Traffic 2012; 13: 273–291 © 2011 John Wiley & Sons A/S

doi:10.1111/j.1600-0854.2011.01306.x

β2 Integrin Adhesion Complexes Maintain the Integrityof HIV-1 Assembly Compartments in PrimaryMacrophages

Annegret Pelchen-Matthews, Sebastian Giese,

Petra Mlcochova, Jane Turner and Mark

Marsh∗

Cell Biology Unit, Medical Research Council Laboratoryfor Molecular Cell Biology, University College London,London WC1E 6BT, UK*Corresponding author: Mark Marsh,m.marsh@ucl.ac.uk

In human monocyte-derived macrophages (MDM),

human immunodeficiency virus type 1 (HIV-1) assembly

takes place primarily on complex intracellular plasma

membrane domains connected to the cell surface by

closely apposed membrane sheets or narrow channels.

Some of the membranes associated with these compart-

ments are decorated by thick (≈30 nm), electron-dense,

cytoplasmic coats. Here we show by immunolabelling

of ultrathin cryosections that the β2 integrin CD18,

together with the αM and αX integrins (CD11b and

CD11c), is clustered at these coated domains, and that

the coats themselves contain the cytoskeletal linker

proteins talin, vinculin and paxillin that connect the

integrin complexes to the actin cytoskeleton. Intracellu-

lar plasma membrane-connected compartments (IPMC)

with CD18-containing focal adhesion-like coats are

also present in uninfected MDM. These compartments

become more prominent as the cells mature in tissue

culture and their appearance correlates with increased

expression of CD18, CD11b/c and paxillin. Depletion

of CD18 by RNA interference leads to parallel down-

regulation of CD11b and CD11c, as well as of paxillin,

and the disappearance of the adhesion-like coats. In

addition, CD18 knockdown alters the appearance of

virus-containing IPMC in HIV-infected MDM, indicating

that the β2 integrin/focal adhesion-like coat structures

are involved in the organization of these compartments.

Key words: β2 integrin, CD18, HIV assembly, intra-

cellular plasma membrane-connected compartment,

macrophage, paxillin, talin

Received 13 July 2011, revised and accepted for

publication 19 October 2011, uncorrected manuscript

published online 21 October 2011, published online 28

November 2011

CD4-expressing T lymphocytes and cells of the mono-cyte/macrophage lineage are the main in vivo targetsfor infection by human immunodeficiency virus type 1(HIV-1), though the virus interacts with these targets indifferent ways, and infected macrophages and T-cellsmake distinct contributions to the pathophysiology ofAIDS (1). One striking difference between HIV replication

in macrophages and in T-cells is that in T-cells, as wellas in various tissue culture cell lines, HIV particles budat the cell surface, while in human monocyte-derivedmacrophages (MDM), virus particles are frequently seento bud and accumulate in membrane-bounded intracel-lular compartments. These compartments have beenvariously described as Golgi components (2,3) or asendosomal organelles (4–6). However, other studies indi-cated that HIV-1 Gag specifically targets the plasmamembrane, and that HIV assembly at the plasma mem-brane, but not in endosomes, is essential for release ofinfectious viruses, both in model cell lines and in primarymacrophages (7). More recently, the intracellular HIVassembly compartments in cultured MDM have beenshown to consist of cell surface-connected plasmamembrane domains (8–11). Thus, as in T-cells, HIVdoes assemble at the plasma membrane in MDM, butin these cells virus assembly is targeted to specializedintracellular plasma membrane-connected compartments(IPMC). The structure of these IPMC is complex. Some ofthe compartments consist of vacuoles filled with viruses,others contain various internal membrane vesicles orintricate membrane protrusions appearing as finger-likeprojections, membrane-sheets or lamellae resemblingthe microvilli or membrane ruffles seen at the cellsurface. The membranes bear clathrin-coated pits and arelinked to complex interconnected membrane meshworkscontaining the tetraspanins CD9 and CD81, as well as thecell adhesion protein/hyaluronic acid receptor CD44 (9,12).In addition, some of these membranes are decorated ontheir cytoplasmic surface by a characteristic thick layer ofelectron-dense material (5,9); these coated membranesdo not label for tetraspanins or CD44 and their identity hasbeen unclear. There has been a suggestion that they maycontain components of the endosomal sorting complexrequired for transport (ESCRT) machinery that is requiredfor the scission of assembled HIV virions (13), thoughvirus budding sites are usually only seen adjacent to thecoated domains.

Here we show that CD18, the leukocyte-specific β2integrin, is associated with the IPMC and appears tobe clustered at the electron-dense coated domains in anactivated conformation. The αM and αX integrin chains(CD11b and CD11c), as well as the cytoskeletal proteinstalin, vinculin and paxillin are also located at these coateddomains, where they may connect the integrin complexesto the actin cytoskeleton. IPMC bearing CD18-containingthick electron-dense coats can be seen in uninfectedMDM and become more prominent as these cells maturein tissue culture. Depletion of CD18 by RNAi results inthe disappearance of the coated domains and changes

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Figure 1: Morphology of HIV-containing IPMC in infected MDM. MDM infected with HIV-1 for 14 days were fixed and embeddedin Epon for transmission EM. A–C) Virus-containing IPMC bear thick electron-dense coats (black arrowheads), either on the surface ofthe compartments or as closely apposed double membranes on either side of a narrow gap. The coats are thicker and more uniformthan the radially stippled clathrin-coated pits (black arrows). The asterisk in (A) marks a pair of grazingly sectioned coated membranes.On the cytoplasmic aspect, the coated membranes link to a layer of fine filaments (marked by brackets in B and the inset to C) that areprobably actin filaments. The compartment in (C) is in contact with the cell surface. The boxed area is shown at higher magnificationin the inset. L: lipid droplet; M: mitochondrion; Su: cell surface plasma membrane ruffles/microvilli; V: virus particles. Representativeimages from one of two experiments with different donors. Scale bars: A, B, 500 nm, and C, 200 nm.

the distribution of HIV-containing compartments. Thus,we have identified and extensively characterized anintegrin/focal adhesion-like coat structure that is involvedin the organization and integrity of the IPMC where HIVassembles in mature macrophages.

Results

Morphology of virus-containing IPMC in cultured

human MDM

In MDM, HIV-1 assembles predominantly in IPMCof complex morphology (8–11). We noted that themembranes of HIV-containing IPMC are often coated,

on their cytoplasmic aspect, by extensive electron-densecoats (5,9). Morphologically similar coats can be seenon electron micrographs from other studies of theintracellular HIV assembly compartment in humanmacrophages [see e.g. Figure 2B in (3), Figure 6B in (14),Figure 1F in (10), Figure 2F in (15), and Figure 3 in (8)],including on an intracellular HIV-containing vacuole froma Pneumocystis carnii-infected lymph node biopsy (16).Further examples of the coats on IPMC in MDM areshown in Figure 1. Typically, the coats are thicker thanthose on clathrin-coated pits and vesicles (28 ± 5 nm onEpon sections, compared to 17 ± 3 nm for clathrin coats),and have a more uniform electron density, lacking the

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Figure 2: The thick coats do not contain clathrin or the adaptor protein AP-2. Immunogold labelling of cryosections from HIV-infected human MDM. A) MDM infected with HIV-1 for 14 days. Double labelling with mouse anti-p24 mAb (PAG 10 nm) and a rabbitpolyclonal antiserum against the clathrin light chain (PAG 15 nm). B–D) MDM infected with HIV-1 for 7 days. B) Double labelling withrabbit UP595 anti-HIV-1 MA (PAG 10 nm) and mouse mAb Clone 23 against the clathrin heavy chain (PAG 15 nm). Note that the areashown does not contain any labelled virions. C) Labelling with the clathrin heavy chain mAb X22 and 10 nm PAG. D) Double labellingwith rabbit UP595 anti-HIV-1 MA (PAG 10 nm) and mouse mAb AP.6 against the clathrin adaptor protein AP-2 (PAG 15 nm). The clathrinand AP-2 antibodies label typical clathrin-coated pits (black arrows, see also insets in C and D), while areas of the thick electron-densecoats remain unlabelled (arrowheads). In B, the double-coated membranes are not labelled, but two 15 nm gold particles are seen on ashallow coated pit continuous with the top right membrane, where the coat has a reduced thickness (arrow with *). V marks labelledHIV particles, while the white arrow in A indicates a budding virus particle. Images are from immunolabelled MDM sections from twoindependent donors. Scale bars: A–D, 200 nm, or 100 nm in the insets in C and D.

