Date sent: Mon, 14 Aug 2000 Source: The American Journal of Medicine Volume 108, Issue 2, pages 99-105 Elsevier - ScienceDirect Date: February 2000 URL: http://www.sciencedirect.com/science/journal/00029343 [Clinical Studies] A 37 kDa 2-5A binding protein as a potential biochemical marker for chronic fatigue syndrome(*1) ------------------------------------------------------------------- Kenny De Meirleir MD, PhD(a), Catherine Bisbal PhD(b), Isabelle Campine MD(a), Pascale De Becker, PT(a), Tamim Salehzada PhD(b), Edith Demettre MSc(b) and Bernard Lebleu PhD(b) a Department of Human Physiology and Medicine (KDM, IC, PDB), Vrije Universiteit Brussel, Brussels, Belgium b Molecular Genetics Institute (CB, TS, ED, BL), Universite Montpellier II, Montpellier, France Received 3 August 1998; revised 15 July 1999; accepted 15 July 1999. Available online 2 February 2000. Abstract PURPOSE: Recent studies have revealed abnormalities in the ribonuclease L pathway in peripheral blood mononuclear cells of patients with the chronic fatigue syndrome. We conducted a blinded study to detect possible differences in the distribution of 2-5A binding proteins in the cells of patients with chronic fatigue syndrome and controls. PATIENTS AND METHODS: We studied 57 patients with chronic fatigue syndrome and 53 control subjects (28 healthy subjects and 25 patients with depression or fibromyalgia). A radioactive probe was used to label 2-5A binding proteins in unfractionated peripheral blood mononuclear cell extracts and to compare their distribution in the three groups. RESULTS: A 37 kDa 2-5A binding polypeptide was found in 50 (88%) of the 57 patients with chronic fatigue syndrome compared with 15 (28%) of the 53 controls (P <0.01). When present, the amount of 37 kDa protein was very low in the control groups. When expressed as the atio of the 37 kDa protein to the 80 kDa protein, 41 (72%) of the 57 patients with chronic fatigue syndrome had a ratio >0.05, compared with 3 (11%) of the 28 healthy subjects and none of the patients with fibromyalgia or depression. CONCLUSION: The presence of a 37 kDa 2-5A binding protein in extracts of peripheral blood mononuclear cells may distinguish patients with chronic fatigue syndrome from healthy subjects and those suffering from other diseases. ----------------------------------------------------------------- Chronic fatigue syndrome is characterized by disabling fatigue of definite onset that persists for at least 6 months. It is accompanied by nonspecific symptoms including low-grade fever, myalgias, neurocognitive dysfunction, and sleep disturbance [1, 2 and 3]. The prevalence of chronic fatigue syndrome has been estimated from 0.1% to 2.6% [4, 5, 6 and 7]. Several studies have reported nonspecific abnormalities, such as immune activation, reactivation of viruses, and abnormal cytokine production, in these patients [8, 9 and 10]. A diagnostic test is not yet available, and the diagnosis is by excluding other potential explanations, often after extensive clinical evaluation. The cause of this disease is unknown, and there is no effective treatment. Chronic fatigue syndrome is often associated with an infectious disease; 66% to 90% of patients report an onset following a viral infection [11]. Viruses such as Epstein-Barr virus [12, 13 and 14], enteroviruses [15 and 16], retroviruses [17], and human herpes virus type 6 [6 and 9] have been suggested as possible causes. However, several studies have found no differences in antibody titers to a variety of viruses when comparing patients with chronic fatigue syndrome and controls [18]. The viral hypothesis has led several groups to investigate the possibility that there is dysregulation of antiviral responses in chronic fatigue syndrome. Defective or overreactive interferon responses could be responsible for some of its symptoms. In particular, the 2-5A synthetase/ribonuclease L pathway (Figure 1) is central to the antiviral activity of interferon [19]. Interferons increase the expression of 2-5A synthetase genes, which increase the production of 2-5A oligonucleotides. These oligonucleotides bind to ribonuclease L, which degrades viral RNA, leading to an antiviral effect [20]. Ribonuclease L is also regulated by ribonuclease L inhibitor [21, 22 and 23]. Increased levels of bioactive 2-5A oligonucleotides and ribonuclease L activity have been found in peripheral blood mononuclear cell extracts from patients with chronic fatigue syndrome [24, 25 and 26]. [Figure 1. Outline of the ribonuclease L/ribonuclease L inhibitor pathway.] More recently, the labeling of peripheral blood mononuclear cell proteins with a 2-5A photoaffinity probe and their immunoprecipitation have revealed a novel low molecular weight form of ribonuclease L in a small group of severely disabled patients with chronic fatigue syndrome [24 and 27]. In this study, we investigated the distribution of 2-5A binding proteins in a large cohort of patients with chronic fatigue syndrome as well as in control groups consisting of healthy subjects and of patients with fibromyalgia [28] or depression [29]. Methods SELECTION OF THE STUDY GROUPS The patient group consisted of 57 patients with persistent fatigue who fulfilled the standard criteria for chronic fatigue syndrome [1 and 2]. Fifty-three patients had a history of acute or subacute postviral onset; in 4 the syndrome had started gradually. The mean (p/m SD) duration of illness was 7 p/m 6 years. Patients with chronic fatigue syndrome were recruited from a university-based fatigue clinic (Vrije Universiteit Brussel) in Brussels, Belgium. The control groups consisted of healthy subjects and patients with fibromyalgia or depression. Subjects selected for the control groups did not fulfill the criteria for chronic fatigue syndrome [1 and 2]. The 28 healthy subjects were recruited from the hospital and university staff, their acquaintances, and students from nonmedical faculties. They were generally "noncontact controls," except for 2 subjects who had frequent contact with either chronic fatigue syndrome patients or their blood specimens. The criteria for the classification of fibromyalgia (n = 11) were widespread pain in combination with tenderness at 11 or more of 18 specific tender point sites [28]. The depressed patients (n = 14) fulfilled the DSM-IV criteria, axis I [29]. The fibromyalgia and depressed patients were recruited from the Free University Hospital in Brussels. All subjects gave informed consent. All subjects underwent an extensive medical evaluation, consisting of a standard physical examination and medical history, a symptom checklist, routine laboratory tests, and a structured psychiatric interview. The laboratory tests included a complete blood cell count, determination of the erythrocyte sedimentation rate, a serum electrolyte panel, measures of renal, hepatic, and thyroid function, and rheumatological and virological screenings including C-reactive protein levels. These biochemical parameters were within normal range for chronic fatigue syndrome patients, healthy controls, and patients suffering from depression or fibromyalgia. Patients diagnosed with disorders such as malignancy, autoimmune disease, inflammatory disease, or other pathological conditions were excluded from the study. ISOLATION OF PERIPHERAL BLOOD MONONUCLEAR CELLS Venous blood samples were drawn between 8 AM and 9 AM. The samples were stored, and the procedure for isolating peripheral blood mononuclear cells was started within 2 hours. Heparinized whole blood was diluted 1:1 with phosphate-buffered saline. Two volumes of diluted blood were overlaid on 1 volume of Ficoll Hypaque (Sigma-Aldrich, St. Louis, Missouri) (density of 1.080) and centrifuged at 20 C at 500g for 30 minutes. The peripheral blood mononuclear cell layer was removed and washed with 5 volumes of phosphate-buffered saline (centrifuged at 20 C at 500g for 15 minutes). The isolated peripheral blood mononuclear cell pellets were resuspended in 5 mL red blood cell lysing buffer (155 mM NH4Cl, 10 mM NaHCO3, pH 7.4, 0.1 mM EDTA), kept on ice for 5 minutes, and centrifuged (20 C, 500g, 10 minutes). After repeating the last step, the peripheral blood mononuclear cells were washed again with phosphate-buffered saline and centrifuged in a microcentrifuge tube (20 C, 8,000g, 5 minutes). The peripheral blood mononuclear cells were frozen at -80 C until use. Coded samples were forwarded on dry ice to the Molecular Genetics Institute in Montpellier and analyzed for 2-5A dependent binding proteins as described below. PERIPHERAL BLOOD MONONUCLEAR CELL EXTRACTS Extracts of peripheral blood mononuclear cells from patients with chronic fatigue syndrome and controls were prepared [30]. Briefly, the cells were resuspended in 2 volumes of hypotonic buffer (20 mL Hepes pH 7.5, 10 mM K