1
Glucosamine

Grants and funding

This work was supported by grants from the Deutsche Forschungsgemeinschaft (RTG1949/2 and Transregio TRR60); Deutsche Forschungsgemeinschaft (DFG) [Transregio TRR60]; Deutsche Forschungsgemeinschaft (DFG) [RTG1949/2].