Development of a Novel Human Hepatoma Cell Line Supporting the Replication of a Recombinant HBV Genome with a Reporter Gene

Response to Reviewer 1 Comments

1. Summary

We sincerely appreciate the time and effort you have dedicated to reviewing our manuscript. Detailed responses to your comments are provided below. All corresponding revisions and corrections have been highlighted or shown in track changes in the re-submitted files.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Yes

Is the research design appropriate?

Yes

Are the methods adequately described?

Can be improved

Are the results clearly presented?

Can be improved

Are the conclusions supported by the results?

Yes

Are all figures and tables clear and well-presented?

Can be improved

3. Point-by-point response to Comments and Suggestions for Authors

Comment 1: The asterisk is missing after the corresponding author´s name.

Response 1: Thank you for pointing this out. We have added an asterisk after the corresponding author’s name.

Comment 2: Figure 3b. The size of the letters are too small. In general for all the figures, the size of the letters is too small.

Response 2: We agree that the font size in the figures was too small. Therefore, we have increased the font size of all labels in Figure 3b as well as in all other figures in the revised manuscript to improve readability.

Comment 3: Figure 3b. The authors mention that except for adefovir, a significant inhibition was observed for the three other drug used. However, for all the drugs, a 50% signal was observed even at the highest concentration. Is this a basal signal? In such case, it would be useful to mention it, and to evaluate the real % inhibition for each drug concentration, including ADV. The mean signal is a mean of how many replicas?

Response 3: We appreciate the reviewer’s insightful comment regarding the residual HiBiT signal observed even at the highest concentrations of nucleos(t)ide analogs. In the three drugs other than ADV, we observed a significant inhibition of HiBiT activity; however, as correctly pointed out by the reviewer, approximately 50% of the signal still remained even at the highest concentrations, which we consider to represent a basal HiBiT signal. This remaining basal signal is likely due to HiBiT expression that cannot be eliminated by nucleos(t)ide analog treatment, most probably attributable to the integration of HBV DNA into the genome of HepG2-derived cells and the consequent expression of integrated viral sequences, as originally described by Edman et al., “Integration of hepatitis B virus DNA in a human hepatoma cell,” Nature, 1980, and Brechot et al., “Presence of integrated hepatitis B virus DNA sequences in cellular DNA of human hepatocellular           carcinoma” Nature, 1980. Therefore, the apparent percentage inhibition at each drug concentration should be interpreted relative to this basal level. We described that the possibility that the basal HiBiT signal could be due to the integration of HBV DNA by referring to these two papers. Indeed, the basal signal by the integration of HBV DNA should be subtracted to assess the inhibitory effect of nucleos(t)ide analogs, however, it is difficult to define correct basal HiBiT signal due to HBV DNA integration. Therefore, we kept the possible basal HiBiT signal in the revised manuscript. The results represent the mean values obtained from experiments performed using three wells per condition. Taking these points into consideration, we have revised the Results section and the Figure 3B legend accordingly.

Comment 4: No explanation is given to the weak inhibitory activity of ADV. Could it be the time (25 days) when the cells were assayed?

Response 4: In our assay, ADV indeed showed only a modest reduction of HiBiT activity compared with the other three nucleos(t)ide analogs, even after 25 days of treatment. We do not believe that the assay duration (25 days) is the main reason for this weaker effect, but rather that it reflects the intrinsically lower in vitro antiviral potency of ADV against HBV. Although there is no single study directly comparing the in vitro activities of all four nucleos(t)ide analogs under identical experimental conditions, several reports have evaluated each drug separately and consistently indicate that ADV has a much higher EC₅₀ than the others. For example, Langley et al. (“Inhibition of hepatitis B virus polymerase by entecavir,” Journal of Virology, 2007) reported an EC₅₀ of approximately 5 nM for entecavir, Delaney et al. (“Intracellular metabolism and in vitro activity of tenofovir against hepatitis B virus” Antimicrobial Agents and Chemotherapy, 2006) reported EC₅₀ of 20 nM for tenofovir alafenamide (TAF), and Schinazi et al. (“Evaluation of Single and Combination Therapies with Tenofovir Disoproxil Fumarate and Emtricitabine In Vitro and in a Robust Mouse Model Supporting High Levels of Hepatitis B Virus Replication”, Antimicrobial Agents and Chemotherapy, 2012) reported an EC₅₀ of about 10 nM for tenofovir (TDF). In contrast, Qi et al. (“In vitro susceptibility of lamivudine-resistant hepatitis B virus to adefovir and tenofovir ” Antiviral Therapy, 2007) reported an EC₅₀ of 1,170 nM for adefovir, indicating substantially lower potency. These published data are in line with our observation that ADV shows only weak inhibition of HiBiT activity in HepG2‑B4 cells, and we have added a brief explanation of this point to the Results section and the Figure 3b legend.

