HIV-1-Infected Macrophage Giant Cells Cytoskeletal Protein

Cytoskeletal Protein Transformation in HIV-1-Infected Macrophage Giant Cells Irena Kadiu, Mary Ricardo-Dukelow, Pawel Ciborowski and Howard E. Gendelman This information is current as of September 25, 2021. J Immunol 2007; 178:6404-6415; ; doi: 10.4049/jimmunol.178.10.6404 http://www.jimmunol.org/content/178/10/6404 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Cytoskeletal Protein Transformation in HIV-1-Infected Macrophage Giant Cells1

Irena Kadiu,*§ Mary Ricardo-Dukelow,*§ Pawel Ciborowski,*†§ and Howard E. Gendelman2*‡§

The mechanisms linking HIV-1 replication, macrophage biology, and multinucleated giant cell formation are incompletely understood. With the advent of functional proteomics, the characterization, regulation, and transformation of HIV-1-infected macrophage-secreted proteins can be ascertained. To these ends, we performed proteomic analyses of culture fluids derived from HIV-1 infected monocyte-derived macrophages. Robust reorganization, phosphorylation, and exosomal secretion of the cytoskeletal proteins profilin 1 and actin were observed in conjunction with productive viral replication and giant cell formation. Actin and profilin 1 recruitment to the macrophage plasma membrane paralleled virus-induced cytopathicity, podosome formation, and cellular fusion. Poly-L-proline, an inhibitor of profilin 1-mediated actin polymerization, inhibited cytoskeletal transformations and Downloaded from suppressed, in part, progeny virion production. These data support the idea that actin and profilin 1 rearrangement along with exosomal secretion affect viral replication and cytopathicity. Such events favor the virus over the host cell and provide insights into macrophage defense mechanisms used to contain viral growth and how they may be affected during progressive HIV-1 infection. The Journal of Immunology, 2007, 178: 6404–6415.

3 ononuclear phagocytes (MP; monocyte, dendritic astrogliosis, myelin pallor, and neuronal dysfunction. These are http://www.jimmunol.org/ cells, tissue macrophages, and microglia) are principal pathological signatures of HIV encephalitis and are linked to cog- M target cells and vehicles for HIV dissemination. Virus nitive and motor deficits in susceptible people (6). The process of can persist within MP for prolonged time periods despite robust MGC formation is thought to occur as a consequence of interac- innate immune responses that include phagocytic, microbicidal, tions between the viral envelope glycoprotein, CD4 and other cell and secretory activities (1, 2). Common morphological and func- surface proteins (7–10). tional changes linked to productive viral replication include altered Despite more than two decades of research into the interactions cell motility, Ag presentation, and cellular differentiation (3, 4). between HIV and the macrophage, how profound changes in MP Such changes occur as monocytes leave the bloodstream and enter secretory, chemotactic, microbicidal, and Ag presentation activi- by guest on September 25, 2021 infected tissue where they undergo transformation to tissue mac- ties during viral infection remain incompletely understood. Likely, rophages and as a consequence of activation acquire proinflam- such events affect MP responses to viral infection and a broad matory secretory phenotypes (5). Such processes are typical of range of virus-induced inflammatory responses and are not specific what occurs within the CNS during progressive HIV replication. for a single microbe (11). Indeed, morphological and functional Indeed, during advanced disease, monocyte-macrophages enter the cell changes elicited as a consequence of HIV infection are not brain within sites of inflammation and active viral replication and virus specific and are operative during interactions with a broad commonly form multinucleated giant cells (MGC) in attempts to range of infectious processes that include, but are not limited to, contain infection. The formation of MGC occurs with profound mycobacterial, fungal, protozoan, and bacterial pathogens (12). In all, the outcome of microbial-MP interactions serves as a means to control or eliminate pathogen growth while in many cases, simul- *Laboratory of Neuroregeneration, Department of Pharmacology and Experimental Neuroscience, †Department of Biochemistry and Molecular Biology, ‡Department of taneously enhancing dissemination and subsequent tissue damage Internal Medicine, and §Center for Neurovirology and Neurodegenerative Disorders, (13). Thus, elucidating the mechanisms underlying the molecular University of Nebraska Medical Center, Omaha, NE 68198 and biochemical changes in MP would have broader implications Received for publication December 9, 2006. Accepted for publication March in infection and immunity. 13, 2007. Viruses as obligate cell parasites have evolved into manipulators The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance of host cell functions. Accordingly, viruses often remodel the cy- with 18 U.S.C. Section 1734 solely to indicate this fact. toskeleton of target cells to convert one of the cellular barriers to 1 This work was supported, in part, by National Institutes of Health Grants 2 R37 viral replication into an advantageous vehicle for the virus which NS36126, 1 P01 NS31492, 1 P01 NS043985-01, 5 P01 MH64570-03, P20 RR15635 facilitates the generation of infectious progeny virions (14). Sur- (to H.E.G.), and R21 MH75489 (to P.C.). prisingly, little is known about the mechanisms used by HIV to 2 Address correspondence and reprint requests to Dr. Howard E. Gendelman, Center for Neurovirology and Neurodegenerative Disorders, University of Nebraska Medical exploit host cell cytoskeletal dynamics. However, HIV is known to Center 985880, Nebraska Medical Center, Omaha, NE 68198. E-mail address: polarize actin and control microtubule organization. This enables [email protected] spread of virus from donor to target cells in close cell contacts 3 Abbreviations used in this paper: MP, mononuclear phagocyte; GCI, Giant Cell termed “virological synapses” in T cells (15). We now have in- Index; LC-MS/MS, liquid chromatography mass spectrometry; MDM, monocyte-derived macrophage; MGC, multinucleated giant cell; MOI, multiplicity of infection; vestigated the influence of HIV-1 infection for its ability to re- P-L-P, poly-L-proline; RT, reverse transcriptase; SELDI-TOF, surface-enhanced laser model cytoskeletal structures and affect macrophage MGC forma- desorption ionization time-of-flight; WCX2, weak cation exchange. tion. In the course of proteomic analyses of the HIV-1-infected, Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 monocyte-derived macrophage (MDM) secretome, we observed www.jimmunol.org The Journal of Immunology 6405

