Open Collections

Intracellular inhibition of immune dysfunction induced by HIV-I NEF protein

University of British Columbia

Current 'cocktail-therapy' toward HIV-1 infection using reverse transcriptase and protease inhibitors have been successful in controlling the viral growth, but not very effective in eradicating the reservoir of HIV-1 infected cells. It is a new challenge for H IV therapy to find ways to remove the virus reservoir that is composed of latently infected CD4+ T cells carrying integrated provirus. A potential new therapeutic target is Nef, a HIV-1 viral protein that downregulates class I M H C and by doing so it enables infected cells to elude killing by cytotoxic T lymphocytes. In this thesis research, intracellular inhibition of Nef-mediated downregulation of CD4 and MHC-1 molecules was studied using recombinant single-chain antibodies (ScFvs) and a dominant-negative Hck. Several anti-Nef single-chain antibodies were first constructed. A l l retained the binding activity of their corresponding parental monoclonal antibodies when expressed intracellularly. However, ScFv expression was unable to inhibit CD4 or MHC-1 downregulation induced by Nef. This indicated that the intracellular binding of ScFv with Nef and the following Nef sequestration may not be sufficient to prevent the receptor downregulation events induced by Nef. The expression of molecules capable of binding to epitopes in Nef, that are implicated specifically in receptor modulation, may be required for these effects. A dominant-negative form of Hck protein-tyrosine kinase, DN-Hck, composed of the Hck amino terminal region and its SH3 and SH2 domains, was then studied as a potential candidate for preventing MHC-1 downregulation; it is known that the Hck SH3 domain binds Nef with a very high affinity (Kd=0.25 uM). In addition, the SH3-binding motif in Nef, PXXP78 is also a major determinant in downregulation of MHC-1. It was demonstrated that DN-Hck was able to block Nef-induced downregulation of class I M H C surface expression in human cells. This effect required a functional SH3 domain, as it was not evident in cells that expressed DN-Hck-W93F, an SH3 domain mutation that results in diminished binding affinity for Nef. The results in this thesis research thus support a model that DN-Hck prevents Nef-induced class I downregulation by blocking the interaction between Nef and an as yet unidentified SH3- containing cellular protein that is capable of coupling Nef to the MHC-1 molecule. Upon binding with Nef, this cellular protein might recruit class I M H C molecules via a specific interaction with their cytoplasmic motifs, which in turn routes these molecules towards an intracellular degradation pathway. The SH3- binding region of Nef therefore represents a new target for therapeutic intervention in individuals infected with HIV-1.

Thesis/Dissertation

application/pdf

2009-07-20

For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.

http://hdl.handle.net/2429/11048

Pathology

University of British Columbia

UBCV

DSpace