radial stipples that are apparent on clathrin-coated pitsand vesicles. Overall, the coats are relatively flat or showa gentle curvature, and they can extend over severalhundred nanometres. Analysis of serial section imagesshows that the coated membranes are present in multipleadjacent sections, covering extended areas of membrane(Figure S1). On the cytoplasmic aspect, the coats appearto connect with a meshwork of fine filaments (Figure 1C,inset), which are likely to be actin (see also below).Occasionally two regions of coated membranes can beseen closely apposed, and separated by a narrow gap of12–20 nm that appears to be filled with fine fibrous mate-rial (Figure 1). In some situations, these double-coatedmembranes are seen between virus-containing IPMC and

the cell surface, and appear to restrict release of the virusinto the extracellular milieu; staining with ruthenium redindicates that most of the intracellular compartments areaccessible to small molecule tracers (9–11). While coatedpatches can be found on virus-containing IPMC, HIV doesnot appear to bud directly from the coated areas, thoughbudding figures can be seen on adjacent membranes.

EM immunolabelling studies to characterize the thick

electron-dense coats

To understand the nature of these electron-densecoats, we immunostained ultrathin cryosections of HIV-1-infected MDM with antibodies against known coat

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Figure 3: The thick coats contain the leukocyte-specific β2 integrin, CD18. Immunogold labelling of cryosections from humanMDM infected with HIV-1 for 7 days. A–D) Immunolabelling with mouse mAb MEM-48 against CD18/β2 integrin. E–G) Immunolabellingwith mouse mAb MEM-148, which recognizes the activated conformation of CD18/β2 integrin. Gold particles are concentrated on theectodomain of membranes bearing the thick electron-dense coats (arrowheads). These coats can be found at the cell surface (D) or onthe surface of IPMC, including virus-containing compartments (A, E, G). Sometimes two coated membranes are closely apposed, withgold particle labelling over the narrow gap between the two membranes (B, F). In C, a thin cytoplasmic process bears electron-densecoats on both membrane surfaces, and both of these are strongly decorated with gold particles. V marks virus particles. The blackarrows in D indicate unlabelled clathrin-coated pits. H and I) Immunofluorescence double staining of semi-thin (0.5 μm) cryosections witha mAb against HIV-1 p17 (red) and with mouse mAb MEM-48 against CD18/β2 integrin (H) or mouse mAb MEM-148, which recognizesthe activated conformation of CD18/β2 integrin (I), in green. Virus particles (red) assemble in intracellular compartments that co-labelfor CD18. Some CD18 staining is seen in bright foci (white arrows), presumably representing CD18 clustered against the thick coats.Phase images of the cryosections are shown on the right. Representative images from immunolabelling experiments with MDM fromfour independent donors. Scale bars: A–G, 200 nm; H and I, 10 μm.

proteins. Although we have previously shown that theIPMC are not endosomal (9), there is a precedent forflat clathrin coats on endosomes; we therefore stainedsections with antibodies against clathrin or the clathrin-adaptor proteins AP-1, AP-2 and AP-3 and protein A-gold(PAG). A rabbit polyclonal antiserum against the clathrinlight chain labelled the numerous clathrin-coated pitsassociated with the IPMC, but the thick electron-densecoated membranes were not labelled (Figure 2A). As the

anti-clathrin light chain antiserum was raised against apeptide from a domain that is involved in the interactionwith the Huntingtin-interacting protein 1-related protein(Hip1R) (17), and may be masked when Hip1R is bound,we also used two monoclonal antibodies (mAbs) tothe clathrin heavy chain. Like the clathrin light chainantiserum, these antibodies labelled clathrin-coated pits,but failed to stain the more extended coats associatedwith IPMC (Figure 2B,C). An antibody to the clathrin

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adaptor protein AP-2 also stained clathrin-coated pits, butnot the thick coats (Figure 2D). Antibodies against theAP-1 and AP-3 adaptor proteins labelled a sub-populationof clathrin-coated pits, but not the pits associated withthe IPMC (Figure S2) and also failed to label the thickcoats. These observations indicate that the thick coatsare distinct from AP-2-containing clathrin coats or thebilayered endosomal clathrin coats containing Hrs (18).Nevertheless, the presence of AP-2 on clathrin-coatedpits associated with the virus assembly compartments,but lack of AP-1 and AP-3 labelling on these structuresis in agreement with the notion that these are plasmamembrane compartments.

In areas where the coated membranes are closelyapposed, the thick electron-dense coat structures havesome morphological similarities to cell–cell junctioncomplexes that are expressed by epithelial cells. Wetherefore tested antibodies against a number of adhesionjunction proteins, including E-cadherin, β-catenin andZO-1. None of these antibodies labelled the coats abovethe background observed with a control antibody or inthe absence of primary antibody (data not shown). Wealso failed to obtain labelling with antibodies against arange of ESCRT proteins, though it is not clear whetherthe epitopes for these antibodies would be exposed inmembrane-bound ESCRT complexes (data not shown).

The leukocyte integrin CD18 is present in the thick

electron-dense coats

Integrins are also known to be involved in adhesionstructures. In particular, the integrin LFA-1 and its receptorICAM-1 have been shown to play roles in HIV binding,fusion and infectivity (19). Indeed, ICAM-1 and theleukocyte-specific integrins LFA-1 (CD11a/CD18), MAC1(CR3, CD11b/CD18) and p150/95 (CR4, CD11c/CD18)are among the host cell proteins incorporated intothe viral membrane (for reviews, see (20,21)) andhave been detected in HIV purified from macrophages(22). The antibody MEM-48 directed against the β2integrin chain CD18 labelled cryosections of MDM(Figure 3A–D). In particular, CD18 labelling appearedto be enriched or clustered on the external side ofmembranes covered on their cytoplasmic aspect withthe thick electron-dense coats, both on single coats onthe surface of IPMC (Figure 3A) and on closely apposedcoated membranes (Figure 3B). Occasionally, membraneprocesses protruding into the compartments appeared tobe coated on both surfaces (inverted double coats) andwere strongly labelled for CD18 (Figure 3C). In addition,patches of electron-dense coat at the cell surface werelabelled for CD18, while clathrin-coated pits remainedunlabelled (Figure 3D). We also tested the antibody MEM-148, which recognizes an epitope that is only exposedon the high-affinity activated conformation of β2 integrins(23,24) and free CD18 not associated with CD11. Thisantibody also stained the IPMC and again the labellingappeared to be enriched over the coated membranedomains (Figure 3E–G).

To obtain an overview of the distribution of CD18 in relationto HIV, we stained semi-thin cryosections (0.5 μm) forimmunofluorescence (Figure 3H,I). CD18 was observedat the cell surface of HIV-infected MDM, and labellingoften appeared stronger over the membrane elaborationsnear the top of the cells (Figure 3H). A major amount ofthe CD18 staining was associated with virus-containingintracellular compartments, and we also detected somebright foci of CD18 staining, presumably representingCD18 clustered against the thick coats (arrows in Figure3H). A similar distribution was observed on cells stainedwith the mAb MEM-148 (Figure 3I).

To identify the integrin α chains associated with CD18,cryosections were stained with antibodies against CD11a,CD11b or CD11c (Figure 4). As expected, cells did not stainsignificantly with antibodies against CD11a, the α chain ofthe T-cell integrin LFA-1, and at the electron microscope(EM) level the coated membranes were essentially unla-belled (Figure 4A). Staining of IPMC could be observed forthe myeloid α integrins CD11b and CD11c (Figure 4B,C).At the EM level, many of the coated membranes alsoremained unlabelled, although some were marked by clus-tered gold particles; overall staining for CD11c appearedstronger and more frequent than for CD11b. Immunofluo-rescence double staining with CD18 and CD11b or CD11cshowed significant overlap for these proteins (Figure4D,E). The presence of the CD11b and CD11c proteinsin the same location as CD18 indicates that these MDMexpress mature integrins containing both α and β chainsand thus represent functional integrin molecules. Further-more, the close overlap in the staining patterns of MEM-48and MEM-148 suggests that a major proportion of theCD18 molecules are in the open, activated conformation.

