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Basic and Translational Science

Cell Surface Downregulation of NK Cell Ligands by Patient-Derived HIV-1 Vpu and Nef Alleles

Galaski, Johanna; Ahmad, Fareed PhD; Tibroni, Nadine; Pujol, Francois M. PhD; Müller, Birthe MA; Schmidt, Reinhold E. MD; Fackler, Oliver T. PhD

Author Information
JAIDS Journal of Acquired Immune Deficiency Syndromes: May 1, 2016 - Volume 72 - Issue 1 - p 1-10
doi: 10.1097/QAI.0000000000000917

Abstract

Erratum

In the article by Galaski et al, appearing in the Journal of Acquired Immune Deficiency Syndromes , Vol. 72 No. 1, pp. 1-10, entitled “Cell Surface Downregulation of NK Cell Ligands by Patient-Derived HIV-1 Vpu and Nef Alleles”, reference 37 was incorrect; the correct reference 37 is:

37. Matusali G, Potestà M, Santoni A, et al. The human immunodeficiency virus type 1 Nef and Vpu proteins downregulate the natural killer cell-activating ligand PVR. J Virol. 2012; 86:4496-4504.

JAIDS Journal of Acquired Immune Deficiency Syndromes. 73(4):e66, December 1, 2016.

INTRODUCTION

The clinical course of HIV infection is determined by the interplay between immune responses of the host, viral evasion strategies thereof and intrinsic replication fitness of the virus.1–4 Typical immune responses to HIV infection include innate recognition events resulting in the release of antiviral cytokines during acute infection, rapid mounting of cytotoxic T cell responses, and subsequent production of virus-specific antibodies.5 Despite the potency of these measures, immunological control of virus spread is not achieved in most HIV patients. This reflects the relative resistance of transmitted founder viruses to antiviral cytokines such as interferons,6,7 the genetic plasticity of HIV that allows evasion from immune recognition by selection of virus variants that lack predominant antigenic epitopes,2 or shielding mechanisms that prevent efficient recognition of target structures by neutralizing antibodies.8,9

The accessory proteins Vpu and Nef make important contributions to the ability of HIV-1 to evade host immune responses. Dispensable for virus replication in most ex vivo cell culture systems, they optimize virus replication in the infected host and this activity is thought, at least in part, to mirror direct effects of Vpu and Nef on immune recognition of productively infected cells.10–14 Even though Vpu and Nef do not share any amino acid homology, differ in their membrane topology (Vpu is a transmembrane and Nef a peripheral membrane protein), and are expressed to peak levels early (Nef) or late (Vpu) in the replication cycle,15 many of their functions are remarkably redundant; although via distinct molecular mechanisms, a major activity of Vpu and Nef is to alter the surface exposure of a large and overlapping set of host cell receptors. This includes the viral entry receptor CD4 for prevention of superinfection, major histocompatibility complex class 1 (MHC-I) molecules for evasion of CD8+ cytotoxic T cells, and many other receptors for which the functional consequences have yet to be established.16–22 Cell surface levels of the antiviral restriction factor CD317/tetherin are also reduced by HIV-1 Vpu and Nef, however, only Vpu is able to counteract the particle release restriction imposed by CD317/tetherin.23–25 Cell surface downregulation of CD4 and CD317/tetherin by Vpu and Nef, also prevent antibody-dependent cell-mediated cytotoxicity.26–29

In addition to the host immune reactions described above, the importance of natural killer (NK) cells especially at early stages of HIV infection is increasingly recognized as an important mechanism of host defense.30–32 The NK cell response is determined by the balance of activating and inhibitory signals after interaction of NK cell receptors with their ligands on target cells.33,34 In case the specific receptor signature encountered triggers NK cell activation, target cells are killed by initiating apoptosis of the target cell.35 Activating signals are delivered mainly upon engagement of the NK cell activating receptors natural killer group 2, member D and DNAX accessory molecule-1 by UL16-binding proteins (ULBPs) and MHC class-I chain related molecules A and B (MICA and MICB) or poliovirus receptor (PVR), respectively, on target cells. Besides the engagement of activating NK cell receptors, lysis of infected cells by NK cells requires coactivating receptors such as NK-T-B-antigen (NTB-A). NK cell activity can also be negatively regulated by NK cell inhibitory receptors such as inhibitory killer cell immunoglobulin-like receptors that protect target cells from lysis upon engagement by their respective human leucocyte antigen (HLA) class I ligand.

