Effects of the SIV(mac239) NEF protein in human CD4(+) T lymphocytes

The CD4 co-receptor functions as a signalling molecule important for the activation of T helper cells and is the viral receptor for the human and simian immunodeficiency viruses (HIV, SIV). Cell surface levels of CD4 are down-regulated on cells expressing the HIV or SIV encoded Nef protein. The nef gene of the simian immunodeficiency virus encodes a 32 kD myristylated protein that is expressed at high levels early after infection. The results presented in this thesis demonstrate that expression of SIV{dollar}sb{lcub}rm mac239{rcub}{dollar} Nef induces the degradation of endogeneous CD4. To examine the mechanism by which Nef mediates the down-modulation of CD4, the human CD4{dollar}sp+{dollar} T cell line CEM-SS was stably transduced with SIV{dollar}sb{lcub}rm mac239{rcub} nef.{dollar} CEM-SS cells expressing SIV Nef display dramatically reduced levels of cell surface CD4. CD4 is translated and transported through the ER and Golgi compartments at comparable rates in Nef-expressing and control cells. CD4 also arrives at the cell surface with similar kinetics in the absence or presence of Nef. However, following the rapid internalization of CD4 in Nef-expressing cells, CD4 accumulates in acidic late endosomal and lysosomal vesicles, as determined by indirect immunofluorescence staining and confocal microscopy. The degradation of CD4 is demonstrated by metabolic labelling and pulse-chase experiments. CD4 degradation in Nef-expressing cells occurs at acidic pH, and is dependent on the lysosomal hydrolase cathepsin D as well as other proteases. Electron microscopy studies reveal that the subcellular morphology of Nef-expressing cells is altered as compared to control cells. Specifically, the cytoplasm contains abundant levels of vesicles surrounded by membrane in the presence of Nef. These granules function along the endosomal/lysosomal pathway as defined by the vesicular protein contents as well as the uptake of nonspecific membrane tracer. The upregulation of acidic vacuoles in the presence of Nef is not dependent on expression of CD4. The Nef-induced endosomal and lysosomal structures are found in all human T cell lines examined, and occur in CEM-SS cells expressing SIV or HIV-1 Nef proteins.