Published online before print January 6, 2005
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
,1
* Department of Pathology, Vanderbilt University, Nashville, Tennessee;
Genpathway Inc., San Diego, California; and
Department of Veterans Affairs Medical Center, Nashville, Tennessee
1 Correspondence: VA Medical Center/Research Service, 1310 24th Ave. S., Nashville, TN 37211. E-mail: Virginia.l.shepherd{at}vanderbilt.edu
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
50% in the presence of Nef and that the MR cytoplasmic tail can confer susceptibility to Nef in the CD4/MR chimera. These data suggest that the MR is a potential intracellular target of Nef and that this regulation may represent a mechanism to further cripple the host innate immune system.
Key Words: macrophages • trafficking • HIV-derived proteins
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
At least five HIV-derived proteins are known to contribute to host protein regulation, including Tat, Nef, Vpu, Vpr, and Env. Each of these proteins acts at a specific point in the host protein life-cycle. For example, the Tat and Vpr proteins regulate host protein expression at the level of transcription [10 11 12 13 ]; Env and Vpu regulate protein expression by slowing the movement of newly synthesized molecules in the endoplasmic reticulum and facilitating movement of the proteins into the proteosome pathway for degradation [14 , 15 ]; and Nef modulates several host surface proteins through interference with normal intracellular trafficking [3 , 16 ]. Of these five proteins, Nef plays a major role in regulation of two important immune molecules: CD4 and MHC I [3 , 16 ]. In the case of CD4, Nef acts as a bridge between a conserved amino acid sequence in the CD4 cytoplasmic tail and adaptor protein-2 (AP-2) of the clathrin machinery, facilitating rapid removal of CD4 from the cell surface followed by delivery to lysosomes for subsequent degradation [17 ]. Nef facilitates removal of MHC I from the surface through a clathrin-independent pathway, resulting in sequestration in the trans-Golgi network (TGN) [2 , 18 ].
Another surface protein, the mannose receptor (MR), is found predominantly on macrophages and dendritic cells (DC) and performs a variety of functions related to innate immunity. The MR has recently been shown to be regulated following HIV infection. Koziel et al. [19
] demonstrated that alveolar macrophages isolated from HIV-infected patients demonstrated an 80% reduction in MR-dependent pathogen clearance, and studies from our laboratory have shown that HIV-1 Tat mediates down-regulation of the MR at a transcriptional level [12
]. In the latter study, Tat decreased MR transcription by 40%, suggesting that additional HIV proteins may contribute to the 80% reduction in the presence of intact virus. Therefore, as a result of similarities in CD4, MHC I, and the MR with respect to cytoplasmic tail sequences, trafficking pathways, and/or function in host immune responses, we investigated the role of Nef in regulating expression of the MR.
The MR is a 180-kDa transmembrane C-type lectin and was first described as a clearance receptor for extracellular hydrolases [20
] and peroxidases [21
]. In more recent work, the function of this receptor has been expanded to include ingestion of a variety of pathogens (reviewed in ref. [22
]) and delivery of soluble antigens to MHC II-containing compartments for subsequent antigen presentation [23
]. These functions have been verified by loss of these activities in mice deficient in MR expression [24
, 25
]. Expression of the MR is tightly linked to the functional state of macrophages and DC. Levels are highest on resident and nonactivated macrophages and immature DC and lowest on cells that have been exposed to activating agents such as interferon-
[26
, 27
] or tumor necrosis factor [28
]. In addition to regulation by HIV, it has been reported that exposure of macrophages to Leishmania, bacillus Calmette-Guerin, or Candida results in down-regulation of MR expression [29
30
31
], suggesting that pathogens have the ability to alter the available entry pathway on the host cell, which could potentially affect the fate of that pathogen.
Studies have suggested that the MR resembles a classic recycling receptor, moving into the clathrin pathway after ligand binding [32 ]. Ligand and receptor separate in the acidic endosomal compartment followed by return of the receptor to the surface and delivery of the ligand to the lysosomal compartment. At least three conserved sequences have been implicated in linking the cytoplasmic tail of surface receptors to intracellular proteins involved in endocytosis [33 ]. A tyrosine residue within a conserved NPXY appears to be required for appropriate trafficking of a number of receptors. The MR contains a similar NTLY sequence (Fig. 1A ), and mutation of the tyrosine residue reduces MR-mediated endocytosis and phagocytosis [34 , 35 ]. An additional, conserved sequence containing a dileucine sequence (SDXXXLL) has been reported to mediate interaction with the µ2 component of AP-2 within the clathrin complex. A similar sequence (SQIKRLL) is involved in CD4 internalization, and mutation of the dileucine reduces internalization [36 , 37 ]. The MR also contains a sequence similar to this motif (SDTKDLV), which could potentially serve as a connector to clathrin (Fig. 1A) . Finally, the motif YSQA has been reported to be involved in appropriate sorting of MHC I, and the MR contains a similar sequence (YFNS).