that both actin and profilin 1 were secreted in abundance and pre- For suspension cell analyses, MDM were stained with allophycocyanin- ceded macrophage cytopathology. Confocal microscopy analysis conjugated anti-CD14 mAb (BD Biosciences) and FITC-conjugated IgG2a showed recruitment of profilin 1 and actin from the cytoplasm to mouse anti-HIV-1 p24 (KC57 FITC; Beckman Coulter) as described above, and data acquisition was performed on a FACSCalibur flow cytom- the cellular membrane during the early stages of MGC and in- eter (BD Biosciences) using CellQuest software (BD Biosciences). Live creased levels within the cytoplasm following cell fusion events. cells were gated by forward and side scatter using FCS Express V3 soft- Most importantly, inhibitors of cytoskeletal rearrangements di- ware (De Novo Software) (see Fig. 1). Data are shown as density plots of rectly affect profilin 1-mediated actin stress filament/podosome uninfected and infected cells at days 3, 5, and 10 after infection. For confocal microscopy examinations, cells were stained with mouse formation, cell-cell contacts, and HIV replication. Based on these anti-profilin 1 mAb (clone 2H11; Synaptic Systems) and FITC-conjugated observations, we posit that actin and profilin 1 cellular transfor- (goat) anti-mouse IgG mAb (Invitrogen Life Technologies), rhodamine- mation is triggered by HIV-1 infection. Modulations of the cy- conjugated phalloidin (Invitrogen Life Technologies) to detect F-actin, and toskeleton are critical events required for the virus to complete its To-PRO as a nuclear stain for 3 h, 30 min, and 20 min, respectively. Each life cycle and lead to an attempt by the macrophage to contain treatment was followed by triplicate 5-min washes with 0.1% saponin in PBS. Cells analyzed for HIV-1 p24 and profilin 1 or F-actin intracellular ongoing viral growth. localization were stained with FITC-conjugated mouse anti-HIV-1 p24 Ab (KC57 FITC; Beckman Coulter), goat anti-profilin 1 mAb (Alexis Phar- Materials and Methods maceuticals) and Alexa 598-conjugated (sheep) anti-goat mAb (Invitrogen Life Technologies), and rhodamine-conjugated falloidin (Invitrogen Life Isolation, cultivation, and HIV-1 infection of human MDM Technologies), respectively. Cells were mounted using anti-fade Pro-Long PBMC from HIV-1, HIV-2, and hepatitis seronegative human donors were Gold mounting reagent (Invitrogen Life Technologies) and examined with obtained by leukophoresis in full compliance and approval of the Univer- a Zeiss LSM 510 META NLO microscope (Zeiss MicroImaging). sity of Nebraska Medical Center Institution Review Board. Monocytes Downloaded from were purified by countercurrent centrifugal elutriation (16) then seeded Culture supernatant and cell lysate preparations onto T-75 or 250-ml Teflon flasks for adherent and suspension cultures, respectively. Cells were cultured in DMEM (Invitrogen Life Technologies) On days 3, 7, 10, and 12 after viral infection, culture fluids and MDM from ϫ g supplemented with 10% heat-inactivated pooled human serum, 1% glu- infected and control cultures were centrifuged for 10 min at 2000 and tamine, 10 ␮g/ml ciprofloxacin (Sigma-Aldrich), and 1000 U/ml highly treated with lysis/deactivation buffer (0.1% Triton X-100, 50 mM Tris- purified M-CSF (a gift from Wyeth, Cambridge, MA) and maintained in a HCl, and protease inhibitor mixture (Sigma-Aldrich)). The supernatants were concentrated using Amicon Centriplus centrifugal filter devices (Mil- 37°C and 5% CO2 atmosphere. On days 2 and 5, half of the medium was http://www.jimmunol.org/ exchanged. After seven days in culture, MDM were infected with HIV- lipore) with a membrane cutoff of 5000 Da molecular mass and dialyzed for 18 h against water at 4°C using QuixSep microdialyzers (Membrane 1ADA (a macrophage tropic viral strain) at a multiplicity of infection (MOI) of 1, or left untreated (controls). On day 1 after infection, a full medium Filtration Products). Protein concentrations were determined using Bio- exchange was performed followed by a half medium exchange every 2–3 Rad DC Protein Assay (Bio-Rad). days thereafter in medium devoid of M-CSF. Medium was exchanged on day 2 after infection without phenol red. On days 3, 5, 7, 10, and 12 after Virus and vesicle preparations infection, cells were exchanged with additive-free DMEM for an additional Infected and control MDM culture fluids were pooled, clarified (10 min at 24 h of incubation. Reverse transcriptase (RT) activity was determined in 2,000 ϫ g), filtered through 450- and 200-nm pore-size sterile filter (Beck- culture supernatant fluids as previously described (17). man Coulter), and concentrated using Amicon centrifugal filter devices

(Millipore) with a 100,000 molecular mass membrane cutoff. Fluids were by guest on September 25, 2021 Cell viability assays depleted of vesicles using anti-CD45 paramagnetic microbeads (Miltenyi Cell viability (mitochondrial activity) in control, HIV-1-infected, and poly- Biotec) as described previously (19). Brieﬂy, culture ﬂuids were treated ␮ L-proline (P-L-P)-treated cells grown in adherence was measured by MTT with 2 l/ml anti-CD45 microbeads for1hatroom temperature with reduction (18). In addition Live/Dead cell assay (Invitrogen Life Technol- shaking and then placed in magnetic separators (Invitrogen Life Technol- ogies) was used to assess cellular viability in adherent cultures at days 3, ogies) at 4°C for 20 h. The vesicles bound to microbeads were recovered 5, 7, and 10 after infection in control, HIV-1 infected, and P-L-P-treated and retained for further analysis, and supernatants were layered on 20% ϫ cells. Monocytes were cultured for 7 days in 90-well plates. Cells were (w/v) sucrose-PBS cushion and centrifuged for4hat120,000 g at 4°C. treated with P-L-P 1 h before infection or left untreated and infected with

