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Originally published online as doi:10.1189/jlb.0505237 on July 6, 2005

Published online before print July 6, 2005
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(Journal of Leukocyte Biology. 2005;78:675-685.)
© 2005 by Society for Leukocyte Biology

HLA-A2 down-regulation on primary human macrophages infected with an M-tropic EGFP-tagged HIV-1 reporter virus

Amanda Brown*,{dagger},1, Suzanne Gartner*, Thomas Kawano{dagger}, Nicole Benoit* and Cecilia Cheng-Mayer{dagger}

* Johns Hopkins University School of Medicine, Department of Neurology, Baltimore, Maryland; and
{dagger} Aaron Diamond AIDS Research Center, The Rockefeller University, New York, New York

1Correspondence: Johns Hopkins University School of Medicine, Department of Neurology, Meyer 6-181, 600 North Wolfe Street, Baltimore, MD 21287. E-mail: abrown76{at}jhmi.edu


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ABSTRACT
 
Multiple mechanisms are used by the human immunodeficiency virus type 1 (HIV-1) to interfere with host-cell immune effector functions. The 27-kD Nef protein has been shown to down-modulate specific genes of the major histocompatibility complex class I (MHC-I) on the surface of infected primary T cells, facilitating their escape from lysis by cytolytic T lymphocytes. Macrophages, as the other major immune cell type targeted by the virus, also contribute to the transmission, persistence, and pathogenesis of HIV-1. Yet, whether Nef modulates MHC-I expression on HIV-infected primary macrophages remains unclear. Currently available infectious HIV-1 molecular clones, which express a reporter gene, only infect T cells and/or do not express Nef. To overcome these limitations, we generated macrophage-tropic green fluorescent protein (GFP)-tagged HIV-1 viruses, which express the complete viral genome, and used these to assess the expression of human leukocyte antigen (HLA)-A2 on the surface of productively infected macrophages. The reporter viral genomes were replication-competent and stable, as Nef, p24 antigen, and GFP expression could be detected by immunostaining of infected, monocyte-derived macrophages (MDM) after more than 2 months postinfection. Fluorescence-activated cell sorter analyses of infected macrophages and T cells revealed that although wild-type reporter virus infection induced a statistically significant decrease in the density of surface HLA-A2, down-regulation of HLA-A2 was not seen in cells infected with reporter viruses encoding a frameshift or a single point mutation in Nef at prolines 74P and P80. The impact of Nef on HLA-A2 surface expression in MDM was also confirmed by confocal microscopy. These results suggest that the mechanisms of HLA-A2 down-modulation are similar in primary T cells and macrophages.

Key Words: Nef • MHC • confocal microscopy • immunofluorescence


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INTRODUCTION
 
Early in the study of human immunodeficiency virus type 1 (HIV-1), macrophages were recognized to be an important target cell in viral pathogenesis [1 2 3 ]. Indeed, most infected patients harbor M-tropic viruses that predominate early and persist during the course of disease [4 5 6 ]. In the brain and intestine, which are major sites of HIV-1 replication early after infection, macrophages are present in abundance [7 8 9 ]. Within the brain, infection of resident macrophages and microglia induces the secretion of neurotoxic factors that contribute to the development of AIDS-associated dementia [10 ]. More recent work in the macaque model suggests that viral particle release from infected macrophages contributes to high plasma viremia during the late stages of infection when all CD4+ T cells have been depleted [11 ]. In addition, specific properties of macrophages and characteristics of HIV-macrophage biology, such as the assembly of viral particles in multivesicular bodies [12 , 13 ], macrophage resistance to the cytopathic effects of HIV-1 replication [1 , 3 , 14 ], as well as their relatively long lifespan of weeks to months [15 16 17 ] and the high level of resistance to antiretroviral drugs [18 ], make macrophages prime candidates as viral reservoirs within patients receiving highly active antiretroviral therapy (HAART). In this regard, several laboratories have detected infected monocytes in the peripheral blood of patients receiving HAART [19 20 21 ].

Macrophages are part of the innate defenses and mediate phagocytosis, clearance of opsonized bacteria, release of reactive oxygen intermediates, secretion of immunomodulatory cytokines, and presention of antigens to T cells and are thus linked to the adaptive arm of the immune system that is responsible for inducing antibody production and cell-mediated responses. To survive and replicate in this cell type, it is likely that HIV-1 has evolved mechanisms to alter macrophage effector functions [22 23 24 25 26 ]. One such mechanism could be the down-modulation of surface molecules that are required for the generation of effective immune responses, a strategy that may be important for viral pathogenesis [28 ]. In fact, in the case of infected T cells, the ability of HIV-1, through the action of the virally encoded Nef protein to down-regulate the major histocompatibility complex class I (MHC-I), has been shown to protect them from cytolytic T lymphocyte (CTL) lysis [28 ]. Whether the same is true of HIV-infected macrophages is not known.