Reflecting the high antiviral potential of NK cells, HIV-1 has evolved several strategies to evade NK cell recognition that (when studied using lab-adapted HIV strains) rely on receptor downregulation by Vpu and/or Nef.36 Both viral proteins act in concert to downregulate cell surface levels of the NK cell activating receptor PVR.37,38 For additional NK cell ligands, only individual viral gene products have been analyzed. Downregulation of the coactivation receptor NTB-A by Vpu is a conserved activity of chronic as well as transmitted founder HIV-1 strains,39 and prevents NK cells from lysing their target cells, and thus protect HIV-infected cells from the NK-mediated immune response.40,41 HIV-1 Nef restricts cell surface expression of natural killer group 2, member D ligands by downregulating ULBP-1, ULBP-2, and MICA.42 Finally, cell surface downregulation of MHC-I by Nef is selective for HLA-A and HLA-B and spares HLA-C, a ligand for inhibitory killer cell immunoglobulin-like receptors on NK cells, to prevent NK cell-mediated lysis.43

Although cell surface downregulation of NK cell ligands is thus an established activity of Vpu and Nef proteins encoded by lab-adapted HIV-1 strains, it is unclear for most NK cell ligands if this activity is exerted by primary HIV-1 isolates. We therefore set out here to study how well-conserved NK cell ligand downregulation is among Vpu and Nef variants isolated from chronic HIV patients.

MATERIALS AND METHODS

For additional Materials and Methods, see Supplemental Digital Content (http://links.lww.com/QAI/A783).

Study Subjects

This study reports data on 27 HIV-infected individuals of a cohort of 162 HIV patients that has been previously described.44 Median [interquartile range (IQR)] plasma viral load and CD4 count were 22,100 copies per milliliter (3780–67,700) and 382 cells per microliter (330–586), respectively. Fourteen patients had low NK cell counts less than 160 cells per microliter [median 67 cells/μL (IQR 52–103)] and 13 patients had high NK cell counts greater than 190 cells per microliter [median 320 cells/μL (IQR 222–344)]. The treatment status of patients at the time of sample donation is indicated in Table 1. Subjects were enrolled at the HIV outpatient clinic of the Medizinische Hochschule Hannover. The study was approved by the local ethics committee and all subjects gave written informed consent for participation in the study.

TABLE 1
TABLE 1:
Summary of Clinical Parameters of the Patients From Which Vpu and Nef Sequences Were Amplified

Statistical Evaluation

All statistical analyses were performed using GraphPad Prism 5. Data were statistically analyzed with one-way analysis of variance followed by Dunnett multiple comparison test. The Mann–Whitney test was used to determine statistical significance of differences between 2 groups. Correlations were statistically evaluated by Spearman rank correlation test. P-values less than 0.05 were considered statistically significant (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001).

RESULTS

Amplification and Sequences of Patient-Derived Vpu and Nef Variants

The goal of this study was to assess whether cell surface downregulation of NK cell ligands is conserved among patient-derived Vpu and Nef variants and if these activities are correlated to clinical parameters of these patients. To this end, proviral vpu and nef sequences were isolated from peripheral blood mononuclear cells of a patient cohort for which information for NK cell counts and NK cell activation as well as viral load and CD4 counts were available (Table 1 and Table S1). Up to 10 viral sequences were amplified from 27 chronic HIV patients. Out of 27 patients, one patient (UV072) was on antiretroviral treatment and one was (AA052) long-term nonprogressor.44 In total, 126 vpu and 157 nef sequences were amplified, of which 61 or 88 were unique nucleotide and 56 or 79 unique amino acid sequences for Vpu and Nef, respectively (Table S2). For 10 patients we were able to obtain vpu and nef sequences, additionally we obtained vpu or nef sequences only from 7 and 10 patients, respectively. Vpu and nef sequences were organized into 2 separate maximum likelihood phylogenetic trees (Figs. 1A, B). Sequences derived from a single patient clustered into monophyletic groups, consistent with their close genetic relationship. Vpu and nef sequences derived from the same patient were organized in similar proximity to each other in the respective phylogenetic trees, reflecting their common origin. All sequences were classified as HIV-1 subtype B according to the Los Alamos HIV database except for samples AA017 and AA057, that represent HIV subtype A1 and the circulating recombinant form CRF-AE, respectively (Fig. 1B). A vpu and/or nef with an intact open reading frame representative of the predominant patient's bulk sequence was selected for every HIV-1 infected patient and subcloned for expression as green fluorescent protein (GFP) fusion protein and subsequent functional analysis. The 17 Vpu and 20 Nef amino acid sequences were aligned with the Vpu and Nef proteins of HIV-1 NL4-3 and HIV-1 SF2 Nef as reference sequence (Fig. S1).

