NgAgo-gDNA system efficiently suppresses hepatitis B virus replication through accelerating decay of pregenomic RNA

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1 Accepted Manuscript NgAgo-gDNA system efficiently suppresses hepatitis B virus replication through accelerating decay of pregenomic RNA Zhuanchang Wu, Siyu Tan, Leiqi Xu, Lifen Gao, Haizhen Zhu, Chunhong Ma, Xiaohong Liang PII: S (17) DOI: /j.antiviral Reference: AVR 4105 To appear in: Antiviral Research Received Date: 10 February 2017 Revised Date: 21 June 2017 Accepted Date: 10 July 2017 Please cite this article as: Wu, Z., Tan, S., Xu, L., Gao, L., Zhu, H., Ma, C., Liang, X., NgAgo-gDNA system efficiently suppresses hepatitis B virus replication through accelerating decay of pregenomic RNA, Antiviral Research (2017), doi: /j.antiviral This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

2 Short Communication NgAgo-gDNA system efficiently suppresses hepatitis B virus replication through accelerating decay of pregenomic RNA Zhuanchang Wu a,#, Siyu Tan a,#, Leiqi Xu a,c, Lifen Gao a, Haizhen Zhu d,e, Chunhong Ma a,b and Xiaohong Liang a,b,* a Key Laboratory for Experimental Teratology of Ministry of Education and Department of Immunology, School of Basic Medical Sciences, Shandong University, Jinan, China; b Key Laboratory of Infection and Immunity of Shandong Province, School of Basic Medical Sciences, Shandong University, Jinan, China; c Department of Gastroenterology, the Qilu Hospital, Shandong University, Shandong, China; d Department of Molecular Medicine of College of Biology, State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha , China; e Research Center of Cancer Prevention & Treatment, Translational Medicine Research Center of Liver Cancer, Hunan Provincial Tumor Hospital (Affiliated Tumor Hospital of Xiangya Medical School of Central South University), Changsha , China. # Zhuanchang Wu and Siyu Tan contributed equally to this work *Correspondence: Xiaohong Liang, Key Laboratory for Experimental Teratology of Ministry of Education and Department of Immunology, School of Basic Medical Sciences, Shandong University, 44 Wenhua Xi Road, Jinan, Shandong, P.R. China liangxiaohong@sdu.edu.cn.

3 Abstract Covalently closed circular DNA (cccdna) in the hepatocytes nucleus is responsible for persistent infection of Hepatitis B virus (HBV). Current antiviral therapy drugs nucleos(t)ide analogs or interferon fail to eradicate HBV cccdna. Genome editing technique provides an effective approach for HBV treatment through targeting viral cccdna. Natronobacterium gregoryi Argonaute (NgAgo)-guide DNA (gdna) system with powerful genome editing prompts us to explore its application in inhibiting HBV replication. Preliminary function verification indicated that NgAgo/EGFP-gDNA obviously inhibited EGFP expression. To further explore the potential role of NgAgo in restricting HBV replication, 10 of gdnas targeting the critical region of viral genome were designed, only S-142, P-263 and P-2166 gdnas led to significant inhibition on HBsAg, HBeAg and pregenomic RNA (pgrna) level in Huh7 and HepG2 cells tranfected with pcdna-hbv1.1 plasmid. Similar results were also found in HBV infected HLCZ01 cells and Huh7-NTCP cells. However, we failed to detect any DNA editing in S-142, P263 and P-2166 targeting region through T7E1 assay and Sanger sequencing. Remarkably, we found that NgAgo/P-2166 significantly accelerated the decay of viral pgrna. Taken together, our results firstly demonstrate the potential of NgAgo/gDNA in inhibiting HBV replication through accelerating pgrna degradation, but not DNA editing. Keywords : Hepatitis B virus; NgAgo-gDNA; Viral replication; DNA editing; pregenomic RNA degradation