HIV-1ADA at MOI of 1. At days 3, 5, 7, and 10 after infection, cells were Surface-enhanced laser desorption and ionization time-of-flight rinsed with PBS and treated with 0.5 ␮M calcein AM and 4 ␮M ethidium (SELDI-TOF) homodimer-1 in PBS for 45 min, rinsed three times with PBS, followed by microplate centrifugations at 500 ϫ g to avoid loss of dead cells by floa- The protein profiles of culture supernatants were analyzed by SELDI-TOF tation. Cells permeabilized with 0.1% saponin were used as controls for ProteinChip assays (Ciphergen Biosystems). The chip types tested included both ethidium homodimer-1 and calcein AM. Live cells were distinguished weak cation exchange (WCX2), strong anion exchange (SAX2), normal from dead cells (red fluorescence; ex/em 495/635 nm) by the uptake of phase (NP20), and immobilized metal affinity capture (IMAC30). Based on calcein AM to acquire a green fluorescence (ex/em 495/515 nm). Cell sensitivity and specificity of profile predetermination, the WCX2 Protein- enumerations were performed using fluorescence microscopy and a M5 Chip was selected to profile the culture supernatants. The ionized proteins microplate fluorometer (Molecular Devices) (limit 1 ex/em 490/522 nm; and their molecular mass/charge (m/z) ratios were assessed by SELDI-TOF limit 2 ex/em 530/645 nm) and normalized as the percentage of dead cells as described previously (20). Spectra were collected and analyzed using to uninfected controls. PBS II ProteinChip Biosystems (Ciphergen Biosystems). All tests were performed in triplicate, in three independent experiments, and from Immunohistochemical tests three separate cell donors. The ProteinChip Reader was externally cal- ibrated for each analysis for both low and high molecular mass regions For determinations of numbers of HIV-1 p24 positive cells in adherent using standard proteins. Peaks were automatically detected with Bio- MDM, monocytes were grown in 4 well Lab-tech chamber slides (Invitro- marker Wizard for ProteinChip software 3.2. Parameters for peak de-

gen Life Technologies), infected with HIV-1ADA at a MOI of 1 or left tection were first pass signal/noise (S/N) ratio 5, second pass S/N ratio uninfected. At days 3, 5, and 10 after infection cells were collected. MDM 2, and mass tolerance 0.5%. were stained with allophycocyanin-conjugated mouse anti-CD14 mAb (BD For the profilin 1 immunodepletion, 200 ␮g of protein from culture Biosciences) in 3% BSA in PBS for 30 min at room temperature. For fluids was incubated with 20 ␮g of anti-profilin 1 rabbit polyclonal Ab for HIV-1p24 staining and florescent microscopic examinations, cells were 24 h at 4°C (Novus Biologicals), followed by a 2-h incubation at room rinsed with PBS and fixed with 4% paraformaldehyde and 0.01% saponin temperature with 100 ␮l of protein A/G agarose gel slurry (Pierce). Fol-

(pH 7.4) for1hatroom temperature, 50 mM NH4Cl to quench autofluo- lowing incubation, the agarose beads were precipitated by centrifugation at rescence, and permeabilized in 100% methanol for 15 min at 4°C and 1% 1000 ϫ g for 5 min, and 5 ␮g of protein was collected from the profilin Nonidet P-40 (Sigma-Aldrich). Cells were incubated with FITC-conju- 1-immunodepleted fluids. Affinity-purified crystallography-grade profilin 1 gated mouse anti-HIV-1 p24 in 3% BSA (KC57 FITC; Beckman Coulter) (provided by Dr. G. Borgstahl, Eppley Institute, University of Nebraska for 45 min. A NIKON E-800 fluorescent microscope (Nikon) was used to Medical Center, Omaha, NE) and nondepleted culture fluids were used as evaluate HIV-1 p24-positive cells. positive controls. 6406 CYTOSKELETON AND MACROPHAGE GIANT CELL FORMATION

One-dimensional SDS-PAGE One-dimensional SDS-PAGE separation used a NOVEX (Invitrogen Life Technologies) system and 4–12% gradient gel. For protein identiﬁcation by liquid chromatography mass spectroscopy (LC/MS-MS), 20 ␮g of protein was loaded per lane, and protein bands were detected using a Typhoon 9410 PhosphorImager (Amersham Biosciences-GE Health Care). Gel pieces excised from one-dimensional were cut into 2- to 4-mm fragments ␮ and destained in 200 l of 50% acetonitrile and 50 mM NH4HCO3 at room temperature for 1 h, then dried, and 2 ␮l of 0.5% trypsin (Promega) and 50 ␮ lof10mMNH4HCO3 were added to each sample overnight at 37°C. Peptides were extracted by washing gel pieces twice with 0.1% triﬂuoro- acetic acid and 60% acetonitrile. Samples were transferred to vials, dried, and resuspended in 15 ␮l of water with 0.1% formic acid for automated injections.

Nanoelectrospray ionization-LC/MS-MS Sequencing was performed using ProteomeX LC-MS/MS system (Ther- moElectron) in nanospray conﬁguration from bands excised from one-di-

mensional SDS-PAGE and loaded onto a microcapillary C18 column. Pep- tides were eluted and sprayed directly into the mass spectrometer using a

gradient of water/acetonitrile and 0.1% formic acid. The spectra obtained Downloaded from were searched against the NCBI.fasta protein database using Bioworks 3.1 SR1 software provided by ThermoElectron.

Western blot assays For these assays, 10 ␮g of protein from the cell lysates and 20 ␮g from the culture ﬂuids were loaded per lane for one-dimensional gel electrophoresis.

Human platelet protein (Novus Biologicals) was used as a positive control. http://www.jimmunol.org/ Blots of samples from culture supernatants, vesicles, virus, and cell lysates were probed with rabbit anti-proﬁlin 1 and anti-actin Abs (Novus Biologi- cals) and HRP-conjugated anti-rabbit IgG (Novus Biologicals). HIV-1 p24 from blots of viral extracts was detected using mouse mAb anti-p24 and HRP-conjugated polyclonal (goat) anti-mouse IgG (Novus Biologicals). Vimentin was detected using mouse anti-vimentin Ab and HRP-conjugated polyclonal (goat) anti-mouse IgG (Novus Biologicals). Signal intensities were determined by densitometry analysis using Gel-Expert software (Nucleotech).

Cytoskeletal inhibitors by guest on September 25, 2021

MDM were treated with P-L-P to assess proﬁlin 1 function. Before infection, cells were treated for 1 h with 3 ␮M solution of P-L-P or left untreated as controls. The optimal concentration for P-L-P was determined from previous dose-response experiments and assessing cell viability and function in MDM cultures for toxic effects. Following incubation, cells were rinsed

