The role of superoxide anions in the establishment of an interferon-alpha-mediated antiviral state.
It has been suggested that CuZn-superoxide dismutase (CuZnSOD) is required for the establishment of an interferon (IFN)-mediated antiviral state. To investigate this possibility further, a panel of 6 stably transfected HeLa clones, expressing CuZnSOD activity from 1.6 to 7.3 times the normal level, were treated with different concentrations of recombinant human interferon alpha A (rHuIFN-alpha A) followed by challenge with vesicular stomatitis virus (VSV). A biphasic response curve was generated (r = 0.87, p less than 0.025). Clones with up to 3-fold basal level CuZnSOD activity exhibited an inverse relationship between their ability to generate an IFN-alpha-mediated antiviral state and CuZnSOD activity: the higher the CuZnSOD activity, the lower the sensitivity to IFN-alpha and the more IFN-alpha required for antiviral defense. Clones with between 4 to 7.3 times higher CuZnSOD activity than the non-transfected HeLa control showed a direct relationship between the CuZnSOD activity and the sensitivity to IFN-alpha. Furthermore, in agreement with the results obtained with the SOD1-transfected HeLa cells with up to 3 times the basal SOD activity, fetal fibroblasts derived from SOD1-transgenic mouse strains, TgHS-229 and TgHS-218, which also express 3 times the basal CuZnSOD activity, required higher IFN-alpha to achieve 50% protection. These results suggest a possible role for superoxide anion in the establishment of IFN-mediated antiviral effect, especially in the dose-response region in which the inverse relationship between the generation of the IFN-alpha-mediated antiviral state and CuZnSOD activity was observed. To assess this possibility, allopurinol was used as a xanthine oxidase inhibitor and hydroxyl radical scavenger in the IFN-alpha-mediated antiviral assay. Addition of 3 mM allopurinol diminished the IFN-mediated antiviral effect by between 40 and 50% (p less than 0.01), and there was a reduction in superoxide generation (p less than 0.05). The degree of reduction caused by allopurinol treatment was higher at an IFN-alpha concentration of 10 U/ml than at 100 U/ml, and there was no correlation between CuZnSOD activity and the degree of reduction. To establish further the role of superoxide as an antiviral agent, paraquat was used as a superoxide generator in the absence of IFN-alpha in the antiviral assay. Although paraquat at high concentrations is toxic to the cells, it actually showed a protective effect against VSV infection, and an inverse relationship (r = 0.79, r less than 0.025) between cell survival and CuZnSOD activity was observed with 150 mM paraquat treatment.(ABSTRACT TRUNCATED AT 400 WORDS)
Huang
T T
TT
Department of Pediatrics, University of California, San Francisco 94143.
Carlson
E J
EJ
Epstein
L B
LB
Epstein
C J
CJ
D000493:Allopurinol / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007370:Interferon Type I / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D010269:Paraquat / Q000494:pharmacology; D011994:Recombinant Proteins; D013482:Superoxide Dismutase / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism*; D014162:Transfection / Q000235:genetics
D007370:Interferon Type I; D011994:Recombinant Proteins; D013481:Superoxides; D000493:Allopurinol; D013482:Superoxide Dismutase; D010269:Paraquat