Nef is a multifunctional, "accessory" protein encoded by HIV-1 and simian immunodeficiency virus (SIV), which is required for pathogenesis in vivo and for efficient replication, as observed in infected patients [29 30 31 ], in the SIV model [32 ], and in several in vitro culture models [33 34 35 36 37 ]. The known molecular functions of Nef include the down-regulation of the CD4 receptor [38 , 39 ] and the MHC-I molecule [40 ], activation of signal-transducing proteins [41 42 43 44 45 46 47 48 49 50 51 ], infectivity enhancement [52 , 53 ], and impairment of Fas and tumor necrosis factor receptor-mediated apoptosis [54 ]. Nef lacks enzymatic activity and relies on its ability to recruit specific cellular factors to mediate its downstream effects.

Although the molecular mechanisms by which Nef alters MHC-I surface expression remain incompletely characterized, some details are known. Nef was shown to induce the endocytosis of MHC-I and direct the molecule to the trans-Golgi network [40 , 55 , 56 ]. The human leukocyte antigen (HLA) alleles targeted by Nef possess a tyrosine-based sorting signal in the cytoplasmic tail [56 , 57 ], which may serve as a binding site for Nef and perhaps other factors that may stabilize the interaction [58 ]. Nef selectively down-regulates HLA-A and HLA-B but not HLA-C or HLA-E alleles [56 , 57 ], a strategy that protects infected T cells from natural killer (NK) cell lysis [57 ]. In addition, Nef, through a conserved acidic cluster, EEEE65, has been shown to interact with the trans-Golgi sorting protein, phosphofurin acidic cluster sorting protein-1 [59 ], supporting a model in which Nef acts as an adaptor protein linking MHC-I molecules to cellular factors involved in protein trafficking [55 , 56 , 59 , 60 ]. Two other sites in Nef, the 72PXXP78 motif and an N-terminal region (amino acids 17–26), have also been implicated in MHC-I down-regulation [55 , 61 ], and recent studies have pinpointed single residues in these regions (P78, M20), which when mutated, disrupt Nef function [62 63 ]. Lastly, Nef can also disrupt MHC-I trafficking by blocking the transport of newly synthesized MHC-I to the cell surface through a mechanism involving phosphatidylinositol 3-kinase [60 ].

Although the immunological importance of Nef-mediated MHC-I down-regulation in vivo remains a matter of debate, studies in the SIV-rhesus model [64 ] and of sequential Nef alleles from infected patients [65 ] suggest that it may have significant consequences early in infection. It has been shown that SIV, encoding a mutation in Nef that specifically inactivates its MHC-I down-regulation function, reverted in infected rhesus macaques 4 weeks postinoculation, a time corresponding to the development of antiviral immunity. It is interesting that for up to 56 weeks postinfection, the animals did not develop disease [64 ]. These findings suggest that blunting the ability of Nef to mediate down-regulation of MHC-I surface molecules during the first weeks of infection allowed the development of effective immune responses, tipping the balance toward virus containment. Following the development of antiviral immune responses, however, Nef-mediated MHC-I down-modulation appears to be important in facilitating viral escape. This latter possibility is supported by another study that analyzed Nef alleles from infected patients in different stages of disease. It was found that early in infection, MHC-I down-regulation activity was maintained. However, with progression to disease, Nef functions that enhance viral replication and infectivity were selected for, and MHC-I reduction function was lost [65 ].

It is not known whether HIV-1 Nef can down-regulate MHC-I in the context of primary macrophage infection. This is likely a result of technical limitations related to the difficulties of introducing large plasmids by DNA transfection into primary macrophages and the low level of infection that is often obtained. To study receptor modulation on HIV-1-infected primary human macrophages, we developed enhanced green fluorescent (EGFP) reporter viruses that can infect this cell type. The reporter viruses were used to infect in vitro-cultured macrophages derived from monocytes of normal blood donors expressing the HLA-A2 allele. Primary macrophages infected with EGFP reporter viruses expressing wild-type or a Nef mutant unable to down-modulate MHC-I were analyzed by confocal microscopy to determine the localization of HLA-A2. Our results reveal that Nef can modestly down-modulate the HLA-A2 allele on HIV-infected primary macrophages.