with PBS and infected with HIV-1ADA at a MOI of 1. Supernatants and cells were collected for analysis by Western blotting, confocal, and cell FIGURE 1. HIV-1ADA replication in MDM. MDM were infected with viability assays. The Giant Cell Index (GCI; numbers of nuclei per cell) HIV-1ADA at a MOI of 1 after 7 days in suspension (A) or adherent (B) and the percentage of giant cells (ratio of cell number containing more than cultures. A, HIV-1 p24 and CD14 expression in control and infected human three nuclei to the overall cell population) were determined. MDM were MDM. Cells were collected days 3, 5, and 10 after infection and stained plated for 7 days in 4-well Lab-Tek chamber slides (Invitrogen Life Tech- with anti-CD14 allophycocyanin-conjugated (extracellular) and anti-HIV-1 ϫ 5 nologies) at a concentration of 5 10 cells/well with or without P-L-P and p24 FITC-conjugated Ab (intracellular). FACS analysis determined in per- virus infected at a MOI of 1. At days 3, 5, 7, and 10 after infection, slides centage of virus-infected MDM. B, HIV-1 p24 expression was assayed in were fixed and stained with Giemsa as described by the manufacturer (In- vitrogen Life Technologies). infected MDM grown in adherence. HIV-1-infected cells were stained against CD14 and HIV-1 p24 (as above) at days 3, 5, and 10 after infection Protein modifications and counted by fluorescent microscopy. Levels of infection are presented as the ratio of HIV-1 p24 to the overall CD14ϩ population. Results are Pooled protein (200 ␮g) from culture fluids of infected cells collected at representative of experiments with four separate donors (n ϭ 4; error ␮ ␮ ␮ days 3 and 5 after infection was incubated with 20 lof0.1 g/ l mouse bars ϭϮSEM). anti-phosphotyrosine mAb (Invitrogen Life Technologies), followed by a 2-h incubation at room temperature with 100 ␮l of protein A/G gel slurry (Pierce). Sample was supplemented with 500 ␮l of BupH TBS (Pierce) immunoprecipitation buffer centrifuged at 2500 ϫ g for 5 min, and the Results agarose beads were precipitated. Supernatant was recovered and retained for immunoblotting. The pellet was treated with 100 ␮l of IgG elution HIV-1ADA MDM infection buffer (Pierce) to elute the immune complex. Sample was centrifuged at To provide a biological framework for subsequent proteomic anal- 2500 ϫ g for 5 min, and the supernatant was retained. Sample fractions and nontreated culture fluids were used for immunoblotting and stained against yses, we first investigated both replication and cytopathic effects of profilin1 and actin using rabbit anti-profilin 1, anti-actin, and HRP-conju- HIV-1ADA infection in MDM of cells grown in suspension (Fig. gated anti-rabbit polyclonal Abs (Novus Biologicals). 1A) and as adherent cultures (Fig. 1B). Measurements included the percentage of cells positive for HIV-1 p24 at days 3, 5, and 10 after Statistical analyses viral exposure at an MOI of 1. Progeny virion production was All resulting data were analyzed for statistical significance by both Stu- measured by RT activity in culture fluids, and GCI and fusion dent’s t tests and a one-way ANOVA with Newman-Keuls posttest. activity were examined by morphology. These findings paralleled The Journal of Immunology 6407

FIGURE 2. SELDI-TOF analysis of HIV-1-infected MDM culture fluids shows profilin 1 secretion. MDM were infected with HIV-1ADA at a MOI of 1 after 7 days in culture. A, Representative spectra of control (top) and HIV-1-infected (bottom) culture fluids are shown within Downloaded from the 10- to 20-kDa range. Arrows indicate nine differentially expressed proteins. B, Representative spectra on culture fluids of control (dashed line) and HIV-1 infected (thick line) obtained by SELDI-TOF analysis on day 7 after infection using a WCX2 chip. Arrow indicates putative peak for profilin 1, which is present in http://www.jimmunol.org/ HIV-1-infected MDM culture fluids. C, Spectral peak is identified as profilin 1. Top panel, SELDI-TOF profile of affinity-purified profilin 1. Spectra of the crude cul- CF) collected from HIV-1-infected MDMء) ture fluids at day 5 after infection, and immunodepleted fluids for profilin 1 are shown in the middle and bottom panels, respectively. Spectra shown are representative of data obtained from four different donors. by guest on September 25, 2021

one another (see Fig. 7, A–C) and allowed correlations between 20-kDa region are shown in Fig. 2A. Arrows indicate 9 of 21 measures of HIV-1 replication and cytopathicity with levels of differentially expressed proteins in infected MDM vs the controls. secreted cytoskeletal proteins (see below). An enlarged version of the marked peak at the 15- to 16-kDa range in Fig. 2A is shown in Fig. 2B. Arrow indicates one differentially SELDI-TOF fingerprints expressed peak in the HIV-1-infected MDM culture fluids. Com- To determine optimal protein binding conditions and spectral ac- parison of the molecular mass against the Swiss-Prot protein da- curacy, the affinities of culture fluid proteins to chromatographic tabase matched profilin 1 as the “putative” protein to the up-reg- surfaces were tested. Based on these analyses, the WCX2 was ulated ϳ15-kDa peak. Spectral analysis of the 40- to 50-kDa shown to be most sensitive and specific and thus used for SELDI- region revealed a 43-kDa peak correlating with increased levels of TOF screening of MDM-secreted proteins. SELDI-TOF spectra of actin (data not shown). To determine whether the 15-kDa peak was culture fluids at days 7, 9, and 12 after infection were obtained indeed profilin 1, HIV-1-infected fluids were immunodepleted within a 5- to 150-kDa (m/z) range. This revealed 21 differentially with Abs to profilin 1 and spotted on a WCX2 chip. Moreover, expressed (up- or down-regulated or uniquely expressed) peaks parallel spectra were obtained with affinity-purified profilin 1 and among the infected and uninfected MDM culture fluids. Represen- crude culture fluids in which profilin 1 was not depleted (Fig. 2C). tative spectra of culture fluids at day 7 after infection in the 10- to The absence of the 15-kDa peak in the profilin 1-immunodepleted 6408 CYTOSKELETON AND MACROPHAGE GIANT CELL FORMATION

Table I. Cytoskeletal proteins identiﬁed in HIV-1-infected MDM culture ﬂuids

Molecular Mass No. of Accession Protein Name (Da)a Peptidesb XC Scorec No.d

Proﬁlin 1 15,054 3 19330 Q53Y44 Coﬁlin 1 18,371 2 6427 P23528 Ezrin 19,237 2 13306 Q9UJZ6 Tropomyosin 3 27,175 3 23425 Q5VU59 Tropomyosin 4 28,391 4 44046 P67936 Cap-G 38,518 3 19658 P40121 Macrophage-capping protein 38,524 2 4019 P40121 Actin ␥ 41,412 2 10507 Q6PJ43 Actin-related protein 2/3 complex 41,569 2 8325 Q92747 Actin ␤ 41,737 2 16565 P60709 Vimentin 53,520 4 9235 P08670 L-Plastin 70,158 6 66331 P13796 Gelsolin isoform b 85,698 3 16174 P06396 Actinin ␣ 2 103,058 2 23798 P12814 Filamin A 278,226 5 19059 Q60FE5

a Theoretical molecular mass for the primary translation product calculated from DNA sequences protein. b

