PMC

Expression of SAMHD1 and P-SAMHD1 was determined in whole cell lysates prepared from a panel of human hepatoma lines by Western blot. HLE, HLF, Huh-1, Huh-7, and KMCH1 cells originate from hepatocellular carcinoma, Huh6 cells originate from hepatoblastoma and Mzcha1 cells are a gall bladder cell line.

HBV infection of SAMHD1 KO clone U12. Parental and Samhd1 KO-U12 cells were infected with HBV at an MOI of 200 for 3 d. The cells were harvested and cccDNA was quantified by qPCR. Data were generated from three independent biological replicates and presented as mean ± SD.

HepG2-NTCP cells were transduced with Ad-HBV-GFP at two different MOIs with or without treatment with 1 μM ETV. Extracellular HBV was quantified by qPCR at the indicated time points. Data are representative of two independent experiments presented as mean ± SEM.

pgRNA was measured in de novo infections of parental and Samhd1 KO cells at 6 d postinfection from . Data are representative of two independent experiments, mean ± SEM. Each experiment consisted of three replicates per condition. Statistical analysis was performed using a Mann–Whitney U test.

HepG2.2.15 cells were treated with ETV (1 μM) and extracellular HBV DNA levels measured every 24 h. After 3 d of treatment, the ETV was removed by extensive washing and the recovery of secreted HBV DNA monitored by qPCR for a further 3 d. Data represent two independent experiments that comprised three technical replicates and are presented as mean ± SD.

Phosphorylated SAMHD1 plays a key role in HBV rcDNA to cccDNA conversion in the nucleus of an infected cell as well as regulating innate immune activation genes such as MxA and APOBECS. In addition, SAMHD1 dNTPase restricts the ability of viral polymerase to reverse transcribe pgRNA-rcDNA, required for the genesis of new infectious particles.

HepAD38 cells were treated with 1 μM ETV for 72 h, drug was removed by washing, and the cells were transduced with Vpx-VLPs. The cells were cultured for 72 h and extracellular HBV DNA was quantified by qPCR. Data represent two independent experiments that comprised three technical replicates and are presented as mean ± SEM. SAMHD1 expression is confirmed by Western blotting.

dNTP levels were quantified in HBV-infected HepG2-NTCP cells at 3 d postinfection. RNR-R2 RRM2 expression was assessed by Western blot. Data are representative of two independent experiments and are presented as mean ± SEM. Each experiment consisted of two replicates per condition. Statistical analysis was performed using a Mann–Whitney U test (*P value ≤ 0.05).

HepG2-Wt and SAMHD1 KO cells were infected with VSV G–pseudotyped HIV NL4.3-Luc for 24 h and luciferase activity was quantified. Data are representative of two independent experiments presented as mean ± SEM. Each experiment consisted of three replicates per condition. Statistical analysis was performed using a Mann–Whitney U test (**P value ≤ 0.01).

HepG2-NTCP Wt and Samhd1 KO clone U8 were treated with CPT (1 μM) for 4 h and immunostained for RPA2 and phospho-RPA2 S4/S8. The number of phospho-RPA2–positive cells was enumerated by fluorescence microscopy and is displayed as a percentage of the total cells. Data represent the analysis of at least 200 cells from two independent experimental repeats.

MxA, ISG20, Apobec 3A, 3B, 3C, 3D, 3F, and 3G RNA levels were measured by RT-qPCR in Wt and Samhd1 KO U8 cells that were infected with HBV for 6 h using a synchronized infection protocol (). As a control, transcript levels were measured in heparin-treated cells that blocked HBV internalization. Data represent two independent experiments, each comprising three replicates per condition and are presented as mean ± SEM. Statistical analysis was performed using a Mann–Whitney U test (*P ≤ 0.05, **P ≤ 0.01).

(A) Immunofluorescent images of Samhd1 KO clone U8 cells transduced with lentivirus expressing HA-tagged SAMHD1 Wt or K11A mutant . Images were taken using an X63 objective; scale bars indicate 50 μm. (B) HepG2-NTCP Samhd1 KO U8 cells were transduced with lentivirus expressing HA-tagged Wt or SAMHD1_K11A for 24 h. Expression was confirmed by Western blotting and dNTP levels quantified. dNTP levels were normalized to total cellular protein. Data from two independent experiments are shown as mean ± SEM. Statistical analysis was performed using a Mann–Whitney U test (*P ≤ 0.05). (C) HepG2-NTCP Samhd1 KO U8 cells were transduced with lentivirus expressing Wt or SAMHD1_K11A and 24 h later infected with HBV at an MOI of 200. cccDNA was quantified by qPCR at 3 d postinfection and SAMHD1 expression confirmed by Western blotting. Data represent two independent experiments, each comprising three replicates per condition and are presented as mean ± SEM. Statistical analysis was performed using a Mann–Whitney U test (*P ≤ 0.05).
Source data are available for this figure.