FIGURE 1
FIGURE 1:
Phylogenetic analysis of HIV-1 Vpu and Nef sequences and expression of Vpu.GFP and Nef.GFP fusion constructs. A and B, Phylogenetic trees of viral sequences used in this study. The same color was assigned to Vpu and Nef sequences if both could be amplified from the same sample. Branch lengths are proportional to the number of nucleotide substitutions per site. A, Unrooted maximum likelihood phylogenetic tree of 126 Vpu nucleotide sequences derived from 17 HIV-1 infected patients. B, Unrooted maximum likelihood phylogenetic tree of 157 Nef nucleotide sequences derived from 20 HIV-1 infected patients. C and D, HeLa TZM-bl cells were transfected with GFP or the indicated expression constructs for (C) Vpu.GFP or (D) Nef.GFP. Twenty-four hours post transfection, cells were lysed and analyzed by Western blotting using the indicated antibodies. The expression levels of transferrin receptor (TfR) served as loading control.

Vpu sequences were 80–89 amino acids long and contained without exception the DS52XXES56 di-serine motif which, in its phosphorylated state, recruits β-transducin repeat-containing protein to target cargo such as CD4 and CD317/tetherin for proteasomal degradation.45–47 Additional amino acid motifs previously identified as determinants for aspects of Vpu's biological activity such as interaction with and antagonism of CD317/tetherin,48,49 localization of lipid raft microdomains,50 trans-Golgi network localization,51 interaction with the endocytic machinery of the host cell (ExxxLV motif),49 or Vpu stability (S61)52 were also generally well conserved across these Vpu variants.

With the exception of AA052, all nef sequences contained intact full length open reading frames and encoded for 197 to 216 amino acid-long proteins. In these full length proteins, functionally relevant Nef motifs mediating membrane association, downregulation of cell surface MHC-I and CD4, as well as interference with host cell signal transduction and actin dynamics were strongly conserved.53 In contrast, all nef sequences amplified from patient AA052 displayed a 5-nucleotide deletion leading to a frame shift at amino acid position 134 and a premature termination codon at position 160, resulting in a protein predicted to lack key determinants for hijacking host cell signaling and vesicular transport. This defect was observed when nef sequences were amplified from peripheral blood mononuclear cells obtained at 4 different time points, with an additional stop codon emerging in some clones amplified from samples from the latest time point (Fig. S2). Consistently, this Nef protein lacked activity in any of the functional tests conducted in this study and was therefore excluded from all analyses described below. Notably, patient AA052 displayed very low viral load (397 copies/mL) and high CD4 counts (1259 CD4+ cells/μL) at the time of analysis and thus resembles the long-term nonprogressor status described for individuals infected with nef-defective HIV-1.54,55

