Frontiers in cellular and infection microbiology · 2026
nc886 noncoding RNA regulates hepatitis B virus replication via PKR-dependent eIF2α phosphorylation.
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Piracha Zahra Zahid, Saeed Umar
Аннотация
OBJECTIVES: Hepatitis B virus (HBV) replication is tightly controlled by host stress and innate immune pathways. The small noncoding RNA nc886 (vtRNA2-1) is a known endogenous inhibitor of protein kinase R (PKR), but its role in HBV biology remains unclear. This study aimed to define the function of the nc886-PKR-eIF2α axis in HBV-replicating hepatoma cells and to determine whether nc886 depletion suppresses HBV replication via PKR-dependent translational control.
MATERIALS AND METHODS: Huh7 cells and Huh7 cells stably harboring a 1.3-mer HBV replicon were used. Endogenous nc886 and PKR expression was assessed by RT-qPCR and Western blot. Loss-of-function experiments employed two independent siRNAs against nc886 and one siRNA against PKR, alone or in combination, with scramble siRNA as control. PKR activation was induced by Poly(I:C); PKR and integrated stress response (ISR) were pharmacologically modulated using C16 (PKR inhibitor) and ISRIB (eIF2B activator), at non-toxic doses defined by MTT assay. Intracellular HBV DNA was measured by Southern blot, HBV pgRNA and subgenomic RNAs by Northern blot and RT-qPCR, and secreted HBsAg/HBeAg by ELISA. PKR-eIF2α-ATF4 signaling was evaluated by Western blot.
RESULTS: nc886 and PKR were efficiently and specifically knocked down without affecting cell viability. nc886 silencing in Huh7-HBV cells increased PKR-dependent eIF2α phosphorylation and ATF4, reduced HBV pgRNA and subgenomic RNAs, and decreased intracellular HBV DNA and secreted HBsAg/HBeAg. PKR knockdown alone slightly enhanced HBV readouts and completely rescued nc886-mediated inhibition of HBV replication and ISR activation in dual-knockdown cells. C16 or ISRIB restored HBV DNA, RNA and antigen production in nc886-silenced or Poly(I:C)-treated cells, while having no effect in control cells, indicating that rescue depended on ISR modulation.
CONCLUSION: nc886 acts as a critical negative regulator of PKR-dependent ISR signaling during HBV replication in Huh7 cells. Its depletion activates PKR and eIF2α, imposing a translational block that suppresses HBV gene expression. The nc886-PKR-eIF2α module represents a novel host regulatory axis with potential relevance for host-directed HBV therapies.
MATERIALS AND METHODS: Huh7 cells and Huh7 cells stably harboring a 1.3-mer HBV replicon were used. Endogenous nc886 and PKR expression was assessed by RT-qPCR and Western blot. Loss-of-function experiments employed two independent siRNAs against nc886 and one siRNA against PKR, alone or in combination, with scramble siRNA as control. PKR activation was induced by Poly(I:C); PKR and integrated stress response (ISR) were pharmacologically modulated using C16 (PKR inhibitor) and ISRIB (eIF2B activator), at non-toxic doses defined by MTT assay. Intracellular HBV DNA was measured by Southern blot, HBV pgRNA and subgenomic RNAs by Northern blot and RT-qPCR, and secreted HBsAg/HBeAg by ELISA. PKR-eIF2α-ATF4 signaling was evaluated by Western blot.
RESULTS: nc886 and PKR were efficiently and specifically knocked down without affecting cell viability. nc886 silencing in Huh7-HBV cells increased PKR-dependent eIF2α phosphorylation and ATF4, reduced HBV pgRNA and subgenomic RNAs, and decreased intracellular HBV DNA and secreted HBsAg/HBeAg. PKR knockdown alone slightly enhanced HBV readouts and completely rescued nc886-mediated inhibition of HBV replication and ISR activation in dual-knockdown cells. C16 or ISRIB restored HBV DNA, RNA and antigen production in nc886-silenced or Poly(I:C)-treated cells, while having no effect in control cells, indicating that rescue depended on ISR modulation.
CONCLUSION: nc886 acts as a critical negative regulator of PKR-dependent ISR signaling during HBV replication in Huh7 cells. Its depletion activates PKR and eIF2α, imposing a translational block that suppresses HBV gene expression. The nc886-PKR-eIF2α module represents a novel host regulatory axis with potential relevance for host-directed HBV therapies.