Multilayered Mechanism of CD4 Downregulation by HIV-1 Vpu Involving Distinct ER Retention and ERAD Targeting Steps

Abstract

A key function of the Vpu protein of HIV-1 is the targeting of newly-synthesized CD4 for proteasomal degradation. This function has been proposed to occur by a mechanism that is fundamentally distinct from the cellular ER-associated degradation (ERAD) pathway. However, using a combination of genetic, biochemical and morphological methodologies, we find that CD4 degradation induced by Vpu is dependent on a key component of the ERAD machinery, the VCP-UFD1L-NPL4 complex, as well as on SCFβ-TrCP-dependent ubiquitination of the CD4 cytosolic tail on lysine and serine/threonine residues. When degradation of CD4 is blocked by either inactivation of the VCP-UFD1L-NPL4 complex or prevention of CD4 ubiquitination, Vpu still retains the bulk of CD4 in the ER mainly through transmembrane domain interactions. Addition of a strong ER export signal from the VSV-G protein overrides this retention. Thus, Vpu exerts two distinct activities in the process of downregulating CD4: ER retention followed by targeting to late stages of ERAD. The multiple levels at which Vpu engages these cellular quality control mechanisms underscore the importance of ensuring profound suppression of CD4 to the life cycle of HIV-1.

The p97 ATPase associates with EEA1 to regulate the size of early endosomes

“…We used a siRNA to deplete Npl4 ( Figure 3C), a p97 cofactor essential for ERAD. Consistent with previous reports [36,37], knockdown of Npl4 inhibited ERAD, as indicated by the stabilization of the model ERAD substrate TCRα (Supplementary information, Figure S3B). Immunostaining showed that EEA1-positive early endosomes in Npl4 knockdown cells were indistinguishable from those in control cells ( Figure 3D, panels 2, 5 versus panels 1, 4), whereas p97 depletion performed in parallel gave rise to obviously enlarged and clustered early endosomes ( Figure 3D, panels 3, 6).…”

Ubiquitination of BST-2 Protein by HIV-1 Vpu Protein Does Not Require Lysine, Serine, or Threonine Residues within the BST-2 Cytoplasmic Domain

“…Targeting of non-lysine resides has been shown for the mK3 Ub ligase encoded by the mouse ␥-herpesvirus 68 (43) and the K3 Ub ligase encoded by KSHV (49,55), each of which targets MHCI for degradation. Vpu itself provides an even more relevant example here, as Vpu has been shown to lead to the ubiquitination of CD4 on serine and/or threonine residues (11). To probe this question further, we next generated a BST-2 protein with substitutions for all of the cytoplasmically exposed lysine, serine, and threonine residues and found that it was still down-regulated (surface and total cellular levels) by Vpu as well as specifically ubiquitinated in the presence of Vpu.…”

“…This leads to the ubiquitination of CD4 (8,9) on both lysine and serine/threonine residues located within its cytoplasmic tail. CD4 is subsequently degraded by the proteasome (10) via what appears to be a noncanonical ER-associated degradation (ERAD) pathway (11). The fate of Vpu in this process is the subject of some debate, as it has been reported to either be exceptionally stable or to be ubiquitinated and degraded (12)(13)(14)(15)(16).…”

How Viruses Use the Endoplasmic Reticulum for Entry, Replication, and Assembly

“…Similarly, the murine g herpesvirus encodes a membranebound E3 ubiquitin ligase called mK3 that promotes MHC class I degradation via the ERAD pathway (Wang et al 2006). Another salient example is observed in HIV, where its Vpu protein also coopts the ERAD machinery to down-regulate the host entry receptor CD4 (Willey et al 1992;Magadan et al 2010). CD4 down-regulation leads to a series of events including disruption of T cell activation (Lanzavecchia et al 1988), ultimately contributing to robust HIV infection.…”