Expression of Patient-Derived Vpu.GFP and Nef.GFP Fusion Proteins

We next evaluated whether the patient-derived vpu and nef sequences resulted in the expression of stable full length protein when fused to enhanced GFP. Western blot analysis of transiently transfected TZM-bl cells revealed that similar levels of full length Vpu.GFP protein were detected with the anti-GFP antibody for all patient-derived alleles (Fig. 1C, α-GFP). NL4-3 Vpu.GFP was only weakly detected with this antibody. Use of a Vpu-specific antibody revealed robust expression of full length NL4-3 Vpu.GFP protein whereas due to variation in the epitope recognized by this antibody,56 some patient-derived Vpu variants were not detected (Fig. 1C, α-Vpu). Full length GFP fusion proteins were also expressed for all patient-derived nef alleles, however to varying expression levels (Fig. 1D). This variability in Nef.GFP expression levels reflected differential transfection efficiency that was not specific to individual alleles in independent experiments (data not shown). Importantly, virtually no degradation products were observed for any of the patient-derived Vpu and Nef proteins, indicating that the expressed GFP fusion proteins allowed for reliable correlation of GFP fluorescence with protein function in flow cytometry-based cell surface receptor down-modulation analyses.

Downregulation of CD4, CD317/Tetherin and MHC-I by Natural Vpu and Nef Variants

To compare natural Vpu and Nef variants in their ability to modulate cell surface receptor levels, patient-derived Vpu.GFP and Nef.GFP proteins were transiently expressed in HeLa TZM-bl cells and subjected to quantitative analysis by flow cytometry. Well characterized Vpu and Nef proteins of lab-adapted HIV-1 strains (NL4-3 for Vpu and SF2 for Nef) and specific mutants thereof served as controls. Cell surface receptors were detected using allophycocyanin-conjugated antibodies and the relative receptor surface expression was calculated as the ratio in APC mean fluorescent intensity (MFI) between GFP-positive and -negative cells. Values obtained for control cells expressing GFP were arbitrarily set to 100% and values obtained for Vpu and Nef variants were normalized accordingly.22,56 We first analyzed cell surface exposure of CD4, CD317, and MHC-I (Fig. 2). Expectedly, NL4.3 Vpu.GFP efficiently reduced cell surface levels of CD4 (Figs. 2A, B) and CD317 (Figs. 2E, F) in a manner dependent on its di-serine motif that allows the viral protein to target interacting proteins to degradation (see S2/6A mutant in which serines 52 and 56 are mutated to alanine).57 NL4.3 Vpu.GFP had only moderate effect on MHC-I cell surface exposure (Figs. 2I, J). NL4-3 Nef more efficiently reduced CD4 (Figs. 2C, D) and MHC-I (Figs. 2K, L) cell surface density and, as recently reported,23 also reduced cell surface levels of CD317 (Figs. 2G, H). Expectedly,53 CD4 downregulation depended on Nef's di-leucine motif (see Nef LLAA mutant) whereas downregulation of MHC-I and CD317 was di-leucin motif independent and required the proline-rich SH3 domain binding motif in Nef (see AxxA mutant).

FIGURE 2
FIGURE 2:
CD4, CD317, and HLA-I downregulation by patient-derived Vpu or Nef constructs. HeLa TZM-bl cells were transfected with GFP, Vpu.GFP, or Nef.GFP fusion proteins. Forty-eight hours post transfection, cell surface levels of CD4, CD317, and HLA-I were analyzed by flow cytometry. A, Representative flow cytometry dot plots of gated living cells transfected with GFP and NL4-3 Vpu.GFP expression plasmids are shown. The CD4.allophycocyanin fluorescent intensity is displayed on the y-axis and the GFP fluorescent intensity on the x-axis. Mean fluorescent intensities are indicated. B, The CD4 surface expression relative to GFP-transfected cells is shown for all patient-derived Vpu alleles. The relative surface expression for each allele was calculated by dividing the MFI of Vpu.GFP expressing cells by the MFI of Vpu.GFP negative cells. For control cells transfected with GFP this value was arbitrarily set to 100% and all other values were normalized accordingly. Bars represent means of 3 independent experiments ± SD. Stars indicate the minimal statistically significant difference to the GFP control (one-way analysis of variance with Dunnett multiple comparisons test; *P ≤ 0.05; ***P ≤ 0.001). C and D, As in (A, B), but for Nef.GFP fusion proteins. E–H, As in (A–D), but for CD317. I–L, As in (A–D), but for MHC-I.