4 Chronic hepatitis B virus (HBV) infection is a major cause of liver cirrhosis and hepatocellular carcinoma with more than 250 million chronically infected individuals worldwide (Trepo et al., 2014). Acting as the template for viral mrna and pre-genomic RNA synthesis, the covalently closed circular DNA (cccdna) residing in the nucleus of infected cells as a minichromosome is the reservoir for HBV persistence and the key obstacle for the cure of chronic hepatitis B (Nassal, 2015). Current antiviral therapy drugs, including nucleos(t)ide analogs (NAs) or interferon (IFN), fail to eradicate HBV cccdna (Shlomai and Rice, 2014). Genome editing techniques targeting cccdna provide an inspiring hope to eliminate HBV infection, and favorable effects has been observed harnessing CRISPR/Cas9 system and TALEN (Bloom et al., 2013; Lin et al., 2014; Ramanan et al., 2015). Recently, Natronobacterium gregoryi Argonaute (NgAgo) with guide DNA (gdna) system published in Nature Biotechnology shows powerful genome editing ability (Gao et al., 2016), which promotes us to explore its application in restricting HBV replication. To evaluate the effectiveness of our NgAgo/gDNA system, we firstly cotransfected NgAgo-encoding plasmid pnls-ngago-gk obtained from Addgene (Cambridge, MA, USA) and EGFP-gDNA into HEK293 cells. Fluorescent imaging and western blot analysis found that EGFP expression was significantly downregulated, while transfection of EGFP-gDNA alone showed no such inhibitory effect (Fig. S1A), which was similar to the result reported by Gao et al. (Gao et al., 2016). Given the protokaryotic origin of pnls-ngago-gk and lack of detecting antibody, we then constructed a mammalian codon-optimized NgAgo with Flag tag

5 and double nuclear localization signal (NLS) (p3xflag-nls-congago, congago sequence seen in Data S1). Flag-coNgAgo protein mainly localized into nucleus (Fig. S1B) and possessed similar efficiency with NgAgo in inhibiting EGFP expression (Fig. S1C), which was utilized to further experiment. In order to explore the potential role of NgAgo in restricting HBV replication, 10 of 5 -phosphorylated gdnas targeting the conservative region of S, core, polymerase, X ORFs and BCP, DR1 regulatory elements were designed and one HBV noncomplementary guide (HBV-NCG) was used as negative control gdna (Fig. 1A and gdna sequences seen in Data S1). Then, p3xflag-nls-congago, gdnas and HBV expression plasmid pcdna-hbv1.1 was cotransfected into Huh7 cells, ELISA and qpcr analysis indicated that HBV-gDNA S-142, P-263 and P-2166 all exhibited strong inhibition in HBsAg, HBeAg and pgrna level comparing with HBV-NCG control group (Fig. 1B). Similar results were also found in HepG2 cell (Fig.S2A). Western blot showed Flag-coNgAgo protein expression was approximate in each group, which excluded the interference of different transfection efficiency (Fig.1C and S2B). In HLCZ01 cells with persistent HBV infection (Yang et al., 2014), gdnas S-142, P263 and P-2166 also showed inhibitory effects on HBeAg and pgrna level (Fig. 1D). Moreover, in order to further estimate the role of NgAgo/gDNA in regulating HBV replication, we established Huh7-NTCP cell line with stable expression of HBV receptor NTCP (Fig.S2C). S-142, P263 and P-2166 also significantly restricted HBsAg secretion, pgrna level, viral DNA and HBV core protein (HBc) expression in HBV infected Huh-NTCP cells (Fig. 1E and 1F).