Number of peptides identified for each protein selected based on the abovementioned criteria. Downloaded from c The CID spectra were compared against those of the EMBL nonredundant protein database by using SEQUEST (ThermoElectron). After filtering the results based on cross-correlation Xcorr (cutoffs of 2.0 for [M ϩ H]1ϩ, 2.5 for [M ϩ 2H]2ϩ, and 3.0 for [M ϩ 3H]3ϩ), peptides with scores Ͼ3000 and meeting ␦ cross-correlation scores (⌬Cn) Ͼ0.3, and fragment ion numbers Ͼ60% were deemed valid by these SEQUEST criteria thresholds, which have been determined to afford Ͼ95% confidence level in peptide identification (20). d Accession numbers for Swiss-Prot (accessible at http://ca.expasy.org/sprot/).

culture fluids provided the first confirmation for the identity of dimensional SDS-PAGE. The band fragments, including those corre- http://www.jimmunol.org/ profilin 1 and its up-regulation in HIV-1-infected MDM cultures. sponding to the molecular mass of 15 and 43 kDa, were excised, digested, and sequenced by LC-MS/MS. The tandem mass spectra Cytoskeletal proteins were compared against spectra of the European Molecular Biology To provide verification of our SELDI-TOF analyses, replicate HIV-1 Laboratory (EMBL) nonredundant human protein database by using and control MDM culture supernatants were fractionated by one- a SEQUEST search program. After filtering the results based on by guest on September 25, 2021

FIGURE 3. HIV-1-infected MDM secrete profilin 1 and actin. Cells and secreted fluids collected from control (Ϫ) and HIV-1-infected MDM (ϩ) were examined from days 5 to 12. A, Intracellular profilin 1 and actin expression throughout infection. Western blot analyses for both profilin 1 and actin in cell lysates revealed no changes between infected and control MDM in the course of infection. B, Profilin 1 and actin are secreted. Immunoblot assays for profilin 1, actin, and vimentin on culture fluids collected at parallel time points showed increased levels of both of these proteins from virus-infected MDM as compared with controls. C, Secreted profilin 1 and actin are phosphorylated. Immunoprecipitation using anti-phosphotyrosine mAb, followed by immunoblotting of unaltered (control) and phosphotyrosine-depleted culture fluids, as well as immunoprecipitate (IP) against profilin 1 and actin, shows these proteins are tyrosine phosphorylated. D, Profilin 1 and actin packed inside exosomes. Immunoblotting against profilin 1 and actin of CD45ϩ vesicles isolated from pooled culture fluids of infected MDM at day 3, 5, and 7 postinfection demonstrates presence of these proteins in the CD45ϩ vesicle fraction. E, Thin section transmission electron microscopy imaging of isolated progeny HIV-1 non-CD45 depleted (control; arrows indicate vesicles), recovered CD45ϩ vesicles, and HIV-1 depleted of CD45ϩ vesicles. Images show each fraction is organelle free and that CD45 depletion of vesicles from the culture fluids is efficient. Bars, 500 and 100 nm; images were obtained at ϫ31,000 and ϫ153,000, respectively. The Journal of Immunology 6409 cross-correlation Xcorr (cutoffs of 2.0 for [M ϩ H]1ϩ, 2.5 for [M ϩ 2H]2ϩ, and 3.0 for [M ϩ 3H]3ϩ), peptides with scores Ͼ3000 and meeting ␦ cross-correlation scores (⌬Cn) Ͼ0.3 and fragment ion numbers Ͼ60% were deemed valid by these SEQUEST criteria thresholds. These were determined to afford Ͼ95% confidence for peptide identification (21). The identities of sequenced proteins were confirmed in samples obtained from three independent experiments. The peptide identifications were segregated into cytoskeletal and other proteins. The identified cytoskeletal proteins, including actin and profilin 1, which met the selection criteria are shown in Table I. Other proteins identified were reported elsewhere (22). In agreement with the SELDI-TOF results, profilin 1 and actin were identified. Quanti- tative immunoblots were subsequently required for verification and substantiation of differential expression. Quantitation of actin and profilin 1 To quantify cytoskeletal proteins in culture fluids, Western blot analyses for profilin 1 and actin in HIV-1 infected and control

MDM cell lysates and culture fluids were performed (Fig. 3, A and Downloaded from B). Intracellular profilin 1 and actin levels were unchanged throughout the course of viral infection. In contrast, these proteins were found increased in culture fluids of HIV-1-infected MDM through day 7 after viral exposure. Interestingly, levels of profilin 1 and actin were decreased by days 9 and 12, corresponding to

maximum MGC activity and cytopathicity. The increases in pro- http://www.jimmunol.org/ filin 1 and actin secretion at days 5 and 7 suggest close associations between cytoskeletal secretion and the early stages of virus-induced MDM fusion events and were not related to cell death. To preclude presence of these cytoskeletal proteins in the culture fluids as a result of dead or dying cells, we monitored, in parallel, cell viability in infected and control MDM grown in adhesion by performing live/dead cell assays at these time points (see Fig. 7E). Cells were stained with calcein AM (live cells-stained green) and Eth-D1 (dead cells stained red) and read on a fluores- by guest on September 25, 2021 cent microplate reader. Readings from cells used as positive and FIGURE 4. Dynamics of actin and profilin 1 protein and progeny virion secretion. MDM were infected with HIV-1 at a MOI of 1 after 7 days negative controls for each dye were included in calculating the ADA in culture. Infected and control MDM were incubated in serum-free me- percentage of dead cells using the formula provided by the man- dium, and culture fluids were collected in three 8-h intervals (collection ufacturer. Live/dead cell assays revealed a low cell death activity points) starting at day 3 after infection. A and B, Dynamics of profilin 1 and compared with the uninfected cells at days 3 and 5, which mark actin secretion and progeny virion production. RT assays were performed maximal levels of extracellular profilin 1 and actin. These data, parallel to densitometry analysis (intensity of band/area) on Western blots taken together, provide clear evidence that HIV-1-induced actin of HIV-1-infected MDM culture fluids for profilin 1 (A, bottom panel) and skeleton transformation can affect the viral infection and cellular actin (B, bottom panel) and normalized to uninfected control of similar fusion and that the secretion of cytoskeletal proteins are not as a time points. consequence of cell death or injury. MTT assays supported these findings (see Fig. 7D). Next, we evaluated whether the secreted profilin 1 and actin ϩ were posttranslationally modified. MALDI-TOF and LC/MS-MS progeny virus. We isolated progeny virions and CD45 vesicles of trypsin digested actin and profilin revealed phosphorylation ac- from HIV-1-infected MDM culture fluids. Fluids pooled in the tivity on amino acid tyrosine (data not shown). Immunoprecipita- course of viral infection were cleared from cellular debris by cen- tion assays using Abs to phosphorylated tyrosine performed on trifugation followed by two filtrations through 400- and 200-nm culture fluids from infected MDM and followed by Western blot filters, immunodepleted of CD45-expressing vesicles and isolated assays for profilin 1 and actin showed that both proteins were in as pellets following ultracentrifugation on a 20% sucrose cushion. fact tyrosine phosphorylated (Fig. 3C). These findings, together Western blot tests performed on virion-enriched fractions and re- ϩ with previous reports regarding vimentin, actin, and profilin 1 se- covered CD45 vesicles demonstrated that actin and profilin 1 cretion by MDM (19, 23), support the presence of phosphorylated were not packed within progeny virions; however, they were ϩ actin and profilin 1 proteins in the culture fluids as a result of readily detected in CD45 vesicles (Fig. 3D). The purity of prog- ϩ infection and giant cell formation. eny virus and CD45 fractions were confirmed by RT activity, HIV-1 p24-specific immunoblots (data not shown), and thin sec- Profilin 1 and actin exosomal transport tioning of the pellet by transmission electron microscopy (Fig. 3E). We next investigated the mode of extracellular transport of profilin 1 and actin. Based on previous studies demonstrating secreted pro- Viral replication and actin and profilin 1 secretion teins may be packed within distinct vesicle subtypes expressing the To assess the dynamics of actin and profilin 1 secretions and its CD45 receptor (24–26), we hypothesized that the cytoskeletal pro- relationship to progeny virion production and MGC formation, teins could be either secreted within exosomes or packaged with serial collections of culture fluids were performed during the early 6410 CYTOSKELETON AND MACROPHAGE GIANT CELL FORMATION

