Abstract
HIV-1 protein Vpu is expressed in the later stages of viral replication, and it plays a very important role in HIV-1 pathology. Vpu misdirects the cellular trafficking of several host immune proteins, disrupting the host’s immune response and favoring viral spread and survival. One of Vpu’s main targets is CD4, an integral membrane protein and a receptor for T-cells. CD4 is the entry receptor for HIV. However, after the infection has been established, CD4 becomes problematic to the virus and has to be removed from the cell surface as well as certain intracellular compartments. While the viral accessory protein Nef downregulates CD4 receptors from the cell surface and transports CD4 to the lysosome for degradation, Vpu targets newly synthesized CD4 in the ER and induces its polyubiquitination, which is followed by the proteasomal degradation of CD4 in the proteasome. It has been shown that Vpu binds to the cytoplasmic tail of CD4 and thus brings CD4 to the SCFᵝ⁻ᵀʳᶜᴾᐟˢᵏᵖ¹ E3 ligase complex to be polyubiquitinated. However, the precise mechanism of the interaction between CD4, Vpu and SCFᵝ⁻ᵀʳᶜᴾᐟˢᵏᵖ¹ E3 ligase complex has remained elusive. Previous work in our lab has validated this interaction biochemically. In this work, we optimized the preparation of the protein complex containing Vpu, CD4, β-TrCP, and Skp1. A convenient co-lysing strategy has been established and polished to afford the complete complex in good yield from a single preparation. The work here paved the way for subsequent studies to elucidate the molecular details of the interaction via a high-resolution structure.