Cell Host & Microbe

Article
Degranulation of Natural Killer Cells Following Interaction with HIV-1-Infected Cells Is Hindered by Downmodulation of NTB-A by Vpu
Ankur H. Shah,1 Bharatwaj Sowrirajan,1,5 Zachary B. Davis,1,5 Jeffrey P. Ward,2 Edward M. Campbell,3 Vicente Planelles,4 and Edward Barker1,*
of Immunology and Microbiology, Rush University Medical Center, Chicago, IL 60612, USA of Microbiology and Immunology, State University of New York, Upstate Medical University, Syracuse, NY 13210, USA 3Department of Microbiology and Immunology, Stritch School of Medicine, Loyola University, Chicago, IL 60660, USA 4Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT 84112, USA 5These authors contributed equally to this manuscript *Correspondence: edward_barker@rush.edu DOI 10.1016/j.chom.2010.10.008
2Department 1Department

SUMMARY

Natural killer (NK) cell degranulation in response to virus-infected cells is triggered by interactions between invariant NK cell surface receptors and their ligands on target cells. Although HIV-1 Vpr induces expression of ligands for NK cell activation receptor, NKG2D, on infected cells, this is not sufficient to promote lytic granule release. We show that triggering the NK cell coactivation receptor NK-Tand -B cell antigen (NTB-A) alongside NKG2D promotes NK cell degranulation. Normally, NK cell surface NTB-A binds to NTB-A on CD4+ T cells. However, HIV-1 Vpu downmodulates NTB-A on infected T cells. Vpu associates with NTB-A through its transmembrane region without promoting NTB-A degradation. Cells infected with HIV-1 Vpu mutant elicited at least 50% more NK cells to degranulate than wildtype virus. Moreover, NK cells have a higher capacity to lyse HIV-infected cells with a mutant Vpu. Thus, Vpu downmodulation of NTB-A protects the infected cell from lysis by NK cells.

INTRODUCTION NK cells respond to virus-infected cells without requiring prior exposure to viral antigens. Hence, they play a major role in managing virus-infected cells during the early stages of viral infection before the emergence of the virus-specific adaptive immune responses. The outcome of an NK cell response to a target cell is determined by intracellular signaling cascades initiated by interactions between germline encoded and invariant receptors on the NK cell and their ligands on the target cell (Lanier, 2005, 2008; Moretta et al., 2001; Moretta and Moretta, 2004). These receptor-ligand interactions are divided into three major categories: inhibiting, activating, and coactivating. One set of inhibitory receptors on NK cells (iNKR) interact with the major histocompatibility complex class I molecules (MHC-I)

(Ciccone et al., 1992; Dohring et al., 1996; Moretta et al., 1997; Natarajan et al., 2002). The three major MHC-I-specific families of iNKRs are the killer immunoglobulin (Ig)-like receptors (KIR), whose members have varying numbers of Ig domains and ligand specificities (recognizing HLA-A, -B or -C as ligands); the lectinlike heterodimer of NKG2A and CD94 (recognizing HLA-E); and interleukin-like transcript type 2 (which recognizes multiple MHC class I molecules). Furthermore, NK cells also express various inhibitory receptors that bind non-MHC-I ligands (reviewed in Borrego et al., 2002; Lanier, 2008). While iNKR-ligand interactions provide a fail-safe mechanism by which NK cells avoid killing normal ‘‘self’’ cells, the lack of or impaired expression of iNKR ligands is insufficient to trigger an NK cell cytolytic response. The engagement of NK cell activation receptors (aNKRs) by ligands on infected cells is required to elicit NK cells to lyse their target cells. Triggering a variety of surface aNKR can induce NK cell activation (reviewed in Bryceson et al., 2006a; Lanier, 2008). The natural cytotoxicity receptors (NCRs) containing Ig-like domains; NKp30, NKp44, and NKp46 (Pende et al., 1999; Pessino et al., 1998); and the C-type lectin, NKG2D (Bauer et al., 1999), are the major aNKRs. Additionally KIRs with short intracellular tails (KIR2DS1/2 and -3, KIR3DS1) are also aNKRs (Dohring et al., 1996; Moretta et al., 1995). Another family of aNKRs is a lectin-like heterodimer consisting of CD94 associated with NKG2C, which binds HLA-E (Braud et al., 1998). Although the activating NK receptors are necessary for NKmediated lysis of target cells, they are insufficient to induce degranulation (Bryceson et al., 2006a) and require the concomitant triggering of coactivating receptors (caNKR) (Bryceson et al., 2006a; Moretta et al., 2001). The simultaneous engagement of both aNKR and caNKR by their ligands on target cells prompts resting NK cells to release their lytic granules (Bryceson et al., 2006b, 2009). Earlier studies indicated that NK cells are ineffective at killing autologous, primary HIV-1-infected T cells (Bonaparte and Barker, 2003; Ruscetti et al., 1986; Zheng and Zucker-Franklin, 1992). However, examination of the infected cell surface revealed that virus infection leads to a decrease in surface expression of HLA-A and -B (Bonaparte and Barker, 2004) through the action of Nef (Cohen et al., 1999) and an increase in ligands for NKG2D (Ward et al., 2007) through the action of Vpr protein

Cell Host & Microbe 8, 397–409, November 18, 2010 ª2010 Elsevier Inc. 397 CHOM 500

