Antiviral research · 2026
NSUN2-mediated RNA m5C dysregulation links HBV infection to TFEB silencing and lysosomal impairment.
СтатьяПоддержка исследования: вне правительства СШАНазвание и аннотация: Перевод в очереди
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Dong Xuejiao, Ding Shuang, He Lili, Luo Fuyu, Cui Zhangbo, Tang Shi, Zhan Guanli, Hao Haojie, Guan Wuxiang, Shi Chunwei
Аннотация
BACKGROUND: TFEB acts as a master transcriptional regulator of lysosomal activity, which is central to host antiviral defense. Multiple viruses target TFEB to evade lysosomal degradation in host cells. This study investigated how HBV suppresses TFEB via NSUN2-mediated m5C RNA modification.
METHODS: Huh7 and HepG2 cells were transfected with pCH9-3091 encoding 1.1 × HBV genome, HBx or HBx-null plasmids. HepG2-NTCP cells were infected with HBV. HBV replication was measured using Real-Time PCR or Southern blot. TFEB protein and mRNA levels were detected using Western blot and Real-Time PCR. The proteasome inhibitor MG-132 was used to assess TFEB degradation. TFEB mRNA m5C was analyzed using GSE246879 dataset and m5C-RIP-qPCR. The binding of NSUN2 to TFEB mRNA and the m5C of TFEB mRNA were verified using Formaldehyde crosslinking-RIP and m5C-RIP-qPCR under NSUN2 overexpression, knockdown or enzymatic deficiency. TFEB mRNA stability and translation efficiency were assessed with RNA stability and ribosome-loaded assays. TFEB-mediated lysosomal degradation was evaluated by detecting CTSD and LysoTracker-RED staining. NSUN2 expression in liver tissues of mice injected with the HBx-null mutant was detected with Immunohistochemistry.
RESULTS: HBV, via HBx protein, downregulated the m5C methyltransferase NSUN2 and reduced TFEB mRNA m5C modification, which impaired TFEB mRNA stability and expression independently of proteasomal degradation. NSUN2 installed m5C marks on TFEB mRNA to enhance its stability and maintain lysosomal activity. NSUN2 rescue restored m5C and stability of TFEB mRNA, and lysosomal function.
CONCLUSIONS: The HBx-NSUN2-m5C-TFEB axis silences TFEB, revealing an epitranscriptomic mechanism for HBV immune evasion and a potential target for HBV infection treatment.
METHODS: Huh7 and HepG2 cells were transfected with pCH9-3091 encoding 1.1 × HBV genome, HBx or HBx-null plasmids. HepG2-NTCP cells were infected with HBV. HBV replication was measured using Real-Time PCR or Southern blot. TFEB protein and mRNA levels were detected using Western blot and Real-Time PCR. The proteasome inhibitor MG-132 was used to assess TFEB degradation. TFEB mRNA m5C was analyzed using GSE246879 dataset and m5C-RIP-qPCR. The binding of NSUN2 to TFEB mRNA and the m5C of TFEB mRNA were verified using Formaldehyde crosslinking-RIP and m5C-RIP-qPCR under NSUN2 overexpression, knockdown or enzymatic deficiency. TFEB mRNA stability and translation efficiency were assessed with RNA stability and ribosome-loaded assays. TFEB-mediated lysosomal degradation was evaluated by detecting CTSD and LysoTracker-RED staining. NSUN2 expression in liver tissues of mice injected with the HBx-null mutant was detected with Immunohistochemistry.
RESULTS: HBV, via HBx protein, downregulated the m5C methyltransferase NSUN2 and reduced TFEB mRNA m5C modification, which impaired TFEB mRNA stability and expression independently of proteasomal degradation. NSUN2 installed m5C marks on TFEB mRNA to enhance its stability and maintain lysosomal activity. NSUN2 rescue restored m5C and stability of TFEB mRNA, and lysosomal function.
CONCLUSIONS: The HBx-NSUN2-m5C-TFEB axis silences TFEB, revealing an epitranscriptomic mechanism for HBV immune evasion and a potential target for HBV infection treatment.