Association of the coated membranes with the actin

cytoskeleton

As integrins have been shown to link the extracellularmatrix to the actin cytoskeleton, we suspected that thefine cytoplasmic filaments adjacent to the electron-densecoats (see Figure 1C) might be actin. Indeed, analysis ofHIV-infected MDM by staining with phalloidin Alexa-594and confocal microscopy demonstrated the presence ofactin at virus-containing IPMC (Figure 5A). To analyse thedistribution of actin in more detail, we labelled ultra-thincryosections with antibodies against actin. Two anti-actin antibodies labelled the interface between the thickcoats and the fine filaments (Figure 5B–H), though theclearest filament bundles often showed low levels oflabelling. This could be due to limited antibody access tofilaments deep within the section or issues of epitopeexposure. Double labelling with the anti-actin antibodiesand markers for viral proteins (p17, Figure 5D or p24,Figure 5E) confirmed the presence of actin on virus-containing compartments. Double staining for actin andCD18 (Figure 5F–H) directly demonstrated the presenceof actin near the thick electron-dense coats labelled forCD18, at the cell surface (Figure 5F) or on the IPMC(Figure 5G,H).

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Figure 4: Analysis of α integrin chains associated with IPMC by immunofluorescence and EM. Immunolabelling of cryosectionsfrom human MDM infected with HIV-1 for 7 days. A–C) Cryosections were stained for immunofluorescence (left, panels) or EMimmunolabelling (right panels). A) Staining for CD11a (pseudocoloured green) and the HIV capsid protein p24 (red) to identify virus-containing compartments. We could not detect CD11a by immunofluorescence or on ultrathin cryosections at the EM level. B and C)Staining for CD11b or CD11c (green) and the HIV matrix protein p17 (red) to identify virus-containing compartments. CD11b expressionwas variable between cells, and on ultrathin cryosections, many membranes bearing thick coats were unlabelled, although occasionallylabelled structures were detected (right panels). CD11c was observed on many cells and was usually associated with virus-containingIPMC by immunofluorescence. On ultrathin cryosections, some of the membranes with electron-dense coats were decorated with10 nm gold particles. D and E) Immunofluorescence double staining of semi-thin cryosections with MEM-48 anti-CD18 (green) andrabbit mAbs against CD11b (D) or CD11c (E) in red. The two integrin α chains broadly colocalize with CD18/β2 integrin and stain thesame intracellular compartments. Phase images are shown on the right. Scale bars, 10 μm for the phase images accompanying theimmunofluorescence panels, and 200 nm for the EM images.

As integrins link to the actin cytoskeleton via focal com-plexes, we investigated whether the thick electron-densecoats may represent adhesion plaques. Cryosections werestained with antibodies against the well-characterizedfocal adhesion scaffold/linker proteins talin, vinculin orpaxillin. All three proteins could be detected in the thickcoats (Figure 6). Even though gold labelling with theseantibodies was somewhat sporadic and gold particleswere often observed in small clusters (due to the use ofa rabbit anti-mouse bridging antibody before labelling withPAG), specific labelling was present for all three proteins

(Figure 6). Of note, the gold particles were located directlyover the electron-dense coats, and not on the exter-nal/lumenal surface as seen with antibodies to CD18. Thepresence of these cytoskeletal linker proteins on virus-containing IPMC was confirmed by double labelling withantibodies against the viral protein p17 (Figure 6C,D,G,J).Talin binds to the cytoplasmic tails of activated integrins,while vinculin and paxillin link other adhesion complex pro-teins. The observation that the CD18 is clustered and talinis present in the plaque supports the conclusion that theβ2 integrins are in an activated conformation at these sites.

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Figure 5: Association of actin with the coated membranes of IPMC. A) Immunostaining of HIV-infected MDM for p17 (green) andactin (phalloidin Alexa-594, red). The confocal sections show actin highlighting the cell surface and associated processes, and alsostaining the virus-containing IPMC. B–H) EM immunolabelling of cryosections from human MDM infected with HIV-1 for 10 days (Band C) or 7 days (D–H). B and C show single staining with the anti-β-actin mAb AAN02 and PAG 10 nm. Gold particles are seen on thecytoplasmic interface between the electron-dense coats and the fine filament meshwork. The coated membranes or closely apposedmembranes (double coats) are marked with arrowheads. D) Double staining immunolabelling with the anti-β-actin mAb AAN02/PAG 5 nmand mouse mAb 4C9 anti-p17/PAG 10 nm. Some labelling for actin is seen against the electron-dense coats (arrowheads). Note thatthe anti-p17 antibody only binds to cleaved p17 and that the virus bud containing p55Gag remains unlabelled (white arrow). E) Doublelabelling with goat polyclonal anti-actin/PAG 5 nm and anti-p24/PAG 10 nm. F–H) Double labelling with goat polyclonal anti-actin/PAG5 nm and MEM-48 anti-CD18/PAG 10 nm. CD18 is clustered against the thick coats at the cell surface (F, black arrows), or at intracellularsingle (G) or closely apposed double-coated membranes (H), while 5 nm gold particles indicating actin are found on the cytoplasmic sideof the same coats. V indicates HIV particles. Images are from immunolabelled MDM sections from two independent donors. Scale bars:A, 10 μm; B–H, 200 nm.

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Figure 6: The electron-dense coats contain talin, vinculin and paxillin. Immunolabelling of cryosections from human MDM infectedwith HIV-1 for 7 days. A–D) Immunolabelling for talin (A and B) or double labelling for talin/PAG 10 nm and mouse mAb 4C9 anti-p17/PAG5 nm (C and D). Talin is associated with some of the coated membranes on IPMC (e.g. at the black arrows). E–G) Immunolabelling forvinculin (E and F) or double labelling for vinculin/PAG 10 nm and mouse mAb 4C9 anti-p17/PAG 5 nm (G). Many of the coats were stainedfor vinculin. H–J) Immunolabelling for paxillin (H and I) or double labelling for paxillin/PAG 10 nm and rabbit UP595 anti-HIV-1 MA/PAG5 nm (G). V indicates HIV particles. White arrows mark budding or immature virus particles in C, G and I. Most of the virus particles in Falso have an immature morphology (white V). Scale bars, 200 nm.

Intracellular membrane structures with focal

adhesion-related coats are present in uninfected

MDM

To assess whether the focal adhesion-like coats onIPMC were induced by infection with HIV-1, or werealso present in uninfected macrophages, sections fromMDM 16 days post-isolation were immunolabelled forCD18. Double staining of semi-thin sections for CD18and the tetraspanin CD9, which we have previouslyshown to be associated with the IPMC (9), indicated thatintracellular structures that could be labelled for CD9 wereassociated with CD18 and, notably, some of this CD18

staining was in bright foci, suggesting that the CD18was clustered (Figure 7A). Occasional cells had moreprominent intracellular compartments positive for CD9and CD18 and bearing foci of strong CD18 staining ontheir periphery (Figure 7B). Similar staining patterns wereseen with the antibody MEM-148 against activated CD18(Figure 7C). Immunolabelling of ultrathin cryosections byEM revealed CD18 staining over membranes with thickelectron-dense coats (Figure 7D–F). Coated intracellularmembranes were also seen on cryosections from anotheruninfected MDM preparation fixed at 18 days post-isolation. These structures were labelled with antibodies

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Figure 7: IPMC containing the β2 integrin CD18 are also present in uninfected MDM. Immunostaining of cryosections fromuninfected human MDM, 16 days post-isolation. A–C) Immunofluorescence double staining of semi-thin cryosections with mousemAb MEM-48 against CD18/β2 integrin (A and B) or mouse mAb MEM-148, which recognizes the activated conformation of CD18integrin (C) in green, and the tetraspanin CD9, a marker for the IPMC, in red. Occasionally, the CD18 staining was seen in bright foci(arrows), presumably representing CD18 clustered against the coats. Phase images of the cryosections are shown on the right. D–F) EMimmunolabelling of ultrathin cryosections for CD18 reveals various IPMC with electron-dense coats and strong labelling for CD18/PAG10 nm (arrowheads). Some of the coats are on closely apposed membranes (e.g. in F). Scale bars: A–C, 10 μm; D–F, 200 nm.

against CD18, activated CD18, CD11b, CD11c as well asthe cytoskeletal linker proteins talin, vinculin and paxillin(Figure 8). We have previously shown that uninfectedMDM contain intracellular compartments enriched forvarious tetraspanins (CD9, CD53 and CD81) that areaccessible to the fluid phase tracer horseradish peroxidase(HRP) (9), suggesting that these cells also have IPMC.Together, these studies indicate that MDM IPMCcontaining tetraspanins and β2 integrin adhesion plaqueare not induced by HIV-1 infection, but rather that the

virus exploits a pre-existing intracellular compartment forassembly (see also (11)).

