Isolation of a cDNA Clone Derived from a Blood-Borne Non-A, Non-B Viral Hepatitis Genome Author(s): Qui-Lim Choo, George Kuo, Amy J. Weiner, Lacy R. Overby, Daniel W. Bradley, Michael Houghton Source: Science, New Series, Vol. 244, No. 4902 (Apr. 21, 1989), pp. 359-362 Published by: American Association for the Advancement of Science Stable URL: http://www.jstor.org/stable/1703266 Accessed: 22/04/2009 13:43 Your use of the JSTOR archive indicates your acceptance of JSTOR's Terms and Conditions of Use, available at http://www.jstor.org/page/info/about/policies/terms.jsp. JSTOR's Terms and Conditions of Use provides, in part, that unless you have obtained prior permission, you may not download an entire issue of a journal or multiple copies of articles, and you may use content in the JSTOR archive only for your personal, non-commercial use. Please contact the publisher regarding any further use of this work. 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McKnight, purified by hybridization chromatography on po- than direct iron starvation or administra- Science240, 1759 (1988). ly(A) Sepharose (Pharmacia). RNA was added to a 4. G. Dreyfuss, L. Philipson, I. W. Mattaj, J. Cell Biol. lysate of K562 cells and incubated for 30 min at tion. Finally, treatment of RD4 cells with 106, 1419 (1988). room temperature before addition of 5 mg of sodi- at doses that inhibit 5. M. P. Wickens and J. E. Dahlberg, Cell 51, 339 um heparin (Hynson, Westcott, and Dunning) per puromycin completely This has no effect on the induc- (1987); G. Shaw and R. Kamen, ibid. 46, 659 milliliter. sample was then added to streptavi- protein synthesis (1986); 0. Melefors and A. von Gabain, ibid. 52, din agarose (Bethesda Research Laboratories) and tion of IRE-binding activity in response to 893 (1988); G. Brawerman, ibid. 48, 5 (1987). the mixture was incubated for an additional 15 min 6. M. before the resin was washed five with Df (19). W. Hentze et al., Proc. Natl. Acad. Sci. U.S.A. times 20 84, 6730 (1987); N. Aziz and H. N. Munro, ibid.p. volumes of 40 mM KCI, 25 mM tris-CI, pH 8, and We therefore propose that chelation of 8478. 1% Triton X-100. Elution of IRE-BP was accom- iron by Df results in the activation of the 7. M. W. Hentze et al., Science238, 1570 (1987). plished with 1M KCI, 5 mg of sodium heparin per IRE-BP to the reduction of an 8. S. W. Caughman, M. W. Hentze, T. A. Rouault, J. milliliter, 25 mM tris-CI, pH 8, and 1% Triton X- by leading B. Harford, R. D. Klausner, J. Biol. Chem. 263, 100. Based on recovery of IRE-binding activity and intramolecular disulfide in the IRE-BP. At 19048 (1988). the recovery of labeled K562 protein from a parallel least one of the now free residues is 9. J. L. Casey et al., Science240, 924 (1988). experiment with a [35S]methionine lysate, we esti- cysteinyl A. mate that for a interaction be- 10. T. Rouault, M. W. Hentze, S. W. Caughman, J. the IRE-binding protein was purified required high affinity B. Harford, R. D. Klausner, ibid.241, 1207 (1988). approximately 50-fold by this procedure. tween the protein and the IRE which, in 11. D. M. Koeller et al., Proc.Natl. Acad. Sci. U.S. A., in 18. J. F. Milligan, D. R. Groebe, G. W. Witherell, O. C. NucleicAcids Res. 8783 turn, is responsible for the repression of press. Uhlenbeck, 15, (1987). 12. P. J. Romaniuk and O. C. Uhlenbeck, Biochemistry 19. M. W. Hentze, T. A. Rouault, J. B. Harford, R. D. ferritin mRNA translation. In essence, alter- 24, 4239 (1985). Klausner, unpublished observations. ation in cellular iron status operates a "sulf- 13. R. M. Starzyk, S. W. Koontz, P. Schimmel, Nature 20. M. W. Hentze et al., Gene 72, 201 (1988). switch" reversible oxidation or 298, 136 (1982). 21. E. A. Leibold and H. N. Munro, Proc. Natl. Acad. hydryl by 14. S. W. Koontz and P. R. Schimmel, J. Biol. Chem. Sci. U.S.A. 85, 2171 (1988). reduction of critical sulfhydryl group or 254, 12277 (1979). 22. T. Rouault, K. Rao, J. Harford, E. Mattia, R. D. groups in the IRE-BP. This hypothesis 15. N. S. Kosower and E. M. Kosower, MethodsEnzy- Klausner, J. Biol. Chem. 260, 14862 (1985). raises the of whether such a switch mol. 143, 264 (1987). 