Pharmacologic Inhibition of Nedd8 Activation Enzyme Exposes CD4-Induced Epitopes within Env on Cells Expressing HIV-1

Abstract

HIV-1 Vpu decreases the exposure of epitopes within the viral envelope glycoprotein (Env) on the surface of infected cells by downregulating both BST2 and CD4. To test the hypothesis that inhibiting Vpu activity would increase the exposure of these epitopes and sensitize infected cells to antibody-dependent cellular cytotoxicity (ADCC), we treated cells with the Nedd8 activation enzyme (NAE) inhibitor MLN4924, which inhibits the cullin1-based ubiquitin ligase complex coopted by Vpu to degrade cellular targets. Treatment of HeLa cells with MLN4924 or expression of a dominant negative mutant of cullin1 inhibited the Vpu-mediated downregulation of CD4 but not the downregulation of BST2. NAE inhibition also increased the surface exposure of CD4-induced epitopes within Env on HEK293 cells containing an inducible HIV genome, on infected CEM T cells, and on infected primary T cells. In contrast, the Vpu-mediated downregulation of BST2 was substantially inhibited by MLN4924 only when T cells were treated with alpha interferon (IFN-␣) to induce high levels of BST2 expression. As reported previously, the absence of vpu or nef and even more so the combined absence of these two genes sensitized infected cells to ADCC. However, NAE inhibition affected ADCC minimally. Paradoxically, even in infected, IFN-treated cells in which NAE inhibition substantially rescued the surface level of BST2, the surface level of Env detected with an antibody recognizing a CD4-independent epitope (2G12) was minimally increased. Mutation of the C-terminal Vpu residue W76, which supports the ability of Vpu to stimulate virion release by displacing BST2 from assembly sites on the plasma membrane by a cullin1-independent mechanism, increased the exposure of Env detected by 2G12 on infected T cells. Thus, inhibiting the displacement function of Vpu together with its ability to degrade CD4 and BST2 may be required to sensitize infected cells to ADCC. IMPORTANCEPathogenic viruses encode gene products that enable evasion of host immune surveillance mechanisms. One such mechanism is antibody-dependent cellular cytotoxicity (ADCC), whereby host antibodies bind envelope glycoproteins of the virus that are inserted into the cellular membrane and direct the destruction of infected cells. Targeting pharmacologically the activity of HIV-1 Vpu, which contributes to evasion of ADCC, could potentially sensitize infected cells to this immune surveillance mechanism, an outcome that would have therapeutic implications with respect to the goal of curing HIV-1 infection. The Nedd8 activation enzyme inhibitor MLN4924 blocks the activity of the host ubiquitin ligase that Vpu coopts to direct the degradation of CD4 and BST2. We observed that while MLN4924 partially reverses the activity of Vpu and could become part of a therapeutic approach by virtue of CD4-induced epitope exposure, sufficient Vpu activity as an antagonist of BST2 persists despite this drug to allow escape from ADCC.T he accessory proteins of HIV-1 remain undeveloped drug targets whose inhibition...

An N-Glycosylated Form of SERINC5 Is Specifically Incorporated into HIV-1 Virions

“…HEK293 Tet-On TRE-HIV oNef cells, HEK293 cells containing a doxycycline-inducible HIV-1 genome lacking nef and constitutively expressing CD4, have been described (40); these cells were maintained in Tet-Free Dulbecco's modified Eagle medium (DMEM plus 10% Tet-free fetal bovine serum [FBS] and penicillin-streptomycin [P/S] with 1 g/ml puromycin, 200 g/ml G418, and 200 g/ml Zeocin). HEK293 cells and HeLa P4.R5 cells were maintained in complete DMEM (DMEM with 10% FBS and P/S).…”

Antibody-dependent cellular cytotoxicity in HIV infection

“…Env is then free to traffic to the plasma membrane in a “closed” conformation effectively occluding CD4i epitopes. Hence, several reports have shown that cells infected with viruses defective for Nef and/or Vpu expression are more susceptible to ADCC responses mediated by HIV+ sera or CD4i antibodies [49,57–59,75,92,93,96–100]. Besides its role in CD4-downregulation, Nef also protects infected cells from ADCC responses by downregulating the expression of NKG2D ligands (MICA, ULBP1, and ULBP2) [97,101,102], which otherwise activate NK cells by interacting with the NKG2D receptor [96,97].…”

βTrCP is Required for HIV-1 Vpu Modulation of CD4, GaLV Env, and BST-2/Tetherin

“…Thus, the βTrCP-independent mechanism would seem to be most relevant when BST-2/tetherin is expressed at low levels. In support of this, it was demonstrated previously that treatment of cells with MLN4924 to block Cullin ubiquitin ligases had greater impact on Vpu antagonism of BST-2/tetherin if cells were first treated with interferon, thus upregulating the BST-2/tetherin levels [ 27 ]. In addition to BST-2/tetherin levels, the level of βTrCP expression may also affect the potency of Vpu activity against BST-2/tetherin.…”

“…While some reports suggest that βTrCP-1 and -2 are partially or entirely required for this activity, others suggest that the adapter proteins AP-1 and/or AP-2 are the primary co-factors [ 14 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 ]. In addition, treatment of Vpu expressing cells with MLN4924, a compound that blocks SCF activity, does not restore surface BST-2/tetherin expression unless cells are first treated with interferon, suggesting that the relevant cellular cofactors vary depending on expression level [ 26 , 27 ]. Finally, Vpu targets BST-2/tetherin for degredation.…”