Time–course of expression of IPMC during MDM

differentiation in tissue culture

Although we could detect intracellular compartmentscontaining CD18 clustered against electron-dense coatsin uninfected MDM at 16 or 18 days post-isolation, thesecompartments were not easily detected in MDM at 7 dayspost-isolation. Indeed, on cryosections from a series

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Figure 8: Immunolabelling of IPMC coats in uninfected MDM. Immunolabelling of cryosections from uninfected human MDM at 18days post-isolation. Sections were labelled with mAb MEM-48 (A) or MEM-148 (B) against CD18, with mAbs against CD11b (C), CD11c(D and E) or against talin (F), vinculin (G) or paxillin (H). Antibodies against the ectodomain of the integrins label the external surface ofthe plasma membrane, while talin, vinculin and paxillin appear to be located in the electron-dense coats. Scale bars, 200 nm.

of uninfected MDM preparations fixed at various timespost-isolation, we observed very little CD18 staining of7–10-day-old MDM, but CD18 labelling was apparent atlater times (e.g. 13 days post-isolation) and intracellularcompartments were seen in MDM preparations up to26 days post-isolation (Figure S3). Staining for activatedCD18 (MEM-148) or CD11c showed a similar pattern(Figure S3). We also studied MDM from the same donorthat were cultured for various periods of time beforeimmunofluorescence staining and analysis by confocalmicroscopy. Again, CD18 expression was low at earlytimes in culture (e.g. 5 days post-isolation), but increasedat longer times (Figure 9A, with quantification shownin Figure 9B). In parallel, intracellular compartments

containing CD18 and CD44 only became prominent inMDM that had been cultured for at least 13–15 days post-isolation (Figures 9C and S3) and there were some brightfoci of fluorescence representing the clustered, activatedCD18. On infected MDM, HIV particles were seen inintracellular compartments at this time (see Figure 9C).

To follow protein expression more directly, MDM werecultured for up to 20 days post-isolation, cell lysateswere prepared at various times and equal amounts ofprotein were separated by SDS–PAGE and analysed bywestern blotting (Figure 9D). CD18 staining was weak1 day after monocyte isolation, but a clear band couldbe detected at 3 days post-isolation and levels of the

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Figure 9: β2 integrin expression is

up-regulated as MDM differentiate

in cell culture. A and B) Mono-cytes were isolated from buffy coatsand differentiated into macrophagesin vitro. At 5, 8, 11 and 15 dayspost-isolation, cells were fixed andimmunostained for CD18 (green) andCD44 (red). Single confocal sectionsof whole cells (A) indicate that CD18expression increases over time. B)Quantification shows the MFI ofCD18 and CD44 on three randomconfocal images for each time-point,normalized for the cell number.C) Images of uninfected and HIV-infected MDM at higher resolution.Cells were left uninfected or infectedat 8 days post-isolation, fixed on day15 after isolation and immunostainedfor the indicated proteins. Single con-focal sections of whole cells areshown. IPMC are characterized byCD18/CD44 colocalization. D) West-ern blotting of cell lysates from MDMfrom one donor prepared at differ-ent times after monocyte isolation.Cells were lysed and equal amountsof protein were loaded in each lane.Filters were blotted for CD18, acti-vated CD18, CD11c, vinculin or pax-illin. Molecular weights of markersare indicated on the right. The blotsare representative of samples fromat least three different donors. Scalebars: A, 40 μm and C, 10 μm.

protein increased further at 7 and 10 days post-isolation.A similar pattern was observed with mAb MEM-148and for the α integrin CD11c (Figure 9D) and CD11b(not shown). Interestingly, the levels of paxillin, but notvinculin, also increased with similar kinetics. Expressionof ICAM-1 appeared constant at all times after isolation of

the cells (data not shown). Together, these observationsindicate that the focal adhesion-like structures and IPMCin MDM develop as the cells differentiate in culture;the compartments are rare at 6–7 days post-isolation,the age at which MDM are usually infected with HIV-1 in vitro, but become prominent in older MDM. In

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HIV-infected MDM, which are usually analysed 6–7 dayspost-infection, the virus accumulates in the IPMC; thesecells express high levels of the integrins and cytoskeletallinker proteins. The synchronized appearance of CD18and CD11c suggests that αXβ2 integrin heterodimersare expressed in MDM, while the induction of paxillinwith similar kinetics indicates a functional link betweenthe adhesion plaque-like structures and the clustered β2integrins.

Depletion of CD18 in MDM

To further investigate the role of CD18 in the IPMC inHIV-infected and uninfected MDM, CD18 levels in humanprimary MDM were down-regulated by RNAi. Seven day-old MDM were infected with HIV-1 and transfected withsiRNA against CD18 or control-scrambled siRNA on thefollowing day, and harvested after a further 7–11 days(Figure 10A). Western blot analysis demonstrated morethan 90% reduction in CD18 levels in the knockdowncell lysates (Figure 10B). Similar knockdown efficiencieswere obtained by electroporation or lipofection, and usingthree different oligonucleotide sequences for CD18 (datanot shown). The integrin α chains CD11b and CD11cwere depleted in parallel in CD18 knockdown cells(Figure 10B). By contrast, siRNA depletion of CD11cdid not lead to reduced CD18 levels (data not shown),presumably as CD11b and CD11c can substitute foreach other in binding to CD18, but both require CD18for expression. The cytoskeletal linker protein paxillinwas also down-regulated in CD18 knockdown cells(Figure 10B), but vinculin was not. Co-modulation ofthe β2 integrin chain with the α integrin chains CD11band CD11c and some cytoskeletal linker proteins againdemonstrates coordinated regulation of the expression ofthe integrin/adhesion protein complexes in MDM.

We also investigated the effect of CD18 knockdownon HIV infection of MDM. In HIV-infected MDM, virusassembly was not affected by CD18 knockdown, andthere was no significant difference in the amount ofHIV released from infected CD18-depleted or controlMDM over a 24-h window (Figure S4A). The virusesproduced by CD18 knockdown MDM were as infectiousas viruses produced in cells treated with control siRNA(Figure S4B). Immunofluorescence staining indicated thatin cells lacking CD18, HIV still accumulated mainlyin intracellular compartments, although the distributionof virus-containing structures was altered. This wasparticularly striking in situations where CD18-expressingand CD18-depleted cells were observed together inthe same cultures (Figures 10C,D and S4C). In CD18-expressing MDM, staining with a mAb against p17 thatonly detects mature virions after Gag cleavage, the virus-containing structures appeared brighter and virus wasmore concentrated in a few large IPMC. By contrast,cells lacking CD18 showed weaker p17 staining, andthe p17-containing compartments appeared as smaller,more scattered structures, with some staining for p17very close to or at the cell surface. To analyse theeffect of CD18 knockdown more quantitatively, MDMwere immunolabelled for CD44 and the tetraspanin CD81,imaged by confocal microscopy and the number ofcells with at least one intracellular compartment withCD44/CD81 co-staining was counted. This indicated thatthe proportion of MDM that contained at least oneCD44/CD81-positive intracellular structure was reducedfrom about 60–70% in control cells to 40–50% of cellsin the CD18 knockdown MDM, independent of HIVinfection (Figure S4D). Note that this analysis probablyunderestimates the effect of CD18 knockdown as it didnot account for changes in the sizes of the compartmentsand does not distinguish cells with a single internal

