Volume 42, Issue 2, Pages 180-187 (February 2005) Sequence-specific inhibition of duck hepatitis B virus reverse transcription by peptide nucleic acids (PNA)  Magdalena Robaczewska, Ramamurthy Narayan, Beatrice Seigneres, Olivier Schorr, Alexandre Thermet, Anna J. Podhajska, Christian Trepo, Fabien Zoulim, Peter E. Nielsen, Lucyna Cova  Journal of Hepatology  Volume 42, Issue 2, Pages 180-187 (February 2005) DOI: 10.1016/j.jhep.2004.10.010 Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 1 (A) Secondary structure of DHBV RNA encapsidation signal ε. The positions of 2 overlapping antisense PNAs, PNA 2053 and PNA 2052, are indicated in black and grey, respectively. Reverse transcription initiation site at nucleotide 2576 is indicated. (B) Schematic representation of in vitro coupled translation-RT assay for the expression of enzymatically active DHBV reverse transcriptase as detailed in Section 2. Unless otherwise stated, PNAs/S-ODNs were added at the beginning of the translation reaction (0min) (indicated by arrow) and remained in the reaction mixture for 180min. Journal of Hepatology 2005 42, 180-187DOI: (10.1016/j.jhep.2004.10.010) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 2 Inhibitory effects of PNAs and S-ODNs on viral (−) strand DNA synthesis. The DHBV polymerase was expressed in the presence of different doses of antisense PNAs and S-ODNs 2052 and 2053 and incubated with (α-32P) TTP and remaining cold dNTPs. (A) Viral nascent (−) strand DNA covalently linked to the viral polymerase was analyzed through 0.1% SDS-10% polyacrylamide gels, and the representative results of one experiment are shown. Control corresponds to the RT activity in the absence of inhibitors. The position of DHBV polymerase (90kDa) covalently linked to (−) strand DNA is indicated by an arrow. (B) The mean percentage of inhibition of (α-32P) TTP incorporation in viral (−) strand DNA, analyzed by dot assay is plotted on the graph with logarithmic scale (mean of four experiments). SD are indicated. Journal of Hepatology 2005 42, 180-187DOI: (10.1016/j.jhep.2004.10.010) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 3 Inhibition of the RT initiation step by PNAs and S-ODNs. The DHBV polymerase was expressed in the presence of different concentrations of PNA 2053 and S-ODN 2053 which were added at the beginning (0min) of the translation reaction and incubated with (α-32P)dGTP alone and the results from one experiment are represented. Control correspond to the (α-32P)dGTP incorporation by DHBV polymerase translated for 180min in the absence of inhibitors. The numbers below the gel correspond to the percentage of inhibition of (α-32P)dGTP incorporation in viral (−) strand DNA, quantified by dot assay. Journal of Hepatology 2005 42, 180-187DOI: (10.1016/j.jhep.2004.10.010) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 4 Time course of co-translational ODN addition and viral (−) strand DNA elongation inhibition. The antisense PNA 2053 (0.5μM final concentration in RT reaction) and S-ODN 2053 (5μM) were added at 30min interval to the translation reaction and remained in the reaction mix until the end of the assay followed by RT elongation reaction. (A) Incorporation of (α-32P(TTP into viral nascent (−) strand DNA. Control corresponds to the (α-32P) TTP incorporation by DHBV polymerase translated for 180min in the absence of inhibitors. The position of DHBV polymerase (90kDa) covalently linked to (−) strand DNA is indicated by an arrow. (B) The viral polymerase expression after 180min translation reaction in the presence of (35S(methionine and PNA 2053 or S-ODN 2053 added at the time of reaction indicated in panel A. (C) The percentage of inhibition of (α-32P) TTP incorporation in viral (−) strand DNA, quantified by dot assay. The results from one representative experiment are shown. Journal of Hepatology 2005 42, 180-187DOI: (10.1016/j.jhep.2004.10.010) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 5 Effect of 5-day treatment with S-ODN 2053 or PNA 2053 on viral DNA synthesis in PDH cultures. Parts A and B show effect of S-ODN 2053 and control 2 nt mismatched S-ODN 2089. Parts C and D show effect of PNA 2053 (alone or peptide-conjugated) and control 4 nt and 2 nt mismatched P1-PNA 2537 and P1-PNA 2089 respectively. (A and C) Southern blots of total intracellular DNA (5μg/lane) of one representative experiment are shown. The positions of relaxed circular (RC), linear (L) and single-stranded (SS) viral DNAs are indicated. (C) Cloned DHBV DNA linearized by EcoR1 (DHBV EcoRI) was used as a linear, 3Kb viral DNA control. DNA extracted from the inoculum shows RC viral DNA in the input virus. Lower part shows gel analysis of GAPDH RNA detection by real time RT-PCR in PDH treated with PNAs alone or PNA peptide-conjugates and in untreated controls. (B and D) PhosphorImager quantifications of all DHBV DNA replicative forms from Southern blots from a total of two and five experiments for S-ODNs and PNAs respectively are represented. The mean relative band intensity, relative to untreated control cells (set as 100%) and standard deviations are shown. Journal of Hepatology 2005 42, 180-187DOI: (10.1016/j.jhep.2004.10.010) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions