These investigations will further characterize the significance and role of an endogenous HTLV-I-related viral element termed HRES 1/1. HRES was recently discovered in our laboratory and isolated from the cloned T-cells of a patient with type II mixed cryoglobulinemia by low stringency hybridization to the HTLV-I gag region pMA-I. Based on the presence of a transcriptionally active ORF which codes for putative p25 or p15 gag-like proteins, the significance of this genomic locus and the potential for its activation in autoimmune diseases and the dysproteinemias will be studied.
The first aim will be to determine the transcriptional and translational state of the 1/1 locus. The mRNA transcripts and the transcriptional activity of the locus will be analyzed in human lymphocytes or commercially available (ATCC) cell lines representing different states of cellular activation and/or stages of differentiation by Northern blotting, and nuclear run-on and run-offs. Attempts will be made to determine the function of the p25 and p15 proteins encoded by the ORFS or HRES 1/1. Segments of the 1/1 ORF encoding p15 and p25 will be cloned into prokaryotic expression vectors (pEV), and the produced proteins identified by SDS-PAGE. Micro amino terminal amino acid sequence analysis will be used to confirm that the protein produced is the same as that deduced from the gene sequence. In addition, pEV peptides corresponding to selected regions of p25 and p15 will be synthesized and used to raise murine monoclonal and rabbit polyclonal antibodies specific for these domains. In order to determine if the 1/1 locus has pathophysiological significance, the state of activation and demethylation of the HRES 1/1 locus and the production of p25 and/or p15 will be studied in cloned subsets of B-cells with and without autoantibody activity and in cloned T-cells, from patients with diseases such as rheumatoid arthritis, Sjogren's and Felty's syndromes, types I and II essential cryoglobulinemia, and common variable immunodeficiency, at different phases during these illnesses. Conversely, in order to determine if the p25 and p15 proteins serve as autoantigens, the patient sera will be tested for antibody activity to these proteins. Finally, the presence of other HRES 1/1 endogenous viral sequences will be searched for using 1/1 probes and screening human genomic DNA under conditions of varying stringency. The goal is to determine the biological and pathological significance of this newly defined genomic locus.
| Leddy, J P; Wilkinson, S L; Kissel, G E et al. (1994) Erythrocyte membrane proteins reactive with IgG (warm-reacting) anti-red blood cell autoantibodies: II. Antibodies coprecipitating band 3 and glycophorin A. Blood 84:650-6 |
| Leddy, J P; Falany, J L; Kissel, G E et al. (1993) Erythrocyte membrane proteins reactive with human (warm-reacting) anti-red cell autoantibodies. J Clin Invest 91:1672-80 |
| Whartenby, K A; Insel, R A; Freeman, S M et al. (1993) Oncogene elements within an endogenous retrovirus. Acta Virol 37:113-22 |
| Dragone, L; Pallant, A; Insel, R et al. (1992) CD43 is expressed normally on Wiskott-Aldrich-derived lymphocytes. Exp Clin Immunogenet 9:130-40 |
