title: 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-α) 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.
  
  
  OBJECTIVE
  Pathogenic 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.
authors.affiliation: University of California-San Diego, La Jolla, California, USA.
authors.forename: Andrey
authors.initials: A
authors.lastname: Tokarev
authors.affiliation: VA San Diego Healthcare System, San Diego, California, USA.
authors.forename: Charlotte
authors.initials: C
authors.lastname: Stoneham
authors.affiliation: University of California-San Diego, La Jolla, California, USA VA San Diego Healthcare System, San Diego, California, USA.
authors.forename: Mary K
authors.initials: MK
authors.lastname: Lewinski
authors.affiliation: VA San Diego Healthcare System, San Diego, California, USA.
authors.forename: Amey
authors.initials: A
authors.lastname: Mukim
authors.affiliation: VA San Diego Healthcare System, San Diego, California, USA.
authors.forename: Savitha
authors.initials: S
authors.lastname: Deshmukh
authors.affiliation: VA San Diego Healthcare System, San Diego, California, USA.
authors.forename: Thomas
authors.initials: T
authors.lastname: Vollbrecht
authors.affiliation: University of California-San Diego, La Jolla, California, USA VA San Diego Healthcare System, San Diego, California, USA.
authors.forename: Celsa A
authors.initials: CA
authors.lastname: Spina
authors.affiliation: University of California-San Diego, La Jolla, California, USA VA San Diego Healthcare System, San Diego, California, USA jguatelli@ucsd.edu.
authors.forename: John
authors.initials: J
authors.lastname: Guatelli
mesh_terms: D000920:Antibody-Dependent Cell Cytotoxicity; D015496:CD4-Positive T-Lymphocytes / Q000187:drug effects* / Q000276:immunology / Q000821:virology*; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000187:drug effects / Q000276:immunology / Q000821:virology; D000939:Epitopes / Q000276:immunology*; D015497:HIV-1 / Q000276:immunology*; D006801:Humans; D000075687:NEDD8 Protein; D014452:Ubiquitins / Q000037:antagonists & inhibitors*; D054299:env Gene Products, Human Immunodeficiency Virus / Q000276:immunology*
chemical_list: D000939:Epitopes; D000075687:NEDD8 Protein; C082860:NEDD8 protein, human; D014452:Ubiquitins; D054299:env Gene Products, Human Immunodeficiency Virus