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MATERIALS AND METHODS
 
Reporter virus construction
The first approach to obtain a full-length macrophage-tropic reporter virus construct places the EGFP gene under the control of an internal ribosomal entry sequence (IRES) element inserted downstream of nef (pSF162EGFP; see Fig. 1A ). As the nef gene overlaps with the viral promoter, an intact 3' long terminal repeat (LTR) was reconstructed. However, the reporter cassette was rapidly lost from this recombinant virus after a few rounds of replication, most likely as a result of homologous recombination between the duplicated U3 regions of the viral promoter (data not shown). Therefore, a second approach, similar to the one used by others to construct T tropic reported viruses [66 , 67 ], was undertaken. For this, the EGFP gene in the vector pIRES2-EGFP (Clontech, Palo Alto, CA) was removed by double digestion with MscI and XbaI and replaced with a blunt-ended nefSF162 gene (NheI-SmaI fragment) from the plasmid pNef-IRES2-EGFP to generate pIRES2-Nef. The EGFP fragment isolated above was cloned into the SalI site of pIRES2-Nef to generate pEGFP-IRES2-Nef. To insert the gene cassette into the start site of nef in the plasmid pSF163-3' encoding env, nef, and flanking chromosomal DNA [68 ], site-directed mutagenesis was performed to introduce a unique SnaBI site and mutate the ATG codon of nef. Into the resulting plasmid, pSF162-3'NefATA-SnaBI, the EGFP-IRES2-Nef cassette was inserted into the unique SnaBI and BspEI sites. A full-length reporter virus (p162EGFP, Fig. 1A ), in which nef is in its native position followed by IRES2-EGFP and the 3' LTR, was used to introduce the new gene cassette at unique StuI and XbaI sites, resulting in pSF162R3. The unique XhoI site in nef of pSF162R3 was ablated by treatment with the Klenow enzyme to generate pSF162R3 Nef–. All other nef mutants contained in pSF162-3' [69 ] were introduced into SF162R3 using unique XhoI and XbaI sites. At appropriate steps during cloning, plasmids were transfected into human embryonic kidney (HEK)-293T cells, and total lysates were analyzed for the expression of Nef by Western as described previously [70 ], and live cells were examined by fluorescence microscopy to detect EGFP. To generate virus, proviral DNA (10 µg) was transfected into HEK-293T cells using DMRIE-C (Gibco-BRL, Grand Island, NY), as recommended by the manufacturer. Three days later, viral supernatants were clarified by centrifugation, filtered (0.45 µm pore size), and stored in aliquots at –70°C until use. The p24 antigen was quantitated by the kinetic enzyme immunoassay (Beckman-Coulter, Miami, FL) method by the Aaron Diamond Core Facility (New York, NY).



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  		Figure 1. Construction and replication of M-tropic HIV-1SF162 reporter viruses. (A) Schematic diagram of viral constructs. (B) Western analyses of cell lysates from 293T cells transfected with no DNA (M) or the indicated proviral DNAs. The first panel was probed with anti-HIV-1 human sera, the second panel was probed with a polyclonal anti-Nef antibody, and the last panel was blotted with a polyclonal anti-GFP antibody. (C) Relationship between p24 production (left and right panels) and the number of GFP+ PBMCs (middle panel). One of three representative donors is shown. The replication of the pSF162R3 viruses was plotted separately in the left panel to highlight differences in their growth kinetics. The symbols represent R3 Nef+ ({blacksquare}), R3 Nef– (•), R3 Nef 74PXXP80 (*), and R3 Nef W59 (x). The replication of the reporter viruses is compared with SF162 full-length Nef+ (FL Nef+, {blacktriangleup}) and Nef– (FL Nef–, {diamondsuit}) viruses in the right panel.

Cell culture and infection
Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats of healthy donors by Ficoll gradient centrifugation and cultured as described previously [70 ]. Mitogen-stimulated PBMCs were infected with 50 ng viral p24, and every 2–3 days, 2 ml cultures were removed for quantitation of p24 antigen, and the percentage of GFP+ cells was determined by flow cytometry using a FACSCalibur flow cytometer (BD Biosciences, Palo Alto, CA). Data were analyzed with CellQuest software (BD Biosciences). Monocytes were isolated by the adherence method [71 ]. This method was used unless otherwise indicated in the text. Alternatively, gradient centrifugation was used to enrich for monocytes as described previously [70 ]. Briefly, after isolation by Ficoll gradient centrifugation, the PBMCs were further fractionated on Percoll gradients to selectively isolate the monocyte population. Monocytes (3x106) were plated in T-25 flasks (Corning, Corning, NY) in 3 ml RPMI-1640 complete medium supplemented with 20% heat-inactivated fetal bovine serum (FBS) and 5% heat-inactivated human AB serum. The percentage of CD11b+ in these cultures on the day of infection was routinely 85–90%, and no significant contamination with CD3+ T cells was detected. Following infection of adherent monocyte-derived macrophages (MDM) on day 7 postdifferentiation with 0.2–1 µg viral p24 produced from transfected HEK-293T cells, supernatant was harvested every 3–4 days for p24 antigen quantitation, and cells were inspected for GFP expression by microscopy. To test the infectivity and replication stability of the reporter viruses, CEM x 174/CC chemokine receptor 5 (CCR5) cells (Nathaniel Landau, Salk Institute, San Diego, CA) were infected with 50 ng viral p24 in RPMI 1640, supplemented with 10% heat-inactivated FBS, 1 µg/ml puromycin, and 1% glutamine, streptomycin, and pennicillin. As a result of the cytopathic nature of HIV-1 infection in these cells, every 3–4 days, half of the medium (10 ml) was replaced, and fresh cells (1x105) were added to the T-75 culture flask. Viral p24 was quantitated, and EGFP expression was monitored.