All patient-derived Vpu proteins significantly downregulated cell surface CD4 levels [median remaining CD4 levels relative to GFP 31.7% (IQR 28.5%–38.5%); P ≤ 0.0001; Dunnett test for multiple comparisons] (Figs. 2A, B). Similarly, the ability to reduce cell surface CD4 levels was highly maintained across all natural Nef variants [median remaining CD4 levels relative to GFP 14.0% (IQR 9.3%–20.2%), P ≤ 0.0001; Dunnett test for multiple comparisons] (Figs. 2C, D). Downregulation of cell surface CD317 was also conserved among the patient-derived Vpu and Nef proteins. Vpu variants were less effective than the NL4.3 Vpu.GFP control [median remaining CD317 levels relative to GFP 57.3% (IQR 53.2%–64.5%)] but all significantly reduced CD317 cell surface exposure (P ≤ 0.0001; Dunnett test for multiple comparisons) (Figs. 2E, F). An analogous analysis for nef alleles revealed CD317 cell downregulation activities comparable to SF2 Nef [median remaining CD317 levels relative to GFP 58.2% (IQR 48.2%–63.4%)] that was significant for all alleles analyzed (P ≤ 0.0001; Dunnett test for multiple comparisons) (Figs. 2G, H). Analysis of MHC-I cell surface levels revealed only moderate but statistically significant effects for all natural Vpu variants tested except AA015 [median remaining MHC-I levels relative to GFP 74.7% (IQR 71.2%–77.8%); P ≤ 0.05; Dunnett test for multiple comparisons], which were however more pronounced than MHC-I downregulation by the NL4.3 Vpu control (Figs. 2I, J). In contrast, Nef variants more robustly reduced cell surface MHC-I levels by 1.3 to 2-fold and this activity was conserved among all Nef variants [median remaining MHC-I levels relative to GFP 65.5% (IQR 57.6%–70.3%); P ≤ 0.0001; Dunnett test for multiple comparisons] (Figs. 2K, L). Together, downregulation of CD4, CD317, and MHC-I were overall conserved properties of the patient-derived Vpu and Nef alleles analyzed.

Downregulation of NK Cell Ligands NTB-A and PVR by Natural Vpu and Nef Variants

We next analyzed the ability of patient-derived Vpu and Nef proteins to downregulate NK cell ligands from the cell surface. Because they were reported to be downregulated by Vpu and by Nef, we first focused on NTB-A and PVR (Fig. 3). NL4.3 Vpu.GFP (Figs. 3A, B) and SF2 Nef.GFP (Figs. 3C, D) controls revealed moderate but statistically significant reduction in NTB-A levels (P ≤ 0.001; Dunnett test for multiple comparisons). In contrast, PVR cell surface exposure was robustly reduced by Nef (Figs. 3G, H) but not Vpu (Figs. 3E, F). The effects of SF2 Nef.GFP on NTB-A and PVR required its proline-rich SH3 binding motif but not the di-leucine motif. The moderate NTB-A downregulation observed by Vpu was independent of the di-serine motif. All natural Vpu variants analyzed significantly reduced cell surface NTB-A levels with alleles such as AA041, AA051, or UV266 displaying enhanced, up to 2-fold, downregulation of cell surface NTB-A as compared with the NL4-3 Vpu control [median remaining NTB-A levels relative to eGFP 75.6% (IQR 70.0%–78.3%), P ≤ 0.01; Dunnett test for multiple comparisons] (Figs. 3A, B). In turn, patient-derived nef alleles had negligible effects on cell surface NTB-A levels [median remaining NTB-A levels relative to GFP 89.4% (IQR 85.9%–92.2%)] that reached statistical significance (P ≤ 0.05; Dunnett test for multiple comparisons) for only 9 of the 19 alleles tested (Figs. 3C, D). Analyzing PVR cell surface levels revealed a robust downregulation to about 40% reduction of cell surface levels for some patient-derived Vpu proteins (eg, AA051, AA074) whereas others had no effect (eg, AA085, AA038) [median remaining PVR levels relative to GFP 86.7% (IQR 80.2%–95.0%)] (Figs. 3E, F). Overall PVR downregulation was statistically significant (P ≤ 0.05; Dunnett test for multiple comparisons) for 11 of 17 alleles tested. In contrast, all patient-derived Nef proteins significantly reduced cell surface PVR levels [median remaining PVR levels relative to GFP 54.0% (IQR 48.0%–61.7%), P ≤ 0.0001; Dunnett test for multiple comparisons] (Figs. 3G, H). Overall, natural Vpu variants reduced cell surface levels of NTB-A, but had variable effect on PVR expression levels. In contrast, most Nef variants failed to affect NTB-A cell surface levels, but all reduced cell surface exposure of PVR.

