1. 1 Molecular Mechanisms for gp160-Enhanced Apoptosis Keith J. Micoli, Olga A. Mamaeva, George Pan, Eric Hunter and Jay M. McDonald University of Alabama at Birmingham, Departments of Pathology and Microbiology, Birmingham, AL 35294, USA Veteran’s Administration Medical Center, Birmingham, AL 35233, USA

2. 2 AIDS and Apoptosis HIV-1 infection is characterized by a progressive loss of immunocompetent cells. One proposed mechamism for this is accelerated apoptosis J Clin Immunol 15:217 (1995) Lymphocytes from infected individuals express elevated levels of Fas and Fas ligand J Clin Immunol 102: 79 (1998) Peripheral blood mononuclear cells (PBMCs) from HIV-infected patients are more sensitive to Fas- mediated apoptosis in vitro than PBMCs from healthy donors J Molec Med 73: 591 (1995)

3. 3 Fas receptor Fas is a cell surface receptor in the tumor necrosis factor (TNF) superfamily. Fas induces apoptosis when it binds Fas ligand. The physiological role of Fas-induced apoptosis is maintenance of tissue homeostasis.

4. 4 Calmodulin/Calcium and Fas- mediated Apoptosis Calmodulin is a multifunctional Ca 2+ modulated protein known to regulate the cell cycle, related cytoskeletal functions, and ion channel activity Apoptosis 5:133 (2000) Calmodulin is involved in regulation of apoptosis via Ca 2+ -dependent enzymes, such as calcineurin, death associated (DAP)- kinase, and the calcium-binding protein, ALG- 2 EMBO J 16:998 (1997) Calmodulin binds Fas directly JBC 279:5661 (2004)

5. 5 HIV-1, Calmodulin and Apoptosis The gp160 contains two calmodulin-binding sites in its c-terminus and co-immunoprecipitates with calmodulin. Calmodulin binding function is conserved among all known sequence variants of HIV-1 H9 cells transfected with gp160 show increased levels of calmodulin protein and mRNA gp160 with deleted calmodulin binding sites (gp160  147) does not increase levels of calmodulin and does not co-immunoprecipitate with calmodulin Transfection of gp160 but not gp160  147 results in increased Fas-mediated apoptosis, an effect blocked by calmodulin antagonists

6. 6 Hypothesis HIV-1 enhanced Fas-mediated apoptosis is dependent upon Env binding to calmodulin HIV-1 enhanced Fas-mediated apoptosis is dependent upon Env binding to calmodulin

7. 7 768 788 826 855 transmembrane domain calmodulin binding domain 826- DRVIEVVQGACRAIRHIPRRIRQGLERILL-855  SD1 (819-838)

8. 8 Prior Findings All point mutations in the CaM-binding domain result in decreased Fas- mediated apoptosis J. Vir, manuscript in review A peptide corresponding to the A835W mutation does not bind calmodulin JBC 275 p.1233, 2000 A835W mutation in stably-transfected cells fails to bind calmodulin or enhance Fas-mediated apoptosis JBC 275 p.1233, 2000

9. 9 Effect of gp160 point mutations on viral replication, calmodulin binding and apoptosis

10. 10 Methods Three point mutations A835W, A838I, and I842R and the corresponding region of HIV-1 HXB2 were engineered into the HIV-1 proviral clone, pNL4.3. Recombinant viruses were obtained after transfection into 293T cells.

11. 11 Methods H9 cells were infected with the env point mutants A835W, A838I, and I842R. Cell culture supernatants were collected at daily intervals following infection and viral p24 levels were determined, in triplicate, by ELISA. Viral proteins were immunoprecipitated with HIV+ patient serum, separated by SDS-PAGE and Western blotted for gp120 and calmodulin. Apoptosis was determined on days 5 and 12 post-infection by Hoechst staining and morphological analysis of nuclei.

12. 12 Effect of gp160 point mutations on HIV production in H9 cells

13. 13 WT A835W A838W Mock CaM gp160 gp120 Env Point Mutants Reduce Binding to Calmodulin IP Ab: HIV + serum WB Ab: gp120 (top) CaM (bottom)

14. 14 Env Point Mutants Reduce Binding to Calmodulin CaM gp160 gp120 A835W A838I I842R HXB2 Mock

15. 15 Apoptotic cells (percent of total)

16. 16

17. 17 Summary None of the point mutations of gp160 impaired viral production in vitro. Mutants A835W and A838W eliminated co- immunoprecipitation of Env and calmodulin. Mutant A838I partially abolished binding with calmodulin, and I842R had no effect on binding with calmodulin. Mutants A835W and A838W significantly decreased Fas-mediated apoptosis, and mutant I842R had no effect on Fas-mediated apoptosis. Importantly, there appears to be a direct link between the ability of gp160/gp41 to bind calmodulin and enhance Fas-mediated apoptosis.

18. 18 Conclusion Calmodulin binding to gp160/gp41 is necessary for enhanced fas-mediated apoptosis. Point mutations in the C-terminus of Env diminish calmodulin binding and apoptosis without enhancing viral replication. Interruption of this interaction by pharmaceutical means could disrupt the sequence of events leading to the CD4+ lymphocyte depletion and immune system failure.