Targeted ribonuclease can inhibit replication of hepatitis B virus

Abstract

Aim: To study the effect of a novel targeted ribonuclease (TN), the fusion protein of HBVc and human eosinophil-derived neurotoxin (hEDN), on the HBV replication in vitro.

Methods: The gene encoding the targeted ribonuclease was cloned into pcDNA3.1 (-) to form recombinant eukaryotic expression vector p/TN. Control plasmids, including p/hEDN, p/HBVc, and p/TNmut in which a Lys113-Arg mutation was introduced by sequential PCR to eliminate the ribonuclease activity of hEDN, were also constructed. Liposome-mediated transfection of 2.2.15 cells by p/TN, p/TNmut, p/hEDN, p/HBVc, and pcDNA3.1 (-), or mock transfection was performed. After that, RT-PCR was used to verify the transgene expression. Morphology of the transfected cells was observed and MTT assay was performed to detect the cytotoxicity of transgene expression. Concentration of HBsAg in the supernatant of the transfected cells was measured using solid-phase radioimmunoassay.

Results: Transgenes were successfully expressed in 2.2.15 cells. No obvious cytotoxic effect of transgene expression on 2.2.15 cells was found. The HBsAg concentration in the p/TN transfected cells was reduced by 58 % compared with that of mock transfected cells. No such an effect was found in all other controls.

Conclusion: The targeted ribonuclease can inhibit HBV replication in vitro while it has no cytotoxicity on host cells. The targeted ribonuclease may be used as a novel antiviral agent for human HBV infection.

Figure 1

Construction of p/TN. cDNA coding for hEDN or HBVc was amplified by RT-PCR, ligated together, and then cloned into pcDNA3.1 (-) to form p/TN.

Figure 2

Generation of hEDNmut gene by sequential PCR. For PCR 1 and 2, pUC18/hEDN was used as template. Primers pair M1 and N2 were used in PCR 1 and N1 and M2 in PCR 2. For PCR 3, the mixture of PCR 1 and 2 product was used as template and M1 and M2 as primers. The black dots in the figure denote the nucleotides introducing the Lys113→Arg mutation which eliminates the ribonuclease activity.

Figure 3

Confirmation of transgenes expression in transfected 2.2.15 cell line. Forty-eight h after the transfection, total RNA was isolated from transfected 2.2.15 cells and RT-PCR was performed to confirm the expression of transgenes. The RT-PCR products were then electrophoresed in 1.2% agarose gel. Lanes 1-5 represent the RT-PCR results for total RNA isolated from 2.2.15 cells transfected by p/TN, p/TNmut, p/HBVc, p/hEDN, and pcDNA3.1 (-), respectively. Lanes 6-10 represent controls corresponding to lanes 1-5, respectively, in which reverse transcriptase was omitted in RT-PCR. M: DNA Marker (2000, 1000, 750, 500, 250, 100bp from top to bottom).

Figure 4

Morphology of 2.2.15 cells 48 h after transfection. A-F represent 2.2.15 cells transfected by p/TN, p/hEDN, p/HBVc, p/TNmut, pcDNA3.1 (-), or mock transfection, respectively. There were no discernible morphological differences of 2.2.15 cells transfected with p/TN, as compared with the controls.

Figure 5

The HBsAg concentration in supernatants of transfected 2.2.15 cells. Groups A-F represent 2.2.15 cells transfected by p/TN, p/hEDN, p/HBVc, p/TNmut, pcDNA3.1 (-), or mock transfection, respectively. The concentration of HBsAg in the supernatant of 2.2.15 cells transfected with p/TN was decreased by 58% as compared with that of mock transfected 2.2.15 cells.