23. R. E. Cappel and H. F. Gilbert, ibid. 263, 12204 question 16. K. Kobashi, Biochim.Biophys. Acta 158, 239 (1968). (1988). could be physiologically relevant in the re- 17. Partial purification of the IRE-binding protein was 24. D. W. Walters and H. F. Gilbert, ibid. 261, 13135 environment of the The achieved by RNA affinity chromatography. An IRE- (1986); ibid., p. 15372; C. J. Clancey and H. F. ducing cytosol. RNA was transcribed in vitro RNA ibid. 13545 redox buffer in the is the containing by Gilbert, 262, (1987). major cytosol polymerase T7 (18) with two synthetic oligodeoxy- 25. We thank O. Uhlenbeck, N. Kosower, and E. glutathione system. The vast excess of re- nucleotides that when hybridized constituted an Kosower for stimulating discussions; D. Haile, D. RNA T7 and a for J. A. and Barriocanal for duced over oxidized is largely polymerase promoter template Koeller, Casey, Dancis, J. glutathione the ferritin IRE (Fig. 1) plus the 3' nucleotides helpful suggestions; and E. Perry for manuscript responsible for the reducing potential of the UCUCUCUCU24. The reaction included 3 mM preparation. cytosol. A study on the reversible oxidation- biotin-11-uridine triphosphate (UTP) (Bethesda reduction of co- Research Laboratories) and 1 mM UTP, in addition 3-hydroxy-3-methylglutaryl to [a-32P]guanosine triphosphate. The RNA was 12 October 1988; accepted 7 February 1989 enzyme A reductase demonstrates that oxi- dized sulfhydrylscan exist and even predom- inate within the cytosolic glutathione redox buffer system (23). Two factors can deter- mine the redox state of a protein sulfhydryl Isolation of a cDNA Clone Derived from a Blood- within the cytosol. One is the ratio of Borne Non-A, Non-B Viral Hepatitis Genome reduced to oxidized glutathione, which can change significantly under physiologic con- ditions (23). The second is the oxidation QuI-LIM CHOO, GEORGE KUO, AMY J. WEINER, LACY R. OVERBY, DANIEL W. MICHAEL HOUGHTON equlibrium constant (Kox) for a particular BRADLEY, sulfhydrylgroup within a protein. Equilibri- um constants for protein sulfhydryls can A random-primedcomplementary DNA librarywas constructedfrom plasmacontaining vary over many orders of magnitude, reflect- the uncharacterizednon-A, non-B hepatitis (NANBH) agent and screenedwith serum ing the effects of the local environment from a patientdiagnosed with NANBH. A complementaryDNA clone was isolatedthat around the cysteinyl moiety on its Kox (24). was shown to encode an antigen associatedspecifically with NANBH infections. This These local effects may reflect the stabiliza- clone is not derived from host DNA but from an RNA molecule present in NANBH tion or destabilization of the thiolate anion. infectionsthat consists of at least 10,000 nucleotidesand that is positive-strandedwith Conformational changes that alter this local respectto the encoded NANBH antigen. These data indicatethat this clone is derived environment can therefore affect the Kox of from the genome of the NANBH agent and are consistentwith the agent being similarto a particular cysteine sulfhydryl group. In the togaviridaeor flaviviridae.This molecularapproach should be of great value in the this way allosteric effectors can perturb the isolationand characterizationof other unidentifiedinfectious agents. Kox of sulfhydryls on specific proteins and thereby alter the redox state of the protein, ITH THE DEVELOPMENTOF SPE- readily transmissible to chimpanzees (7, 8). even in the presence of a constant cytosolic cific diagnostics for the hepatitis A major impediment to progress in studies redox buffer. Our data on the IRE-BP pro- A virus (HAV) and the hepatitis of this virus has been that despite intensive vide an example of the utility of oxidation- B virus (HBV) in the 1970s, it became clear work, conventional immunological methods reduction as a reversible covalent modifica- that most cases of hepatitis arising from have consistently failed to identify specific tion in the regulation of cellular protein blood transfusion were not caused by infec- viral antibodies and antigens (5, 6). Al- function. tions with these or other known viral agents though this failure could be interpreted in (1-4). Despite over a decade of research, the terms of a lack of viral antibody, we consid- REFERENCES AND NOTES agent or agents responsible for this so-called 1. R. Schleif, Science241, 1182 non-A, non-B remains (1988). hepatitis (NANBH) Q.-L. Choo, G. Kuo, A. J. Weiner, L. R. Overby, M. 2. P. J. Grabowski and P. A. ibid. 1294 Sharp, 233, unidentified (5, 6), although there is evi- Houghton, Chiron Corporation, 4560 Horton Street, (1986); V.