acetate, 1.5 mM Mg acetate, 0.5% [v/v] ethylphenylpolyethylenglycol [Nonidet P 40] detergent, 1 mM para-methyl sulfonyl fluoride, 10 mug/mL aprotinin, 150 mug/mL leupeptin), disrupted by repeated pipeting with a micropipet, and centrifuged at 10,000g for 10 minutes at 4 C. The protein concentration in the supernatant was determined by spectrophotometry. SYNTHESIS AND LABELING OF THE 2-5A PROBE 2-5A was synthesized enzymatically from ATP in extracts from human alpha/beta interferon-treated HeLa cells [31 and 32]. Milligram amounts were obtained and fractionated by high-pressure liquid chromatography on a diethylaminoethyl cation exchange column (TSK-DEAE, Toso-Haas), and the purity of each fraction was verified by chromatography on a mu-Bondapak C18 column. The labeled 2-5A probe was synthesized by ligation of (32P) pCp (specific activity: 3000 Ci/mmole) to the 2',3'-terminus of 2-5A4 with T4 RNA ligase. The terminal 3'-phosphate group was removed by alkaline phosphatase. The pH was adjusted to 4.7 and the 3' ribose residue was oxidized with 10 mM sodium metaperiodate. RADIOCOVALENT AFFINITY LABELING AND ANALYSIS OF 2-5A BINDING PROTEINS The 3'-oxidized 2-5A (32P) pCp (3000 Ci/mmole, 20,000 cpm) probe was incubated with peripheral blood mononuclear cells extracts (200 mug protein) for 20 minutes at 4 C and for a further 20 minutes at room temperature with 20 mM sodium cyanoborohydride. The extract was then fractionated on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate as a denaturing agent (PAGE-SDS) [33]. Labeled polypeptides were detected by autoradiography and the autoradiograms were analyzed by scanning densitometry [30 and 31]. ASSAY A sensitive assay allowing the specific identification of 2-5A binding proteins in biological samples without a fractionation step was used. Unfractionated peripheral blood mononuclear cell extracts were prepared in the presence of protease inhibitors to avoid artifacts from differential proteolysis. Extracts (200 mug of protein) were incubated with radiolabeled 2-5A (32p) pCp (specific activity: 3000 Ci/mmole) in conditions allowing the covalent linkage of the probe to all 2-5A binding protein species. Proteins were then fractionated by polyacrylamide electrophoresis using denaturing conditions. The size distribution of 2-5A labeled polypeptides was visualized by autoradiography, and the relative abundance of the various bands was calculated by densitometric analysis using the Intelligent Quantifier Program (Bio Image Systems Corporation, Ann Arbor, Michigan). STATISTICAL ANALYSIS The relative amounts of the 37 kDa, 40 kDa, and 80 kDa 2-5A binding proteins in the control groups and the patients with chronic fatigue syndrome were compared using the Mann-Whitney U Test. Statistical significance was set at P <0.05. The proportions of patients in the different groups with a particular protein fraction were compared with the chi-square test. Results The gender and age distributions of the patients with chronic fatigue syndrome and the controls were similar (Table). Figure 2A shows the 2-5A binding proteins identified in 10 representative healthy subjects and 10 representative patients with chronic fatigue syndrome. Figure 2B illustrates the distribution of 2-5A binding proteins in patients with depression or fibromyalgia. All samples contained the major 80 kDa 2-5A binding polypeptide that is characteristic of ribonuclease L under protein denaturing conditions. Its abundance was not correlated with chronic fatigue syndrome. However, a 37 kDa 2-5A binding protein was more frequently found in peripheral blood mononuclear cell extracts from patients with chronic fatigue syndrome than in controls. In addition, a 40 kDa band, of lower intensity, was detected in greater quantity in patients with chronic fatigue syndrome compared with the controls. Table. Gender and Age Distributions among Study Participants -------------------------------------------------------------------- Characteristic Patienta with CFS Healthy Patiens w. Patients w. with CFS (n=57) Controls Depession FM (n=11) -------------------------------------------------------------------- Female gender 47 (82%) 20(71%) 10(71%) 9(82%) Age in years 36 p/m 11 31 p/m 8 39 p/m 12 42 p/m 7 (mean p/m SD) -------------------------------------------------------------------- [Figure 2. Distribution of 2-5A binding proteins in extracts of peripheral blood mononuclear cells from patients with chronic fatigue syndrome and controls: analysis by polyacrylamide gel electrophoresis. A. Scans of the autoradiograms of a representative cohort of 10 healthy subjects (left panel) and 10 patients with chronic fatigue syndrome (right panel). B. Scans of the autoradiograms of patients with depression (left panel) or fibromyalgia (right panel). 