Le Noury, Denise Anne2016-10-242016-10-242016-10-24http://hdl.handle.net/10539/21252A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, In fulfilment of the requirements for the degree of Doctor of Philosophy Johannesburg, February 2016Viral protein U (Vpu) is a versatile accessory protein of human immunodeficiency virus type 1 (HIV-1) and some simian immunodeficiency virus isolates. Amongst other functions, Vpu targets human CD4 for ubiquitination and proteasomal degradation. This process requires the presence of phospho-serine residues within the Vpu cytoplasmic domain in order to allow for the recruitment of beta-transducin repeat-containing protein (β-TrCP) and ultimately the E3 ligase complex. Similarly, it has been shown that the Vpu cytoplasmic domain also interacts with the human CD74 cytoplasmic region. CD74, the invariant chain of major histocompatibility complex class II (MHCII), is involved in the presentation of foreign antigens to CD4+ T cells. The Vpu/CD74 interaction may prevent the maturation of the MCHII complex resulting in the downmodulation of cell surface levels of MHCII. There are several structural similarities between the human CD4 and CD74 host proteins. Consequently, we hypothesised that HIV-1 Vpu targets human CD74 for proteasomal degradation through the binding of β-TrCP similar to that of Vpu-mediated CD4 proteasomal degradation. In view of this, this study aimed to delineate the binding interaction between HIV-1 Vpu and CD74 and to determine whether Vpu also targets CD74 for proteasomal degradation. An in silico homology study determined that the α-helices found within the cytoplasmic domains of CD4 and CD74 were similar with an RMSD of 1.437 Å. Recombinant Vpu::GFP fusion proteins were expressed in HEK 293T cells and purified in sufficient quantities and purity for use in subsequent in vitro assays. Using ELISAs, it was found that CD74 effectively prevented the binding of an anti-Vpu antibody, but not an anti-GFP antibody, to the Vpu::GFP fusion protein with an IC50 of 888.67 ± 130.08 nM. Further to this, using CD74 overlapping peptides of increasing lengths in combination with a Vpu overlapping peptide set, it was determined that the sequences “RIRERAEDSGNESEG” and “EDQKP” in the Vpu and CD74 cytoplasmic regions, respectively, contain the binding sites for this interaction. While the CD4 and CD74 cytoplasmic peptides yielded different responses after binding Vpu peptides, these two host proteins were shown to compete for binding to the Vpu protein. This provides evidence that steric hindrance has an effect on binding. Finally, total CD74 levels including cell membrane and intracellular fractions were significantly downregulated by Vpu at 24 and 48 hours (p = 0.0270 and 0.0062, respectively) as indicated by immunoprecipitation and flow cytometry in Vpu-transfected U937 cells. Notably, Vpu phosphorylation mutants (S52A and S52/56A) were unable to reduce CD74 levels within U937 cells, indicating that CD74 is not targeted for proteasomal degradation. In conclusion, this study has defined the specific Vpu/CD74 binding site and provides novel evidence that CD74 is downregulated in the presence of Vpu. Moreover, CD74 is not targeted for proteasomal degradation through the binding of β-TrCP and the recruitment of the E3 ligase complex. These findings have future relevance for HIV-1 treatment and management as CD74 downregulation is yet another mechanism by which HIV-1 impairs the host immune response.enCD74CD4 AntigensHIVThesis