An NLR family member X1 mutation (p.Arg707Cys) suppresses hepatitis B virus infection in hepatocytes and favors the interaction of retinoic acid-inducible gene 1 with mitochondrial antiviral signaling protein.

Publisher: Springer-Verlag Country of Publication: Austria NLM ID: 7506870 Publication Model: Electronic Cited Medium: Internet ISSN: 1432-8798 (Electronic) Linking ISSN: 03048608 NLM ISO Abbreviation: Arch Virol Subsets: MEDLINE

Original Publication: Wien, New York, Springer-Verlag.

Hepatitis B virus*/genetics ; Hepatitis B virus*/physiology ; Hepatocytes*/virology ; Hepatocytes*/metabolism; Humans ; Hepatitis B/virology ; Hepatitis B/genetics ; Mitochondrial Proteins/genetics ; Mitochondrial Proteins/metabolism ; Adaptor Proteins, Signal Transducing/genetics ; Adaptor Proteins, Signal Transducing/metabolism ; Signal Transduction ; DEAD Box Protein 58/genetics ; DEAD Box Protein 58/metabolism ; Mutation ; Hepatitis B Surface Antigens/genetics ; Hepatitis B Surface Antigens/metabolism ; Interferon-alpha/genetics ; Interferon-alpha/metabolism ; Hep G2 Cells ; Male

NLR family member X1 (NLRX1) is an important member of the NOD-like receptor (NLR) family and plays unique roles in immune system regulation. Patients with hepatitis B virus (HBV) infection are more likely to have the NLRX1 mutation p.Arg707Cys than healthy individuals. It has been reported that NLRX1 increases the infection rate of HBV in HepG2 cells expressing sodium taurocholate cotransporting polypeptide (NTCP). However, the role of NLRX1 mutation (p.Arg707Cys) in hepatitis remains unclear. We constructed Huh7 cells that stably overexpressed NTCP, using LV003 lentivirus. First, wild-type (WT) and mutant (MT) NLRX1 overexpression plasmids were constructed. The MT plasmid contained a point mutation at position 707 of the WT overexpression plasmid. Then, Huh7-NTCP cells were transfected with the WT or MT NLRX1 overexpression plasmid, and subsequent NLRX1 expression was analyzed using real-time quantitative polymerase chain reaction (RT-qPCR) and western blot. HBV RNA levels were determined using RT-qPCR. HBsAg and HBcAg levels were confirmed immunohistochemically. Interferon alpha (IFN-α), interleukin 6 (IL-6), and type I interferon beta (IFN-β) levels were determined using enzyme-linked immunosorbent assay kits. p-p65, p-interferon regulatory factor (IRF) 3, and p-IRF7 expression levels were examined using western blot. The interaction of NLRX1 and retinoic acid-inducible gene (RIG)-1/mitochondrial antiviral signaling (MAVS) protein was confirmed by coimmunoprecipitation. The interaction of NLRX1 with IFN-α, IL-6, or IFN-β was analyzed by dual luciferase reporter gene assay. The levels of HBV RNA, HBsAg, and HBcAg in infected cells transfected with the WT NLRX1 or MT NLRX1 expression plasmid were higher than those in the untransfected control group; and these levels were lower in the cells transfected with MT NLRX1 than in those transfected with WT NLRX1. The levels of IFN-α, IFN-β, IL-6, p-p65, p-IRF3, and p-IRF7 were lower in cells transfected with WT NLRX1 or MT NLRX1 than in control cells. The levels of IFN-β, p-p65, p-IRF3, and p-IRF7 were higher in cells transfected with MT NLRX1 than in those transfected with WT NLRX1. Moreover, NLRX1 competitively inhibited RIG1 binding to MAVS, but the mutation in MT NLRX1 reduced this inhibitory effect. In addition, NLRX1 decreased the promoter activity of IFN-α, IFN-β, and IL-6. Our findings revealed that NLRX1 is a regulatory factor that inhibits the anti-HBV ability of hepatocytes and that the mutation p.Arg707Cys in NLRX1 suppresses HBV infection and activates the IFN/nuclear factor κB pathway.
(© 2024. The Author(s).)

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20231A011049 Guangzhou Health Technology Project; 82070611 and 81873572 Natural Science Foundation of China; 2020A1515010317 Natural Science Foundation of Guangdong Province; 21202104030000608 Guangdong Basic and Applied Basic Research Foundation; 202102010204 and 2023A04J1193 Guangzhou Science and Technology Plan Projects; 2020007 and 2018009 Sun Yat-Sen University Clinical Research 5010 Program; 82000-18843236 Transformation of scientific and technological achievements project of Sun Yat-sen University; K00006 Five-Year Plan of Third Affiliated Hospital of Sun Yat-sen University; 2024A03J0873 Municipal and University (Hospital) joint funding project of Guangzhou Municipal Science and Technology Bureau

Keywords: Hepatitis B virus; Infection; Mutation; NLR family member X1; Nuclear factor κB; Type I interferon

0 (NLRX1 protein, human)
0 (Mitochondrial Proteins)
0 (MAVS protein, human)
0 (Adaptor Proteins, Signal Transducing)
EC 3.6.4.13 (DEAD Box Protein 58)
0 (Hepatitis B Surface Antigens)
0 (Interferon-alpha)

Date Created: 20241105 Date Completed: 20241105 Latest Revision: 20241128

20241202

PMC11538211

10.1007/s00705-024-06133-0

39499386