1986. In contrast. Our studies therefore illustrate that Vpu downregulates cell surface NTB-A by a mechanism that is distinct from that of CD4 and BST-2 downregulation.. 2009). we asked what effect proteasomal inhibition would have on surface expression of NTB-A. NTB-A is found on all bloodderived NK. Because proteasomal degradation has been implicated in CD4 or BST-2 downmodulation (Douglas et al.. cell surface CD4 expression was downmodulated on Vpu-transduced cells (MFI = 1427) relative to CD4 on untransduced cells (MFI = 6094) (Figure 1C). NTB-A is a type I transmembrane protein of the Ig superfamily with an N-terminal IgV domain and a proximal truncated C-terminal IgC2 domain (Bottino et al. 1992). treatment with 100 nM epoxomicin or 10 mM MG132 abrogated the capacity of Vpu to downregulate CD4 surface expression when compared to that in cells treated with vehicle only (Figure 2C). Margottin et al. but total NTB-A levels remained unchanged (Figures 1A).Cell Host & Microbe Vpu Downmodulates NTB-A to Evade NK Cell Lysis (Richard et al. 2008. Ward et al. As a positive control.. Vpu (S52. 2004)... Zheng and Zucker-Franklin. we generated a mutant of NTB-A lacking a cytoplasmic tail (NTBA D250–331). This indicated that. NTB-A Downregulation by Vpu Is Mechanistically Distinct from that of CD4 and BST-2 Downregulation Vpu enhances virus production by downmodulating CD4 and bone marrow stromal antigen 2 (BST-2) expression. HIV-1 downmodulates NTB-A on the infected cell surface (Fogli et al. In contrast. we found that removal of the cytoplasmic tail did not significantly alter the capacity of Vpu to downmodulate NTB-A (Figure 2B). Vpu serves as an adaptor protein linking CD4 and BST-2 to b-TrCP. HIV-1 infection of primary CD4 T cells led to a decrease in both cell surface and intracellular CD4 expression (Figures 1A). 2001). Margottin et al. relative to NTB-A on untransduced cells (MFI = 2962) (Figure 1C). after permeabilizing the infected cells and then staining them with anti-NTB-A. 1998). we wanted to determine if Vpu was increasing internalization of NTB-A from the cell surface. 1991. NTB-A Is Downregulated by HIV-1 Vpu Since the HIV-1 accessory proteins. Willey et al. 56N). 2010. In contrast. downregulation of NTB-A on the surface by HIV-1 does not coincide with changes in the steady-state levels of NTB-A in the infected cells. Following transfection with Vpu. Nef and Vpu. Vpu Does Not Increase NTB-A Endocytosis or Degradation within Acidified Vesicles In the study shown in Figure 1. 2007. As expected. we demonstrated that Vpu did not change the steady-state levels of NTB-A even though surface levels of NTB-A were decreased.. Ward et al.. after staining the surface of HIV-infected cells with anti-NTB-A. 1998). 397–409. treatment of NTB-A expressing cells with proteasome inhibitors failed to relieve NTB-A downregulation by Vpu relative to vehicle-treated cells (Figure 2C). Vpu-mediated NTB-A downregulation is independent of proteasome activity. we asked whether either or both of the viral gene products may play a role in NTB-A downregulation.. 2004. we have demonstrated that NTB-A facilitates NK cell-mediated killing of HIV-1-infected cells (Ward et al. Flaig et al... CHOM 500 . Ruscetti et al. November 18. The downregulation of inhibitory ligands combined with the upregulation of activating ligands should lead one to predict that HIV-1-infected cells could serve as ideal targets for NK cell-mediated destruction. For this reason. we determined cell surface NTB-A expression on both Vpu-transduced and untransduced cells. 2007).. 2010. and B cells and is a member of the signaling lymphocytic activation molecule (SLAM) family of receptors (Bottino et al. As a positive control. T. 2008. a human F box protein that functions as a substrate recognition receptor for the multisubunit E3 ubiquitin ligase (Douglas et al. One such factor that regulates NK cell cytolytic activity is NTB-A.. 1996. 2010.. 56N]) would be able to downmodulate NTB-A. 2001.. Thus. 2008. These experiments indicate that Vpu is both necessary and sufficient to downregulate NTB-A. We found that HIV1DNef downregulated NTB-A from the infected cell surface to the same extent (MFI = 461 compared with MFI = 2088 for unin- fected cells) (Figure 1B) as wild-type (WT) HIV-1 (MFI = 485 compared with MFI = 2032 for uninfected cells) (Figure 1B). the ability of NK cells from even healthy uninfected individuals to destroy HIV-1-infected cells has been consistently characterized to be weak at best (Bonaparte and Barker. We addressed this question by comparing the levels of NTB-A. To determine whether Vpu is sufficient to downregulate cell surface NTB-A.. As expected. we evaluated CD4 expression.. Kerkau et al. 1997. 56N) was able to downregulate NTB-A to comparable levels as WT Vpu (Figure 2A). 2004). We observed that cell surface NTB-A expression was reduced during infection. we asked if Vpu with both serine residues mutated to asparagines (Vpu [S52.. 2010 ª2010 Elsevier Inc. 2008. To rule out any role of the putative ubiquitination residues in the cytoplasmic tail of NTB-A on Vpu’s ability to downmodulate NTB-A. NTB-A was 398 Cell Host & Microbe 8. unlike CD4.. on and within infected cells. Van Damme et al. Moreover. HIV-1 Does Not Alter the Steady-State Levels of NTB-A It is unclear if the surface downregulation of NTB-A by HIV-1 coincides with changes in the total NTB-A levels of the infected cell. with the levels of NTB-A. As Figure 3A shows.. 1992).. Neil et al. induce cell surface downregulation of a number of host cell proteins (Garcia and Miller. failed to downregulate CD4 as compared to WT Vpu (Figure 2A). Fogli et al. we found that Vpu (S52.. In contrast.. Falco et al. Ward et al. NTB-A functions as a homotypic ligand-caNKR pair (Falco et al. To determine whether b-TrCP association with Vpu is required for NTB-A downmodulation. 2007.. additional factors must be involved in regulating NK activity in the presence of HIV-1 infection. RESULTS Despite NTB-A Surface Downmodulation. 2001).. Moll et al. 2004.. It contains immunoreceptor tyrosine-based switch motifs that are docking sites for the SH2 domain of SLAMassociated proteins and the related Ewing’s sarcoma-associated transcripts (EAT)-2 (Bottino et al. Therefore.. The consequence that decreased NTB-A expression on HIV-1-infected cells will have on the ability of NK cells to lyse the infected target remains to be determined. 2001. Schwartz et al. Flaig et al. We found NTB-A to be downmodulated on Vpu-expressing cells (MFI = 1485). 1998). As expected (Schubert et al.. 2004) and has been observed to induce NK cell cytotoxicity (Bottino et al. Flaig et al. we also evaluated the cell surface expression of CD4. 2003. However. 2010... 2007). HIV-1DVpu was unable to downregulate NTB-A (MFI = 1998 compared with MFI = 2055 for uninfected cells) (Figure 1B). However. Tomescu et al.

