Inhibition of virus production in JC virus-infected cells by postinfection RNA interference

Abstract

RNA interference has been applied for the prevention of virus infections in mammalian cells but has not succeeded in eliminating infections from already infected cells. We now show that the transfection of JC virus-infected SVG-A human glial cells with small interfering RNAs that target late viral proteins, including agnoprotein and VP1, results in a marked inhibition both of viral protein expression and of virus production. RNA interference directed against JC virus genes may thus provide a basis for the development of new strategies to control infections with this polyomavirus.

FIG. 1.

Effects of postinfection RNAi on the abundance of JCV proteins in JCV-infected SVG-A cells. (a) Schematic representation of major early and late mRNAs of JCV. Major early mRNAs encode the small t antigen and T-Ag, which are translated as splicing variants. Two major forms of late mRNA encode either agnoprotein and VP1 or agnoprotein, VP2, and VP3. The regions of the viral RNAs targeted by the siRNAs are indicated by arrows. (b) Immunoblot analysis of the abundance of VP1 and agnoprotein of JCV in SVG-A cells at the indicated times after infection with JCV. (c) Schedule for JCV infection and siRNA transfection in SVG-A cells. (d) Immunoblot analysis of the indicated proteins in JCV-infected cells subjected to transfection with the indicated siRNAs. (e) Immunoblot analysis of the indicated proteins in JCV-infected cells subjected to transfection with the Ag122 siRNA at 60 or 120 pmol/well or with a lamin A/C-specific siRNA. Control (infected) cells were subjected to mock transfection.

FIG. 2.

Indirect immunofluorescence analysis of the effects of RNAi on viral protein expression in JCV-infected SVG-A cells. (a) The proportion of cells that were positive for agnoprotein or VP1 was determined by an indirect immunofluorescence analysis 48 h after transfection with the indicated siRNA(s). The data are expressed as percentages of the proportion determined for JCV-infected cells transfected with the scrambled siRNA (control) and are means ± standard deviations (SD) of values from at least three independent experiments. **, P < 0.02 versus the value for cells transfected with the scrambled siRNA (Student's t test). (b) Immunoblot analysis of VP1, agnoprotein, and actin expression in JCV-infected cells transfected with the indicated siRNA(s). (c) The signals for agnoprotein and VP1 were quantified with an image analyzer and expressed as percentages of the value for cells transfected with the scrambled siRNA.

FIG. 3.

Depletion of viral RNAs by RNAi in JCV-infected SVG-A cells. Total RNAs isolated from JCV-infected cells 12 or 24 h after transfection with the indicated siRNAs were subjected to an RT-PCR analysis of agnoprotein and VP1 mRNAs. The data were normalized to the amount of β-actin mRNA and are expressed as percentages of the normalized value for JCV-infected cells transfected with the scrambled siRNA (control); they are means ± SD of values from at least three independent experiments. *, P < 0.05, and **, P < 0.02 versus the value for cells transfected with the scrambled siRNA.

FIG. 4.

Inhibition of JCV production by RNAi in SVG-A cells. Extracts prepared from JCV-infected cells 36 h after transfection with the indicated siRNAs were assayed for hemagglutination activity (HA). The data are expressed as HA titers per 25 μl of cell extract and are means ± SD of values from at least three independent experiments. **, P < 0.02 versus the value for cells transfected with the scrambled siRNA.