Figure 10: CD18 depletion in MDM disrupts the integrity of the IPMC where HIV-1 assembles. A) Line diagram of the CD18depletion experiments. Monocytes were differentiated into macrophages for 7 days in vitro. Cells were infected with HIV-1, transfectedwith 60 nM control or CD18 siRNA 1 day post-infection (dpi) and harvested 8–12 dpi. Media were changed twice. B) MDM were infectedwith HIV-1 and left untransfected or treated with control or CD18 siRNA, and cultured in the presence of TAK-779 for 7 days. Cells werelysed in non-reducing Laemmli buffer and subjected to immunoblotting for CD18, CD11b, CD11c, vinculin or paxillin, or for α-actin as aloading control. Data are representative of samples from at least three independent donors. C–F) MDM were infected with HIV-1 andtreated with CD18 siRNA for 11 days in the presence of TAK-779. Cells were fixed and immunostained for CD18 (green) and p17 (red).Single confocal sections are shown in C. D shows a snapshot of a 3D reconstruction of the area shown in C. E and F are enlarged 3Dsnapshots of the boxes in D. The images are representative of experiments performed with four independent donors. Scale bar, 20 μm.G–J) CD18 immunogold labelling of cryosections from MDM treated with control or CD18 siRNA. G and H) Images from MDM infectedwith HIV-1 for 11 days, CD18 siRNA for 10 days. Virus still accumulates in intracellular compartments with complex morphology. NoteHIV buds and immature virus particles in H (white arrows). Most of the IPMC on this specimen lacked the electron-dense coats. Vindicates accumulations of virus particles. I) Uninfected MDM from the same donor after 18 days in culture, control siRNA for 10 daysprior to fixation. J) Uninfected MDM from the same donor as in G–I, 18 days in culture, siRNA for CD18 for 10 days prior to fixation.These images are representative of experiments with MDM from two independent donors. K) Quantification of the frequency of thickelectron-dense coats. For one donor, cryosections as in G–J were stained for CD18 and areas of the EM grids scanned systematically.Any membranes bearing thick coats were photographed and measured. The graph shows the total length of membranes with thickelectron-dense coats observed per μm2 of section area scanned (samples contained similar cell densities). For the CD18 knockdowncells, coats clearly labelled with CD18 are shown in the black bars, and coat-like structures in cells lacking cell surface CD18 stainingin light grey. L–N) Cryosections from uninfected MDM after 18 days in culture, treated with control (L and M) or CD18 siRNA (N) for10 days, were immunolabelled for paxillin. Paxillin labelling was seen over the electron-dense coats (L) and occasionally over smallerelectron-dense patches at the plasma membrane at the base of the cells (M). On CD18-depleted cells, the plasma membrane stainingwas still observed (N), but there were no labelled coats. Scale bars: G, 500 nm; H, I, J and L–N, 200 nm.

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Figure 10: Legend on previous page.

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compartment from cells with more numerous smallercompartments. Flow cytometry analysis of HIV-infectedMDM double stained for p17 and CD18 showed thatthe population of cells depleted for CD18 also containedquantitatively less p17 than CD18-expressing cells presentin the population (Figure S4E).

To analyse the effects of CD18 knockdown on the ultra-structure of the IPMC, cells were prepared for cryosectionimmunolabelling EM. Screening of semi-thin cryosectionsshowed that CD18 knockdown was widespread, but virusstill accumulated in intracellular compartments on CD18knockdown cells, and these compartments could still bestained for CD44 or the tetraspanin CD9 (data not shown).At the EM level also, virus was still seen to bud intoand accumulate in intracellular compartments (Figures10G,H and S5) though these were often more dispersedand, where visible, the connections to the cell surfaceappeared less tight. Strikingly, these compartments wereno longer decorated with thick electron-dense coats.Only rare cells contained coated membranes, but theseinvariably labelled for CD18, suggesting that these cellshad escaped the CD18 knockdown. On uninfected MDMtreated with control siRNA, intracellular membranes withthick electron-dense coats, including closely apposedmembranes, were strongly labelled for CD18 (Figure 10I),but only a few coated membranes were detected onCD18 knockdown MDM. One of these is shown in Figure10J, though this is thicker than the CD18-labelled coats,has a more irregular appearance, and the precise natureof these structures is unclear. Analysis of the length ofcoated membranes found during systematic searchingof CD18-labelled cryosections showed that the numberof thick electron-dense coats observed was dramaticallyreduced in CD18 knockdown cells, particularly in infectedCD18-depleted MDM (Figure 10K); membranes withcoats that were not labelled for CD18 were reduced 11-and 40-fold in uninfected and infected MDM, respectively.MDM were also stained for paxillin: some paxillin stainingwas observed over irregular electron-dense patches atflat portions of the plasma membrane as often seen at thebase of cells, and strong paxillin labelling decorated theelectron-dense coated membranes (Figure 10L). On CD18-depleted MDM, the irregular plasma membrane labellingfor paxillin was still seen (Figure 10, compare M and N),but again membranes bearing the thick electron-densecoats were no longer observed on the IPMC.

Together, these observations indicate that the coordinatedexpression of CD18/CD11c and focal adhesion compo-nents correlates with the appearance of IPMC in MDMduring differentiation. CD18/CD11c and the cytoskeletalcomponents, talin and paxillin, are required for the forma-tion of the adhesion plaque structures and are involved inorganizing the integrity of the IPMC where HIV assembles.

Discussion

While it has long been known that HIV assemblesand accumulates in intracellular compartments in cul-tured human MDM, the nature of this compartment hasremained controversial. The compartment has been sug-gested to be related to the Golgi complex/trans-Golginetwork (TGN) (2,3) or to be endosomal (4–6,25), butrecent studies have demonstrated that it consists of anelaborate meshwork of plasma membrane-linked mem-branes within the cells (8–11). Studies with fluid tracersand various EM approaches involving staining with themembrane-impermeant dye ruthenium red, in conjunc-tion with serial sectioning, focussed ion-beam milling, ortomography show that the compartments remain con-nected to the cell surface through narrow gaps betweenmembrane sheets or via channels whose diameter is lessthan that of HIV particles, which are therefore retainedwithin the cells (8–11). Nonetheless, as the membranedomains are open to the extracellular milieu, we presumethe lumen of the compartment is at the extracellular pH,and that the restricted connections to the cell surface maylimit access of extracellular components such as antibod-ies to the compartment. Here we have further character-ized these intracellular compartments by an extensive EMimmunolabelling analysis, focussing in particular on vari-ous coat structures associated with the compartments.We show that the intracellular HIV-containing compart-ments are decorated with clathrin-coated pits containingthe plasma membrane clathrin adaptor protein AP-2, aswell as focal adhesion-like structures, further validatingthe identity of these compartments as intracellular plasmamembrane domains.

We demonstrate that the thick electron-dense coats thatcan frequently be observed on IPMC are related tofocal adhesion complexes. They contain the leukocyteintegrins CD11b/CD18 (Mac1) and, more prominently,CD11c/CD18 (p150,95). CD18 (β2 integrin) is clusteredagainst the electron-dense coat structures that containvarious cytoskeletal scaffold proteins including talin,vinculin and paxillin that link to the actin cytoskeleton. Theintracellular compartments decorated by focal adhesion-like domains are particularly prominent in HIV-infectedMDM, where the accumulating virus particles appearto expand the internal compartments (see also 9,11),but similar intracellular membrane structures with focaladhesion-related coats are also present in uninfectedMDM (9). Indeed, the intracellular compartments andthe associated focal adhesion domains appear as MDMdifferentiate in culture and become prominent in cells after10–14 days in vitro. Although depletion of CD18 by RNAidoes not affect HIV assembly in IPMC, virus release orthe infectivity of the released viruses, CD18 knockdownin MDM disrupted the focal adhesion-like plaques and, ininfected cells, changed the arrangement of the intracellularvirus-containing compartments, suggesting that the β2integrin-containing adhesion structures play a role in theorganization and regulation of the IPMC.