FIGURE 5. Profilin 1 and actin distribution in HIV- 1-infected MDM. Human MDM were propagated as suspension (G–O) or adherent cultures (A–F) for 7 days then infected with HIV-1ADA at a MOI of 1. Uninfected (A–C and J–L) and infected (D–F, G–I, and M–O) MDM were collected on days 5 (A–I) and 10 (J–O) after infection. MDM grown in suspension were sedimented onto slides before analysis. Cells were stained against profilin 1 with mouse anti-profilin-1 (mAb) and FITC- Downloaded from conjugated anti-mouse IgG (green), F-actin by rhodamine-conjugated phalloidin (red), and nuclei by To-PRO (blue). Images in G–I indicate increased recruitment of profilin 1 and actin to the contact interface between infected MDM at the initiation of cellular fusion (indicated by arrows). Profilin 1 and actin colocalization in a http://www.jimmunol.org/ fully formed MGC at day 10 is shown in M–O. D–F, Profilin 1 and F-actin colocalization in the podosomes of infected MDM at day 5 postinfection in adherent cells. Arrows indicate profilin 1-mediated actin podosome formation and establishment of intercellular contacts between cells. Images reveal newly reorganized profilin 1 and actin from the cortical region of the cellular membrane to the perinuclear region of MGC (indicated by arrows). The merged images are presented in differential interference contrast (DIC). Images were ob- by guest on September 25, 2021 tained at ϫ100. Bars, 25 ␮m.

and later stages of viral infection. MDM were infected with HIV- trols. Fig. 4 compares dynamics of proﬁlin 1 and actin secretion

1ADA at a MOI of 1. Cells were washed with serum-free medium, and progeny virion production. Interestingly, virus production pre- and culture fluids were sampled every 8 h for 24 h on days 3, 5, 10, ceded profilin 1 release into the culture fluids (Fig. 4A). The dy- and 12 after viral exposure. After the last sampling at 24 h, cells namics of actin secretion were shifted, yet similar, to that observed were maintained in serum containing medium until the next col- for profilin 1 (Fig. 4B). Western blots analyzed by densitometry lection. Serum deprivation during sampling was performed to 1) are shown in the panels below A and B of Fig. 4. These results enhance the outcome of infection causing cell stress by serum support distinct patterns of profilin 1 and secretion in the course of starvation, 2) avoid spurious results because the gene that codes for infection, as well as the basis for protein-virus interactions in re- actin contains a serum response element and could be induced as gards to critical cellular response to HIV-1 infection. such through engagement of its serum response factor (27), and 3) to maximize proteomic analyses as high albumin content in the Cytoskeletal transformation and giant cell formation supernatants masks the presence of low abundance proteins and Based on the data obtained and the dynamics of protein secretion, limits their detection. Quantitative assessment for profilin 1 and we reasoned that profilin 1 and actin secretion may be due to actin actin by Western blot assays and RT were performed simulta- cytoskeleton reorganization by HIV-1. At days 5 and 10 after viral neously. RT and densitometry data shown were previously nor- infection, MDM were fixed and stained for profilin 1 (Fig. 5, A, D, malized to values obtained by similar analysis on uninfected con- G, J, and M) and F-actin (Fig. 5, B, E, H, K, and N), and images The Journal of Immunology 6411

FIGURE 6. Proﬁlin 1, actin, and HIV-1 distribution in infected MDM. Human MDM were propagated as adherent cultures, then infected with HIV-

1ADA at a MOI of 1, and cells were im- munostained at days 5 (A–C) and 10 (G–I) for proﬁlin 1 with goat-anti- proﬁlin mAb and anti-goat Alexa 533 (red). For HIV-1 detections, FITC-conjugated anti-HIV-1 p24 (green) were

used. Cells at day 5 (D–F) and 10 (J–L) Downloaded from were stained for F-actin and HIV-1p24 with rhodamine-phalloidine (red) and FITC-conjugated anti-HIV-1 p24 (green). To-PRO (blue) was used as a nuclear stain. Images demonstrate colocalization of HIV-1 with cytoskeletal

proteins during MDM fusion. Robust http://www.jimmunol.org/ HIV-1 staining is noted along the actin stress ﬁlaments (arrows; A–F) and at the point of contact between the podosome from one cell and the cell membrane of the next (shown by top arrow A–C). At the completion of fusion (day 10; G–L), images reveal a distinct colocalization of HIV, proﬁlin 1, and F-actin in the perinuclear region. Images were obtained at ϫ60. Bars, 50 ␮m. by guest on September 25, 2021