Analysis of cell-surface markers
The following conjugated antibodies were used in this study: CD14-allophycocyanin (APC; clone Mfp9), CD4-APC (clone SK3), CD3-peridinin chlorophyll protein (PerCP; clone SK7), anti-mouse immunoglobulin G1 (IgG1)-phycoerythrin (PE), IgG2a-PE isotype controls (BD Biosciences), and HLA-A2/A28 (One Lamba Inc., Canoga Park, CA). Infected PBMCs were stained for surface molecules near the peak of viral replication between days 7 and 10 postinfection. Cells were washed once in phosphate-buffered saline containing 10 mM EDTA (PBS-EDTA) and then with PBS-EDTA-10 mM sodium azide (PBS-EDTA-NaN3). The cells were stained under low light conditions with saturating amounts of the conjugated antibodies at room temperature for 20 min, washed as described above, and resuspended in PBS-1% formaldehyde and stored at 4°C in the dark until analyzed by fluorescence-activated cell sorter (FACS). For PBMCs the minimum number of events collected was 50,000. For viruses of lower infectivity, 100,000 events were collected. To prepare macrophages for flow cytometric analyses, the supernatant was aspirated and replaced with 3 ml PBS-EDTA. The cells were detached completely using a cell scraper. The cells were washed once in PBS-EDTA-NaN3, and Fc receptors were blocked by incubation in PBS-EDTA-NaN3 containing 2% human serum for 10 min at room temperature. Cells were incubated with saturating amounts of conjugated antibodies at room temperature for 20 min, washed as described above, and resuspended in PBS-1% formaldehyde. For macrophages, a minimum of 100,000–250,000 events was collected and analyzed as given above.

Fluorescence and confocal microscopy
To visualize macrophage plasma membrane and nuclei, live cell cultures were treated with Image-iT (Molecular Probes, Junction City, OR), as specified by the manufacturer. Viral protein expression was detected at the end of the culture period as follows: MDM monolayers were washed with PBS and fixed in 4% paraformaldehyde, permeabilized with PBS-0.2% Triton X-100 for 5 min, washed, and then blocked for 1 h in PBS-5% goat serum before immunostaining with antibodies recognizing HIV p24 [Dako or anti-p24, #183-H12-5c, National Institutes of Health (NIH) AIDS Research and Reference Reagent Program, National Institute of Allergy and Infectious Diseases (NIAID), Bethesda, MD; from Bruce W. Chesebro and Herbert Chen] or Nef [72 ]. The appropriate secondary antibodies conjugated to Alexa-568 or -350 (Molecular Probes) were used. Images were acquired on a Nikon E2000U inverted microscope equipped with epifluorescence. Images were processed using Adobe Photoshop software. Specifically, the Photoshop filter unsharp mask was applied equally to each color to sharpen the images. For confocal microscopy, MDM were grown on LabTek slide flasks (Nunc, Rochester, NY), fixed, and immunostained with antibody recognizing HLA-A2/A28 and subsequently reacted with the secondary reagent Cy5-conjugated streptavidin (Jackson ImmunoResearch Laboratories, West Grove, PA). Analyses were performed on a Zeiss LSM 510 Meta confocal, laser-scanning, inverted microscope. Lasers at 488, 543, and 647 nm were used simultaneously to capture images with a 40x oil immersion Plan Neofluar objective lens. Adobe Photoshop imaging software was used as described above for image processing.

Statistical analysis
The Prism 4 (GraphPad software) statistic program was used to determine the P values for experiments involving two comparisons with an unpaired Student’s two-tailed t-test. Significance was determined by P values less than 0.05.


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RESULTS
 
Construction and replication of macrophage-tropic reporter viruses
The existing recombinant HIV-1 molecular clones that express a reporter gene upon infection replicate only in T cells and/or do not express Nef [66 , 67 , 73 74 75 ] or Vpr [76 ], viral proteins that are required for efficient replication in primary macrophages [34 , 77 78 79 80 ]. Studies of the impact of HIV-1 infection on primary macrophage functions could benefit from the availability of a reporter virus that can infect this cell type. Toward this end, a cassette encoding EGFP-IRES2-Nef was inserted into the start site of nef contained in an HIV-1SF162 proviral plasmid to generate pSF62R3 Nef+ (Fig. 1A) . The resulting proviral construct expressed a bicistronic mRNA encoding EGFP-IRES-Nef under the control of the viral promoter, but the translation of Nef was governed by cap-independent ribosomal recognition of the IRES element and Nef ATG codon (Fig. 1A) . The reporter virus encoding all viral proteins will be referred to as pSF162R3 Nef+. To determine if the recombinant viruses expressed all viral proteins and EGFP, plasmids encoding the viral genomes were transfected into HEK-293T cells, and 3 days later, the cell lysates were analyzed by Western blotting. The correct production of the viral proteins, Env and Gag, as well as Nef and GFP was observed (Fig. 1B) . Nef expression from the pSF162R3 Nef+ virus was comparable with the level of protein produced under the control of HIV regulatory mechanisms (pSF162EGFP, Fig. 1B , Nef panel). EGFP expression was similar between pSF162R3 Nef+ and pSF162R3 Nef– but reduced compared with that of the 162EGFP virus (Fig. 1B , EGFP panel). Long-term passage of pSF162R3 Nef+- and Nef–-infected CEM x 174/CCR5 cells was performed to assess viral genome stability. EGFP and viral capsid p24 antigen expression were detected during the 4-month culture period (data not shown).