Cell Surface but Not Total NTB-A Expression Is Reduced by HIV-1-Vpu (A) CD4+ T cells were infected with DHIV-3 and were stained for surface (i and iii) or permeabilized and evaluated for total NTB-A or CD4 (ii and iv). internalized to the same extent over a course of 45 min regardless if Vpu was present or not. Bafilomycin A1 inhibited the capacity of Vpu to downregulate BST-2 from the cell surface and enhanced intracellular BST-2 levels by either blocking pH-dependent trafficking to late endosomes or inhibiting Cell Host & Microbe 8. red line) or 104 viable HIV-1 p24 Ag+-infected cells (blue line). cells were stained with anti-CD4 (i) or anti-NTB-A (ii). All samples were stained intracellularly for HIV-1 p24 antigen. (C) Primary CD4 T cell blasts were transduced with a retroviral vector expressing Vpu-GFP (blue line) or GFP (red line). (B) (i) DHIV-3 WT. For untransduced cells. 399 CHOM 500 . Untransduced cells are shown in black. 2010 ª2010 Elsevier Inc. Following transduction. November 18. Acidification of endocytic vesicles is a necessary step in various processes following endocytosis. Histograms were derived following acquisition on a flow cytometer of either 104 viable HIV-1 p24À cells (for uninfected cells.. Uninfected cells were used as a control. including receptor recycling (Tycko et al. HIV-1 p24À (red line). 1983). and (iii) DHIV-3 DVpu-infected primary T cell blasts were surface stained for NTB-A and then stained intracellularly for HIV-1 p24 Ag. (ii) DHIV-3 DNef. The figure is representative of three separate experiments. Histograms were derived following acquisition on a flow cytometer of 104 viable and GFP+ cells.Cell Host & Microbe Vpu Downmodulates NTB-A to Evade NK Cell Lysis Figure 1. 397–409. and uninfected cells (black line) is presented. This figure is representative of two separate studies. A comparison of HIV-1 p24+ (blue line). 104 viable cells were collected.

56N)-GFP (iii and vi). (B) HeLa cells expressing NTB-A or NTB-A D250–331 were transfected with GFP (i and ii) or Vpu-GFP (iii and iv) and evaluated for NTB-A. or Vpu(S52. The MFI for NTB-A on Vpu-transfected cells treated with 100 nM bafilomycin was 5. 2009).. epoxomicin.309 compared with an MFI of 22.121 compared with 23.578 for NTB-A on untransfected cells. lysosomal degradation (Mitchell et al. We treated HeLaNTB-A cells with 100 nM bafilomycin A1 and found no effect on Vpu’s ability to modulate NTB-A (Figure 3B). or MG-132. November 18.Cell Host & Microbe Vpu Downmodulates NTB-A to Evade NK Cell Lysis Figure 2. Transfected cells were evaluated for CD4 (i–iii) or NTB-A (iv–vi). NTB-A Downmodulation by Vpu Does Not Involve Ubiquitination (A) HeLa cells expressing CD4 or NTB-A were transfected with GFP (i and iv). (C) HeLa cells expressing CD4 (i–iii) or NTB-A (iv–vi) cells were transfected with Vpu-GFP before treatment with DMSO. The MFI of NTB-A on Vpu-transfected cells was 5. CHOM 500 . Transfected cells were evaluated for CD4 (i–iii) or NTB-A (iv–vi). Vpu-GFP (ii and v). This same concentration of bafilomycin A1 prevented Nef-dependent downmodulation of CD4 (see Figure S1 available online). 2010 ª2010 Elsevier Inc. 400 Cell Host & Microbe 8.143 for bafilomycin A1-treated untransfected cells. 397–409.

we first asked if NTB-A binds directly to Vpu. we proceeded to determine the region of Vpu that was critical for its interaction with NTB-A. all other lanes did not contain this band. The Transmembrane Region of Vpu Is Required for NTB-A Downregulation Although NTB-A downregulation by Vpu is mechanistically distinct from that of CD4 and BST-2. At least 104 GFPÀ (red) and 104 GFP+ (blue) viable cells were collected by flow cytometry. 401 CHOM 500 . and 45 min post-Ab incubation at 37 C. 5. the above findings indicated that Vpu binds to NTB-A. 397–409.Cell Host & Microbe Vpu Downmodulates NTB-A to Evade NK Cell Lysis Figure 3. Taken together. Results are representative of five different experiments. To evaluate this. At least 104 GFP (red) and Vpu-GFP (blue) expressing viable cells were collected by flow cytometry. we observed a 37 kD band corresponding to Vpu-GFP only in the lane corresponding to NTB-A expressing cells transfected with Vpu. and then fixed and stained with an APC-conjugated secondary antibody. As shown in Figure 4A. November 18. 2010 ª2010 Elsevier Inc. At 0. 15. (B) HeLa-NTB-A cells were transfected with either GFP (i and iii) or Vpu-GFP (ii and iv) before treatment with either DMSO or bafilomycin A1. Since Vpu binds to NTB-A. 30. 10. Vpu-mediated downregulation of BST-2 points to Vpu’s Cell Host & Microbe 8. it remains to be determined if Vpu binds to NTB-A as it does to CD4 or BST-2. Transfected cells were evaluated for NTB-A. Vpu Does Not Increase NTB-A’s Endocytosis or Lead to Its Degradation in Acidified Vesicles (A) HeLa-NTB-A cells transfected with either GFP or Vpu-GFP were surface stained with unconjugated anti-human NTB-A.