 View larger version (21K): [in a new window] |
Figure 1. Schematic illustration of cytoplasmic tail sequences and chimeric molecule. (A) Sequences in shaded regions of the CD4 and MHC I molecules are those known to participate in the regulation by HIV-1 Nef. The shaded regions in the MR are those with sequence similarity to CD4 and MHC I and represent putative sites of interaction with Nef. (B) A schematic representation of the chimeric CD4/MR with the sequences postulated to interact with HIV-1 Nef shaded.
|
In the current study, we have used a reporter chimeric molecule consisting of the extracellular and transmembrane domains of CD4 linked to the cytoplasmic tail of the MR (CD4/MR; Fig. 1B ) to examine the mechanism of Nef-mediated MR regulation. We report that the MR tail region contains sequences that allow for Nef-mediated modulation. The mechanism of down-regulation appears to be more similar to that described for MHC I in the presence of Nef, where surface CD4/MR is removed through a direct interaction with Nef, followed by potential sorting to the TGN.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
For purposes of performing the studies involving the CD4/MR mutant chimeras, the epithelial cell line 293T (obtained from Dr. Christopher Aiken, Vanderbilt University, Nashville, TN) was maintained in Dulbecco’s modified Eagle’s medium with 10% FBS and antibiotics. For transient and stable transfectants, the HeLa continuous cell line, obtained from the American Type Culture Collection (Manassas, VA), was maintained in RPMI-1640 medium with 10% FBS, 100 U/ml penicillin and streptomycin, and 100 ng/ml gentamycin.
Viruses
The viruses used for the various studies were prepared in a bio-safety level 3 laboratory in the Department of Microbiology, Vanderbilt University, under the direction of Dr. Christopher Aiken. The macrophage-tropic laboratory strain NLHX-ADA, used for infection of cells, was provided by Dr. Paul Spearman (Vanderbilt University). Determination of infectivity and titer for all viruses was accomplished using an enzyme-linked immunosorbent assay-based assay for p24 levels, and titers were ascertained via a P4.R5-Magi cell assay [49
]. Adenovirus constructs expressing the full-length Nef and green fluorescent protein (GFP) were obtained from Dr. Mario Stevenson (University of Massachusetts, Amherst) [50
].
Plasmids and antibodies
Plasmids CMX-CD4, CMX-CD8, and CMX-Nef were obtained from Dr. Christopher Aiken. CD4, CD8, and Nef were further subcloned into the pcDNA 4.0 expression vector (Invitrogen, Carlsbad, CA). Rat bone marrow macrophage (RBM) cDNA and the CMX-CD4 vector were used to generate the CD4/MR chimeric protein (Fig. 1B)
in an overlapping polymerase chain reaction (PCR) strategy. The CD4 extracellular and transmembrane regions were amplified from CMX-CD4, and the MR tail region was amplified from RBM cDNA. The chimera was made by combining the two products in a reaction containing a sense CD4 primer and an antisense MR tail primer. The product was then cloned into the CMX vector using the restriction sites BamHI and EcoRI. The pCG-Nef-GFP expression vector was obtained from Jacek Skowronski (Cold Spring Harbor Laboratories, NY). The DsRed-tagged HIV-1 Nef was obtained from Kathleen Collins (University of Michigan, Ann Arbor) [51
]. Antibodies used in immunoblots for detection of CD4 and Nef were obtained from the National Institutes of Health (NIH) AIDS Research and Reference Reagent Program (Rockville, MD). Polyclonal antibody used for detection of the MR was prepared in our laboratory [52
]. Monoclonal antibody against the human MR was obtained from PharMingen (San Diego, CA). Antibodies conjugated to Alexa dyes (488, 568, and 647) for use in confocal studies were obtained from Molecular Probes (Eugene, OR). Phycoerythrin (PE)-conjugated anti-MR, PE- and fluorescein isothiocyanate (FITC)-conjugated anti-CD4, and PE- and FITC-conjugated anti-MHC I for use in fluorescent-activated cell sorting (FACS) were obtained from PharMingen. Alexa-488-conjugated anti-CD4 was obtained from Caltag Laboratories (Burlingame, CA).
