MEMBRANE ASYMMETRY IN APOPTOTIC CELLS, SIGNALS ASSOCIATED WITH PHAGOCYTOSIS AND THEIR IMPACT ON HIV TRANSCRIPTION

Open Access
- Author:
- Gekonge, Bethsebah Nyankomba
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- November 30, 2005
- Committee Members:
- Andrew Thomas Henderson, Committee Chair/Co-Chair
Robert Allen Schlegel, Committee Chair/Co-Chair
Andrea Marie Mastro, Committee Member
Pamela Hankey Giblin, Committee Member - Keywords:
- MACROPHAGE
PHOSPHATIDYLSERINE
APOPTOSIS
PHAGOCYTOSIS
HIV - Abstract:
- Transbilayer membrane phospholipid asymmetry is the defining feature between healthy viable cells and their apoptotic counterparts. Healthy cells maintain all their phosphatidylserine (PS) and a large proportion of their phosphatidylethanolamine (PE) in the inner leaflet of the plasma membrane. Loss of asymmetry and exposure of PS on the cell surface targets apoptotic cells for destruction by macrophages. An aminophospholipid translocase is responsible for maintaining this asymmetry. Since all cells maintain this asymmetric distribution, all cells must have a plasma membrane aminophospholipid translocase; however, the protein responsible for this activity has never been identified. Subfamily IV P-type ATPases comprise amphipathic transporters whose members have yet undetermined function. If a type IV P-type ATPase were the plasma membrane aminophospholipid translocase, it should be expressed in all cells. Using RT-PCR to measure gene expression, I demonstrate that multiple subfamily IV P-type ATPase members are simultaneously expressed in a single mammalian cell type. Of the ten genes analyzed, 1c, 1d, 2a, 2b, 5c, 6f, 6g and 6h were expressed in J774 cells, NIH 3T3 cells, and PC12 cells, suggesting a more general or housekeeping role for these genes; however, these genes also stand as candidates for the plasma membrane aminophospholipid translocase. 1a was expressed in J774 cells and PC12 cells and not in the NIH 3T3 cells, suggesting that 1a may have a specialized function in these cells. PS expression on the surface of apoptotic cells is involved in their removal from tissue in order to maintain overall homeostasis. The engagement of phagocytic receptors by ligands such as PS found on the apoptotic cell surface results in the activation of signaling cascades that facilitate engulfment. Macrophages and CD4+ T cells are the primary targets of HIV infection; however, unlike HIV infected macrophages which are invulnerable to the cytopathic effects of HIV, infected CD4+ T cells as well as uninfected bystander T cells undergo apoptosis. Consequently, these infected apoptotic cells as well as the HIV virions that are released from them have external PS. I examined how PS associated with virions and apoptotic cells influences HIV replication. Using annexin V and PS vesicles to mask, or compete with virus-associated PS, respectively, I show that PS inhibits HIV infection at a step prior to integration but following the generation of early reverse transcription products such as strong stop DNA. These observations suggest that PS initiated signals participate in HIV infection. Moreover, apoptotic cells inhibited HIV transcription in macrophages that have an established infection. This ability of apoptotic cells to suppress transcription was independent of PS, signifying that signals triggered following the interaction between an undetermined recognition ligand on the apoptotic cell surface and phagocytic receptor on the macrophage surface might affect HIV infection of monocytic cells. Furthermore, Elmo, a key signaling molecule that is associated with phagocytosis inhibited HIV transcription in infected macrophages, demonstrating that signaling events associated with phagocytosis of apoptotic cells participate in the establishment of HIV infection and provirus transcription.