Thus the transmembrane portion of Vpu is critical for downmodulation of NTB-A but not of CD4.. and URD-GFP. On WT-Vpu-infected cells. As controls. As a control. NTB-A Is Required for NK Cells to Degranulate When the Activation Receptor NKG2D Is Triggered Since NTB-A was downregulated by Vpu. Lysates were analyzed by western blot and probed for Vpu and b-actin.01. and HIV-1 p24+ is represented in blue. As a control. November 18. this does not appear to be the case. The cells were lysed and immunoprecipitated using anti-HA Ab. in the 402 Cell Host & Microbe 8. transmembrane region as being important for BST-2 downmodulation (Van Damme et al. since URD-GFP did not bind to NTB-A even though the Vpu Ab used in our study detected URD-GFP in the lysates. Thus. (B) HeLa-HA-NTBA cells were transfected with GFP. we asked whether NTB-A engagement. CHOM 500 . As shown in Figure 4C. To determine if Vpu interacted with NTB-A through the transmembrane region. Hence. HIV-1 p24À is represented in red. we probed the input lysates with anti-Vpu and anti-b-actin and probed the IP with anti-NTB-A. Infected cells were stained with anti-NTB-A (i and ii) or anti-CD4 (iii and iv). both NTB-A and CD4 are downregulated. Vpu Binds to NTB-A through Its Transmembrane Region (A) HeLa and HeLa-HA-NTB-A cells were transfected with GFP or GFP-Vpu. 1996).Cell Host & Microbe Vpu Downmodulates NTB-A to Evade NK Cell Lysis Figure 4. it may be possible that detection of Vpu when immnoprecipitating NTB-A could be due to the GFP binding to NTB-A. (C) CD4+ T cell blasts were infected (blue line) with either Vpu transmembrane domain mutant encoding HIV-1NL4/3 (URD) (i and iii) or WT HIV-1NL4/3 (ii and iv) at an moi of 0.. We immunoprecepitated NTB-A from lysates of NTB-A transfected with Vpu-GFP or URD-GFP followed by immunoblotting with anti-Vpu. 2010 ª2010 Elsevier Inc. Immunoprecipitates were analyzed by western blot for NTB-A (60 kDa) and Vpu-GFP (37 kDa) using antibodies directed to NTB-A and Vpu. Immunoprecipitates were analyzed by western blot and probed for NTB-A and Vpu. Next we infected primary CD4 T cells with HIV-1NL4/3 and HIV-1NL4/3-URD. Figure 4B illustrates that NTB-A does not interact with the transmembrane scrambled Vpu mutant URD but does bind to the WT Vpu. 397–409. uninfected cells (red) were also stained with anti-NTB-A or anti-CD4. Results are representative of three different experiments. Vpu-GFP. we next wanted to determine the consequences modulating this ligand on the infected cells surface had on the NK cells’ cytolytic function. However. 2008). Vpu interacts with NTB-A through the transmembrane portion of Vpu. However. Vpu used in our study is part of a GFP fusion protein. we found that URD-infected cells expressed NTB-A on primary T cells to a similar extent as that on uninfected cells (MFI of 8960 for HIVinfected cells and 7313 for uninfected cells). we utilized a Vpu mutant that encodes a scrambled amino acid sequence of the transmembrane domain (URD) (Schubert et al. To begin. URD downregulated CD4 indistinguishably from WT Vpu. untransfected cells were used. Lysates were analyzed by western blot for b-actin (42 kDa) by loading 20 mg of lysate per sample.

Numbers represent the percentage of cells in that quadrant. Results are representative of three separate studies. could trigger NK cells to degranulate. November 18. However. However.. (iii) anti-NTB-A. or purified.3% (Figure 5Aiv). and numbers in parentheses represent the percentage of NK cells that express CD107a. B cells.. NKT. 2006b). it has also been demonstrated that triggering NKG2D alone on primary NK cells is insufficient to induce optimal degranulation (Bryceson et al. only 1. 2010 ª2010 Elsevier Inc. Similarly. the percentage of NK cells expressing both CD69 and CD107a was determined after exposure to (ix) K562 cells or (x) purified DHIV-3 WT-infected autologous T cells. the percent of degranulating NK cells increased to 19. are able to trigger NK cell lysis of HIV-infected cells (Ward et al. CD107a. engagement of NTB-A alone caused only 1. K562 cells (iii and vii). Cell Host & Microbe 8.6%) fresh blood derived-NK cells spontaneously express CD107a (Figure 5Ai).3% of NK cells degranulated (Figure 5Aii). 397–409. (B) Freshly isolated human peripheral blood mononuclear cells (PBMCs) were incubated alone (i and v) or together with either uninfected autologous CD4 T cells (ii and vi). Results are representative of three different experiments. Twenty-thousand viable cells were collected per sample. When NKG2D was triggered alone. We found that very few (1. NTBA Is Involved in NK Cell Degranulation When NKG2D Is Also Triggered (A) Freshly isolated human PBMCs were labeled with antibodies specific for NK cells (CD56) and the degranulation marker. and CD14-negative population. VSV-G-pseudotyped DHIV-3 WT (infected at an moi of 5) autologous T cells (iv and viii). 403 CHOM 500 . B cells. after a 4 hr exposure to P815 cells with either (i) nonspecific Ab. These results demonstrate that triggering NTB-A together with NKG2D induces the release of granules from NK cells even though triggering either receptor alone has a minimal effect. and CD14 with fluorochromeconjugated Ab and gating on the negative cells for NK cells. such as ULBP-1 and -2. To address this issue. Numbers in dot plots represent percentage of CD69+ CD56+ cells (i–iv) or CD107a+ CD56+ cells (v–viii). (ii) anti-NKG2D. CD20. Numbers in (ix) and (x) represent the percentage of cells within the quadrant. -CD20. and (iv) antiNKG2D and anti-NTB-A. NK cells were identified following staining with anti-CD56. Following the coincubation the cells were stained with fluorochrome-conjugated CD69 or CD107a. T cell. Whether NTB-A is required for triggering NK cell degranulation when NKG2D is also triggered remains to be determined. and -CD14. NK cells were detected by CD56 in the CD3-. Ligands for the activation receptor NKG2D.8% of NK cells to express CD107a (Figure 5Aiii). In a similar experiment. T cells. we utilized a redirected degranulation assay in which only these two specific receptors can be triggered using agonist antibodies as ligand mimics.Cell Host & Microbe Vpu Downmodulates NTB-A to Evade NK Cell Lysis Figure 5. context of NKG2D engagement. 2007). CD20-. NKTs. and macrophages/monocytes were excluded by staining with anti-CD3. and macrophages/monocytes were excluded by staining for CD3. and number in parentheses represent the percentage of CD69+ NK cells that are either CD107aÀ or CD107a+. when NKG2D and NTB-A were triggered together.

