HIV-1 Vpr Triggers Natural Killer Cell–Mediated Lysis of Infected Cells through Activation of the ATR-Mediated DNA Damage Response

Abstract

Natural killer (NK) cells are stimulated by ligands on virus-infected cells. We have recently demonstrated that NK cells respond to human immunodeficiency virus type-1 (HIV-1)-infected autologous T-cells, in part, through the recognition of ligands for the NK cell activating receptor NKG2D on the surface of the infected cells. Uninfected primary CD4pos T-cell blasts express little, if any, NKG2D ligands. In the present study we determined the mechanism through which ligands for NKG2D are induced on HIV-1-infected cells. Our studies reveal that expression of vpr is necessary and sufficient to elicit the expression of NKG2D ligands in the context of HIV-1 infection. Vpr specifically induces surface expression of the unique-long 16 binding proteins (ULBP)-1 and ULBP-2, but not ULBP-3, MHC class I-related chain molecules (MIC)-A or MIC-B. In these studies we also demonstrated that Vpr increases the level of ULBP-1 and ULBP-2 mRNA in primary CD4pos T-cell blasts. The presence of ULBP-1 and ULBP-2 on HIV-1 infected cells is dependent on the ability of Vpr to associate with a protein complex know as Cullin 4a (Cul4a)/damaged DNA binding protein 1 (DDB1) and Cul4a-associated factor-1(DCAF-1) E3 ubiquitin ligase (Cul4aDCAF-1). ULBP-1 and -2 expression by Vpr is also dependent on activation of the DNA damage sensor, ataxia telangiectasia and rad-3-related kinase (ATR). When T-cell blasts are infected with a vpr-deficient HIV-1, NK cells are impaired in killing the infected cells. Thus, HIV-1 Vpr actively triggers the expression of the ligands to the NK cell activation receptor.

HIV-1 Vpr degrades the HLTF DNA translocase in T cells and macrophages

“…The Δ-Env HIV-1 viruses (DHIV NL4.3 viruses) were produced by using pNL4.3 deltaEnv HIV-1 constructs lacking the gene encoding Vpr (DHIVΔVpr) or encoding WT Vpr (DHIV WT) along with a plasmid encoding the vesicular stomatitis virus glycoprotein G (VSV-G). The proviral plasmids were a kind gift from Vincente Planelles, University of Utah, Salt Lake City (89). HIV-1 VLPs were produced by using psPAX2 lentiviral packaging plasmid along with the plasmid encoding VSV-G and a plasmid encoding either HA-tagged WT or mutant Vpr.…”

Distinct Roles for Human Cytomegalovirus Immediate Early Proteins IE1 and IE2 in the Transcriptional Regulation of MICA and PVR/CD155 Expression

“…As previous studies reported that HCMV manipulates the DDR (26,27,(29)(30)(31)(32)(33)(34), a pathway able to stimulate NKG2DL and DNAM-1L expression as well (35)(36)(37)(38)(39)(40)(41)(42)(43), we examined the involvement of DDR signaling in the HCMV-mediated upregulation of activating ligands using genetic and pharmacological approaches.…”

“…DDR is activated by many viruses, including HCMV, and although its functional relevance in HCMV infection has not been clarified, this virus induces DDR, including activation of ataxia telangiectasia mutated (ATM), ataxia telangiectasia and Rad3-related protein (ATR), and the downstream protein H2AX (26,27,(29)(30)(31)(32)(33)(34). Interestingly, expression of some NKG2DL and DNAM-1L can be dependent on the activation of DDR and on ATM/ATR kinases (35)(36)(37)(38)(39)(40)(41)(42)(43).…”

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

“…Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4 . Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells [3][4][5][6] . The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population [5][6][7] .…”

“…Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells [3][4][5][6] . The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population [5][6][7] . Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules".…”