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A number of observations indicate that the thick coatson the IPMC in MDM represent functional structures.The structures can be stained with two antibodies againstCD18, the pan-CD18 antibody MEM-48, and MEM-148,which binds the high-affinity activated conformation of β2integrin as well as unassociated CD18. The presenceof the α integrins CD11b and CD11c in the coateddomains indicates that a proportion of the β2 integrinin the complexes consists of mature αMβ2 and/or αXβ2integrins in the activated conformation. This is furthersupported by the observation that the β2 integrins areclustered in these structures, and by the presence of talin,which binds to activated integrin chains (26). Analysis ofMDM at different ages post-isolation shows that, whileintegrin expression is low on freshly adhered MDM, CD18levels gradually increase in the cultures, plateauing 10–14days after isolation, and are maintained until at least 26days post-isolation. Immunofluorescence staining withantibodies to CD44 indicated that the IPMC in MDMappeared with a similar time–course, and after 10–12days bright patches of CD18 staining were detected,consistent with clustering of CD18 into adhesion plaquedomains. Western blotting showed a similar time–courseof expression for the α integrin chains CD11b andCD11c. Notably, paxillin expression increased with asimilar time–course, while vinculin expression did not varysignificantly with the age of MDM in culture. When CD18was depleted by siRNA, the cellular levels of paxillin alsodecreased. Indeed, siRNA knockdown of paxillin induceda reciprocal decrease in CD18 levels (data not shown),supporting a close association and co-regulation of thesefocal-adhesion-associated proteins.

Immunofluorescence staining for the plasma membraneproteins CD44 and CD9 also demonstrates that the IPMCin MDM develop as the cells mature. This may alsoclarify the question of where the bulk of HIV assemblytakes place in macrophages. Indeed, studies tracingthe assembly of HIV-1 Gag virus-like particles in Gag-GFP model systems, which tend to involve transfectionof 6–7-day-old MDM and analysis within 6–24 h, havedocumented significant particle assembly at the cellsurface with comparatively little budding at intracellularsites (7,27); IPMC are not well developed at this age.In contrast, experiments with infected macrophages areoften analysed at later times, after around 2 weeks inculture, when intracellular accumulations of HIV becomepredominant (3,5,6,8–10,14). In our studies, virus particleswere only rarely detected at the cell surface and almostall HIV budding profiles and immature virions were seenin the IPMC. However, at this point, it is not clear whatproportion of HIV buds from the cell surface proper andis released into the medium, compared to how muchbudding occurs at the IPMC where virus is retained,how this varies with the age of the cells in culture, orhow viral components are targeted to the intracellularplasma membrane domains. The lipid phosphatidylinositol4,5-bisphosphate [PI(4,5)P2], which is required for themembrane targeting of Gag via the MA domain and

which also plays a role in the activation of focal adhesioncomplexes, appears to be present on the intracellularmembranes and may contribute to targeting of the viralcomponents (P.M. et al., manuscript in preparation).

While the appearance of the IPMC and intracellular HIVassembly in MDM corresponds in time with an increasein the expression of CD18 and focal adhesion plaquecomponents, depletion of CD18 by siRNA indicates thatCD18 is not required for the formation of the intracellularplasma membrane domains per se, or for assembly of HIVat intracellular sites. Cells depleted of CD18 lacked theelectron-dense adhesion plaque coats, demonstrating thatCD18 and/or the other associated components (CD11b,CD11c and paxillin) are required for the formation of thesestructures. Although CD18-depleted MDM supportedreplication by an R5 HIV strain and released infectiousvirus particles, immunofluorescence staining revealed areduction in the number of cells with clear intracellularcompartments and an altered appearance of the IPMC,with many smaller, more dispersed compartments, somevery close to the cell surface proper. In addition, flowcytometry analysis of infected cells suggested that theamount of HIV associated with CD18 knockdown cellsis reduced. These observations indicate that the β2integrins and adhesion plaque proteins may be involved inorganizing the intracellular compartments, in particular thenarrow connections between the intracellular vacuole-like domains or between the compartments and thecell surface, and suggest that the integrins may affectthe ability of MDM to accumulate and release HIVparticles. However, the current lack of tools to rapidly andsynchronously manipulate the CD18 domains associatedwith the IPMC limits our ability to analyse the impactof these structures on HIV release or cell-to-celltransmission.

The presence of β2 integrins may hint at the normalfunction of the IPMC in uninfected MDM. In macrophages,β2 integrins are involved in various functions such asextravasation and migration to sites of inflammation,phagocytosis, cell–cell and cell–matrix adhesion andthe cell–cell interactions that take place during antigenpresentation to T-cells. Expression of β2 integrins mayincrease macrophage adhesion and decrease migration,and could thus facilitate the retention of these cells at sitesof inflammation (28). The CD18 integrins also function asthe complement receptors CR3 (with CD11b) and CR4(with CD11c), which are involved in the uptake of iC3b-opsionized pathogens. In addition, signalling via integrinslinks interactions with the extracellular environment tomodulation of the intracellular cytoskeleton (29). Infectionby HIV would affect these signalling pathways and, as aresult, macrophage behaviour. This may be particularlyrelevant for HIV-infected tissue macrophages, e.g. inthe brain, where microglia are the main infected cellpopulation and are involved in HIV-associated dementiaand neuropathy.

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Virus-containing IPMC have been observed in a numberof different MDM preparations (3,5,6,8–10,14), and theappearance of these structures appears to be largelyindependent of the method by which the cells are isolatedor by culture conditions such as serum, growth factortreatments or the surfaces that the cells are culturedon. Furthermore, HIV has been shown to be present inintracellular compartments in macrophages in vivo, e.g. ininfected lymph nodes or in microglial cells in post-mortembrain tissue from infected patients (16,30–32). This sug-gests that IPMC may be present in macrophages in vivo.Indeed, CD11c, the integrin α chain most prominentlyassociated with CD18 on MDM, has been describedas a marker for tissue macrophages in humans and isprominently expressed on macrophage sub-populationsin tissues such as spleen, thymus and tonsil, as well ason alveolar macrophages and brain microglial cells (33).Cell-surface-connected intracellular compartments thatcan accumulate HIV particles have been described indendritic cells (34–36). These structures resemble theIPMC where HIV assembles in MDM. In dendritic cells,these compartments have been shown to be involvedin the formation of virological synapses and in the trans-infection of T-cells (34–38). As HIV-infected macrophagescan also transfer HIV to uninfected macrophages orT-cells (39,40), the virus-containing IPMC in MDM anddendritic cells may be related and have a similar function.

In vivo, macrophages play a central role in HIVpathogenesis. They have important roles in the immuneactivation and inflammation that accompany HIV disease.Furthermore, as CD4 and chemokine-receptor-expressingcells, they can be infected by HIV and may constitute areservoir of virus (41,42), which they are able to transmitto T-cells via virological synapses (43). The identificationof β2 integrins in the IPMC in MDM, together withother proteins such as CD44, increases the repertoireof markers for these compartments and will help in thefurther analysis of the structure and function in infectedand uninfected cells. In addition, the β2 integrins maybe involved in regulating the structure of the IPMCand thereby influence transmission of HIV from infectedmacrophage reservoirs.

Materials and Methods

Antibodies and staining reagentsmAbs to HIV p17 (4C9, ARP342 obtained from R.B. Ferns and R.S.Tedder, Middlesex Hospital Medical School, London, UK) and p24 (38:96Kand EF7, ARP365 and 366, respectively, from B. Wahren, NationalBacteriological Laboratory, Stockholm, Sweden) were obtained throughthe National Institute for Biological Standards and Control Centre forAIDS Reagents (CFAR), which is supported by the EU Program EVA andthe UK Medical Research Council. Rabbit antiserum UP595 against HIVp17 was provided by M. Malim (Guy’s, King’s and St Thomas’ Schoolof Medicine, London, UK). Rabbit antiserum against clathrin light chainwas provided by F. Brodsky (University of California San Francisco, SanFrancisco, CA), and the mAbs Clone 23 and X22 against the clathrin heavychain were purchased from BD Transduction Laboratories and Abcam,respectively. The anti-α-Adaptin AP-2 antibody AP.6 was purchased from

Calbiochem (Merck). Mouse mAbs against CD44 (MEM-85) and CD18(MEM-48 and MEM-148) and rabbit mAbs against CD11b and CD11c(EP1345Y and EP1347Y) were from Abcam. mAbs to CD9 (MRP-1),CD11a (Clone 38) and CD11c (Bu15) were purchased from AbD Serotec.Mouse anti-CD11b (VIM12) and goat polyclonal anti-actin (I-19) were fromSanta Cruz Biotechnology, Inc. and mouse anti-β-actin (AAN02) was fromCytoskeleton. Mouse mAbs against talin (clone 8D4), vinculin (hVIN-1) andγ-adaptin AP-1 (100/3) were from Sigma-Aldrich, and anti-paxillin (clone349) was from BD Transduction Labs. Antibodies against AP-3 (mousemAb SA4 and a rabbit polyclonal antibody to the AP-3 δ hinge region)were from Andrew Peden. Anti-CD54 (ICAM-1) (Clone 15.2) was from AbDSerotec, and the mAb to CD81 (M38) was provided by F. Berditchevski(University of Birmingham, UK). Alexa Fluor®-488 or -594-labelled secondantibody reagents against rabbit or mouse immunoglobulins, includingisotype-specific antibodies against mouse IgG1, IgG2a or IgG2b, AlexaFluor®-594 phalloidin and Hoechst 33258 reagent were from Invitrogen.

