Antiviral effects against influenza A virus infection by a short hairpin RNA targeting the non-coding terminal region of the viral nucleoprotein gene

Abstract

RNA interference (RNAi) can inhibit Influenza A virus (IAV) infection in a gene-specific manner. In this study, we constructed a transgene expressing a short hairpin RNA (shRNA) that targets the noncoding region of the IAV RNA gene encoding nucleoprotein (NP). To investigate the antiviral effects of the shRNA, we generated two transgenic mouse lines with this transgene. Unfortunately, there was no apparent difference in IAV resistance between transgenic and non-transgenic littermates. To further investigate the antiviral effects of the shRNA, we prepared mouse embryonic fibroblasts (MEFs) from transgenic and non-transgenic mice. In experimental infections using these MEFs, virus production of mouse-adapted IAV strain A/Puerto Rico/8/1934 (PR8) in the transgenic MEFs was suppressed by means of the down-regulation of the viral RNA gene transcription in the early stages of infection in comparison with non-transgenic MEFs. These results indicated that expression of the shRNA was able to confer antiviral properties against IAVs to MEFs, although the effects were limited. Our findings suggest that the shRNA targeting the noncoding region of the viral RNA (vRNA) of NP might be a supporting tool in developing influenza-resistant poultry.

Keywords: RNA interference; antiviral effect; influenza A virus; short hairpin RNA; transgenic mouse.

Fig. 1.

Schematic representation of the shRNA expression vector. This vector includes a β-actin promoter driving shRNA expression. The arrows denote the PCR primers used to identify transgene integration in the transgenic mice.

Fig. 2.

Experimental infections in transgenic mice expressing shRNA. Transgenic (TG) and non-transgenic (non-TG) mice were challenged intranasally with IAV strain PR8. Mice body weight during the challenge phase (14 days) was calculated relative to their starting body weight. Error bars indicate standard deviation.

Fig. 3.

Viral replication in transgenic mouse embryonic fibroblasts (MEFs) expressing shRNA. MEFs were infected with PR8 at an MOI of 0.01. The culture supernatants were collected at 8 and 24 hr post-inoculation for virus titration in MDCK cells. The mean values of the virus titers were determined in triplicate. Error bars indicate standard deviation. MEF cell lines A–D and F–H were prepared from transgenic mouse embryos of lines 6522 and 6837, respectively. MEF cell lines E, I, and J were prepared from non-transgenic mouse embryos. Statistical analysis was performed using Student’s t-test, and * indicates P<0.01; significant differences in transgenic MEFs versus non-transgenic MEFs (A–D vs. E in cell line 6522, F–H vs. I in cell line 6837).

Fig. 4.

Expression levels of a viral gene in transgenic MEFs expressing shRNA after infection with strain PR8. Total RNA was extracted from MEFs infected with PR8 at an MOI of 0.01 after 8 and 24 hr post-inoculation. To determine the expression level of mRNA gene of the viral NP, reverse transcription and semi-quantitative real-time PCR were carried out. The results are presented in relative value to the expression level in non-transgenic MEFs (cell lines E and I for transgenic lines 6522 and 6837, respectively), and error bars represent the mean ± SEM of duplicate experiments. MEF cell lines A–D and F–H were prepared from transgenic mouse embryos of lines 6522 and 6837, respectively. MEF cell lines E, I, and J were prepared from non-transgenic mouse embryos. Statistical analysis was performed using Student’s t-test, and * indicates P<0.01; significant differences in transgenic MEFs versus non-transgenic MEFs (A–D vs. E in cell line 6522, F–H vs. I in cell line 6837).