Transfection of cells
HeLa and 293T cells were transfected using Polyfect transfection reagent (Qiagen, Valencia, CA), according to the manufacturer’s directions. Briefly, cells were cultured at a concentration of 2 x 106 cells per P-100 dish overnight. Plasmid DNA (10 µg) was transfected into cells using 80 µl Polyfect reagent, and cells were incubated overnight at 5% CO2 and 37°C. Cells were cotransfected with CD4 or CD4/MR and Nef-GFP or a control vector. In cases where Nef-GFP was not used, CD8 was cotransfected for the purposes of gating positively transfected cells. Following incubation, the cells were washed, and fresh RPMI with 10% FBS was added. Cells were collected 48 h post-transfection for flow cytometry or immunoblot analysis. Transfection of cells for confocal experiments was performed in 35 mm glass-bottom dishes (MatTek, Ashland, MA). Briefly, 5 x 105 cells were seeded into each dish and incubated overnight. Plasmid DNA (2 µg) was transfected into cells using 20 µl Polyfect reagent. The cells were incubated overnight at 5% CO2 and 37°C. Following incubation, the cells were washed, and fresh RPMI with 10% FBS was added. Cells were collected 24 h and 48 h post-transfection. Stable transfectants were generated by transiently transfecting the pcDNA 4.0 vector expressing CD4 or CD4/MR into HeLa cells, which were incubated overnight at 5% CO2 and 37°C. Following overnight incubation, stable transfectants were selected by continuous culture in media containing 125 µg/ml Zeocin. Following confluent growth of cells in Zeocin-containing media, the cells were analyzed by flow cytometry to confirm expression of CD4 or CD4/MR.
Flow cytometry
Flow cytometry was performed using a FACSCaliber (Becton Dickinson, San Jose, CA) bench-top analyzer in the Vanderbilt University Howard Hughes Medical Institute core facility. Cells were stained with FITC-, Alexa-488-, and PE-conjugated antibodies against CD4 and MR as follows: Cells were collected by centrifugation, followed by suspension in staining buffer [1% bovine serum albumin (BSA), 0.1% sodium azide, 0.5% normal goat serum in PBS]. Normal goat serum was included in the stain buffer to saturate the Fc receptor (FcR) and minimize autofluorescence. The appropriate concentrations of FITC-, Alexa-, or PE-conjugated antibodies were diluted in staining buffer and added to the cells. In addition, an isotype-matched control was performed to account for nonspecific fluorescence, and an unstained sample was included to account for autofluorescence. The cells were incubated for 20 min in the dark at 4°C. The cells were washed twice with staining buffer followed by fixation in 500 µl 2% paraformaldehyde. The modulation of cell-surface receptor density was represented as the percent change in mean fluorescence intensity (MFI) as compared with the control. The number of events acquired for each sample was 3 x 104, and cells were analyzed on a Becton Dickinson FACScan flow cytometer using CellQuest software.
Confocal microscopy
Confocal microscopy was performed using a Zeiss LSM 510 confocal laser-scanning inverted microscope in the Cell Imaging Core Laboratory at Vanderbilt University. Cells were stained in a two-step method using monoclonal anti-CD4 or monoclonal anti-Nef followed by Alexa-488, Alexa-568, or Alexa-647 secondary dyes. Cells were grown on MatTek 35 mm glass-bottom dishes, and following transfection, the cells were fixed in ice-cold methanol. The cells were blocked in PBS with 5% BSA for 15 min. Following one wash in PBS, the primary antibody was added at the appropriate dilution and incubated for 1 h at room temperature. The cells were then rinsed twice in PBS, and the appropriate secondary antibody was added and incubated for 1 h at room temperature. The cells were rinsed twice with PBS and mounted with Aqua-Polymount mounting media (Polysciences, Warrington, PA). Cells were imaged, and serial Z-sections were taken of cells. Images were detected at 488, 543, and 647 nm using a 63x oil immersion objective lens and collected simultaneously on a Z-plane of focus 3–4 µ from the upper edge. Images were collected with a pinhole of one, and each of the lasers was optimized independently with single dye-stained control cells to prevent bleed-through of signal from one channel to the next. Image processing was performed using Adobe Photoshop imaging software.