2-5A (32P) pCp labeled protein bands are indicated at the left side of the autoradiograms. Molecular masses have been estimated with reference to molecular weight markers (not shown).] The 37 kDa 2-5A binding protein band was seen in 50 (88%) of 57 patients with chronic fatigue syndrome, in 9 (32%) of 28 healthy controls, in 2 (14%) of 14 depressed patients, and in 4 (38%) of the 11 patients with fibromyalgia (Figure 3). The 2 identified contact controls had elevated 37 kDa values. The 40 kDa band was also more common in patients with chronic fatigue syndrome. The quantities of the 37 kDa and 40 kDa bands were greater (P <0.0001 for both) in the patients with chronic fatigue syndrome than in the control groups. In addition, the quantity of the 80 kDa band was greater in the patients with chronic fatigue syndrome (P <0.05). [Figure 3. Distribution of 2-5A binding protein in patients with chronic fatigue syndrome and controls. A. The amounts of 80 kDa, 40 kDa, and 37 kDa labeled material were plotted in arbitrary units for each sample in patients with chronic fatigue syndrome and in controls (healthy, depressed, fibromyalgia). Each point represents a study subject. The 2 contact controls in the healthy group are identified by square symbols. B. A 37 kDa/80 kDa x 10 ratio was calculated and plotted for each sample. The dotted line corresponds to the 0.5 threshold value. The 2 contact controls in the healthy group are identified by a square symbol.] The ratio of the 37 kDa band to the 80 kDa band, multiplied by 10, was calculated for each sample. Values in the control groups were =<0.5, except for the 2 contact controls, while 41 (72%) of the patients with chronic fatigue syndrome had values >0.5. The three polypeptides identified by this assay (the 80 kDa, 40 kDa, and 37 kDa bands) were authentic 2-5A binding proteins (Figure 4). Indeed, binding of the radioactive 2-5A probe could be reduced by unlabeled 2-5A, but not by ATP. Incubations of peripheral blood mononuclear cell extracts with recombinant ribonuclease L inhibitor prevented the binding of the radioactive 2-5A probe to all three proteins (as shown for one sample in Figure 4). As an additional control, peripheral blood mononuclear cell extracts from 3 patients with chronic fatigue syndrome and 1 control were incubated under various conditions before 2-5A covalent binding. As shown in Figure 5 for 1 representative patient (lane 2 in Figure 2A, right panel), all three 2-5A binding proteins disappeared upon incubation of the extract. The 80 kDa 2-5A binding protein appears to be more sensitive to proteolytic degradation than the 40 kDa and 37 kDa species (Figure 5). The disappearance of the 80 kDa protein band was not accompanied by the concomitant accumulation of the 37 kDa protein band. [Figure 4. 2-5A binding specificity. Peripheral blood mononuclear cell extracts (200 mug protein) were incubated with the radioactive 2-5A(32P)pCp probe and either unlabeled 2-5A (p3A4) (100 nM), ATP (1 muM), or recombinant ribonuclease L inhibitor (54 pg) as indicated.] [Figure 5. Stability of 2-5A binding proteins to proteases. Peripheral blood mononuclear cell extracts (200 mug protein) from a representative patient with chronic fatigue syndrome (corresponding to lane 2 in Figure 2A, right panel) were incubated at 30 C (panel A) or at 37 C (panel B) for the indicated times before the addition of the radioactive 2-5A(32P)pCp probe.] Discussion Previous studies have shown that there is altered expression of several components of the interferon-regulated antiviral pathways in blood cells isolated from patients with chronic fatigue syndrome. In particular, a previously unknown low molecular weight form of ribonuclease L, a key enzyme in the interferon-mediated antiviral response, has been detected in some patients [27]. In keeping with these observations, we found a low molecular weight (37 kDa) 2-5A binding protein in patients with chronic fatigue syndrome, along with 80 kDa and 40 kDa 2-5A binding proteins. These results, using a different