A small yeast RNA inhibits HCV IRES mediated translation and inhibits replication of poliovirus in vivo

Abstract

Aim: To investigate the anti-virus infection activity of internal ribosome entry site (IRES) specific inhibitor RNA (IRNA).

Methods: IRNA eukaryotic vector pcRz-IRNA or mIRNA eukaryotic vector pcRz-mIRNA was transfected into human hepatocarcinoma cells (HHCC), then selected with neomycin G418 for 4 to 8 weeks, and then infected with polio virus vaccines line. The cytopethogenesis effect was investigated and the cell extract was collected. At last the polio virus titer of different cells was determined by plaque assay.

Results: Constructive expression of IRNA was not detrimental to cell growth. HCV IRES-mediated cap-independent translation was markedly inhibited in cells constructively expressing IRNA compared to control hepatoma cells. However, cap-dependent translation was not significantly affected in these cell line. Additionally, HHCC cells constitutively expressing IRNA became refractory to infection of polio virus.

Conclusion: IRES specific IRNA can inhibit HCV IRES mediated translation and poliovirus replication.

Figure 1

Detection IRNA or mIRNA in the total RNA of HHCCs which constitutionally expressing IRNA or mIRNA. 1: DNA Marker DL2000; 2 and 3: RT-PCR of cell lines expressing IRNA; 4: RT-PCR of cell lines expressing mIRNA; 5 and 6: PCR of cell lines expressing IRNA or mIRNA.

Figure 2

The translation level of β-actin in HHCC and IRNA-expressing lines. (Laser confocal, 400 × 1.00). A: IRNA-express-ing cell lines, B: Control cell lines.

Figure 3

The effect of long-term expressing IRNA on cap-independent and cap-dependent protein translation.

Figure 4

The cytopathic effect of different HHCC cells infected with PV. A: control HHCC cells; B: mIRNA cells; C: pcDNA3 cells; D: IRNA cells.

Figure 5

Plaque characteristic of different HHCC infected of PV. A: control HHCC; B: mIRNA cells; C: pcDNA3 cells; D: IRNA cells.

Figure 6

IRNA forms a gel-retarded complex which is inhibited by 5’ UTR of HCV RNA. ³²P-labeled IRNA was incubated with RRL extract in the absence (lane 2) or presence of an unla-beled 25- or 50-fold molar excess of IRNA (lane3 and lane 4, respectively) or 5’ UTR (lane 5) or of a 50-fold molar excess of nonspecific RNA (lane 6). Protein-RNA complexes were analyzed on a nondenaturing gel. In lane 1, RRL extract was not added. FP: free probe; C: complexes.