were merged (Fig. 5, C, F, I, L, and O). Confocal images of MDM port the notion that profilin 1 and actin translocation to the plasma grown in suspension revealed massive profilin 1 and F-actin reor- membrane, as well as podosome formation, are necessary events ganization and recruitment to the cell membrane in association preceding formation of cell-cell contacts and intercellular mem- with fusion events in HIV-1-infected MDM (Fig. 5, G–I; arrows brane fusion. indicate profilin 1 and actin localization at the fusion interface). Unlike the infected MDM, images of the uninfected cells at a similar time point (day 5) revealed profilin 1 and actin were dis- Actin and profilin 1 transformation, viral replication, cell fusion, tributed homogeneously throughout the cytoplasm (Fig. 5, A–C). and cytopathicity At the latter stages of infection (day 10) and completion of cel- Confocal analysis performed on days 5 (Fig. 6, A–F) and 10 (Fig. lular fusion (ϳ100 nuclei/cell), profilin 1 and actin showed reor- 6, G–L) after viral infection revealed significant colocalization of ganization from the membrane back to the cytoplasm and more HIV-1 p24 with profilin 1 (Fig. 6, A–C and G–I) and actin (Fig. 6, distinctly localized in the perinuclear region as indicated by the D–F and J–L). This was seen at day 5 in MDM throughout the arrows (Fig. 5, M–O), whereas the uninfected cells maintained a length of cell processes with intense staining at the area of focal homogeneous distribution of actin and profilin 1 (Fig. 5, J–L). In contact with the next cell (as indicated by arrows in Fig. 6, C and replicate experiments performed on adherent MDM, images re- D). At the completion of cellular fusion at day 10, HIV-1 was vealed profilin 1-mediated actin stress filament formation at the colocalized with profilin 1 and actin in the perinuclear region of active edges of the membrane in the form of podosome-like ex- the MGC (Fig. 6, G–I and J–L). Fig. 5, M–O, showed a similar tensions and establishment of focal contacts with the adjacent cells pattern of profilin 1 and actin colocalization in the perinuclear as early as day 5 after infection (Fig. 5, D–F). These results sup- region of the giant cells. These data suggest that HIV-1 may use 6412 CYTOSKELETON AND MACROPHAGE GIANT CELL FORMATION Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 7. Cytokeletal inhibitors affect HIV-1 replication and MDM fusion. Monocytes were propagated as adherent cells for 7 days, then treated for ␮ 1 h with 3 MP-L-P before infection with HIV-1ADA at a MOI of 1. HIV-1-infected and -uninfected MDM without or with P-L-P served as controls. A, Profilin 1 inhibitor P-L-P suppresses productive infection. P-L-P significantly reduces progeny virus formation as shown by levels of RT activity. B and C, P-L-P suppresses cellular fusion. To generate indices of cell fusion, control and treated cells were stained with Giemsa, and numbers of nuclei per cell were counted for GCI measurements (B). Fusion activity was presented as a ratio (in %) of number of cells containing more than three nuclei to the total cell counts (C). D and E, Cell viability and P-L-P toxicity. To determine whether secretion of cytoskeletal proteins in the culture fluids could be due to cell death, both MTT (D) and live dead assays (E) were performed. On days 3 and 5 when maximal secretion of actin and profilin 1 were observed, cell death was limited. P-L-P showed minimal cytotoxicity. F, Disruption of actin filament formation and fusion. P-L-P-treated infected MDM were harvested on day 5. Cells were stained for profilin 1 (green), F-actin (red), and nuclei (blue) as described in Fig. 5 and analyzed by confocal microscopy. Panels show lack of actin filaments, disruption of profilin 1 recruitment to the plasma membrane, and no fusion activity. Arrows indicate G-actin aggregates randomly distributed throughout the cytoplasm. G,P-L-P reduces profilin 1 and actin secretion. Western blot analysis of culture fluids from untreated and P-L-P-treated, HIV-1-infected MDM against profilin 1 and actin revealed a reduction of profilin 1 and actin in the P-L-P-treated cells. Images were obtained at ϫ100. Bars, .(p Ͻ 0.001; error bars, Ϯ SEM, n ϭ 4 ,ءء p Ͻ 0.05 and ,ء) .␮m 25

the actin cytoskeleton intercellular network to increase the effi- HIV-1ADA at a MOI of 1. The inhibitor for profilin 1, P-L-P, ciency of infection and cellular fusion. binds with high affinity to the phosphorylated form of profilin 1 After day 7 in culture, uninfected MDM were treated with 3 and suppresses its interaction with G-actin (monomeric form) ␮MP-L-P. Following 1-h incubation, cells were infected with and consequently the process of actin filament formation The Journal of Immunology 6413

(28–30). Escalating concentrations of these drugs were tested on Profilin 1 and actin translocation at the active membrane, along both HIV-1-infected and control MDM to determine an optimal with the formation of podosome-mediated intercellular contacts therapeutic index (drug efficacy with limited toxicity; Fig. 7E). and exosomal secretion, likely affect the high efficiency of HIV-1 Cell viability assays showed treatment with 3 ␮M solution of infection that occurs with cell-cell fusion. In addition, increased P-L-P exerted maximal suppression of viral progeny production secretion of profilin 1 in exosomes preceding syncytium formation (Fig. 7A) and cell fusion (Fig. 7, B and C) with minimal associated indicates a possible role of the protein as a regulator of focal ad- cytotoxicity (Fig. 7, D–F). Suppression of productive infection hesions, actin stress filament orientation, and facilitation of giant along with cellular fusion appears to be a direct reaction to the cell formation. Similar profilin 1 secretion dynamics and RT ac- disruption of actin filament formation by P-L-P because phagocytic tivity in the HIV-1-infected culture fluids further support this hy- activity and reactive oxygen species production were not affected pothesis. During infection of HEp-2 cells by respiratory syncytial in inhibitor-treated MDM (data not shown). Along with signifi- virus, profilin 1 plays a central role in virus-mediated stress fiber .(p Ͻ 0.01) reduced RT activities at days formation, as well as virion maturation and cellular fusion (49 ,ءء p Ͻ 0.05 and ,ء) cantly 3–10 postviral exposure, MDM treated with P-L-P showed com- Although occurrence of giant cells has been described in gran- plete disruption of the F-actin intercellular network and reduced ulomas and other inflammatory reactions since the middle of last profilin 1 and actin secretion (Fig. 7, F and G). These data suggest century (50), their role in disease remains undefined. Cellular fu- HIV-1-induced actin and profilin 1 alterations and reorganization sion has been shown to occur in the course of chronic inflamma- play important roles in productive infection and cellular fusion. tion mediated by IL-4 and IL-13 through a macrophage mannose receptor pathway. This model proposes cellular fusion and MGC