Site-directed mutagenesis
Mutations in the cytoplasmic tyrosine and dileucine sequences were engineered into the chimeric CD4/MR by alanine substitution using the Quickchange PCR site-directed mutagenesis kit from Stratagene (La Jolla, CA), according to the manufacturer’s instructions. The desired mutations were inserted using the 5' and 3' primers as shown in Table 1
. Verification of successful mutagenesis was performed by sequence analysis in the Vanderbilt University DNA sequencing core facility.
|
View this table: [in a new window] |
Table 1. Oligonucleotides Used in Mutagenesis Reactions
|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
 View larger version (16K): [in a new window] |
Figure 2. Infection of MDM with adenovirus expressing HIV-1 Nef. MDM (5x106) were plated into six-well dishes. The cells were washed, and 1 ml serum-free media containing adenovirus (Ad) GFP or adenovirus Nef [multiplicity of infection (MOI)=10] was added to the cell monolayer and allowed to adsorb for 1 h. Following adsorption, the cells were washed, and fresh media with serum was added for 72 h. Following incubation, the cells were harvested for FACS and immunoblot analysis. Cells were harvested and stained with PE-conjugated anti-MR antibody. Cells were also harvested for unstained and negative-control samples, which were incubated with PE-conjugated isotype-matched control. The number of events acquired for each samples was 3 x 104. Marker gates were established for positive cells based on the fluorescence of isotype-matched controls. (A) Relative change in mean fluorescence as compared with mock-infected for MR and CD4 following infection with adenovirus. Data are expressed as a representative experiment of two independent experiments. (B) Immunoblot analysis of total CD4 protein (upper panel) and MR protein (lower panel) present in Nef-expressing cells. Data are representative of three independent experiments.
|
 View larger version (14K): [in a new window] |
Figure 3. Infection of a macrophage hybridoma cell line with macrophage-tropic HIV-1 virus or Nef-expressing adenovirus. MR-positive 43 cells (1x106) were seeded into T-25 flasks in minimal media. Cells were infected with NLHX-ADA macrophage-tropic virus (100 ng/p24; A) or adenovirus (Ad) expressing Nef (MOI=10; B) and incubated for 72 h. Following incubation, the cells were harvested and stained with PE-conjugated anti-MR antibody. Cells were also harvested for unstained and negative-control samples, which were incubated with PE-conjugated, isotype-matched control. The number of events acquired for each samples was 3 x 104. Marker gates (PE+) were established for positive cells based on the fluorescence of isotype-matched controls. Histograms of representative experiments show a reduction in MFI of MR in the presence of HIV-1 Nef (bold curve) as compared with mock-infected cells (shaded curve) and isotype-matched control (dotted curve). Data shown are the relative change in MFI of MR as compared with the mock-infected control. The data are the mean ± SD of three independent experiments. **, P < 0.01, versus control.
|
50% reduction in the surface levels of CD4 and CD4/MR in the presence of HIV-1 Nef (Fig. 4 A and B
). Cell lysates were examined by immunoblot assay to assess the level of degradation following receptor internalization. In agreement with results using primary macrophages and MR-positive 43 cells, we found that Nef facilitated removal of CD4 from the surface followed by degradation (Fig. 4C
, left panel). In contrast, the chimeric molecule was not degraded, suggesting a sequestering of the receptor in an intracellular compartment (Fig. 4C
, right panel).
 View larger version (25K): [in a new window] |
Figure 4. Effect of HIV-1 Nef on CD4 and CD4/MR stably expressed in HeLa cells (2x106), which were plated in P-100 tissue-culture dishes and transiently transfected with a pcDNA 4.0 vector expressing Nef. Cells were also transfected with an empty CMX or pcDNA 4.0 vector as controls. Cells were harvested for FACS and Western blot at 48 h and were harvested and stained with Alexa-488- or PE-conjugated anti-CD4 antibody. Cells were also harvested for unstained and negative-control samples, which were incubated with Alexa-488- or PE-conjugated isotype-matched control. The number of events acquired for each sample was 3 x 104. Marker gates (M1) were established for positive cells based on the fluorescence of isotype-matched controls. (A) Histograms of representative experiments showing a reduction in the MFI of CD4 and CD4/MR in the presence of HIV-1 Nef (bold curve) as compared with control-transfected cells (shaded curve). Data are representative of three experiments. (B) Surface levels of CD4 and CD4/MR following transfection with control vector or Nef-expressing vector. After 48 h, surface expression was measured by flow cytometry. The data are the mean ± SD of three independent experiments. (C) Immunoblot analysis of total protein for CD4 (left panel) or CD4/MR (right panel) following transfection with control vector or Nef-expressing vector. Data are representative of three independent experiments. **, P < 0.01, versus control.
