Application of RNA interference for inhibiting the replication of feline immunodeficiency virus in chronically infected cell lines

Abstract

RNA interference (RNAi) is a process in which double-stranded RNA induces the post-transcriptional sequence-specific degradation of homologous messenger RNA. The present study was carried out to apply the RNAi technology to inhibit the replication of feline immunodeficiency virus (FIV). Four small interfering RNAs (siRNAs) homologous to the FIV gag gene were synthesized and transfected into a feline fibroblastic cell line chronically infected with FIV (CRFK/FIV). These synthetic siRNAs efficiently inhibited the replication of FIV. Next, we examined the effect of retroviral vector-mediated transfer of FIV-specific short hairpin RNA (shRNA) on the replication of FIV in a feline T-cell line chronically infected with FIV (FL4). The retroviral vector-mediated transfer of FIV-specific shRNA was shown to markedly inhibit the replication of FIV in the FL4 cells. These results provide useful information for the development of RNAi-based gene therapy strategy to control FIV infection.

Fig. 1

Effect of siRNAs on the replication of FIV in CRFK/FIV cells. The RT activity in the culture supernatants was measured 48 h after transfection with each siRNA at a concentration of 200 nM. The columns and bars show means and standard deviations, respectively, obtained from the data in triplicate samples. Asterisks indicate statistically significant difference between transfection with anti-FIV siRNAs and Luc-siRNA as a control (P < 0.05).

Fig. 2

Dose-dependency of the effect of FIV-specific siRNA on the amount of FIV RNA in CRFK/FIV cells. The amount of FIV RNA in the cells (a) and RT activity in the culture supernatants (b) were measured 48 h after transfection with G4-siRNA or Luc-siRNA (control) at concentrations as indicated. The amount of FIV RNA was measured by a real-time sequence detection system and the relative FIV RNA expression values ($2^{-\Delta C_{\text{T}}}$) were calculated in comparison to the amount of β-actin mRNA. The amounts of FIV RNA are shown as percentages of the amount of FIV RNA in mock-transfected cells. The columns and bars show means and standard deviations, respectively, obtained from the data in triplicate samples. Asterisks indicate statistically significant difference between transfection with G4-siRNA and that with Luc-siRNA at same concentration (P < 0.05).

Fig. 3

Time-course analysis of the inhibitory effect of FIV-specific siRNA on the replication of FIV in CRFK/FIV cells. RT activity in the culture supernatants was measured at several time points as indicated after transfection with G4-siRNA at concentrations of 0.04 μM (♦), 0.2 μM (●) and 1 μM (▴). RT activity in the culture supernatants are shown as percentages of RT activity in the mock-transfected cells (□). The symbols and bars show means and standard deviations, respectively, obtained from the data in triplicate samples. Asterisks indicate statistically significant difference between transfection with G4-siRNA and mock-treated cells (P < 0.05).

Fig. 4

Effect of retrovirus vector-mediated transfer of FIV-specific shRNA on the replication of FIV in FL4 cells. FL4 cells were infected with the retrovirus vector, which express shG4 or shNC and then selected under puromycin. The amount of FIV RNA in the untreated, shG4-transduced, and shNC-transduced FL4 cells (a) and RT activities in the culture supernatants (b) were measured 2 weeks after retrovirus vector-mediated transduction. The amount of FIV RNA was measured by a real-time sequence detection system and the relative FIV RNA expression values ($2^{-\Delta C_{\text{T}}}$) were calculated. The columns and bars show means and standard deviations, respectively, obtained from the data in triplicate samples. The asterisk indicates statistically significant difference between cells transduced with shG4 and shNC (P < 0.05).