6 To further investigate whether the inhibition of NgAgo on HBV replication is dependent on its genome editing function, T7E1 assay was carried out to detect nucleotide insert or deletion (Indel) in S-142, P-263 and P-2166 targeting region. Unfortunately, we failed to detect any cleavage in these sites (Fig. 2A). Further Sanger sequencing also uncovered any DNA sequence mutation (Fig. 2B). Similarly, we did not detect expected cleavage band by T7E1 assay (Fig. S1D) and sequence alternative at EGFP-gDNA targeting region by Sanger sequencing (Fig. S1E), However, mrna level of GFP was obviously down-regulated after cotransfection of Flag-coNgAgo and EGFP-gDNA (Fig. S1F). Thus, based on the protocol from Gao et al., we did not found that NgAgo-gDNA system has strong DNA editing ability. This phenomenon was highly similar with recent report that NgAgo/ fabp11a-gdna without DNA editing ability still induces eye developmental defects in zebrafish through inhibiting fabp11a transcription (Qi et al., 2016). However, the exact reason for this divergence with Gao s report remains to be an open question. Due to failure in detecting DNA editing ability of NgAgo-gDNA, we further investigated whether NgAgo/HBV gdna affect the turnover rate of the pregenomic RNA. As shown in Fig. 2C, after treatment with ActD, the pgrna showed time-dependent decrease in pcdna-hbv1.1-transfected Huh7 cells either cotransfected with HBV noncomplementary control guide DNA (HBV-NCG) or HBV specific gdnas P-2166, S-751 due to mrna degradation. However, in pcdna-hbv1.1 transfected Huh7 cell, transfection of P-2166 gdna together with congago led to an obvious decline of pgrna half-life (6 h in P-2166 group versus 10

7 h in HBV-NCG group), while transfection of S-751 had no significant effect on pgrna degradation. Our results are consistent with the published data from Sunghyeok Ye et al, in which they found NgAgo induced DNA-guided gene knockdown through its DNA-dependent RNA cleavage activity in a targeted manner not DNA editing ability (Sunghyeok Ye, et al., 2017). Thus, it was speculated that NgAgo-gDNA might restrict HBV replication mainly through gdna mediated RNA interference of pgrna. In summary, these results firstly demonstrate the potential of NgAgo/gDNA in inhibiting HBV replication. The inhibitory effect is obviously associated with gdna targeting region. Despite of significant decrease of HBsAg, HBeAg and pgrna level, we failed to detect any DNA editing ability of NgAgo. Finally, we demonstrated that NgAgo/gDNA significantly shortened the half-life of HBV pregenomic RNA. Our results demonstrate that NgAgo/gDNA exhibited inhibition in HBV replication mainly through gdna-induced pgrna degradation based on the protocol from Gao et al. (Gao et al., 2016), which might provide the interesting clues for the application of NgAgo/gDNA system in controlling virus infection. REFERENCE Bloom, K., Ely, A., Mussolino, C., Cathomen, T., and Arbuthnot, P. (2013). Inactivation of hepatitis B virus replication in cultured cells and in vivo with engineered transcription activator-like effector nucleases. Molecular therapy : the journal of the American Society of Gene Therapy 21, Gao, F., Shen, X.Z., Jiang, F., Wu, Y., and Han, C. (2016). DNA-guided genome editing using the Natronobacterium gregoryi Argonaute. Nature biotechnology 34, Lin, S.R., Yang, H.C., Kuo, Y.T., Liu, C.J., Yang, T.Y., Sung, K.C., Lin, Y.Y., Wang, H.Y., Wang, C.C.,