technique in a larger group of patients and controls, confirm those of Suhadolnik et al [27]. The 37 kDa 2-5A binding protein that we described probably corresponds to the previously described low molecular weight ribonuclease L species [27]. This 37 kDa 2-5A binding protein has not been reported in healthy subjects or in other diseases. Because protease inhibitors were included at the time of peripheral blood mononuclear cell lysis, it is unlikely that the 37 kDa 2-5A binding protein arises from the increased breakdown of the 80 kDa ribonuclease L during preparation and processing of the extracts. Incubating the extracts at 37 C did not result in the conversion of the 80 kDa species to the 37 kDa or 40 kDa species (Figure 5). The 80 kDa species rapidly disappeared upon incubation, whereas the low molecular weight forms were more stable. Therefore, the 37 kDa 2-5A binding protein does not arise from a protease-mediated degradation of the 80 kDa protein in the laboratory. Nevertheless, cellular or viral proteases that could accumulate or be activated in chronic fatigue syndrome might be responsible for the conversion of the 80 kDa ribonuclease L to a lower molecular weight form. Other possibilities, such as a defect of mRNA processing, are also possible. This low molecular weight 2-5A binding protein may be useful as a biological marker for chronic fatigue syndrome, perhaps helping to distinguish it from other disorders, including fibromyalgia and severe depression, in which it appears to be uncommon (Figure 2 and Figure 3). However, the specificity of the 37 kDa 2-5A binding protein cannot be estimated precisely because we studied a relatively small number of subjects. The biological significance of these 2-5A binding protein in patients with chronic fatigue syndrome is not known. Additional studies are under way to establish whether the ribonuclease L enzyme dysfunction in patients with chronic fatigue syndrome is associated with a particular stage of the illness or if it fluctuates over time. Acknowledgements The authors thank Dr. Christian Demanet, Prof. Micheline Kirsch-Volders, Dr. Elke Van Steenberge, Prof. Krista Vandenborne, Lieve De Hauwere, and Angeline De Troyer for use of laboratory facilities and for research assistance in the preparation of this report. We also would like to thank Prof. Robert Suhadolnik and Dr. Dan Peterson for communicating and discussing unpublished data. References 1. G.P. Holmes, J.E. Kaplan, N.M. Gantz et al., Chronic fatigue syndrome: a working case definition. Ann Intern Med 108 (1988), pp. 387-389. 2. K. Fukuda, S.E. Straus, I. Hickie et al., The chronic fatigue syndrome: a comprehensive approach to its definition and study. International Chronic Fatigue Syndrome Study Group. Ann Intern Med 121 (1994), pp. 953-959. 3. P.O. Behan, W.M. Behan and E.J. Bell, The postviral fatigue syndrome - an analysis of the findings in 50 cases. J Infect 10 (1985), pp. 211-222. 4. D. Buchwald, P. Umali, J. Umali et al., Chronic fatigue and the chronic fatigue syndrome: prevalence in a Pacific Northwest health care system. Ann Intern Med 123 (1995), pp. 81-88. 5. D.W. Bates, W. Schmitt, D. Buchwald et al., Prevalence of fatigue and chronic fatigue syndrome in a primary care practice. Arch Intern Med 153 (1993), pp. 2759-2765. 6. S. Wesseley, T. Chalder, S. Hirsch et al., The prevalence and morbidity of chronic fatigue and chronic fatigue syndrome: a prospective primary care study. Am J Pub Health 87 (1997), pp. 1449-1455. 7. A.R. Lloyd, I. Hickie, C.R. Boughton et al., Prevalence of chronic fatigue syndrome in an Australian population. Med J Aust 153 (1990), pp. 522-528. 8. A.L. Landay, C. Jessop, E.T. Lennette and J.A. Levy, Chronic fatigue syndrome: clinical condition associated with immune activation. Lancet 338 (1991), pp. 707-712. 9. D. Buchwald, P.R. Cheney, D.L. Peterson et al., A chronic illness characterized by fatigue, neurologic and immunologic disorders, and active human herpesvirus type 6 infection. Ann Intern Med 116 (1992), pp. 103-113. 10. C.C. Chao, E.N. Janoff, S.X. Hu et al., Altered cytokine release in peripheral blood mononuclear cell cultures from patients with the chronic fatigue syndrome. Cytokine 3 (1991), pp. 292-298. 11. A. Schluederberg, S.E. Straus, P. Peterson et al., NIH conference. Chronic fatigue syndrome research. Definition and medical outcome assessment. Ann Intern Med 117 (1992), pp. 325-331. 