|
 View larger version (18K): [in a new window] |
Figure 5. Confocal analysis of HIV-1 Nef transient transfection in HeLa cells stably expressing CD4/MR (5x104), which were plated into 35 mm MatTek glass-bottom, tissue-culture dishes and transiently transfected with a vector expressing DsRed tagged-Nef. Cells were also transfected with a pcDNA 4.0 control vector. Cells were stained for confocal analysis with an Alexa-488-conjugated anti-CD4 antibody. (A–D) Fluorescent staining of HeLa cells stably expressing CD4/MR following transfection with DsRed-Nef at 48 h; (E–H) fluorescent staining of HeLa cells stably expressing CD4/MR following transfection at 48 h with TGN46 marker staining. Cells were imaged, and serial Z-sections were taken of cells. Cells were determined to be 6–8 µ in thickness, and representative images were collected simultaneously on a Z-plane of focus. Images are presented at the midpoint of the cell (3.0–4.0 µ). Cells were imaged under oil immersion at 63x original optical with additional 2x original digital zoom. Data are representative of at least three independent experiments.
|
 View larger version (19K): [in a new window] |
Figure 6. Confocal analysis of HIV-1 Nef transient transfection in HeLa cells stably expressing CD4 (5x104), which were plated into 35 mm MatTek glass-bottom, tissue-culture dishes and transiently transfected with a vector expressing DsRed tagged-Nef. Cells were also transfected with a pcDNA 4.0 control vector. Cells were stained for confocal analysis with an Alexa-488-conjugated anti-CD4 antibody. (A–D) Fluorescent staining of HeLa cells stably expressing CD4 following transfection with DsRed-Nef at 48 h; (E–H) fluorescent staining of HeLa cells stably expressing CD4 following transfection at 48 h with TGN46 marker staining. Cells were imaged, and serial Z-sections were taken of cells. Cells were determined to be 6–8 µ in thickness, and representative images were collected simultaneously on a Z-plane of focus. Images are presented at the midpoint of the cell (3.0–4.0 µ). Cells were imaged under oil immersion at 63x original optical with additional 2x original digital zoom. Data are representative of at least three independent experiments.
|
 View larger version (14K): [in a new window] |
Figure 7. Effect of HIV-1 Nef on transient transfection of 293T cells (2x106), which were plated in P-100 tissue-culture dishes and transiently transfected with CMX or pcDNA 4.0 vectors expressing Nef. Cells were also transfected with an empty CMX or pcDNA 4.0 vector as a control. Cells were harvested and stained with Alexa-488- or PE-conjugated anti-CD4 antibody and were also harvested for unstained and negative-control samples, which were incubated with Alexa-488- or PE- conjugated, isotype-matched control. The number of events acquired for each sample was 3 x 104. Marker gates were established for positive cells based on the fluorescence of isotype-matched controls. (A) Surface levels of CD4 and CD4/MR following transfection with control vector or Nef-expressing vector. After 48 h, surface expression was measured by flow cytometry. The data are the mean ± SD of three independent experiments. (B) Immunoblot analysis of total protein for CD4 (left panel) or CD4/MR (right panel) following transfection with control vector or Nef-expressing vector. Data are representative of three independent experiments. **, P < 0.01, versus control.