Vpu’s Downmodulation of NTB-A Prevents NK Cell Degranulation and Lysis of HIV-Infected Cells (A) Peripheral blood NK cells were incubated at a effector cell to target cell ratio of 1:2 with (i) no targets. This is a representative of two separate studies. Results are representative of three different experiments. Cells were incubated in the presence of anti-CD107a for 4 hr. Numbers in quadrant 4 are the percent of NK cells lacking inhibitory receptors to HLA-C and -E that express CD107a. or DHIV-3-URD infected cells. (iii) uninfected CD4 primary T cells. (C) Peripheral blood NK cells were incubated at an effector cell to target cell ratio of 1:2 for 4 hr without targets. CHOM 500 . (iv) HIV-1 (DHIV-3) WT-infected primary T cells. Results are representative of three different experiments. or with K562. Numbers in quadrant 2 are the percent of NK cells possessing inhibitory receptors to HLA-C and -E that express CD107a. (B) Peripheral blood NK cells were stimulated overnight with 200 U/mL rhIL-2 and then were incubated with the same targets as well as stained and analyzed in a similar fashion as in Figure 6A. One group of NK cells were pretreated with anti-NTB-A blocking antibody (ON56) for 20 min prior to exposure to DHIV-3 URD infected cells and stained for CD107a expression. or (v) HIV-1 (DHIV-3)-infected primary T cells expressing Vpu in which the amino acid sequence in the transmembrane region is scrambled (URD). 404 Cell Host & Microbe 8. (ii) K562 cells. Twenty-thousand NK cells (CD56+CD3À) were collected for each group. 397–409. Twenty-thousand NK cells (CD56+CD3À) were collected per sample and further analyzed for expression of CD107a.Cell Host & Microbe Vpu Downmodulates NTB-A to Evade NK Cell Lysis Figure 6. November 18. uninfected CD4+ T cells. CD159a (HLA-E-specific iNKRs). DHIV-3 WT-infected cells. and CD158a and CD158b (HLA-C-specific iNKRs). 2010 ª2010 Elsevier Inc.

2005. We found that 66% of the CD69+ NK cells degranulated in response to K562 cells (Figure 5Bix). 2006b. when exposed to HIV-1 WT 15. Mean percent specific lysis ± the standard deviation (SD) is shown for each group. we evaluated the percentages of NK cells degranulating when cultured alone. we blocked the NTB-A on the NK cells with anti-NTB-A antibodies prior to adding URD-infected T cells. Therefore.30% of NK cells degranulated. only 11. When exposed to HIV-1-infected cells. 397–409. Bryceson and Long (Bryceson et al. When exposed to 200 U/ml of IL-2. The level of lysis seen (D) NK cells purified from whole blood were left unstimulated or treated overnight with 200 U/mL rhIL-2 and then used in a 51Cr release assay with K562 cells.. Less than 7% of NK cells degranulated when exposed to uninfected CD4+ T cells. Thus even ‘‘activated’’ NK cells require engagement of two activation receptors in order to achieve maximal degranulation. we wanted to determine if Vpu’s ability to downmodulate NTB-A decreased the responding NK cells’ ability to lyse the infected cells. when exposed to uninfected CD4 T cells.6% of NK cells expressed CD69 and 6. we concluded that the failure of NK cells to lyse HIV-1-infected target cells occurs at or near the stage of degranulation. 2004). CD107a (Peters et al. uninfected CD4+ T cells. 1994). The results in Figures 5Bi–5Bviii were confirmed on a per-cell basis by comparing NK cell activation and degranulation simultaneously. and NTB-A is required for NK cells to degranulate when NKG2D is triggered (Figure 5). would enable NK cells to degranulate in response to HIV-1-infected cells.Cell Host & Microbe Vpu Downmodulates NTB-A to Evade NK Cell Lysis HIV-1-Infected Cells Induce Vigorous Activation of NK Cells but Fail to Trigger Maximal Degranulation Our previous studies demonstrated that HIV-1-infected cells have a modest sensitivity to lysis by NK cells (Bonaparte and Barker. with uninfected CD4 T cells. and with CD4 T cells infected with HIV-1 WT. part of the HLA-E-specific receptor) as well as KIR2DL1 and 2/3+ (CD158a and -b. However. or DHIV-3-URD-infected cells at E:T ratios of 2.. K562 cells. Stimulation with IL-2 leads NK cells to express the activation marker CD69 (see Figure S2) regardless of target cells used to stimulate the NK cells. with K562 cells. 1991). Following exposure to uninfected CD4 T cells. 2007). we utilized the NK-sensitive cell line.21% of NK cells degranulated. however.05 for both E:T ratios. 2004. even though the pattern of degranulation remained similar. Therefore we asked whether stimulation of NK cells with IL-2 prior to exposure to target cells would still require that two activation receptors be engaged to get maximal degranulation. Vpu’s Downmodulation of NTB-A Is Responsible for Preventing NK Cells from Degranulating and Lysing HIV-Infected Cells Since Vpu downmodulates NTB-A (Figure 1). uninfected CD4 T cells. Ward et al.. and that NTB-A is downmodulated by Vpu (Figures 1B and 1C).2% of NK cells expressed CD69 (Figure 5Biv). November 18. In contrast. but not at an earlier activation step.6% of the cells expressed the degranulation marker CD107a (Figure 5Bviii). 7. 405 CHOM 500 . indicating vigorous activation and degranulation. 2009) indicated that ‘‘resting’’ NK cells required engagement of two activation receptors in order to degranulate. we analyzed the capability of NK cells lacking iNKRs to HLA-C and -E to degranulate. As seen in Figure 6C. 1999). 2010 ª2010 Elsevier Inc.0%. As shown in Figure 6A. 4.2% and 1.3% percent of fresh NK cells lacking HLA-C and -E iNKRs degranulate. As seen in Figure 6A. NK cells had a decreased ability to degranulate when exposed to URD cells after they were treated with anti-NTB-A blocking Ab prior to adding the target cells (Figure 6C).8% of NK cells expressed CD69 and 35. indicating potent activation. DHIV-3 WT-infected cells. NK cells had a 2-fold greater capacity to lyse HIV-infected cells with URD compared to target cells infected with WT virus (p < 0. Very few NK cells expressed CD69 or CD107a spontaneously (1. 34. Vpu prevented NK cells expressing iNKRs to HLA-C and -E from lysing infected cells by 2-fold (Figure 6A). We have also performed analysis with NKG2A+ (CD159a. and similarly. 2004). Error bars represent standard deviation of the mean percent specific lysis. after a 4 hr exposure to HIV-infected cells (Figure 5B).. Although the HLA-C and -E on infected cells decreased NK cell degranulation by 2-fold. Fogli et al. Over twice as many NK cells degranulated (39. and as a negative control. For this purpose we evaluated the proportion of NK cells expressing on their surface the activation marker. Since HLA-C and -E remain on the infected cell surface and may suppress NK cell response to the infected cells (Bonaparte and Barker..85% of the cells degranulated. Cell Host & Microbe 8. Studies by Drs. All groups were done in triplicate.3% of NK cells expressed CD107a (Figures 5Bii and 5Bvi). When exposed to K562 cells. As controls. This is representative of two separate studies. Vpu’s ability to downmodulate NTB-A limits the capacity of NK cells to degranulate by at least 2-fold. In Figure 6D. To accomplish this. respectively) when freshly isolated from the blood (Figures 5Bi and 5Bv).89%) when exposed to HIV-1 URD-infected cells. Student’s t test).89% of NK cells degranulated. 43. we predicted that introducing the URD mutation in HIV-1 Vpu. regardless of the iNKRs expressed. Given that NTB-A is required as a costimulatory signal to trigger NK cells to degranulate (Figure 5A). 2004. Next we wanted to determine whether the URD-infected cells triggered NK cell degranulation to a greater extent because of its inability to downmodulate NTB-A. which fails to downregulate NTB-A (Figure 4). Of the NK cells exposed to K562 cells. 2008. This is important since NK cells are variegated with regards to expression of these receptors (Bonaparte and Barker. only 1. overall. Since degranulation of NK cells equates to its capacity to lyse target cells (Alter et al. When exposed to K562 cells.34% of total NK cells evaluated were CD107a+) than against WT infected cells (22.. we wished to determine whether the failure of NK cells to lyse infected cells effectively occurs at the level of degranulation per se or at the prior step of activation. only 19% of CD69+ NK cells degranulated in response to HIV-1-infected T cells (Figure 5Bx). NK cells degranulated to a greater extent than NK cells not exposed to cytokines (Figure 6B). As a positive control target cell. Cohen et al. and the degranulation marker.2% expressed CD107a (Figures 5Biii and 5Bvii). 34. two HLA-C-specific receptors) CD56+ CD3À cells. 71. NK cells degranulated $3-fold higher against URD-infected cells (63. CD69 (Ziegler et al.5:1 and 5:1.6% of total NK cells evaluated were CD107a+).. Our observations demonstrate that NK cells become activated in response to HIV-1-infected T cells but fail to degranulate to the same extent. Thus.