Discussion formation as a necessity for increased phagocytosis of glycopro- Downloaded from Links between HIV-1-induced cytoskeletal alterations, productive teins and microorganisms bearing terminal mannose, fucose, or viral replication, and membrane fusion were shown using combi- glucose residues (51). This idea is complementary to other pro- nations of proteomics and immunologic approaches. HIV-1 infec- posed models that describe cellular fusion as a prerequisite for tion of MDM induced polarized recruitment of actin and proﬁlin 1 increased contact area with the pathogen at the phagosomal site to the plasma membrane, secretion through exosomes, induction of (52). Indeed, giant cells have been recognized previously as an end

podosome-mediated intercellular contacts, and giant cell forma- product of overwhelmed phagocytosis favored by high tempera- http://www.jimmunol.org/ tion. These underlie cellular events necessary for productive tures and acidic pH (53) and vessels for physical containment of HIV-1 replication as inhibition of profilin 1 with P-L-P disrupts the pathogen (54). We propose that HIV-1 accelerates macrophage profilin 1 transformation suppresses progeny virion production and differentiation and giant cell formation at the peak of viral repli- cell fusion. cation and is reflective of a vain attempt of the cell to affect max- Studies by others investigating the interactions between cy- imal pathogen clearance. We demonstrated that MGC possess toskeletal proteins and HIV-1 have shown virus-induced, actin- newly reorganized actin networks and reduced membrane recy- dependent receptor colocalization regulates viral entry, podosome cling. Images reveal elevated recruitment of profilin 1 and actin formation (9), and intercellular virion transfer across a “virological from the plasma membrane to the perinuclear region and profilin 1 synapse” (15). In addition, actin reorganization induced by HIV- and F-actin heavy staining of the nuclei. Based on these data, it is by guest on September 25, 2021 1/coreceptor interactions and Rac-1 GTPase are events demon- likely that profilin 1 along with actin, recently shown to participate strated to precede cellular fusion and MGC formation (31). Inves- in gene transcription and mRNA splicing (55, 56), may represent tigations in various model systems have shown profilin 1 to be a another check point in expression of viral proteins. Actin and pro- common denominator in the intimate cross-talk of small GTPase filin 1 structural modifications leading to their nuclear localization (Rac, ␳, and Cdc42)-mediated polymerization of actin (32, 33), will be thoroughly investigated in future studies. receptor activities, guidance of focal adhesions (34, 35), and mem- Culture fluids of MDM treated with latrunculin A (G-actin se- brane trafficking (36–39). Because focal adhesions and stress fil- questering drug) revealed similar viral load compared with the ament formation is often an artifact of cell growth on adhesive infected controls (data not shown). The efficacy of its action, as substrate, we performed replicate experiments of MDM infection well as the simplicity and specificity of latrunculin A interaction in suspension and observed a consistent and similar pattern of actin with G-actin, has made it a compound of choice, supplanting the and profilin 1-polarized recruitment at the fusion interface of ad- classic actin-depolymerizing drug cytochalasin-D. A subpool of jacent cells. cytoplasmic G-actin may also be sequestered by latrunculin A, Other intercellular pathogens such as Listeria monocytogenes which explains the disruption of actin recruitment to the cellular (40, 41) and Shigella flexneri (42) use profilin 1 and actin to affect interface and delayed MGC formation. Others have reported sim- its propulsion across the cytoplasm and spread into neighboring ilar effects of actin depolymerizing drugs in cells infected with cells without entering the extracellular space. Moreover, envel- equine infectious anemia virus (57). Sequestration of actin by la- oped viruses use the cortical actin network as a scaffold to orches- trunculin A may exert its effects at multiple stages of the HIV-1 trate the lateral mobility of the virion-receptor complex in the life cycle as the actin network is closely involved in viral entry and plane of the plasma membrane, as well as their dissemination transport of the reverse transcription complexes to the nucleus, as across cells (43). Viruses use the actin stress filaments to engage well as progeny virion assembly and release (58). the surface of the cell on lipid rafts in search of internalization or Profilin plays important roles in cell growth and function (59– endocytic sites following receptor binding (44, 45). This is likely 61). Its ability to bind actin (62), P-L-P (28, 29), a plethora of other facilitated by coupling of the virus through the cytosolic domain of proteins related to the microfilament system (63, 64), and to its receptor to an actin filament at the base of the filopodium (46, polyphosphoinositides (65) suggests that profilin 1 is involved in 47). In the final stages of the viral life cycle, progeny virions bud- signal transduction and may link transmembrane signaling to the ding from the host’s cytoplasm use pseudopodium-mediated inter- control of the microfilament system. The P-L-P binding site creates cellular contacts as fast tracks for viral dissemination. This process a stearic hindrance for some of profilin 1-binding partners that are is often associated with up-regulation of vesicle recycling to aug- involved in signaling events leading to the translocation of profilin ment the plasma membrane surface area and facilitate focal con- 1 to distinct cellular regions, including the plasma membrane (28– tacts with adjacent cells (48). 30). P-L-P became our inhibitor of choice due to its high binding 6414 CYTOSKELETON AND MACROPHAGE GIANT CELL FORMATION specificity to profilin 1 and minimal toxicity to the MDM. Com- 10. Collman, R. G. 2003. HIV-1 Env-chemokine receptor interactions in primary plete inhibition of profilin 1 expression by siRNA was not pre- human macrophages: entry and beyond. Res. Initiat. Treat. Action 8: 6–9. 11. Kedzierska, K., S. M. Crowe, S. Turville, and A. L. Cunningham. 2003. The ferred due to the suppressive effects of profilin 1 interaction with influence of cytokines, chemokines and their receptors on HIV-1 replication in Ͼ50 profilin 1-binding partners and signal transduction interme- monocytes and macrophages. Rev. Med. Virol. 13: 39–56. 12. Goldberg, M. B. 2001. Actin-based motility of intracellular microbial pathogens. diates (30). Microbiol. Mol. Biol. Rev. 65: 595–626. HIV-1 may control temporal actin and profilin interactions, as 13. Rogan, M. P., P. Geraghty, C. M. Greene, S. J. O’Neill, C. C. Taggart, and well as shuttling to distinct cellular compartments, through the N. G. McElvaney. 2006. Antimicrobial proteins and polypeptides in pulmonary innate defence. Respir. Res. 7: 29. modulation of multiple posttranslational modification tags on these 14. Fackler, O. T., and H. G. Krausslich. 2006. Interactions of human retroviruses proteins. Both profilin 1 and actin posses the structural character- with the host cell cytoskeleton. Curr. Opin. 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