The pSF162R3 reporter virus replicates in primary human T cells and macrophages, and HIV-1 Nef enhancement of infectivity function is conserved
Although insertion of the IRES-EGFP cassette into the pSF162 viral genome did not impair its replication in the CEM x 174/CCR5 T cell line, we wanted to determine whether the reporter viruses were capable of stably replicating in primary target cell types. Second, we wanted to test the ability of Nef to enhance viral infectivity, a phenotype that is best assessed in primary T cells and macrophages. Point mutations (alanine substitutions) in nef, known to affect CD4 surface expression and viral infectivity (Nef W59) [81 , 82 ], and MHC-I down-modulation, residues 74P and 80P within the proline-rich motif (Nef 74PXXP80 corresponds to the 72P and 78P residues of the NL4-3) [55 , 61 ], were also introduced into nef of the pSF162R3 virus to assess Nef functions. We found that virus produced from HEK-293T cells could productively infect and spread in interleukin-2/phytohemagglutinin-stimulated, human PBMCs, as measured by p24 secretion (Fig. 1C , left panel). It is important that the difference in infectivity and spread between the pSF162R3 Nef+ and pSF162R3 Nef– viruses as detected by p24 antigen production was also reflected in the difference observed in GFP quantitation (Fig. 1C , middle panel). Furthermore, the percentage of GFP+ cells increased concomitantly with the rise in p24 antigen. The decrease in GFP+ cells after the peak is likely a result of the combination of cell death induced by HIV-1 and the diminished availability of new target cells (Fig. 1C , middle panel). The MHC-I mutant pSF162R3 Nef 74PXXP80 replicated as well as pSF162R3 Nef+, and pSF162R3 Nef W59 grew with kinetics similar to pSF162R3 Nef–, consistent with reports that Nef-mediated CD4 down-regulation is required for efficient replication in CD4+ T cells (Fig. 1C , left panel) [36 ]. However, the reporter viruses replicated in stimulated PBMCs with slower kinetics and to lower titers compared with their respective non-EGFP entities (Fig. 1C , right panel). This most likely reflects the fact that the 1.7-kb increase in the genome size of the reporter viruses retarded their replication kinetics. Such a phenomenon has also been reported for the T-tropic NL4-3 vpr minus heat-stable antigen [76 ], the dual-tropic GFP-tagged HIV-189.6 Nef+/– [74 ], and NL4-3 Gag-GFP reporter viruses [75 ].

The reporter viruses were then tested for their ability to initiate a spreading infection of human macrophages. MDM were productively infected with the pSF162R3 viruses, as detected by an increase in p24 antigen and the number of GFP-positive MDM with time (Fig. 2A and 2B ). Infection levels, as measured by the percentage of pSF162R3 Nef+ GFP+ cells, ranged from 1% to 7%, 12 days postinfection in cells from 10 independent donors. It is most important that the marked phenotypic difference in infectivity and spread of the Nef+ versus the Nef– viruses that we reported previously [70 ] was maintained in the reporter viruses and observed in three out of 10 donors (Fig. 2B , upper panel, MDM I). In the remaining donor MDMs, however, the replication enhancement effect of Nef was reduced substantially (Fig. 2B , upper panel, MDM II). The slower replication kinetics and lower titers of the pSF162R3 viruses than the non-EGFP-containing viruses seen in PBMCs were also observed in MDM, but the differences in macrophages were greater (Fig. 2B , lower panels). Additionally, the donor effect of Nef seen for the reporter viruses was observed for their non-EGFP counterparts (Fig. 2B , lower panels).



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  		Figure 2. The reporter viruses productively infect primary human macrophages. Monocytes were purified by Percoll gradient centrifugation, differentiated, and infected with 200 ng viral p24 produced from transfected HEK-293T cells. (A) Phase contrast and GFP images of a macrophage monolayer infected with pSF162R3 Nef+ at different times postinfection. Days 4 and 9 were taken at 100x and day 17, at 40x original magnification to illustrate the spreading nature of the infection. The day 16 photograph, taken at 200x original magnification, illustrates the varied morphology of the infected MDM. (B) Replication of the reporter viruses in two representative donor macrophages (MDM I and II). The symbols represent R3 Nef+ ({blacksquare}) and R3 Nef– (•). The replication of the reporter viruses is compared with SF162 full-length Nef+ (FL Nef+, {blacktriangleup}) and Nef– (FL Nef–, {diamondsuit}) viruses in the lower panels.