we demonstrate that HLA-C and -E control NK cells ability to degranulate. it has never been reported that NTB-A is capable of triggering degranulation in combination with NKG2D engagement. Moreover. Although they reduce the ability of NK cells to release lytic granules by 2-fold. This was important to note.B. Van Damme et al. Following HIV entry. The latter two HLAclass I molecules were believed to prevent NK cell killing of HIV-infected cells (Cohen et al. 2010 ª2010 Elsevier Inc. 2009). we have found that HIV-2-infected cells do not downregulate NTB-A and are killed more efficiently by NK cells than HIV-1-infected cells (J. 1999).1 for both E:T ratios. there are a couple of notable differences. In fact. K562 cells (Bonaparte and Barker. By downregulating NTB-A. and E. 2007). in the destruction of HIV-infected cells by NK cells (Ward et al. 397–409.W. we have found that NK cells are variegated with regards to expression of HLA-C and -E iNKRs. In this study. relative to the ability of the NK cells to lyse the NKsensitive cell line. Although our studies centered specifically on Vpu derived from HIV-1NL4/3. 2004). the level of lysis of URD-infected cells approached the level of NK cell lysis of K562 cells. they still do not prevent degranulation entirely. CHOM 500 . This is not only true of ‘‘resting’’ NK cells but of activated ones as well. 2004). aside from this similarity in Vpu-mediated downmodulation of BST-2 and NTB-A. in that the transmembrane region of Vpu is important for not only BST-2 downmodulation (Van Damme et al. DISCUSSION We had previously demonstrated the importance of NKG2D and NTB-A. these studies indicate that Vpu’s ability to prevent NTB-A downmodulation protects the infected cell from lysis by NK cells. In the present study.Cell Host & Microbe Vpu Downmodulates NTB-A to Evade NK Cell Lysis Figure 7. downmodulation of NTB-A does not require the phosphorylation of serines in the 52nd and 56th positions of Vpu while it appears to be required for BST-2/tethein downmodulation (Mitchell et al. Moreover. 2008).. we demonstrate that simultaneous engagement of NKG2D and NTB-A is essential to elicit NK cell degranulation.P. One of the earliest observations regarding HIV-1 evasion of NK cell responses made by Drs. since K562 cells are sensitive to NK cellmediated lysis..B.. Thus... triggering NKG2D and the activation coreceptor DNAM-1 simultaneously was insufficient to induce degranulation even after exposure of NK cells to cytokines (data not shown). the connection between these receptors in the NK cell cytolytic response has never been made... We have noted that the URD-infected cells were lysed by NK cells 2-fold higher than lysis of HIV-1-infected cells expressing WT Vpu. we have observed NTB-A downmodulation on cells infected with other strains such as HIV-1SF162 and HIV1SF128A (Ward et al.. 2008). Vpu-mediated downmodulation of BST-2 is sensitive to proteasome inhibitors and inhibitors of endosomal/lysosomal acidification (bafilomycin A1) (Mitchell et al. since the proper activating receptor pairings are essential for NK cells to degranulate (Bryceson et al. Second. unpublished data). Model of Vpu Modulation of NTBA in the Context of Vpr and Nef and Their Impact on NK Cell Lytic Response to HIVInfected Cells against URD-infected cells was similar to the capability of NK cells to lyse the NK-sensitive K562 cell line (Figure 6D) (p > 0. 1999). 406 Cell Host & Microbe 8. but Vpu-mediated downmodulation of NTB-A is not. and a significant number of NK cells could not be controlled by HLA-C or -E (Bonaparte and Barker. 2008. 2007). unpublished data). Student’s t test). This is important to know.. though modestly. This is important since HIV-infected cells express CD155. separately. November 18.. and furthermore. our current study shows that Vpu’s impact on the ability of NK cells to degranulate appears to be far greater than HLA-C and -E. 1996) even though it does encode Vpr and Nef. 2006b). David Baltimore’s and Jack Strominger’s laboratories over 10 years ago was that HIV-1 Nef downmodulated HLA-A and -B but left -C and -E on the infected cell surface (Cohen et al. It is noteworthy that HIV-2 does not encode a Vpu protein (Bour and Strebel. Moreover. and here we begin to evaluate which activation/coactivation receptor pairs may be functional against HIV-1infected cells. However. However. Vpu may protect HIV-1-infected cells from NK killing. From our studies a model is generated showing the steps leading to NK cells’ response to HIV-infected cells and how the HIV-infected cell evades lysis (see Figure 7). we found that NK cells lacking HLA-C and -E iNKRs lysed HIV-infected cells.B.D.. and E. Furthermore. NTB-A downmodulation by Vpu reveals a similarity with Vpumediated downmodulation of BST-2 (Neil et al. In contrast. First. the ligand for DNAM-1 (Z.. we have even observed downmodulation of NTB-A on infected cells derived from HIV1-infected patients (Fogli et al. 2008) but also for NTB-A downmodulation. However. 2009)..