(A) HepG2-NTCP cells were arrested with 2% DMSO 3 d before treating with IFNa (1,000 IU/ml) for 24 h. Levels of total and phosphorylated SAMHD1 were assessed by Western blot. (B) HepG2-NTCP cells were transduced with Vpx-LVPs and infected with HBV at an MOI of 200 for 6 d. DNA extracted after HIRT lysis was analyzed by Southern blotting using an HBV-specific probe. Size markers of a known length were run as a control. Numbers below the blot indicate densitometric values for cccDNA ± Vpx-LVP transduction. Vpx knockdown of SAMHD1 was confirmed by Western blotting. (C) HepG2-NTCP cells were transduced with Vpx-LVPs or supplemented with 0.5 mM dN 24 h before infection with HBV at an MOI of 200 genome equivalents (GEs). cccDNA levels were quantified at 3 d postinfection by qPCR. In addition, HepG2-NTCP cells were infected as before with Vpx-LVPs delivered 3 d postinfection. The cells were cultured for a further 3 d and harvested for cccDNA quantification by qPCR. Data represent two independent experiments, each comprising three replicates per condition and are presented as mean ± SEM. Statistical analysis was performed using a Mann–Whitney U test (*P ≤ 0.05).
Source data are available for this figure.

(A) HepG2.2.15 cells were pretreated with ETV (1 μM) or vehicle control for 72 h, drug was removed by washing, and cells transduced with Vpx-LVPs. The cells were cultured for 72 h and extracellular HBV DNA quantified by qPCR. Data represent three independent experiments that comprised three technical replicates and are presented as mean ± SEM. SAMHD1 expression is confirmed by Western blotting. (B) HepG2-NTCP cells were transduced with Vpx-LVPs 24 h before infecting with HBV at an MOI of 200. Secreted HBV DNA was measured at 6 and 10 d postinfection. As before, to control for nascent particle production, the cells were treated with 1 μM of entecavir (ETV). Data represent three independent experiments that comprised three technical replicates and are presented as mean ± SEM. Statistical analysis was performed using a Mann–Whitney U test (*P value ≤ 0.05, **P value ≤ 0.01). (C) HepG2-NTCP cells were transduced with Vpx-LVP and 24 h later, infected with Ad-HBV-GFP at an MOI of 20. After 3 d, secreted HBV DNA was measured and SAMHD1 expression assessed by Western blot. Transduced cells were treated with 1 μM of ETV as a control for nascent particle production (data not shown). Data represent three independent experiments and are presented as mean ± SEM. Statistical analysis was performed using a Mann–Whitney U test (**P value ≤ 0.01). (D) Wt and Samhd1 KO cells were transduced with Ad-HBV-GFP at an MOI of 20 and extracellular HBV DNA was measured at indicated time points. Particle production per hour was estimated and values are shown below the graph. Data represent three independent experiments that comprised three technical replicates and are presented as mean ± SEM relative to the Wt control. Statistical analysis was performed using a Mann–Whitney U test (**P value ≤ 0.01). Source data are available for this figure.

(A) HepG2-NTCP cells were transfected with guide RNAs for CRISPR-Cas9 KO of Samhd1 and single colonies obtained by limiting dilution. The colonies were amplified and screened for SAMHD1 expression by Western blot. (B) Extracts were prepared from parental and Samhd1 KO clones U8 and U12 and dNTP levels measured. Data are shown relative to total cellular protein and represent two independent experiments, each comprising three replicates per condition and presented as mean ± SEM. (C) NTCP expression in Samhd1 KO HepG2-NTCP clones U8 and U12 was tested using Myrcludex B (MyrB) tagged with an Alexa 488 fluorophore. Huh-7.5 cells (NTCP negative) were used as a control. Images were taken on a 20× objective; scale bar depicts 20 μm. (D) HBV uptake in Wt and Samhd1 KO U8. HepG2-NTCP Wt and Samhd1 KO U8 cells were infected with HBV for 6 h using a synchronized infection protocol (see the Materials and Methods section) and total HBV DNA levels quantified by qPCR. Data are shown relative to Wt cells. As a control to block HBV entry, the cells were treated with heparin (50 IU/ml) for 1 h before infection. Data represent two independent experiments, each comprising three replicates per condition and are presented as mean ± SEM. Statistical analysis was performed using a Mann–Whitney U test. (E) HepG2-NTCP Wt and Samhd1 KO U8 cells were infected with HBV at an MOI of 200 for 6 h and cccDNA levels determined at 3 d postinfection by qPCR. Data are shown relative to Wt cells and represent three independent experiments, each comprising three replicates per condition and are presented as mean ± SEM. Statistical analysis was performed using a Mann–Whitney U test (***P ≤ 0.001). DNA extracted from infected Wt and SAMHD1 KO cells was analyzed by Southern blotting using an HBV-specific probe. Size markers of a known length were run as a control. Numbers below the blot indicate densitometric values for cccDNA in Wt and KO cells. (F) Parental and Samhd1 KO U8 cells were transduced with Vpx-LVP 24 h before HBV infection. The cells were infected with HBV at an MOI of 200 for 3 d after which cccDNA was quantified by qPCR. Data represent two independent experiments, each comprising three replicates per condition and presented as mean ± SEM. Statistical analysis was performed using a Mann–Whitney U test (*P ≤ 0.05). (G) Infection of Wt and Samhd1 KO U8 cells with a single-cycle HBV-Gaussia reporter virus (HBV-Gluc). The cells were infected with HBV-Gluc at an MOI of 200 and luciferase activity measured 24 h postinfection. The cells treated with the entry inhibitor MyrB (1 μM) acted as a control for background luciferase signal. Data represent two independent experiments, each comprising three replicates per condition and are presented as mean ± SEM. Statistical analysis was performed using a Mann–Whitney U test (**P ≤ 0.01).
Source data are available for this figure.