We next wanted to determine whether GFP positivity could be used as a surrogate marker for Nef expression. Macrophages in culture represent a heterogeneous population with varied cell morphology. To identify cellular organelles, MDM infected with pSF162R3 Nef+ on D14 postinfection were stained for the plasma membrane and nuclei, and GFP, a protein known to be expressed in the cytoplasm, served as a marker for this compartment. As shown in Figure 3A , GFP+-infected MDM are often multinucleated (Fig. 3A , *), and uninfected cells, irrespective of their size or shape, tend to have fewer or only a single nucleus (Fig. 3A) . In more rounded MDM, the boundary of the plasma membrane is discerned readily; however, in cells that are larger and more spread out, the cell body, in addition to a thin, transparent lamellapodium that extends out from the cell body, also labeled diffusely with the plasma membrane tag (Fig. 3A) . To determine whether in MDM infected with pSF162R3 Nef+, GFP-positive MDM also expressed Nef and p24, long-term cultures were fixed and double-labeled for these viral antigens. In a monolayer stained at D78 postinfection, the majority of GFP+ MDM coexpressed p24 and Nef (Fig. 3B) . Although p24 and Nef staining were excluded from the nuclei, GFP appeared to accumulate in the cytoplasm as well as in the numerous nuclei of these MNGC, which represented ~46% of the culture (Fig. 3B , arrows). Of these MNGC, 25% were GFP+. These results suggest that the reporter virus can productively infect primary MDM and that the viral genome can be maintained in a stable form in MDM cultured for more than 2 months.



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  		Figure 3. (A) Immunofluorescent images of pSF162R3 Nef+-infected macrophages at day 14 postinfection. Cells were stained live with Image-iT, in which the plasma membrane is labeled with wheat germ agglutinin (WGA)-Alexa Fluor 594 (red), and the nuclei, with Hoechst 33342 (blue), are labeled simultaneously. The infected MDM that expresses GFP are also multinucleated (*). In contrast, the uninfected MDM each have a single nucleus (blue). MDM, which are larger and more spread out, exhibit a more diffuse plasma membrane staining, except at the boundary of the cell body, which labels more intensely (arrows). (B) Immunofluorescent image of pSF162R3 Nef+-infected multinucleated giant cells (MNGC; green) at day 78 postinfection. Cells were fixed and immunostained for viral antigens p24 (red) and Nef (blue) and labeled with goat anti-mouse Alexa-568 and goat anti-rabbit Alexa-350 secondary antibodies, respectively. A few of the numerous nuclei are indicated by the arrows.

Nef in the context of the macrophage-tropic reporter virus down-modulates HLA-A2 and CD4 surface levels in infected primary T cells
To determine whether the reporter viruses could be used to study Nef-mediated receptor down-modulation in primary cells and that such Nef function was conserved in the context of the reporter viruses, we first tested them in PBMCs isolated from seven independent, serotyped donors. Stimulated PBMCs were infected with pSF162R3 Nef+ and Nef mutant strains, and the surface expression of HLA-A2 and CD4 on the CD3+ population was analyzed by flow cytometry. We chose to examine a HLA-A allele, as Nef expressed in a NK tumor cell line was shown to have stronger down-regulation activity on HLA-A than on HLA-B alleles [57 ]. HLA-A2 surface density was decreased approximately threefold (P=.0001) or 4.7-fold (P=.0014) on pSF162R3 Nef+ compared with mock or Nef-infected PBMCs, respectively (Fig. 4A and 4B ). As expected, T cells infected with the pSF162R3 Nef 74PXXP80 mutant failed to down-modulate HLA-A2 (Fig. 4A and 4B) . The MFI for the CD3 receptor was similar on control (80.9±14.1) and infected T cells (101.8±7.2, Nef+; 134.7±48 Nef–), confirming the selectivity of Nef function (Fig. 4A) . Analyses were performed to determine the ability of Nef to down-regulate the CD4 receptor on infected PBMCs. Because of the combined effects of Nef, Env [83 ], and Vpu [84 ], a more dramatic effect on CD4 down-regulation was seen in infected cultures at day 7 postinfection (Fig. 4A) . Nevertheless, although nearly all of the pSF162 R3 Nef-infected T cells had CD4 on their cell surface, only 23% and 35% of Nef+- and Nef 74PXXP80-infected cells, respectively, expressed CD4 (Fig. 4A) . Further, in comparison with T cells infected with pSF162 R3 Nef–, CD4 levels were twofold lower (P=.0139) on pSF162 R3 Nef+- and Nef 74PXXP80-infected cells. These results demonstrate that Nef function is conserved in the context of the reporter virus and expressed at sufficient levels during viral infection to modestly decrease surface MHC-I expression, a phenotype known to require greater quantities of Nef than CD4 down-regulation [56 , 85 ].