Preparation of MDM and infection with HIV-1BaL

MDM were prepared from peripheral blood mononuclear cells (PBMC)isolated from buffy coats from healthy donors (National Blood Service,Essex, UK) and differentiated with 10 ng/mL of M-CSF (R&D Systems) for2 days. The cells were infected after 6–7 days in culture with HIV-1BaL asdescribed (5,9,44).

EM of Epon-embedded MDMHIV-infected MDM were fixed for 4 h in 2% paraformaldehyde (PFA)/3%glutaraldehyde in 0.1 M NaPi buffer, pH 7.4, post-fixed for 1 h on ice in 1%OsO4/1.5% K3[Fe(CN)6], and treated with 1.5% tannic acid, as described(9). Samples were dehydrated in graded ethanols and embedded in Epon812 (TAAB Laboratories). Ultrathin sections were cut on an Ultracut UCTultramicrotome (Leica) and stained with lead citrate.

Preparation of cells for cryosectioningHIV-infected MDM or uninfected control cells were fixed by adding anequal volume of pre-warmed double-strength fixative (8% PFA in 0.1 M

NaPi buffer, pH 7.4) directly into the culture medium. After 10 min, themedium was replaced with 4% PFA and fixation continued for 90 min. Cellswere rinsed in 20 mM glycine in PBS, embedded in 12% gelatine, infiltratedwith 2.3 M sucrose, and frozen in liquid nitrogen as previously described(9,44). Semi-thin (0.5 μm) or ultrathin (50–60 nm) cryosections were cuton an Ultracut UCT microtome equipped with an EM FCS cryochamber(Leica).

Immunolabelling of cryosectionsFor immunofluorescence staining, semi-thin cryosections (0.5 μm) on glassslides were quenched in 50 mM glycine/50 mM NH4Cl and, where stated,extracted for 6 min in 0.1% TX-100. Sections were stained with antibodiesdiluted in PBS 1% BSA, and mounted in Mowiol (Merck BiosciencesLtd.). Sections were examined at room temperature with a microscope(Axioskop; Carl Zeiss Ltd) fitted with Plan Neofluar oil immersion objectives[63×/1.25 numerical aperture (NA) or 100×/1.30 NA]. Images wererecorded with a charge-coupled device camera (Orca-ER; Hamamatsu)controlled by OPENLAB 5.0.2 software (Improvision, Perkin Elmer), falsecoloured, converted to tiff files, adjusted for brightness and contrast andassembled into montages with Photoshop CS (Adobe).

For EM immunolabelling, ultrathin cryosections (50–60 nm) on formvar-coated grids were quenched in 50 mM glycine/50 mM NH4Cl and labelledwith primary antibodies and PAG (5, 10 or 15 nm, the EM Lab, UtrechtUniversity). Sections stained with mouse mAbs were incubated witha rabbit anti-mouse bridging antibody (DakoCytomation) before labellingwith PAG. For double labelling, cells were stained sequentially with a firstprimary antibody and PAG and fixed in 1% glutaraldehyde for 10 min beforerequenching and staining with the second antibody reagent and PAG of adifferent size. Sections were examined with a Tecnai G2 Spirit transmission

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EM (FEI) and digital images were recorded with a Morada 11 MegaPixelTEM camera (Olympus Soft Imaging Solutions) and the ANALYSIS softwarepackage. Images were adjusted for brightness and contrast and figureswere assembled with Photoshop CS.

Immunofluorescence staining of whole-cell

preparationsImmunofluorescence staining was essentially as described (9). MDMwere washed with PBS and fixed by adding 4% PFA for 20 min at roomtemperature. After washing, free aldehyde groups were quenched with50 mM NH4Cl, and the cells were incubated for 1 h in permeabilizationbuffer (0.1% Triton-X-100 and 0.5% BSA in PBS) containing 6 μg/mLpurified human IgG. Cells were stained with primary antibodies diluted inblocking buffer (6 μg/mL human IgG, 0.5% BSA in PBS) for 1 h, washed,and incubated for 45 min with appropriate combinations of fluorescentsecondary antibodies. For actin staining, Alexa Fluor-594-labelled phalloidinwas added to the fluorescent secondary antibody. Cells were washedand DNA stained for 10 min with 1 μg/mL Hoechst 33258 diluted inPBS. Coverslips were mounted in Mowiol, and stored at 4◦C. Stainingwas analysed using a Leica TCS SPE confocal microscope and LEICA

APPLICATION SUITE (LAS) software. Images were processed using Photoshopor IMAGEJ (National Institute of Health). The mean fluorescence intensities(MFI) of confocal images were quantified using IMAGEJ. Cell numbers weredetermined by counting the number of nuclei identified by Hoechst stainingand used to normalize the MFI.

Western blottingFor western blot analysis, MDM were lysed in NP40 buffer [2% NP40,150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA) in 20 mM

Tris–HCl pH 7.8, with 1 mM PMSF and 5 μg/mL each of chymostatin,antipain and pepstatin and 10 μg/mL leupeptin, Sigma] for 10 min andnuclei removed by centrifugation for 10 min at 12 000 × g at 4◦C. Theprotein content of the lysates was determined with the DC protein assay(Detergent-compatible protein assay kit, BioRad), and equal amounts ofprotein for each sample were separated on 8% SDS–polyacrylamide gels,and transferred to Immobilon-P Polyvinylidene fluoride (PVDF) membranes(Millipore) at 0.4 A for 1 h under wet blotting conditions. Alternatively,cells were lysed directly in Laemmli sample buffer (10% SDS, 15%glycerol, 0.2 M Tris–HCl pH 6.8, bromophenol blue in H2O) for 10 min at95◦C. The lysates were separated on 10% SDS–polyacrylamide gelsunder non-reducing conditions, and transferred to Immobilon-P PVDFmembranes (Millipore) at 20 V for 1 h under semi-dry blotting conditions.Blots were quenched with blocking buffer (0.1% Tween, 5% non-fatmilk in PBS) for 1 h at room temperature, and immunoblotted withprimary antibodies at 4◦C overnight. The membranes were washedthree times with PBS–0.1% Tween and incubated with the appropriatesecondary antibodies conjugated to HRP (Thermo Scientific) for 1 h at roomtemperature. After four washes with PBS–0.1% Tween, membranes werebriefly incubated in SuperSignal West Pico Luminol (Thermo Scientific).Signals were detected with Hyperfilm ECL (Amersham Biosciences).Time–course blots were detected using SuperSignal West Femto Luminol(Thermo Scientific) and signals were detected using the ImageQuantLAS4000 mini system (GE Healthcare).

CD18 RNAiStealth Select RNAi targeting CD18 (oligo ID HSS105563) and a non-targeting control oligonucleotide (Stealth RNAi siRNA Negative ControlMed GC) were purchased from Invitrogen. MDM were transfectedwith 60 nM control or CD18-specific siRNAs 1 day after infectionusing Lipofectamine RNAiMAX Reagent (Invitrogen) according to themanufacturer’s instructions. To prevent multiple rounds of infection,cells were cultured in the presence 50 nM TAK-779 (NIH AIDS Researchand Reference Reagent Program, Division of AIDS, NIAID, USA) whereindicated (45).

p24 ELISA assaySupernatants from HIV-1-infected cells were centrifuged for 5 min at1000 × g to remove cell debris. p24 levels in the supernatants wereanalysed by enzyme-linked immunosorbent assay (AIDS Vaccine Program,NCI-Frederick Cancer Research and Development Center, Frederick, MD)following the manufacturer’s instructions (44).