FIGURE 3
FIGURE 3:
NTB-A and PVR downregulation by patient-derived Vpu or Nef constructs. A3.01 T cells or HeLa TZM-bl cells were transfected with GFP, Vpu.GFP or Nef.GFP fusion proteins. Forty-eight hours post transfection, cell surface levels of NTB-A or PVR were determined by flow cytometry and analyzed as described in legend of Figure 2. A, Representative flow cytometry dot plots of gated living cells transfected with GFP and NL4-3 Vpu.GFP expression plasmids. NTB-A. Allophycocyanin and GFP fluorescent intensities are displayed on the y axis and x axis, respectively. Mean fluorescent intensities are indicated. B, NTB-A surface expression relative to GFP-transfected cells for all patient-derived Vpu alleles. Bars represent means of 3 independent experiments ± SD. Stars indicate the minimal statistically significant difference to the GFP control (oneway analysis of variance with Dunnett multiple comparisons test; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001). C and D, As in (A, B), but for Nef fusion proteins. E–H, As in (A–D) but for PVR.

Downregulation of NK Cell Ligands ULBP-2,5,6 and MICA

Since Nef was also described to reduce cell surface levels of the NK cell ligands ULBP and MICA42 and Vpu and Nef display a significant functional overlap,22 we also analyzed all patient-derived Vpu and Nef variants for downregulation of these receptors. MICA cell surface levels were moderately downregulated by the NL4.3 Vpu and SF2 Nef controls (85% and 83% of cell surface molecules remaining). This weak activity was not well conserved among natural variants with only 7 of 17 Vpu alleles and 0 of 19 Nef variants displaying statistically significant activity (P ≤ 0.05; Dunnett test for multiple comparisons) (Fig. S3). In contrast, significant downregulation of ULBP-2,5,6 cell surface was observed for NL4-3 Vpu (Figs. 4A, B) and SF2 Nef (Figs. 4C, D) (44% and 56% of cell surface molecules remaining). All natural Vpu variants significantly reduced cell surface levels of ULBP-2,5,6 [median remaining ULBP-2,5,6 levels relative to GFP 49.7% (IQR 44.1%–52.7%), P ≤ 0.0001; Dunnett test for multiple comparisons] (Figs. 4A, B). This activity was less pronounced and conserved among natural Nef variants [median remaining ULBP-2,5,6 levels relative to GFP 81.0% (IQR 72.7%–84.1%)], with 9 of 19 Nef variants displaying statistically significant activity (P ≤ 0.05; Dunnett test for multiple comparisons) (Figs. 4C, D). Because downregulation of cell surface ULBP-2,5,6 by Vpu had not been reported before, and the molecular determinants of HIV-1 Nef mediating this effect had only partially been identified,42 we used a mutant panel to map motifs in both viral proteins required for ULBP-2,5,6 downregulation (Figs. 4E–H). In the case of Vpu (Figs. 4E, F), not only the di-serine motif (S2/6A mutant) but also residues required for interaction with CD317/tetherin (A14L/W22A mutant) were dispensable. Residues governing subcellular localization51 (R30K31A mutant) and membrane microdomain association50 (V25GY29G mutant) of Vpu or the identity of Vpu's transmembrane (mutant TM vesicular stomatitis virus G glycoprotein in which the transmembrane domain of Vpu is replaced with that of vesicular stomatitis virus G glycoprotein) contributed to ULBP-2,5,6 downregulation. Most importantly, residues that couple Vpu to the clathrin adaptor protein complex 1 (AP-1)58 were essential for this activity (see H2ELV Vpu mutant). In contrast, not only association with host cell endocytic machinery but also signal transduction complexes was dispensable for ULBP-2,5,6 downregulation by Nef as the mutants LLAA, EDAA (both deficient in interaction with endocytic machinery), E4A4 (lacks interaction with the sorting adaptor PACS), AxxA (SH3 domain binding site disrupted), and Δ12-39 (lacks binding site for the Nef-associated kinase complex) retained almost full downregulation activity (Figs. 4G, H). In contrast, determinants for membrane association (G2A Nef mutant without N-terminal myristoylation site), segregation into membrane microdomains (KKAA mutant) and interaction with the host cell kinase PAK2 (F195A mutant) were required for efficient ULBP-2,5,6 downregulation by Nef. These results suggest that Vpu and Nef downregulate cell surface ULBP-2,5,6 by distinct mechanisms and identify ULBP-2,5,6 downregulation as a novel conserved activity of HIV-1 Vpu.