Comment 5: Figure 4. Again, a (possible) basal signal of HiBiT is observed but not mentioned. Why this basal extracellular activity?

Response 5: Similar to Figure 3b, a residual extracellular HiBiT activity was observed even under conditions where nucleos(t)ide analog treatment markedly reduced the signal, and we agree that this represents a basal HiBiT signal. We consider this basal extracellular activity to reflect HiBiT expression that cannot be eliminated by nucleos(t)ide analogs, most likely due to HBV DNA integrated into the genome of HepG2-derived cells and the consequent expression of integrated viral sequences, as reported previously in human hepatoma cell lines. We have already discussed these points at the answer to comment 3 above. We have now added an explanation of this basal HiBiT signal to the Results section and clarified that the degree of inhibition should be interpreted relative to this basal level.

Comment 6: Figure 5. The cccDNA band is lower than the one of the control.

Response 6: Thank you for this insightful comment. In Figure 5, the cccDNA band in the HepG2-B4 samples was detected at a slightly lower position than the corresponding band in the control lane. While the precise reason for this shift is not fully clear, we occasionally observe fluctuations in band migration in samples extracted by the Hirt method, particularly when a relatively high amount of DNA is loaded onto the gel. Crucially, we have confirmed that this signal remains after heat denaturation—a condition under which relaxed circular (rcDNA) and linearized DNA are lost—supporting our identification of this band as cccDNA. To avoid potential confusion, we have removed the molecular weight marker lane from the revised Figure 5. Furthermore, as shown in the attached figure from our previous study using the same protocol (Sumiyadorj A, et al., Hepatol Commun., 2022;6(9):2441-2454, "A single hepatitis B virus genome with a reporter allows the entire viral life cycle to be monitored in primary human hepatocytes"), the cccDNA bands were in good agreement with the markers and controls. Therefore, we consider the shift observed in this instance to be an experimental variation rather than a structural change in the cccDNA itself.

Comment 7: Lines 437-440. Can the authors ascertain that full HBV replication is obtained with ETV, to mention this point? This related to the basal signal observed.

Response 7: The sentences in lines 437–440 were not intended to imply that full HBV replication is maintained in the presence of ETV. In our system, ETV treatment clearly reduces extracellular HBV DNA, HBsAg, HBcAg, and HiBiT activity, however, we cannot ascertain that complete inhibition of HBV replication is obtained under ETV treatment. The basal signals observed (including residual HiBiT activity) are more likely to reflect a combination of partial residual replication and expression from HBV sequences integrated into the genome of HepG2‑derived cells, rather than complete inhibition of HBV replication. To avoid misunderstanding, we have revised the text in lines 437–440 to state more cautiously that gene expression alterations persist despite antiviral treatment and may contribute to an unresolved risk of hepatocellular carcinoma, without suggesting that complete inhibition of HBV replication is preserved with ETV.

Comment 8: In Discussion, it would be advisable that the authors mention as perspective the possibility of cloning and establishing other hepatoma cell line supporting other HBV genotype replication.

Response 8: We appreciate the reviewer’s valuable suggestion. In response, we have expanded the Discussion to mention, as a future perspective, the possibility of cloning and establishing additional hepatoma cell lines that support the replication of other HBV genotypes. We agree that generating such genotype‑diverse cell lines would further increase the utility of our HiBiT‑based system and provide a broader platform for studying genotype‑specific viral biology and for evaluating antiviral agents across different HBV genotypes.

Response to Reviewer 2 Comments

1. Summary

We sincerely appreciate the time and effort you have dedicated to reviewing our manuscript. Detailed responses to your comments are provided below. All corresponding revisions and corrections have been highlighted or shown in track changes in the re-submitted files.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Yes

Is the research design appropriate?

Yes

Are the methods adequately described?

Yes

Are the results clearly presented?

Yes

Are the conclusions supported by the results?

Yes

Are all figures and tables clear and well-presented?

Can be improved

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: All figures with quantified data should have appropriate statistics to enhance the impactfulness of the conclusions.