Infectivity assaySingle-cycle infectivities were determined by challenging TZM-β-galactosidase (β-gal) indicator cells (obtained from J. Martin-Serrano, Guy’s,King’s and St. Thomas’ School of Medicine, London, UK) with cell-freevirus-containing supernatants, as described previously (44). Briefly, 105

cells/well in 24-well plates were infected with virus preparations contain-ing 0.5–2 ng of p24, and the induced expression of β-galactosidase activityin cell lysates was measured 24 h later by using the Galacto-Star systemfrom Applied Biosystems. The infectivities of the supernatants from controlsiRNA-treated MDM were set to 100%.

Flow cytometry analysisMDM were washed with PBS and fixed by adding 4% PFA for 20 min atroom temperature. After washing, the cells were scraped off the culturedish and incubated in blocking buffer (0.1% saponin, 1% FCS, 6 μg/mLhuman IgG, 2 mM EDTA in PBS) for 30 min at 4◦C. Cells were stainedwith primary antibodies diluted in blocking buffer for 1 h at 4◦C, washedthree times in washing buffer (0.1% saponin, 1% fetal calf serum (FCS),2 mM EDTA in PBS) and incubated for 30 min at 4◦C with appropriatecombinations of Alexa Fluor-conjugated fluorescent secondary antibodies.Cells were washed three times in washing buffer and analysed on aFACSCalibur flow cytometer (BD Biosciences). Data were analysed usingCELLQUEST software (BD Biosciences).

Acknowledgments

This work was supported by UK Medical Research Council funding to theCell Biology Unit, by a PhD studentship from Boehringer Ingelheim Fondsto S. G., and the EU HIV-ACE research network grant HEALTH-F3-2008-201095. A.P.-M., S. G. and M. M. conceived and designed the experiments.A.P.-M. performed all the EM and associated analyses, S. G. performedthe RNAi experiments and associated analyses, P. M. analysed actindistributions and J. T. performed biochemical analyses and macrophagepreparation. We thank M. Malim, F. Brodsky, A. Peden, F. Berditchevskiand the National Institute for Biological Standards and Control Centre forAIDS Reagents (CFAR) for antibodies, and the NIH AIDS Research andReference Reagent Program, Division of AIDS, NIAID, NIH, for the TAK-779 reagent. We acknowledge the advice of Dr. Emmanuelle Caron duringthe early stages of this work and would like to dedicate this paper to hermemory.

Supporting Information

Additional Supporting Information may be found in the online version ofthis article:

Figure S1 (related to Figure 1): Serial sections of an IPMC near the

cell surface in an HIV-infected macrophage. Selected serial sectionsof the IPMC shown in Figure 1C demonstrate the 3D structure of thecompartment and the extended areas of coated membranes. Sections4, 6, 8, 9, 10 and 12 of a series of 18 sections (70 nm thickness) areshown. Section 8 is the image shown in Figure 1C, where there appearsto be a connection to the sheet-like processes at the cell surface. Theclosely apposed membranes bearing electron-dense coats separate thevirus-containing IPMC from the cell surface and may retain virus particlesin this compartment. Scale bars, 200 nm.

Figure S2 (related to Figure 2): The clathrin adaptor proteins AP-1 and

AP-3 are not present in the thick electron-dense coats. Immunolabelling

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of cryosections from human MDM infected with HIV-1 for 7 days. A andB) Staining for AP-1. Double labelling with mAb 100/3 anti-AP-1/10 nmPAG and rabbit UP595 anti-HIV-1 MA/15 nm PAG. A) shows a large virus-containing IPMC near the cell surface (cell surface microvilli are markedMV) while (B) shows an area of cytoplasm at higher magnification. AP-1labelling (10 nm gold particles) is seen over certain small clathrin-coatedvesicles (black arrows), while larger coated vesicles closer to the cellsurface remain unlabelled (white arrow). Membranes with thick electron-dense coats are not labelled for AP-1 (arrowheads). C–G) Staining for AP-3.C, D and E) Double labelling with mouse mAb SA4 anti-AP-3/10 nm PAGand rabbit UP595 anti-HIV-1 MA/15 nm PAG. F and G) Double labelling witha rabbit antibody against the AP-3 hinge domain/10 nm PAG and mousemAb 4C9 anti-HIV-1 p17/15 nm PAG. Virus-containing IPMC are markedVCC. AP-3 labelling (10 nm gold particles) is seen over certain small coatedvesicles (black arrows in C, D and F). This coat appears thinner (about16 nm) and more irregular than that of coated vesicles associated with theIPMC (about 20 nm, white arrows). Membranes with thick coats are notlabelled for AP-3 (arrowheads in E and G). Scale bars: A and G, 500 nm;B–F, 200 nm.

Figure S3 (related to Figure 9): Immunofluorescence staining of MDM

preparations fixed at various times after isolation. Uninfected MDMprepared from different donors and maintained in culture for various timeswere fixed and semi-thin cryosections were stained with the mAbs MEM-48 anti-CD18 (A), MEM-148 against activated CD18 (B), Bu15 anti-CD11c(C), or with a mAb against CD54/ICAM-1 (D). Staining for the integrinsCD18, activated CD18 or CD11c was weak on MDM 7 days post-isolation,but appeared stronger on older MDM cultures. The bright foci of clusteredCD18 are first seen at 13 days post-isolation and become stronger on oldermacrophages. ICAM-1 staining did not appear to vary with the age of thecells. All scale bars, 10 μm.

Figure S4 (related to Figure 10): Effects of CD18 depletion on HIV

assembly and the morphology of the IPMC in MDM. Uninfected orHIV-1-infected MDM were left untreated, or treated with 60 nM controlor CD18 siRNA 1 dpi and cultured in the presence of 50 nM TAK-779except where indicated. A) Culture supernatants were collected 24 h aftera medium change 7 dpi, and levels of released virus were determined usinga p24 ELISA assay. The graph shows the mean of three donors ± standarddeviations relative to the control RNAi samples. B) Cells were culturedwithout TAK-779 and cell-free virus particles in culture supernatantscollected 24 h after a medium change 7 dpi were subjected to a β-galactosidase infectivity assay. The average of two donors with standarddeviations is shown. C) MDM were fixed at 8 dpi and immuno-stained forCD18 (green) and p17 (red). Single confocal sections and 3D snapshotsare shown as indicated. The images are representative of experimentsperformed with four independent donors. Bar, 20 μm. D) MDM were fixedat 12 dpi and immuno-stained for CD44 and CD81. The proportion ofcells showing any intracellular CD44/CD81 colocalization was quantifiedfor each condition. Between 88 and 213 cells were counted for eachcondition. E) CD18 siRNA or control siRNA treated cells were fixed at 8dpi, immuno-stained for CD18 and p17 and analysed by flow cytometry.Cell populations were arbitrarily gated into CD18 low and CD18 highpopulations as indicated. The numbers in the boxes denote the p17 MFIfor the respective cell populations. Data are representative of resultsobtained with four different donors.

Figure S5 (related to Figure 10): Analysis of the morphology of IPMC

in MDM after CD18 depletion. A) Line diagram of the CD18 depletionexperiment. Monocytes were isolated from buffy coats of healthy donorsand differentiated into macrophages for 7 days in vitro. Cells were infectedwith HIV-1, transfected with 60 nM control or CD18 siRNA 1 dpi and fixedat 11 dpi. B) Semi-thin (0.5 μm) cryosections were extracted with 0.1%TX-100 and stained for immunofluorescence with mAbs against HIV-1 p17(red) and MEM-48 against CD18/β2 integrin in green. No CD18 stainingwas detected on cells treated with CD18 siRNA. The p17 staining indicatesthat virus is present in smaller compartments in CD18 knockdown cells.C–F) Ultrathin cryosections from the CD18 siRNA-treated cells above werelabelled for CD18 with 10 nm PAG and a rabbit antibody against MA with5 nm PAG. An overview from a CD18-depleted cell is shown in C, while D,E and F are enlarged views of the areas indicated. Representative images

from MDM from two different donors. Scale bars: B, 10 μm; C, 1 μm; D–F,200 nm.

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