and incubated at 37 C with 5% CO2. We then provided the VSV-G glycoprotein in trans to form pseudotyped virions as described (Andersen et al. USA) through the AIDS Research and Reference Reagent Program.3-Urd was used as the template to generate the Urd-GFP fusion protein. that has a deletion in the env gene. HeLa-CD4 cells were obtained from Dr. (2009). Antibodies Pacific Blue-conjugated anti-CD3. and -CD4. NY. Sorted cells were maintained in medium containing 200 ng/ml puromycin. 2009). we used a defective HIV-1 construct.e. PMSF (Sigma) in methanol and a protease inhibitor cocktail (Roche)..56N was constructed by site-directed mutagenesis (Stratagene) using the primer pair shown in Figure S3A. The human codon-optimized Vpu sequence was PCR amplified with appropriate restriction enzyme extensions and cloned into pAcGFP-N1 (Clontech) to generate Vpu with a C-terminal GFP fusion protein.25 3 105 cells/ well in a 6-well tissue culture plate (BD) and incubated overnight at 37 C.. pcDNA-hVpu was from Dr. Division of AIDS. HIV-1NL4/3-URD was a direct gift of Dr. IL. transfected HeLa and HeLaHA-NTB-A cells were harvested 48 hr posttransfection and lysed overnight with 0. The Vpu and NTB-A coding regions of all plasmids were sequenced (University of Chicago Sequencing Facility) to verify accuracy of the coding sequence. the downmodulation of NTB-A on the infected cell decreases NK cells’ ability to degranulate even though NKG2D ligands are present and HLAA and -B are downmodulated. The Vpu sequence from pNL4. NIAID. NIH. P815 cells. Transfection of HeLa Cells HeLa-NTBA or HeLa-CD4 cells were seeded at a density of 1. For inhibition of vesicle acidification. 2006b). NIAID. Klaus Strebel (NCI.. Richard Axel (Columbia University. The HIV-1NL4/3 reagent was obtained through the AIDS Research and Reference Reagent Program. The vector pAcGFP-Vpu S52. NIH. The 293 FT cell line was used in the generation of viruses and vectors and maintained according to the manufacturer’s (Invitrogen) specifications. Immunoblots with mutant Vpu-GFP constructs were done in the absence of epoxomicin. and donkey anti-mouse antibodies were obtained from Jackson Immunoresearch. 5. the activation of ATR also led to the expression of NKG2D ligands (i. DHIV-3. This viral protein downmodulates HLA-A and -B. 1998). BDIS) for cell surface NTB-A expression. Viruses and Vectors To eliminate possible differences in replication kinetics due to the presence or absence of Vpu and Nef in the studies described in Figures 1.. 2009). Vpr. While the decrease of the two MHC class I molecules lowers the capacity of HLA-A and -B restricted HIV-1 peptide antigen specific cytotoxic T-lymphocytes from lysing the infected cells (Collins et al. Klaus Strebel (NIH).05% trypsin (Mediatech) prior to analysis by flow cytometry or immunoblots. the combination of Vpr and Nef activities should increase the susceptibility of infected cells to lysis by NK cells. Retroviral particles were generated according to the manufacturer’s instructions (Clontech). Cell lysates (200 mg) Cell Host & Microbe 8. Division of AIDS. NIH. triggering NKG2D is insufficient for NK cells to degranulate. HRP-conjugated mouse anti-rabbit. 5% CO2. Vpu-GFP and GFP encoding retroviral constructs were generated by subcloning either Vpu-GFP or GFP (from pACGFP-N1) into pQCXIP (Clontech). and labeled with APC conjugated goat anti-mouse IgG2 (Jackson Immunoresearch). the lysis of the target cell does not occur because Vpu prevents the release of lytic granules by NK cells by downmodulating the ligand to NTB-A responsible for the second signal needed for degranulation (Bryceson et al. Without engaging NTB-A on NK cells. Analysis of flow cytometric data was performed with FloJo Software (TreeStar. proteasome inhibitors epoxomicin and MG-132 (Sigma-Alrdrich) were reconstituted in DMSO (Mediatech) prior to its addition to medium 24 hr after transfection. For determination of total NTB-A or CD4 cellular content. rabbit antigoat. Klaus Strebel. they also reduce the threshold for inhibiting KIR3DL1bearing NK cells. AF700-conjugated anti-CD56 and PECy7 anti-CD16 were obtained from Biolegend. Purified anti-NKG2D (BD PharMingen) and anti-NTB-A (R&D Systems) were used in the redirected degranulation assay. -CD14. NTB-A was cloned by RT-PCR of mRNA obtained from Jurkat cells as described (Ward et al. Cells were transfected with 1. However.. EXPERIMENTAL PROCEDURES Primary Cells and Cell Lines All primary cells used in this study were isolated from peripheral blood obtained from healthy HIV-1 uninfected donors after informed written consent was acquired in accordance with the Declaration of Helsinki and the policies of the Institutional Review Board at Rush University Medical Center. Stably transduced cells were selected in 300 ng/ml puromycin (Invitrogen) for 60 days and subjected to three rounds of cell sorting (FACS Aria. only surface antigen staining was performed. Endocytosis Assay HeLa-NTB-A cells were transiently transfected with plasmids encoding either GFP or Vpu-GFP for 24 hr. NTB-A D250–331 was constructed by site directed mutagenesis (Stratagene) using the primer pair shown in Figure S3C. -CD20. aliquoted into sterile tubes containing DMEM complete media. NTB-A encoding retroviral constructs were generated by subcloning NTB-A into pQCXIP (Clontech) bicistronic retroviral vector. HeLa-NTBA and HeLaNTBA D250–331 cells were generated by transduction of HeLa cells with retroviral particles encoding a bicistronic mRNA for NTB-A or NTB-A D250–331 and puromycin resistance. 2010 ª2010 Elsevier Inc. Infections and transductions of primary cells were done as described in Ward et al. Inc). Thus.05% trypsin and labeled with monoclonal anti-human NTB-A (Biolegend). and HeLa and Jurkat E6-1 cell lines were obtained from ATCC and maintained according to distributors’ recommendations. Cells were harvested with 0. and integration into the host chromosome... Protein concentration was determined by performing Bradford assay (Pierce). Samples were acquired using FACS LSRII. However. the virus begins to express one of the early gene products. ULBP-1 and -2) on the infected cells. 2009). Division of AIDS. FITC-conjugated anti-CD69. 50 mM Tris-HCl. K562 cells. Simultaneous detection of surface antigens and intracellular HIV-1 p24 antigen (Ag) was done as previously described (Ward et al. NTB-A primer pair sequence is shown in Figure S3B. 2006. Thus. Although HLA-C and -E remain on the infected cells surface.Cell Host & Microbe Vpu Downmodulates NTB-A to Evade NK Cell Lysis reverse transcription.5% NP40 with 150 mM NaCl. Human codon-optimized Vpu sequence of HIV-1NL4/3 (pcDNA-hVpu) was obtained through the NIH AIDS Research and Reference Reagent Program. CD4 T cells were isolated and stimulated in vitro as described (Ward et al. Nef..5 mg of Vpu expression plasmids using HeLa Monster Trans-IT reagent (Mirus Bio) and incubated for 24–36 hr. Viral particles were produced and titered as previously described (Ward et al. For inhibition of proteasome activity. November 18. NIAID. 407 CHOM 500 . Frank Maldarelli and Dr. enhances HIV virion production through its ability to activate ATR. despite the activation of NK cells by HIV-infected cells. 2009). Bosque and Planelles. Another HIV-1 gene product. Stephan Bour and Dr. and 6. At indicated times. All HeLa cells were harvested with 0. NIAID. FITC. Chicago. APC-conjugated anti-NTBA was obtained from R&D Systems. simultaneous cell surface and intracellular labeling was performed with the same antibody. washed with PBS. NIH (HeLa-T4+) and maintained in medium containing 500 mg/ml G418 (Invitrogen). bafilomycin A1 (Sigma-Aldrich) was reconstituted in DMSO (Mediatech) prior to its addition to medium 24 hr after transfection. NIH. they only affect a subpopulation of NK cells. cells were fixed with 1% paraformaldehyde. Cells were washed with PBS. 2009). Division of AIDS. HIV-1NL4-3 Vpu antiserum was obtained from Dr. NIH) through the AIDS Research and Reference Reagent Program. and PE-conjugated anti-CD107a and staining controls were obtained from BD PharMingen. For GFP-expressing cells or for HeLa-NTBA cell sorting. USA. Immunoblots For coimmunoprecipitation of Vpu and NTB-A. 397–409.and PE-conjugated anti-HIV-1 p24 was obtained from Beckman Coulter.

