The present study showed for the first time that combined siRNAs targeting viral gene and siHsc70 is extremely effective in suppressing ongoing viral gene expression and replication in HepG2.2.15 cells. However, there are some issues that need to be addressed.

General comments:

This is a potentially interesting study tempting to demonstrate that siRNA targeting Hsc70 are valuable tools for HBV replication silencing. There are so many grammatical errors in the manuscript that an English language editing is needed.

Major Compulsory Revisions:

1. As there are drugs targeted at Hsc70 can markedly suppress HBV replication, why not combine the HBV siRNA and drugs targeted at Hsc70? Or carry out two combinations with ‘HBV siRNA + Hsc70 siRNA’, or ‘HBV siRNA + drugs’, and make a comparison.

2. For the transfection: (1) How about the transfection efficiency in your experiments, when you did fluorescence-activated cell sorting, you can get the ratio of EGFP positive cells, so what is the ratio? (2) The authors stated that they did transfection in 96-well plates with 0.6 or 0.3 μg of plasmid, which is too much to be used in such format. Please check whether it is written incorrectly or ……? And are the cells in 96-well plates enough for RNA and protein preparation for realtime PCR and Western blotting?

3. In the combination experiment, siHsc70 and S2 could independently inhibit HBsAg and HBeAg by 60-70%，while the combination of siHsc70 and S2 only inhibited 79% of HBsAg and 76.7% of HBeAg, the effects of combinational RNAi on HBV mRNA and HBVDNA are not satisfying as well. Whether there is statistic significance between combinational RNAi and the siHsc70 or S2 group? Please show the p value in the figures.

Minor Essential Revisions:

1. Have you investigate the effect of RNAi on HBV replicative intermediates by Southern blotting?

2. In the ‘M & M’ part: (1) This part should be revised and rearranged as: Selection of target sequences, Plasmid construction, Cell culture and transfections, EGFP expression assay, RNA preparation and realtime PCR, Quantitation of HBV progeny DNA in culture supernatants. (2) The description of ‘transfection’ is very long and nonsense, this part should be revised. (3) The sentence ‘Three pairs of primers……were used respectively’ is advised to be revised as: ‘Three pairs of primers used for realtime PCR are: ……’

3. In Fig.3C, S1 and S2 showed inhibitory effect on S-EGFP expression but no effect on EGFP expression, exhibiting the sequence-specific effect of siRNAs. The problems are: (1) why siRNA-EGFP exhibits no effect on EGFP expression (pEGFP-N1), (2) Have you test the siHsc70 in this system? Can siHsc70 inhibit S or EGFP expression?

4. Fig.4: Something is wrong with the title with ‘FN- α, FN- β’. The sentence ‘The protein levels of IFN-α, IFN-β and TNF-α, which come from culture medium from transfected cells, were determined by ELISA assay using an IFN-α, IFN-β and TNF-α kits’ is advised to be revised as: ‘The protein levels of IFN-α, IFN-β, and TNF-αin cell culture supernatants from transfected cells were determined by ELISA assay’.

5. Some graphs especially Excel-generated graphs were improperly stretched so that the characters in the graphs are not proportional in shape, such as: , and .

6. The units of cytokines in Fig. 4A and C, why not use ‘ng/ml’ in stead of ‘pg/ml’, so that the ‘.000’ could be omitted.

7. ‘GFP’ or ‘EGFP’? Please keep it consistent.

Discretionary Revisions:

1. In page 9, lane 1, the authors declared that the S1, S2 and siHsc70 could …… inhibit the transcription of HBV S, as far as I know that RNAi is a post-transcriptional process, which has no effect on transcription. In the second line from the bottom, what does ‘in every cells ’ mean.

2. Please note the difference between ‘cell culture medium’ and ‘cell culture supernatant’.