Article Figures & Data
Figures
- Fig. 1.
Inducible expression and enzymatic activation of transfected RNase L. (A) RNase L in 100 μg of postmitochondrial supernatant protein from independent transfected cell lines (LS1, LS2, and LS3) was analyzed on Western blots before and 24 h after treatment with 3 mM IPTG. Blots were reacted with a monoclonal antibody specific for transfected human RNase L and with a polyclonal antibody to the constitutively expressed Lac repressor (Stratagene). A film exposed to an ECL (Amersham)-reacted blot is shown. (B) Total RNA from control (C) and RNase L-inducible LS1 cells which had been transfected with 1 μM 2-5A trimer and treated with 1,000 U of murine IFN-α/β (Lee Biomolecular), as indicated, was analyzed on a glyoxal-agarose gel (15 μg of RNA/lane). A Northern blot of this gel was hybridized with an 18S rRNA probe; an autoradiograph of the blot is shown in the lefthand panel. A photograph of the ethidium-stained gel is shown in the righthand panel. Arrows indicate specific rRNA cleavage products.
- Fig. 2.
RNase L enhances the anti-EMCV, but not the anti-VSV, activity of IFN. LS1 cells were treated with the indicated concentrations of murine IFN-α/β for 24 h, in the presence and absence of IPTG (3 mM), to induce RNase L. Cells were then infected with EMCV (A) or VSV (B) at an MOI of 1.0, as described in Materials and Methods. At 18 h PI, viable cells were stained. Data points on the graphs represent means of quadruplicate samples; for each point, the coefficient of variability is ≤3%.
- Fig. 3.
RNase L-dependent reduction in EMCV RNA in IFN-treated cells. (A) Total RNA isolated from LS1 cells following RNase L induction, IFN treatment, and EMCV infection, as indicated, was analyzed on a glyoxal-agarose gel (5 μg of RNA/lane); IFN and IPTG treatments were as described in Materials and Methods, and EMCV infection was for 6 h. The ethidium-stained gel was photographed (lower panel) or transferred to a nylon membrane and hybridized to EMCV and GAPDH cDNA probes; an autoradiograph of these blots is shown in the upper panel. (B) Total RNA isolated from mutant RNase L (SVT2/ZB1) or vector control (SVT2/pSVL) cells following IFN treatment or EMCV infection, as indicated, was analyzed on a glyoxal-agarose gel (7 μg of RNA/lane). A Northern blot of this gel was hybridized to EMCV (upper panel) and GAPDH (lower panel) cDNA probes; an autoradiograph of this blot is shown.
- Fig. 4.
RNase L inhibits EMCV RNA synthesis. (A) LS1 cells were treated with IFN and IPTG, as indicated, and then infected with EMCV in the presence of [3H]uridine, as described in Materials and Methods. Cells were harvested at the indicated times, and [3H]uridine incorporation was determined by TCA precipitation. (B) At 8 h PI, [3H]uridine was removed from the medium and chased with unlabeled UTP and CTP for an additional 2 h; percent RNA remaining was calculated based on TCA-precipitable counts at the beginning of the chase for each treatment. Data points on both graphs are means of six samples at each time point. UNT, untreated (control) cells.
- Fig. 5.
RNase L induction does not alter the cellular RNA profile. (A) The Northern blot shown in Fig. 3A was rehybridized with c-myc (top panel), ISG15 (middle panel), and GAPDH (bottom panel) cDNA hybridization probes; an autoradiograph of the blot is shown. (B) Total RNA from the EMCV-infected cells shown in Fig. 3 (200 ng/reaction) was analyzed by differential display, as described in Materials and Methods; the reverse primer was oligo(dT)11CA, and the forward primers were EMCV specific (5′GAGTCTGTTCTGG3′ [lanes 1 to 5]) or were arbitrary sequence decamers (5′CTGATCCATG3′ [lanes 6 to 9]; 5′CGTAGATCGT3′ [lanes 10 to 13]). An autoradiograph of the gel is shown.
- Fig. 6.
2-5A transfection induces a widespread reduction in cellular RNA. Total RNA from the control and 2-5A-transfected LS1 cells shown in Fig. 1 (200 ng/reaction) was analyzed by differential display, as described in Materials and Methods; the reverse primer was oligo(dT)12A, and the forward primers were 5′TTTTGGCTCC3′ (lanes 1 and 2) or 5′CTTTCTACCC3′ (lanes 3 and 4). An autoradiograph of the gel is shown.