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  		Figure 4. (A) Nef-mediated down-regulation of HLA-A2 and CD4 on infected T cells. PBMCs were analyzed by flow cytometry at day 7 postinfection and stained indirectly for HLA-A2-PE and directly for CD4-APC and CD3-PerCP. The analyses shown were gated on the CD3+population. The pSF162R3 Nef+ and Nef mutant viruses are indicated. (B) Graphical representation of the data in A. The mean and SE of two independent experiments with six different donors are shown. For HLA-A2, P values, ***, .0001, and **, .0014. For CD4, P values, **, .0025, and *, .0139. MFI, Mean fluorescent intensity.

Down-modulation of HLA-A2 on primary human macrophages infected with pSF162R3 Nef+
Having demonstrated the use of the reporter viruses in analyses of surface protein expression, we then infected four independent MDM donors possessing the HLA-A2 allele and harvested cells for flow cytometric analyses between days 10 and 13, the range of time-points postinfection at which the percentage of GFP+ MDM was maximal. The level of HLA-A2 was determined on the CD14+ MDM population. A statistically significant difference in the density of HLA-A2 on the surface of pSF162R3 Nef+ versus mock (P=.0068) and pSF162R3 Nef– (P=.0377)- or Nef 74PXXP80 (P=.0204)-infected MDM was detected, suggesting that Nef can down-modulate MHC-I molecules on HIV-infected primary macrophages (Fig. 5B ). Nef strongly reduced HLA-A2 levels on the infected GFP+ MDM of Donors 1 and 4 (48% and 83%, respectively) and modestly on Donors 2 and 3 (23% and 38%, respectively).



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  		Figure 5. Nef down-regulates HLA-A2 cell-surface molecule expression on primary human MDM. The cells were analyzed by flow cytometry between days 10 and 13 postinfection. (A) The gated cell population, isotype, and unstained pSF162R3 Nef+-infected (GFP+) and CD14-APC controls are shown (left to right) for a representative donor. SSC, Side-scatter; FSC, forward scatter. (B) Four independent HLA-A2+ MDM donors infected with the indicated reporter viruses are shown. The MFI of HLA-A2 for the relevant quadrant is shown. The percentage of GFP+ cells in the infected cultures ranged from 0.43% to 3.0%. The mean and SE of fluorescent intensity (MFI) of HLA-A2 staining results for four to seven donors are shown in the graph. P values, **, .0068 (Mock vs. Nef+), and *, .0377 (Nef+ vs. Nef–), and *, 0.0204 (Nef+ vs. Nef 74PXXP80).

Confocal microscopy has been used to visualize Nef-mediated receptor down-regulation in various cell lines and T cells. We used this technology to examine the surface expression of HLA-A2 in MDM infected with pSF162R3 Nef+or Nef–. In GFP-expressing MDM infected with pSF162R3 Nef+, little plasma membrane labeling of HLA-A2 (blue, Cy5, Fig. 6 ) was observed throughout multiple optical sections (Fig. 6) . In contrast, in uninfected or GFP+ MDM infected with the Nef– reporter virus, HLA-A2 labeling of the plasma membrane was readily detected (Fig. 6) .



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  		Figure 6. Confocal microscope analyses of HLA-A2 surface expression in primary MDM infected with pSF162R3 reporter viruses (green). Shown is a representative MDM donor (do258, Donor 1, in Fig. 5B ) grown on LabTek flasks at D13 postinfection. Cells were fixed, then stained for HLA-A2, and then treated with the secondary reagent Cy5-streptavidin (Cy5, blue). As a control, MDM were treated as described in the legend to Figure 3A with Image-iT, which labels the plasma membrane (red). Cells were imaged under oil at 40x with 2x digital zoom. Serial Z-sections were taken at 0.5 µm intervals. For analyses, a single, comparable Z-section was examined.


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DISCUSSION
 
In this report, we described the construction and use of an infectious replication-competent, macrophage-tropic, GFP-tagged, HIV-1 reporter virus, which expresses all of the viral proteins including Nef. We showed that the Nef functions of receptor down-modulation and infectivity enhancement were preserved in the pSF162R3 Nef+ reporter virus. The level of Nef expression mediated by the IRES element was similar to that when the gene was in its native position in the viral genome. GFP and Nef are present on the same multiply spliced message, and therefore, detection of GFP is nearly equivalent to visualizing Nef expression. As seen in Figures 4A and 5B , the extent of HLA-A2 down-regulation in primary T cells or macrophages correlated with GFP MFI, as would have been expected with the degree of Nef expression. This observation is in agreement with studies showing that higher levels of Nef expression are required for MHC-I down-regulation [85 ]. Although FACS analyses indicated only a three- to 4.7-fold reduction in surface HLA-A2 expression on T cells and MDM, confocal analyses revealed a clear down-modulation of this molecule on Nef-expressing MDM compared with uninfected or Nef-infected MDM. Differences in the sensitivity of detection of antibody staining by flow cytometry and microscopy could be responsible for these findings. Regardless, the observation that mutation of proline residues P74 and P80 (P72 and P78 NL4-3 numbering) within Nef’s Src homology 3-binding motif abrogated MHC-I down-regulation in macrophages as well as T cells suggests that the molecular mechanisms used by Nef to affect MHC-I surface expression are conserved among different primary cell types.