|
 View larger version (26K): [in a new window] |
Figure 8. Transient transfection of 293T cells with mutant CD4/MR chimera molecules. 293T cells (2x106) were plated in P-100 tissue-culture dishes and transiently cotransfected with pcDNA 4.0 vectors expressing Nef and wild-type CD4/MR or CD4/MR mutants. Cells were also transfected with an empty pcDNA 4.0 vector as a control. Cells were harvested for FACS at 48 h and were harvested and stained with Alexa-488- or PE-conjugated anti-CD4 antibody. Cells were also harvested for unstained and negative-control samples, which were incubated with Alexa-488- or PE-conjugated, isotype-matched control. The number of events acquired for each sample was 3 x 104. Marker gates were established for positive cells based on the fluorescence of isotype-matched controls. (A) Schematic representation of CD4/MR mutants generated by alanine substitution used in transient cotransfections of 293T cells. (B) Percent down-regulation of surface expression of CD4/MR wild-type and CD4/MR mutants following transfection with control vector or Nef-expressing vector. After 48 h, surface expression was measured by flow cytometry. (C) Immunoblot analysis of transient transfection of mutant chimera molecules in the presence or absence of HIV-1 Nef at 48 h. The data are the mean ± SD of three independent experiments *, P < 0.05, versus wild-type CD4/MR; ***, P < 0.001, versus wild-type CD4/MR.
|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
50%. These results are in agreement with previous work from Koziel et al. [55
], who reported that alveolar macrophages isolated from HIV+ individuals demonstrated up to an 80% reduction in MR surface expression and endocytic activity, and in vitro infection of normal alveolar macrophages with HIV reduced MR-mediated endocytosis and phagocytosis by 53% and 67%, respectively. However, only limited information is available concerning the mechanisms involved in this down-regulation. The goal of the current study was to extend the studies on HIV-mediated regulation of the MR and to specifically investigate the role of the HIV-derived protein Nef in MR modulation.
Studies of Nef-mediated down-regulation of CD4 and MHC I expression have shown that in both cases, Nef interferes with normal intracellular trafficking, facilitating removal of the molecules from the surface leading to increased degradation or intracellular sequestration. Comparison of the MR with CD4 and MHC I provided compelling evidence that the MR might also be a target for the Nef protein: All three molecules are involved in the immune response, each is expressed on macrophages and moves into the endocytic pathway once internalized, and all three receptors share classic sorting motifs in their cytoplasmic tail. To examine Nef-mediated regulation of the MR, we used expression of a chimeric molecule constructed from the extracellular and transmembrane N-terminal portion of CD4 and the cytoplasmic tail regions of the MR (CD4/MR). This was necessitated by the technical difficulties experienced by a number of laboratories in expressing a full-length, functional MR in mammalian cells and is similar to the strategies used by Kruskal et al. [34
] and Schweizer et al. [35
] to examine cytoplasmic sequences involved in MR trafficking. Using cells transiently and stably expressing CD4 or CD4/MR, we found that surface expression of both molecules was reduced by 50% following exposure to Nef, in agreement with previous reports on CD4 regulation by Nef and supporting our hypothesis that Nef participates in MR regulation. Furthermore, as previously reported, Nef enhanced degradation of CD4, as shown by decreased cellular CD4 by immunoblot analysis. In contrast, total cellular CD4/MR was not altered by Nef, suggesting that CD4/MR chimeras were removed from the cell surface but targeted for storage within the cell.
A number of studies have provided information concerning mechanisms involved in Nef-mediated CD4 regulation [3
, 4
, 37
, 45
, 56
]. Normally, CD4 internalization follows a clathrin-dependent pathway, where CD4 is delivered to the lysosomal-degradative compartment with a turnover time of 8 h. It has been postulated that Nef interferes with this pathway by providing a key recognition signal for the AP-2 component of the clathrin complex [36
]. CD4 delivery to lysosomes is then accelerated in the presence of Nef through an ARF-1- and ß-COP 1-dependent route [45
, 57
, 58
], decreasing the half-life of the protein to <1 h. In contrast, very little information is available concerning the normal routing of the MR and how Nef might interfere with surface expression of this receptor. It has been suggested that the pathway for MR endocytic trafficking is clathrin-dependent. Harding et al. [32
] reported that ligand-loaded or nonligated MR moves into the cell and associates with clathrin-coated vesicles. Once recruited into clathrin-coated pits, the receptor is internalized into an early endosome. Following acidification, the ligand and receptor separate, and the receptor returns to the plasma membrane, and the ligand moves on to the lysosomal compartment.
Given the differences in the routing pathways for the MR and CD4, the finding in the current study that the MR is not degraded in the presence of Nef suggests that Nef may be interfering with the recycling of the MR to the surface or may be actively redirecting the MR to an intracellular storage location. This latter scenario is similar to the studies that have described Nef-mediated regulation of MHC I expression, where Nef binds to the MHC I cytoplasmic tail and facilitates downstream interaction with cellular molecules such a PACS-1, resulting in sequestration of MHC I in the TGN through a clathrin-independent phosphatidylinositol 3-kinase regulated-ARF-6 pathway [18 ].