8 Shen, Y.C., et al. (2014). The CRISPR/Cas9 System Facilitates Clearance of the Intrahepatic HBV Templates In Vivo. Molecular therapy Nucleic acids 3, e186. Nassal, M. (2015). HBV cccdna: viral persistence reservoir and key obstacle for a cure of chronic hepatitis B. Gut 64, Qi, J., Dong, Z., Shi, Y., Wang, X., Qin, Y., Wang, Y., and Liu, D. (2016). NgAgo-based fabp11a gene knockdown causes eye developmental defects in zebrafish. Cell research 26, Ramanan, V., Shlomai, A., Cox, D.B., Schwartz, R.E., Michailidis, E., Bhatta, A., Scott, D.A., Zhang, F., Rice, C.M., and Bhatia, S.N. (2015). CRISPR/Cas9 cleavage of viral DNA efficiently suppresses hepatitis B virus. Scientific reports 5, Shlomai, A., and Rice, C.M. (2014). Virology. Getting rid of a persistent troublemaker to cure hepatitis. Science 343, Trepo, C., Chan, H.L., and Lok, A. (2014). Hepatitis B virus infection. Lancet 384, Yang, D., Zuo, C., Wang, X., Meng, X., Xue, B., Liu, N., Yu, R., Qin, Y., Gao, Y., Wang, Q., et al. (2014). Complete replication of hepatitis B virus and hepatitis C virus in a newly developed hepatoma cell line. Proceedings of the National Academy of Sciences of the United States of America 111, E Sunghyeok Ye, Taegeun Bae, Kyoungmi Kim, Omer Habib, Seung Hwan Lee, Yoon Young Kim, Kang-In Lee, Seokjoong Kim, and Jin-Soo Kim1 (2017). DNA-dependent RNA cleavage by the Natronobacterium gregoryi Argonaute. BioRxiv, preprint. FIGURE LEGENDS Fig. 1. NgAgo-gDNA efficiently inhibited HBV replication. (A) Schematic representation of HBV genome targeted by gdnas. (B-E) congago-gdna efficiently inhibited HBV replication. Cells were plated at a density cells per well in 24 well plate, for pcdna-hbv1.1 model, 600 ng p3xflag-nls-congago, 600 ng different gdnas targeting HBV genome and 200 ng pcdna-hbv1.1 were cotransfected into Huh7 cells for 96 h, the HBsAg and HBeAg secretion in supernatant were measured by ELISA and pgrna level were analyzed by qrt-pcr

9 (B), Flag-coNgAgo expression was measured using western blot (C). For the persistent HBV infection model, 500 ng p3xflag-nls-congago and 500 ng different gdnas were transfected into HBV infected HLCZ01 cell for 48h, HBeAg in medium was measured by ELISA and pgrna level was analyzed using qrt-pcr (D). For Huh-NTCP model, cells were firstly infected with HBV at MOI of 500, and then transfected with 500 ng p3xflag-nls-congago and 500 ng different gdnas for 48h, HBsAg was measured by ELISA, pgrna and viral DNA was analyzed using qpcr, and HBV core protein expression was measured with western blot (E and F). The data shown are representative of three independent experiments. One-Way ANOVA; * P < 0.05, ** P < 0.01, n=3. Fig. 2. NgAgo-gDNA restricting HBV replication through accelerating pgrna degradation not DNA editing. Analysis of DNA editing ability of NgAgo-gDNA in S-142, P-263 and P-2166 gdna targeting region using T7E1 assay (A) and Sanger sequencing(b). Huh7 cells transfected with pcdna-hbv1.1, HBV infected HLCZ01 and Huh7-NTCP cells were co-transfected with p3xflag-nls-congago and S-142, P-263 or P-2166 gdna were collected to amplify S-142, P-263 and P-2166 flanking regions by PCR, purified PCR products were annealed to cut with T7E1 or cloned into pmd18-t vector to make Sanger sequencing. (C) The half-life of pgrna was determined by qrt-pcr. p3xflag-nls-congago and pcdna-hbv1.1 were cotransfected with gdna P-2166, S-751 or HBV-NCG into Huh7 cells for 48 h, followed by treatment with actinomycin D (ActD, 10 µg/ml) for indicated periods of time. pgrna levels were quantified using qrt-pcr. The data represents mean values

10 ± SEM from three independent experiments. One-Way ANOVA; * P < 0.05, n=3.

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13 Hightlights 1. NgAgo-gDNA system efficiently suppresses hepatitis B virus replication in a gdna sequence-dependent manner. 2. NgAgo-gDNA has no DNA editing ability on HBV genome. 3. NgAgo-gDNA accelerates the decay of HBV pregenomic RNA.