Although insertion of the GFP gene into the full-length viral genome attenuated its replication kinetics, this strategy was preferred over several others to study the impact of HIV-1 infection on primary macrophages for the following reasons: First, intracellular p24 staining, which requires the use of antibody and multiple processing steps for detection, does not allow for the visualization and isolation of live, infected MDM for functional assays. Second, although the use of a vesicular stomatitis virus (VSV)-G pseudotype would increase the frequency of infection, this approach would not allow one to assess the impact of Nef on the enhancement of virion infectivity in primary macrophages. Further, possible effects of envelope/receptor coupling-mediated signaling in HIV-1 infection and replication in primary macrophages could not be studied with the use of a VSV-G pseudotype. In this regard, in spite of the attenuated replication kinetics of the reporter virus, the percentage of infected MDM obtained in different donors ranged from 1% to 7%. This frequency of infection is similar or better than that reported in other studies [86 , 87 ]. The variability and low frequency of infection seen in macrophages are not an intrinsic problem of the reporter virus, as similar variability is seen with full-length, replication-competent viruses, but is rather a general problem connected with the use of primary, physiologically relevant cells. Variations in donor MDM susceptibility to HIV infection have been reported previously [88 ]. Differences in CD4 and coreceptor expression of two- to fivefold between normal donors have been seen and may provide one explanation for differences in infection susceptibility [89 ]. However, other studies have suggested that the block is post-entry at stages including reverse transcription to nuclear import [90 ]. The M-tropic reporter viruses described herein may prove useful in studies aimed at the identification and characterization of the subpopulation of monocyte/macrophages that are susceptible to infection.

The idea that Nef-mediated HLA class I down-regulation function contributes to the ability of HIV to evade the immune response is not fully accepted as a result of the fact that HIV-specific CTL can be detected in infected individuals. However, despite the presence of antiviral cell-mediated immunity, disease progresses in these individuals. Indeed, it is increasingly clear that the inability of the host to control HIV infection does not lie in the frequency but rather the quality of the immune response [91 ]. Thus, it is tempting to speculate that as a result of Nef-mediated HLA down-regulation, the function of antigen-presenting cells such as macrophages will be compromised, resulting in the generation of a T cell immunity that lacks breath and depth, permitting the virus to mutate and escape from immune pressure with relative ease. In this regard, several early studies using HIV-infected cells from normal donors or cells purified from the blood of HIV-infected and uninfected individuals, which examined the impact of HIV infection on the ability of monocyte/macrophages to stimulate T cell effector function, support the notion that macrophage antigen-presenting functions are impaired [92 93 94 95 ]. Other reports, however, dispute this claim and suggest that variations in cell culturing and in the number of HIV-infected macrophages present could account for the differences observed [96 ]. Further studies at the population and single-cell level with the reporter viruses described herein should shed light on whether the modulation of HLA class I surface expression by Nef alters macrophage effector functions.

In summary, the reporter viruses described here permitted the assessment of several Nef functions in primary macrophages. Our findings are important in light of the renewed focus on the role of macrophages in innate immunity and in HIV-1 spread and viral persistence. The selectivity of Nef for specific HLA alleles, such as HLA-A2, may be related to the fact that it is one of the most prevalent worldwide [97 ]. Evolving mechanisms to impede the presentation of viral epitopes within the context of HLA-A2 in primary macrophages and CD4+ T cells are likely to contribute to HIV-1 dissemination [98 ] and persistence by blunting the development of antiviral immunity and allowing for escape from immune recognition, respectively [99 ].


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ACKNOWLEDGEMENTS
 
This work was supported by NIH Grant RO1 AI38532 to C. C-M. We thank Peter Lopez and Khairul-Bariah Abd-Majid for assistance with the flow cytometry, Zhiwei Chen for the anti-HIV antisera, Ashok Chauhan for secondary antibody reagents, the NIH AIDS Research and Reference Reagent Program, NIAID, NIH, anti-p24, #183-H12-5c, from Bruce Chesebro and Kathy Wehrly, and Jeff Rothstein for use of the Nikon E2000U microscope.

Received May 2, 2005; revised May 16, 2005; accepted June 3, 2005.


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A. Brown, H. Zhang, P. Lopez, C. A. Pardo, and S. Gartner
In vitro modeling of the HIV-macrophage reservoir
J. Leukoc. Biol., November 1, 2006; 80(5): 1127 - 1135.
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