Blood 113.F... M.O. Rev. 661–671. Malenfant. a receptor for stressinducible MICA.. Phillips. Science 285. D. CHOM 500 .M. Nature 391. et al.O.. J. Immunoprecipitates and 20 mg of whole-cell lysates were analyzed for the presence of Vpu on a 10% SDS-PAGE gel using a Mini Protean 3 Cell (Bio-Rad) for 2 hr at 100 V and 500 mA in 13 SDS (Fisher Scientific) running buffer. All unbound antibody was removed by washing once with RPMI complete. Zimmerman. Membranes were blocked overnight at 4 C in 5% skim milk (Fisher Scientific) in Tris-buffered saline with 0. B and C.G.....1% Tween 20 (Fisher Scientific) with agitation. B. Degranulation and Activation Assay HIV-infected cells (106) were incubated with fresh PBMC(106) from the same donor for 4 hr at 37 C. Benichou. 159–166. J. Y. O. and Long.5 hr in 5% milk solution. 397–401. B... S. The gel was transferred to a methanol-soaked PVDF membrane in Towbin transfer buffer for 2 hr at 250 Volts and 200 mA. dead cells were selected using the LIVE/ DEAD Fixable Dead Cell Stain Kit (Invitrogen)... Genoa. Marcenaro. 10.chom.. R.B. University of Genoa.. The human immunodeficiency virus (HIV) type 2 envelope protein is a functional complement to HIV type 1 Vpu that enhances particle release of heterologous retroviruses. Soderstrom. E. Chen.. R. 8285–8300. 70. 2087–2094. A. T. Landi. Ogg. (2006a). Membranes were then exposed to autoradiography film (Denville Scientific) and developed in an autoprocessor (Konica). Mandelboim.. E. and Long.T. Viable CD3ÀCD20ÀCD14À and CD56+ cells (2 3 104) were evaluated for the percentage of CD107a and/or CD69. A.. CD14. Biassoni. Lanier. and Barker. 963–971.. Y. S. S. K. Precipitation was accomplished with protein G agarose for 30 min at 4 C. Immunol. (1999). 58–65. 408 Cell Host & Microbe 8. Bell. R..E. 637–660.T...L.. Bryceson. J. 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