FIGURE 4
FIGURE 4:
ULBP-2,5,6 downregulation by patient-derived Vpu or Nef constructs. A3.01 T cells were transfected with expression plasmids for GFP, Vpu.GFP or Nef.GFP fusion proteins. Forty-eight hours post transfection cell surface levels of ULBP-2,5,6 were determined by flow cytometry and analyzed as described in legend to Figure 2. A, Representative flow cytometry dot plots of gated living cells transfected with eGFP and NL4-3 Vpu.GFP expression plasmids. ULBP-2,5,6.allophycocyanin and GFP fluorescent intensities are displayed on the y axis and x axis, respectively. Mean fluorescent intensities are indicated. B, ULBP-2,5,6 surface expression relative to GFP-transfected cells for all patient derived Vpu alleles. Bars represent means of 3 independent experiments ± SD. Stars indicate the minimal statistically significant difference to the GFP control (oneway analysis of variance with Dunnett multiple comparisons test; *P ≤ 0.05; ***P ≤ 0.001). C and D, As in (A, B), but for Nef fusion proteins. E, Representative flow cytometry dot plots of gated living cells transfected with GFP and NL4-3 Vpu S2/6A.GFP expression plasmids. The ULBP-2,5,6.APC fluorescent intensity is displayed on the y axis and the GFP fluorescent intensity on the x axis. F, The ULBP-2,5,6 surface expression relative to GFP-transfected cells for all Vpu mutants. The relative surface expression for each mutant was calculated by dividing the MFI of Vpu.GFP expressing cells by the MFI of Vpu.GFP negative cells. For control cells expressing GFP, this value was arbitrarily set to 100% and all other values were normalized accordingly. Bars represent means of 3 independent experiments ± SD. G and H, As in (E, F), but for Nef proteins.

DISCUSSION

The aim of this study was to analyze HIV-1 Vpu and Nef variants isolated from chronic HIV patients for their ability to downregulate cell surface levels of host cell receptors with a particular focus on NK cell ligands. In agreement with previous reports,56,59,60 downregulation of cell surface CD4, CD317/tetherin and MHC-I were well conserved among patient derived Vpu as well as Nef variants, confirming the previous observation that both viral proteins target an overlapping set of host cell receptors.22 In contrast, NK cell ligand downregulation was more specific with NTB-A and ULBP being more frequently targeted by Vpu and PVR by Nef variants. This study only addressed the general potency of these patient-derived alleles upon ectopic expression and does not take into account all properties of these proteins in the context of HIV primary cell infection. Despite this limitation, the results reveal downregulation of NTB-A, PVR, and ULBP by at least one of the accessory HIV proteins Vpu and Nef, as a conserved activity of HIV isolates from chronic patients and reveal ULBP as a previously unrecognized target for downregulation by Vpu.

