A study of the interferon antiviral mechanism: apoptosis activation by the 2-5A system - PubMed (original) (raw)

A study of the interferon antiviral mechanism: apoptosis activation by the 2-5A system

J C Castelli et al. J Exp Med. 1997.

Abstract

The 2-5A system contributes to the antiviral effect of interferons through the synthesis of 2-5A and its activation of the ribonuclease, RNase L. RNase L degrades viral and cellular RNA after activation by unique, 2'-5' phosphodiester-linked, oligoadenylates [2-5A, (pp)p5' A2'(P5'A2')]n, n >=2. Because both the 2-5A system and apoptosis can serve as viral defense mechanisms and RNA degradation occurs during both processes, we investigated the potential role of RNase L in apoptosis. Overexpression of human RNase L by an inducible promoter in NIH3T3 fibroblasts decreased cell viability and triggered apoptosis. Activation of endogenous RNase L, specifically with 2-5A or with dsRNA, induced apoptosis. Inhibition of RNase L with a dominant negative mutant suppressed poly (I).poly (C)-induced apoptosis in interferon-primed fibroblasts. Moreover, inhibition of RNase L suppressed apoptosis induced by poliovirus. Thus, increased RNase L levels induced apoptosis and inhibition of RNase L activity blocked viral-induced apoptosis. Apoptosis may be one of the antiviral mechanisms regulated by the 2-5A system.

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Figures

Figure 1

Figure 1

Inducible expression of human RNase L in NIH3T3 cells. (A) Western blot analysis was performed for the detection of human RNase L expression in vector control 3T3/neo cells (lane 1) and in NIH3T3 cells transfected with a lac inducible vector containing the human RNase L gene (3T3/RNaseLS) incubated 24 h in the absence (lane 2) or in the presence (lane 3) of 3 mM IPTG. Western blot analysis was performed as previously described (25) using a 1:2,500 dilution of a monoclonal antibody specific for the human RNase L enzyme which did not detect the endogenous murine RNase L (lane 1). Proteins were detected using ECL reagents (Amersham, Arlington Heights, IL). An autoradiograph of a 1-min exposure is shown. RNA degradation after induction of RNase L. (B) 3T3/RNaseLS cells were incubated in the absence (lanes 1 and 2) and presence (lane 3) of 3 mM IPTG for 24 h then transfected with 1 μM of ppp5′(A2′p5′)2A (lanes 2 and 3) by calcium phosphate coprecipitation as previously described (12). (C) Northern blot analysis using an 18S rRNA probe is shown after incubation of cells in the absence (lane 2) and presence (lanes 1 and 3) of 3 mM IPTG for 24 h followed by transfection with 1 μM of ppp5′(A2′p5′)2A (lanes 2 and 3). Cellular RNA was isolated and electrophoresed as described in Materials and Methods).

Figure 1

Figure 1

Inducible expression of human RNase L in NIH3T3 cells. (A) Western blot analysis was performed for the detection of human RNase L expression in vector control 3T3/neo cells (lane 1) and in NIH3T3 cells transfected with a lac inducible vector containing the human RNase L gene (3T3/RNaseLS) incubated 24 h in the absence (lane 2) or in the presence (lane 3) of 3 mM IPTG. Western blot analysis was performed as previously described (25) using a 1:2,500 dilution of a monoclonal antibody specific for the human RNase L enzyme which did not detect the endogenous murine RNase L (lane 1). Proteins were detected using ECL reagents (Amersham, Arlington Heights, IL). An autoradiograph of a 1-min exposure is shown. RNA degradation after induction of RNase L. (B) 3T3/RNaseLS cells were incubated in the absence (lanes 1 and 2) and presence (lane 3) of 3 mM IPTG for 24 h then transfected with 1 μM of ppp5′(A2′p5′)2A (lanes 2 and 3) by calcium phosphate coprecipitation as previously described (12). (C) Northern blot analysis using an 18S rRNA probe is shown after incubation of cells in the absence (lane 2) and presence (lanes 1 and 3) of 3 mM IPTG for 24 h followed by transfection with 1 μM of ppp5′(A2′p5′)2A (lanes 2 and 3). Cellular RNA was isolated and electrophoresed as described in Materials and Methods).

