Adenovirus vectors lacking virus-associated RNA expression enhance shRNA activity to suppress hepatitis C virus replication

Abstract

First-generation adenovirus vectors (FG AdVs) expressing short-hairpin RNA (shRNA) effectively downregulate the expressions of target genes. However, this vector, in fact, expresses not only the transgene product, but also virus-associated RNAs (VA RNAs) that disturb cellular RNAi machinery. We have established a production method for VA-deleted AdVs lacking expression of VA RNAs. Here, we showed that the highest shRNA activity was obtained when the shRNA was inserted not at the popularly used E1 site, but at the E4 site. We then compared the activities of shRNAs against hepatitis C virus (HCV) expressed from VA-deleted AdVs or conventional AdVs. The VA-deleted AdVs inhibited HCV production much more efficiently. Therefore, VA-deleted AdVs were more effective than the currently used AdVs for shRNA downregulation, probably because of the lack of competition between VA RNAs and the shRNAs. These VA-deleted AdVs might enable more effective gene therapies for chronic hepatitis C.

Figure 1. Structures of vectors containing shRNA cassettes and suppression efficiencies.

(a) Structures of AdV-aGFP vectors. The arrow shows the orientation of transcription. Hatched box, shRNA cassette including the human U6 promoter. (b) (Left) Suppression efficiency of GFP RNA expressed from the cell line using anti-GFP vectors. FC-18 cells that constitutively express GFP RNA were infected with the vectors at MOI 50. (Right) Suppression efficiency of Cre RNA expressed from the AdV genome using anti-Cre vectors. FC-18 cells were doubly infected with AdV expressing Cre under the control of the CAG promoter at MOI 10 and anti-Cre vectors at MOI 50. Three days after infection, the amount of cytoplasmic RNA of GFP and Cre were measured using qPCR. The suppression efficiency for vector-infected FC-18 cells was calculated using copy numbers per cell, where uninfected FC-18 cells were denoted as 0% suppression of GFP RNA, while that of CV1 cells, the parent cells of FC-18 that do not contain the GFP gene, is denoted as the control of 100% suppression. Copy number, n = 6. *P < 0.05, **P < 0.01 compared with the E4L vector (unpaired Student's t-test).

Figure 2. Schematic representation of the HCV replicons.

The coding regions in the HCV polyproteins are indicated by the open boxes. The bold lines indicate the HCV 5′-untranslated region (UTR), which is the target of sh277 and sh331 (arrows), and the 3′-UTR. Gray bars, EMCV internal ribosome entry site; dC, 5′-region of Core gene; neo, neomycin-resistance gene; Luc-neo, firefly luciferase gene fused with neo gene.

Figure 3. Suppression of HCV RC RNA by the AdVs expressing sh277 and sh331.

Effects on HCV RNA replication in HuH 5–15 cells (a) and SGR-JFH1 cells (b). The cells were infected with FG AdVs (FG, white bars) and VA-deleted AdVs (VA-del, black bars). NC, AdVs expressing negative-control shRNA. The copy numbers of intracellular HCV RNA were measured at 72 h after infection. The suppression efficiency was calculated relative to the copy numbers in uninfected cells as 0%; the copy numbers of HCV RNA in the control, uninfected cells were 4.0 × 10⁴ copies/cell and 1.1 × 10⁴ copies/cell in HuH 5–15 cells (a) and SGR-JFH1 cells (b), respectively. The suppression efficiencies of NC FG AdV and NC VA-deleted AdV were (a) 3.8 (±6.7) and 15.1 (±4.4), while those were (b) −4.1 (±3.4) and 18.2 (±9.0), respectively. Each data point represents an average of n = 3, mean ± S.D. (error bars). *P < 0.05, **P < 0.01.

Figure 4. Suppression of HCV RC RNA by double infection of the shRNA-expressing AdVs.

HuH 5–15 cells (a) and SGR-JFH1 cells (b) were infected with the VA-deleted AdVs expressing shRNAs and, three days later, the intracellular RNA levels of HCV RC were measured. The copy numbers of HCV RC were 5.7 × 10⁴ copies/cell and 1.4 × 10⁴ copies/cell in the HuH 5–15 cells (a) and the SGR-JFH1 cells (b), respectively. The suppression efficiencies of NC FG AdV and NC VA-deleted AdV were (a) 8.7 (±13.6) and (b) 18.7 (±2.0), respectively. **P < 0.01 against the value of the coinfection (bar 6) (a), (b). The other presentations are the same as in Fig. 3.

Figure 5. Suppression of replicating HCV genome and the expressed proteins in the HCV-infected HuH cells.

(a) Suppression levels of HCV genomes. HCV-infected HuH-7 cells were infected with shRNA-expressing AdVs and interferon-expressing AdVs at MOI 2 (bars 1 to 4, 7, 8, 11 and 12) or at MOI 10 (bars 5, 6, 9 and 10). The copy numbers of HCV RNA were 1.0 × 10³ copies/cell. The suppression efficiencies of NC FG AdV and NC VA-deleted AdV were 3.7 (±5.5) and 27.8 (±9.7), respectively. The other presentations are the same as in Fig. 3. (b) Western blot analysis of HCV NS5A protein expressed in the viral-infected cells. Three days after the AdV infection at MOI 10, HCV NS5A and GAPDH proteins were detected using specific antibodies. CC, Cells infected with HCV but not with AdVs expressing the shRNAs. Arrows for NS5A indicate its hyper- (upper) and hypo- (lower) phosphorylated forms.