Although we did not investigate the molecular mechanisms employed by Vpu or Nef for downregulation of individual receptors, correlating downregulation activities of the patient-derived alleles for individual receptors provided some mechanistic insight. This is of particular interest for downregulation of PVR and ULBP for which such information is currently unavailable. For Vpu, downregulation of PVR correlated with downregulation of MHC-I (P = 0.009) and possibly CD317/tetherin (P = 0.019) (Fig. S5 A–D). In line with the finding that both effects require an interaction motif in the transmembrane domain of Vpu,38,48 this correlation suggests that downregulation of PVR, MHC-I, and CD317/tetherin may be mediated by similar molecular mechanisms. Consistent with results by Pickering et al,60 patient-derived Vpu variants were often more active than the lab-adapted NL4-3 Vpu (eg, for PVR and MHC-I). In contrast, the newly identified downregulation of ULBP did not correlate with any other receptor downregulation by Vpu alleles (Fig. S5 E–I) and was comparable for lab-adapted and patient-derived Vpu variants. The analysis of Vpu mutants suggests that ULBP downregulation by Vpu does not rely on the di-serine motif and is thus distinct from many of the described receptor downregulation mechanisms by the viral protein. Because disruption of Vpu's interaction motif with the AP-1 adaptor abrogates ULBP downregulation, Vpu may affect anterograde transport of this receptor, however through a different mechanism than described for CD317/tetherin or NTB-A.40,41 Similar analysis for the nef alleles analyzed suggested that downregulation of PVR is mechanistically linked to effects on MHC-I, CD317, NTB-A (P < 0.0001), and ULBP (P = 0.0008) but not CD4 (Fig. S6 A–D, I). A similar correlation pattern, however with lower statistical significance, was observed for ULBP (P = 0.007 for MHC-I; P = 0.017 for CD317; P = 0.003 for NTB-A). Among these, downregulation of MHC-I by Nef is studied best and is thought to result from re-routing of the receptor to degradation during anterograde transport in an AP-1 and β-COP dependent manner.61,62

Because of the low numbers of patient-derived Vpu and Nef variants studied, and the analysis of patient samples from one individual time point, this study does not allow drawing firm conclusions on whether downregulation of NK cell ligands by Vpu and/or Nef is linked to the clinical progression of the respective patient. However, these analyses provide first insight into whether the ability of downregulating NK cell ligands is associated with NK cell activation state and number in HIV patients. Vpu or Nef proteins displayed similar downregulation activity for NTB-A, PVR, or ULBP irrespective of whether they were isolated from patients with high or low NK cell activity, as judged by the presence of CD107a-positive NK cells (Fig. S4). Similar results were obtained when using other NK cell activation markers (Table S1 and data not shown) or when correlating NK cell ligand downregulation activity to total NK cell counts (Fig. S4). The magnitude by which the predominant Vpu and Nef variants can downregulate surface levels of NK cell ligand thus does not appear to impact significantly on these parameters in chronic HIV patients. Nevertheless, downregulation from the cell surface by at least Vpu or Nef was conserved for all NK cell ligands analyzed except MICA. This suggests that despite the absence of a clinical signature in chronic HIV patients, this downregulation activity may be relevant to the overall outcome of HIV infection. HIV accessory protein function is subject to remarkable plasticity in the course of HIV infection and rapidly adapts to prevailing selection pressures such as cytotoxic T lymphocytes or antiviral restriction factors.23,63 Considering the role of NK cells as early defense mechanism against HIV, NK cell receptor downregulation may be particularly relevant during the acute phase of infection. In this scenario, the overall conservation of NK cell ligand downregulation by Vpu or Nef in chronic HIV patients would be a footprint of a potent selection pressure for this activity during acute infection. Consistently, downregulation of NTB-A was recently reported to be conserved in Vpu variants of transmitted founder viruses.39 It will therefore be interesting to analyze the full spectrum of NK cell ligand downregulation of Vpu, Nef, and possibly Vpr variants of transmission founder viruses and their evolution during the acute phase of infection. NK cell activation is regulated by the complex interplay of activating, costimulatory, and inactivating ligands. How the specific cell surface expression profile of NK cell ligands induced by individual patient-derived accessory proteins affects the efficiency of NK cells to lyse HIV infected cells thus remains to be determined. To integrate individual effects of all accessory proteins, such studies should address their activity in parallel, preferentially in the context of infectious molecular clones.

In conclusion, this study reveals that NK cell ligand downregulation by Vpu and/or Nef is well conserved in chronic HIV patients and provides a framework for future studies on the underlying molecular mechanisms. These results warrant an in-depth analysis of NK cell ligand downregulation of accessory protein variants during HIV-1 transmission and acute infection.

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Keywords:

HIV; Vpu; Nef; NK cell ligands; cell surface downregulation

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