12. D. Buchwald and A.L. Komaroff, Review of laboratory findings for patients with chronic fatigue syndrome. Rev Infect Dis (1991), pp. S12-S18. 13. J.F. Jones, C.G. Ray, L.L. Minnich et al., Evidence for active Epstein-Barr virus infection in patients with persistent, unexplained illnesses: elevated anti-early antigen antibodies. Ann Intern Med 102 (1985), pp. 1-7. 14. G. Miller, E. Grogan, D. Rowe et al., Selective lack of antibody to a component of EB nuclear antigen in patients with chronic active Epstein-Barr virus infection. J Infect Dis 156 (1987), pp. 26-35. 15. L.C. Archard, N.E. Bowles, P.O. Behan et al., Postviral fatigue syndrome: persistence of enterovirus RNA in muscle and elevated creatine kinase. J R Soc Med 81 (1988), pp. 326-329. 16. G.E. Yousef, E.J. Bell, G.F. Mann et al., Chronic enterovirus infection in patients with postviral fatigue syndrome. Lancet 1 (1988), pp. 146-150. 17. E. DeFreitas, B. Hilliard, P.R. Cheney et al., Retroviral sequences related to human T-lymphotropic virus type II in patients with chronic fatigue immune dysfunction syndrome. Proc Natl Acad Sci USA 88 (1991), pp. 2922-2926. 18. C.M. Swanink, W.J. Melchers, J.W.M. van der Meer et al., Enteroviruses and the chronic fatigue syndrome. Clin Infect Dis 19 (1994), pp. 860-864. 19. G.C. Sen and R.M. Ransohoff, Interferon-induced antiviral actions and their regulation. Adv Virus Res 42 (1993), pp. 57-102. 20. C. Bisbal, RNase L: effector nuclease of an activatable RNA degradation system in mammals. Prog Mol Subcell Biol 18 (1997), pp. 19-34. 21. C. Bisbal, C. Martinand, M. Silhol et al., Cloning and characterization of a RNAse L inhibitor. A new component of the interferon-regulated 2-5A pathway. J Biol Chem 270 (1995), pp. 13308-13317. 22. C. Martinand, T. Salehzada, M. Silhol et al., RNase L inhibitor (RLI) antisense constructions block partially the down regulation of the 2-5A/RNase L pathway in encephalomyocarditis-virus-(EMCV)-infected cells. Eur J Biochem 254 (1998), pp. 238-247. 23. C. Martinand, C. Montavon, T. Salehzada et al., RNaseL inhibitor is induced during HIV-1 infection and down regulates the 2-5A pathway in H9 cells. J Virol 73 (1999), pp. 290-296. 24. Horvath SE, Peterson DL, Suhadolnik RJ. Characterisation of RNase L dysfunction in peripheral blood mononuclear cell extracts from patients with chronic fatigue syndrome. Proceedings of the Fourth International AACFS Research & Clinical Conference on CFS, Boston, 1998:45. 25. R.J. Suhadolnik, N.L. Reichenbach, P. Hitzges et al., Upregulation of the 2-5A synthetase/RNase L antiviral pathway associated with chronic fatigue syndrome. Clin Infect Dis (1994), pp. S96-S104. 26. R.J. Suhadolnik, N.L. Reichenbach, P. Hitzges et al., Changes in the 2-5A synthetase/RNase L antiviral pathway in a controlled clinical trial with poly(I)-poly(C12U) in chronic fatigue syndrome. In Vivo 8 (1994), pp. 599-604. 27. R.J. Suhadolnik, D.L. Peterson, K. O'Brien et al., Biochemical evidence for a novel low molecular weight 2-5A-dependent RNase L in chronic fatigue syndrome. J Interferon Cytokine Res 17 (1997), pp. 377-385. 28. F. Wolfe, H.A. Smythe, M.B. Yunus et al., The American College of Rheumatology 1990 criteria for the classification of fibromyalgia. Arthritis Rheum 33 (1990), pp. 160-172. 29. Diagnostic and Statistical Manual of Mental Disorders (4th ed. ed.), American Psychiatric Association, Washington, DC (1994). 30. C. Bisbal, T. Salehzada, B. Lebleu and B. Bayard, Characterization of two murine (2'-5')(A)n-dependent endonucleases of different molecular mass. Eur J Biochem 179 (1989), pp. 595-602. 31. B. Bayard, C. Bisbal and B. Lebleu, Activation of ribonuclease L by (2'-5')(A)4-poly(L-lysine) conjugates in intact cells. Biochemistry 25 (1986), pp. 3730-3736. 32. D.H. Wreschner, R.H. Silverman, T.C. James et al., Affinity labelling and characterization of the ppp(A2'p)nA-dependent endoribonuclease from different mammalian sources. Eur J Biochem 124 (1982), pp. 261-268. 33. U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227 (1970), pp. 680-685. *1 Supported by grants from ARC and from the CNRS to Dr. Bernard Lebleu and from the CFIDS Association of America to Dr. Bernard Lebleu and Dr. Kenny De Meirleir. Requests for reprints should be addressed to Prof. Dr. K. De Meirleir, V.U.B. KRO gebouw-1, Laarbeeklaan 101, 1090 Brussel, Belgium -------- (c) 2000 Excerpta Medica Inc. (c) 2000 ScienceDirect