To gain some insight into the intracellular localization of the CD4/MR molecule following Nef-mediated internalization, cells stably expressing CD4 or CD4/MR were exposed to Nef and examined by confocal microscopy. Following exposure of HeLa CD4 cells to Nef for 48 h, CD4 levels were diminished on the cell surface with relocation to a perinuclear region. There appeared to be some colocalization of CD4 and Nef, in agreement with previous reports that this interaction is transient [45 ]. In contrast, HeLa CD4/MR cells exposed to Nef showed relocation of the CD4/MR molecule to a perinuclear location with significantly greater colocalization of Nef and CD4/MR. Staining of cells with a marker for the TGN resulted in little or no colocalization of CD4 with TGN46 but significant colocalization of CD4/MR with TGN46. In preliminary studies, we have found that immobilized Nef binds to CD4/MR in cell lysates from stable HeLa transfectants, and Williams et al. [51 ] have reported that MHC-Nef binding is stable enough to be detected. These results suggest that CD4/MR is in part rerouted to the TGN as has been shown with MHC I and that CD4/MR and Nef form a stable interaction.
Amino acid residues within the cytoplasmic tail play a critical role in the regulation of cell-surface receptors by HIV gene products. Specific residues within the cytoplasmic region of CD4 and MHC I have been reported to be involved in Nef-mediated regulation [3
, 38
]. Sorting motifs located in the cytoplasmic domains consist primarily of tyrosine-based motifs (YXX
) or leucine-based motifs (L), where X is any amino acid, and represents an amino acid with a bulky hydrophobic chain such as L, V, M, or I. The regulation of CD4 by Nef depends on a membrane proximal dileucine sequence in the cytoplasmic tail [3
], and regulation of MHC I depends on a membrane proximal tyrosine at position 321 together with two other residues, alanine at position 325 and aspartic acid at position 328 [38
]. Comparison of the tail sequences of the MR with the corresponding regions in CD4 and MHC I revealed the presence of a dileucine-like motif (SDTKDLV) and a tyrosine-based sorting motif (NTLY or YFNS). Two separate studies using an FcR-MR chimeric construct [34
] and M6PR-MR construct [35
] demonstrated that the tyrosine in the YFNS motif was required for MR-mediated endocytosis and phagocytosis and additionally, that the tyrosine-phenylalanine was critical for recycling from endosomes back to the plasma membrane. Mutation of these residues reduced both processes by greater than 50%. Previous studies have reported that mutation of the dileucine residues in the CD4 tail and substitution of the tyrosine residue in the MHC I tail abrogated Nef regulation. To examine the possible role of either of these motifs in Nef-mediated regulation of MR expression, the dileucine residues or the tyrosine residue in CD4/MR were mutated to alanines, and the mutant chimera was expressed in 293T cells in the presence and absence of Nef. It is surprising that mutation of the tyrosine or the LV residues independently did not reduce the regulatory effect of Nef. In fact, removal of the LV in the context of an intact tyrosine motif actually enhanced Nef-mediated MR removal from the cell surface. However, when both sites were mutated, Nef regulation was lost. Based on these results, we would speculate that the MR may contain two active sorting motifs that potentially direct it through different pathways. The LV sequence may interact with the clathrin machinery and facilitate movement into the early endosome pathway. This sequence has been found in a number of other endocytic receptors that are internalized via a clathrin-dependent pathway [59
]. At this point, Nef may bind to the MR tail and block recycling of the MR to the cell surface. Alternatively, the tyrosine residue may be important in normal routing of the MR to an intracellular location, as found with MHC I. It is known that up to 80% of the MR in macrophages is localized to an intracellular compartment [60
]. It is not known if this intracellular pool participates in ligand binding and internalization at the surface or functions as an intracellular trafficking receptor. The tyrosine motif might then serve under normal circumstances as a second binding site for Nef, leading to transport to and sequestration within the TGN. The finding that two separate motifs might function as separate sorting sequences appears to be unique among the known surface endocytic receptors and needs to be more fully investigated to define the tail motifs involved in normal trafficking as well as the motifs involved in Nef-mediated MR modulation.