Figure 2

Figure 2

Induction of RNase L expression decreased protein synthesis and cell viability. After IPTG treatment, cellular protein synthesis was determined in 3T3/neo cells (circles) and 3T3/RNaseLS cells (squares) and plotted as a percentage of untreated cells. The points are mean values from duplicates with the standard deviation shown.

Figure 3

Figure 3

RNase L overexpression caused DNA cleavage characteristic of apoptosis. 3T3/neo (A and C) and 3T3/RNaseLS (B and D) cells were incubated for 24 h in the absence (A and B) and presence (C and D) of 3 mM IPTG. Apoptotic cells were detected in situ by using T7 DNA polymerase with methyl green counterstaining. Experiments were performed in triplicate.

Figure 4

Figure 4

Apoptosis of interferon-treated fibroblasts after activation with dsRNA. In situ DNA end-labeling (A–C) and Hoechst dye no. 33342 staining (D–F) of L929 cells. (A and D) After 48 h incubation in interferon-α/β (1,000 U/ml) (Sigma Chemical Co., St. Louis, MO). (B and E) 24 h incubation in synthetic dsRNA poly (I)·(C) (25μg/ml). (C and F), 24 h preincubation in interferon-α/β followed by an additional 24 h in poly (I)·poly (C). Apoptotic cells were detected in situ by using terminal deoxynucleotidyl transferase with methyl green counterstaining. Cells were stained with Hoechst dye (0.1 mg/ml in PBS for 15 min) and photographed using fluorescence on an Axiovert microscope (Carl Zeiss, Inc., Thornwood, NY). Inhibition of RNase L protects fibroblasts against poly (I)·poly (C)–induced apoptosis. (G) L929 cells were transiently cotransfected with the GFP (green fluorescent protein) gene and either control vector (open circles) or the RNase LZB1 gene (closed squares) then incubated in interferon-α/β (1,000 U/ml) for 24 h. Cells were then treated with poly (I)·poly (C) (25 μg/ml) and green fluorescent cells were quantified as a measure of cell viability at each of the indicated time points. (25).

Figure 4

Figure 4

Apoptosis of interferon-treated fibroblasts after activation with dsRNA. In situ DNA end-labeling (A–C) and Hoechst dye no. 33342 staining (D–F) of L929 cells. (A and D) After 48 h incubation in interferon-α/β (1,000 U/ml) (Sigma Chemical Co., St. Louis, MO). (B and E) 24 h incubation in synthetic dsRNA poly (I)·(C) (25μg/ml). (C and F), 24 h preincubation in interferon-α/β followed by an additional 24 h in poly (I)·poly (C). Apoptotic cells were detected in situ by using terminal deoxynucleotidyl transferase with methyl green counterstaining. Cells were stained with Hoechst dye (0.1 mg/ml in PBS for 15 min) and photographed using fluorescence on an Axiovert microscope (Carl Zeiss, Inc., Thornwood, NY). Inhibition of RNase L protects fibroblasts against poly (I)·poly (C)–induced apoptosis. (G) L929 cells were transiently cotransfected with the GFP (green fluorescent protein) gene and either control vector (open circles) or the RNase LZB1 gene (closed squares) then incubated in interferon-α/β (1,000 U/ml) for 24 h. Cells were then treated with poly (I)·poly (C) (25 μg/ml) and green fluorescent cells were quantified as a measure of cell viability at each of the indicated time points. (25).

Figure 5

Figure 5

Effect of Bcl-xL overexpression on apoptosis induced by dsRNA or poliovirus. After transient cotransfection of the GFP gene with control vector, (gray bars), RNase LZB1 (black bars), or Bcl-xL (hatched bars) genes, L929 cells were treated with (A) interferon-α/β (1,000 U/ml) and poly (I)·poly (C) (25 μg/ml) for 24 h and HeLa cells were treated with (B) poliovirus at 106 PFU for 45 h. Cell viability was assessed by quantitation of green fluorescent cells and presented as a percentage of untreated transfected cells. Experiments were performed in duplicate.

Figure 6

Figure 6

Inhibition of RNase L blocks poliovirus-induced apoptosis. HeLa cells were transiently cotransfected with GFP gene and either control vector (gray bars) or the RNase LZB1 gene (black bars). After incubation in 106 PFU type I poliovirus, GFP-positive cells were quantitated at each of the indicated time points. Cell numbers were calculated as a percentage of uninfected cells transfected with each of the vectors.

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