Yonsei Med J.  2006 Oct;47(5):721-728. 10.3349/ymj.2006.47.5.721.

Intracellular Antibody Fragment Against Hepatitis B Virus X Protein Does Not Inhibit Viral Replication

• Jin YH
• Hong SH
• Kim K
• Shin HJ
• Park S

• KMID: 1381254
• DOI: http://doi.org/10.3349/ymj.2006.47.5.721

Abstract

Replication of the hepatitis B virus is suppressed by deficiency of the X protein. Although several molecules that block cellular targets of X protein reduce the production of hepatitis B virus progeny, the effect of a specific inhibitor of X protein on viral replication has not been investigated. To block X protein specifically, we adopted an intracellular expression approach using H7 single chain variable fragment (H7scFv), an antibody fragment against X protein. We previously demonstrated that cytoplasmic expression of H7scFv inhibits X protein-induced tumorigenicity and transactivation. In this study, intracellular H7scFv expression inhibits reporter gene transactivation but not viral replication determined by endogenous hepatitis B virus polymerase activity assay and real-time PCR. Our findings imply that intracellular expression of antibody fragment against X protein may not be an alternative therapeutic modality for inhibition of hepatitis B virus replication.

Keyword

Antibodies; viral; hepatitis B virus; trans-activators; virus replication

MeSH Terms

Virus Replication/*drug effects
Trans-Activators/*antagonists & inhibitors/immunology
Immunoglobulin Variable Region/genetics/metabolism/*pharmacology
Hepatitis B virus/*drug effects/physiology
Hepatitis B e Antigens/metabolism
Cell Line

Figure

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  Fig. 1 Intracellular expression of H7scFv. (A) HepG2-WT10 cells were transiently transfected with H7scFv-expression plasmid or control plasmid (empty vector or S2E1scFv-expression plasmid). After 48 hours, the lysate of transfected cells was analyzed by Western blotting with anti-Myc-Ab (upper) and anti-tubulin Ab (lower). (B) HepG2-WT10 cells were transfected with H7scFv expression plasmid or control plasmid. One day after transfection, scFv protein in the cytoplasm was visualized by immunofluorescence microscopy using anti-Myc Ab and FITC-conjugated rabbit affinity purified Ab to mouse IgG.

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  Fig. 2 HBeAg production was not affected by intracellular expression of H7scFv. Culture medium was harvested and analyzed by ELISA for HBeAg 48 hours after transfection of HepG2-WT10 cells with pIRES-H7scFv or control plasmid (pIRES-EGFP or pIRES-S2E1).

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  Fig. 3 Expression of intracellular H7scFv did not influence endogenous HBV polymerase activity. Cytoplasmic core particles were isolated from HepG2-WT10 cells transiently expressing H7scv or S2E1scFv, incubated with buffer containing [α-32P]dATP and visualized by gel electrophoresis and autoradiography. Relative band intensity was plotted.

• 

  Fig. 4 Intracellular expression of H7scFv had no effect on HBV replication. HepG2-WT10 cells were transfected with pIRES-H7scFv or control plasmid. Forty-eight hours later, intracellular and extracellular core particles were isolated and viral DNA was amplified by real-time PCR. At the same time, serially diluted HBV plasmid samples were amplified for the construction of a standard curve (correlation coefficient r = 0.98, slope = -3.3). HBV DNA copy numbers were calculated using the standard curve and cycle threshold. The bar indicates the mean value of three different experiments. Similar results were obtained using the pCMV vector series.

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  Fig. 5 Intracellular H7scFv expression suppressed transactivation in HepG2-WT10 cells. (A) To demonstrate transactivation of a gene under the SV40 enhancer/promoter in HepG2-WT10 cells, luciferase activity was measured in cells transfected with the indicated amount of pGL3-Control vector. Increased luciferase activity was observed in HepG2-WT10 cells compared with HepG2 cells. The experiments were repeated three times and similar trends were observed in each case. (B) Luciferase activity was measured at the indicated time after co-transfection of HepG2 cells and HepG2-WT10 cells with pGL3-Control vector and H7scFv expression plasmid, S2E1scFv or empty vector. Relative luciferase activity shows the percentage of relative light units of the sample with respect to relative light units of WT10 cells transfected with empty vector. The mean percentage of triplicates with standard errors is shown. The experiments were repeated two times and similar trends were observed in each case.

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