The current report extends the role of the Nef protein of HIV in regulation of host cell function to include regulation of the MR in addition to CD4 and MHC I. Previous studies have suggested that HIV-mediated decreases in MHC I and CD4 expression may have a profound effects on the host immune system. For example, MHC I is required for identification of virally infected cells by presentation of viral antigens on the surface of infected cells. Decreased MHC I expression protects HIV-infected cells from killing by cytolytic T lymphocytes in vitro [61 ]. Furthermore, macaques infected with simian immunodeficiency virus, which expresses a nef gene mutated to disable MHC I regulation, do not develop simian AIDS [62 ]. Reduction of CD4 in HIV-infected cells may impact viral infectivity [63 ], confer resistance to superinfection [64 ], and protect T cells from the cytopathic effects of the viral envelope glycoprotein [65 ]. Down-regulation of CD4 has also been shown to modulate T cell activation, possibly leading to enhanced virus production and spread.
Regulation of the MR may also have a variety of implications for HIV survival. The macrophage is an important intermediate during HIV infection. There is evidence that the macrophage is infected by HIV and that the macrophage plays a role in the chemotaxis and activation of T lymphocytes [50 , 66 , 67 ]. There is also evidence that macrophages may serve as a reservoir of HIV-1 [68 69 70 ] and that macrophages are among the first cells to encounter HIV-1 in the mucosa. The removal of the MR from macrophages may have a variety of advantages to the virus. First, Nguyen and Hildreth [71 ] have reported that the HIV-1 gp120 envelope glycoprotein can bind to the MR, potentially providing an alternate receptor for entry in macrophages; Liu et al. [54 ] have shown a requirement for the MR in CD4-independent infection of astrocytes. The binding of HIV-1 to the MR may then serve as a mechanism of entry into macrophages via an endocytic mechanism rather than the fusion mechanism described for CD4-dependent entry. Second, as shown previously for CD4, the presence of the MR on the surface of macrophages could result in its incorporation into budding virions, which might inhibit infection of neighboring cells, similar to the mechanism proposed for CD4. Down-regulation of the MR from the cell surface would remove this inhibitor, allowing for successful spread of infection. Third, several groups have reported that the MR functions in antigen-capture [72 , 73 ]. Particles internalized by the MR are delivered to acidic compartments, where antigen peptides are loaded into the binding pocket of MHC II [23 ]. The loss of MR would prevent the identification and processing of viral antigens for MHC II loading. Finally, the MR serves as the primary receptor for a number of opportunistic pathogens found in AIDS patients. Decreased MR expression might reduce infection of macrophages by these pathogens and ultimately reduce their ability to compromise HIV survival in coinfected cells.
Received August 13, 2004; revised November 18, 2004; accepted November 19, 2004.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
occurs at the level of transcription Blood 80,2363-2373 J. Immunol. 151,4973-4981[Abstract]This article has been cited by other articles:
 |
|
 |
|
  J. Sturge, S. K. Todd, G. Kogianni, A. McCarthy, and C. M. Isacke Mannose receptor regulation of macrophage cell migration J. Leukoc. Biol., September 1, 2007; 82(3): 585 - 593. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. J. Vigerust, K. B. Ulett, K. L. Boyd, J. Madsen, S. Hawgood, and J. A. McCullers N-Linked Glycosylation Attenuates H3N2 Influenza Viruses J. Virol., August 15, 2007; 81(16): 8593 - 8600. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. G. Quaranta, B. Mattioli, L. Giordani, and M. Viora The immunoregulatory effects of HIV-1 Nef on dendritic cells and the pathogenesis of AIDS FASEB J, November 1, 2006; 20(13): 2198 - 2208. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Michel, K. Ganter, S. Venzke, J. Bitzegeio, O. T. Fackler, and O. T. Keppler The Nef Protein of Human Immunodeficiency Virus Is a Broad-Spectrum Modulator of Chemokine Receptor Cell Surface Levels That Acts Independently of Classical Motifs for Receptor Endocytosis and G{alpha}i Signaling Mol. Biol. Cell, August 1, 2006; 17(8): 3578 - 3590. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. F. Roeth and K. L. Collins Human Immunodeficiency Virus Type 1 Nef: Adapting to Intracellular Trafficking Pathways Microbiol. Mol. Biol. Rev., June 1, 2006; 70(2): 548 - 563. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
