Abstract
The Human Immunodeficiency Virus (HIV) is a retrovirus that primarily infects CD4⁺ T cells. HIV evades host immune defense mechanisms largely through the actions of its accessory proteins. Among these accessory proteins, Nef and Vpu play particular key roles—they remodel the cell surface of host cells and thereby disable immune factors and/or disrupt immune communications. One significant, common target of Nef and Vpu is the CD4 receptor. CD4 is a co-receptor for T-cell receptors that notifies the host’s immune system in the event of a pathogenic invasion; without CD4 the host’s immune response is impaired. Although CD4 is the primary receptor for viral entry, post entry, however, CD4 interferes with viral transformation and dissemination and is therefore downregulated by HIV. Nef downregulates CD4 from the cell surface, while Vpu targets the newly synthesized CD4 in the ER and reroutes it for proteasomal degradation. This latter route depends on Vpu-mediated polyubiquitination of CD4 by the SCFᵝ-ᵀʳᶜᴾ E3 ligase. However, how Vpu recruits CD4 onto the adaptor β-TrCP remains elusive. The scope of this research is to elucidate, through protein biochemistry and structural biology, the mechanism of Vpu-mediated proteasomal degradation of newly synthesized CD4. Using purified proteins in a MBP pulldown assay, we have shown that the MBP-CD4-ᴾʰᵒˢVpu complex and β-TrCP/Skp1 interact in a cooperative manner and the CD4CD portion of MBP-CD4CD-VpuCD contributes to the overall binding. This result has inspired the complex that is formed between CD4CD-VpuCD and β-TrCP/Skp1 to be an ideal construct for a high-resolution crystal structure to reveal the mechanistic details of this crucial interaction.