Response 1: We appreciate the reviewer’s constructive suggestion regarding statistical analysis. In this study, our main objective was to establish and characterize the HiBiT-based HBV replication system, and we therefore focused on presenting reproducible trends based on multiple independent experiments. Quantified data are shown as mean ± SD to illustrate the variability across experiments, and these consistent patterns support the conclusions drawn from our work.

Comments 2: Fig 1A – it would be useful to show a schematic of the HBV genome with the ORFs indicated and where this fits with the HiBit Tag insertions. This would enhance the accessibility of the image for a more general readership.

Response 2: We have revised Figure 1 to include a schematic representation of the HBV genome to Figure 1b, indicating the major ORFs and showing how the HiBiT tag insertions are positioned relative to these regions. We agree that this modification improves the accessibility and interpretability of the figure for a more general readership.

Comments 3: Fig 1B – What is mock? HiBit inserted into a plasmid? If yes, which plasmid?

Response 3: In this experiment, mock refers to HepG2 cells treated with the transfection reagent alone without any plasmid DNA, and it is included as a negative control for the HiBiT luminescence assay. We have added this explanation to the figure legend to clarify the meaning of the mock condition.​　

Comments 4: Lines 263/264/285 requires proper annotation (e.g. )

Response 4: The annotations at lines 263, 264, and 285 have been corrected as requested.

Comments 5: Fig 3B – Top graph should be labelled to indicate that it is measuring HiBit activity while the bottom graph is showing cell viability for better clarity.

Response 5: The labels in Figure 3B have been revised so that the upper graph is now clearly annotated as HiBiT activity and the lower graph as cell viability, thereby improving the clarity of the figure.

Comments 6: Fig 6 – Images are blurry, but interpretable. This figure would benefit from higher quality images, measurement of signal intensity and statistics of the intensity.

Response 6: We appreciate the reviewer’s comment regarding Figure 6. We have replaced part of the images with higher-resolution versions to improve clarity. However, it is technically difficult to perform a quantitative comparison of signal intensity between HepG2.2.15 and HepG2-B4 cells in this immunostaining, because the staining conditions slightly differed between experiments and signal intensity varied among individual cells. Furthermore, we did not intend to compare signal intensity of HBsAg and HBcAg between HepG2.2.15 and HepG2-B4 cells in this immunostaining, rather, we would like to show the protein expression of both viral proteins in HepG2-B4 cells and HepG2.2.15 cells were used for the positive control. Accordingly, we have revised the text to remove the previous description stating that the signal intensity in HepG2-B4 cells exceeded that in HepG2.2.15 cells.

Comments 7: Fig 7 – Is there an explanation for the noticeable HiBit activity in the MyrB sample in the PXB cells at 6dpi?

Response 7: We hypothesize that the residual HiBiT signal observed at 6 dpi is likely due to a small amount of inoculated viral particles remaining in the wells after infection. However, during subsequent medium replacement and sampling, the HiBiT activity in the MyrB-treated wells decreased, while the activity in the DMSO-treated wells continued to increase. Therefore, we consider that the HiBiT-HBV replication and spread occurred only in the DMSO-treated cells, supporting the hypothesis that the inhibitor MyrB effectively blocked infection.

Comments 8: Line 320 – need a proper citation for Hirt extraction

Response 8: We thank the reviewer for this comment. A proper citation for the Hirt extraction method has already been included in the Methods section (Section 2.6, “Southern Blot Analysis”), where the Hirt procedure is described in detail.

Comments 9: Line 440 – this statement needs a proper reference.

Response 9: We thank the reviewer for this valuable comment. The statement that “the range of insertion sites compatible with robust virus replication and stable cell line establishment remains very restricted due to the compactness of the HBV genome and the presence of essential regulatory elements” is supported by several previous studies. These reports have highlighted that the compact and highly overlapped organization of the HBV genome, together with multiple embedded regulatory elements, severely limits the number of sites that tolerate foreign insertions without impairing viral replication or genome packaging (e.g., Bai et al., “Engineering Hepadnaviruses as Reporter-Expressing Vectors: Recent Progress and Future Perspectives,” Viruses, 2016; Li et al., “Replication-Competent Infectious Hepatitis B Virus Vectors Carrying Substantially Sized Transgenes by Redesigned Viral Polymerase Translation,” PLOS ONE, 2013; Nishitsuji et al., “Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle,” Scientific Reports, 2017). We have added these references in the revised manuscript to support this sentence.