NHERF1 regulates gp120induced ... - Wiley Online Library

Eur. J. Immunol. 2012. 42: 299–310

DOI 10.1002/eji.201141801

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Yi-Qun Kuang1, Wei Pang2, Yong-Tang Zheng 2 and Denis J. Dupre´1 1 2

Department of Pharmacology, Faculty of Medicine, Dalhousie University, Halifax, NS, Canada Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences and Yunnan Province, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan, P. R. China

The scaffolding protein Na1/H1 exchanger regulator factor 1 (NHERF1) plays an important role in the trafficking of G protein-coupled receptors. We previously demonstrated that NHERF1 is involved in chemokine receptor CCR5 homodimer internalization and signal transduction. Given the importance of CCR5 internalization during HIV-1 infection, we evaluated NHERF1’s contribution in HIV-1 infection. We challenged human osteosarcoma cells coexpressing CD4 and CCR5 cells expressing either NHERF1 fragment domains or WT NHERF1 with an HIV-1 strain to examine the effects of NHERF1 on HIV-1 entry and replication. WT NHERF1 potentiates HIV-1 envelope gp120-induced CCR5 internalization, and promotes the replication of HIV-1. In order to better understand how NHERF1 affects signal transduction, different domains of NHERF1 were overexpressed in cells to analyze their effect on the different signaling pathways. Here, we show that NHERF1 can associate with CCR5, and promote activation of the gp120-induced MAPK/ERK, focal adhesion kinase and RhoA (Ras homolog gene family member A) signaling pathways. NHERF1 overexpression also increases HIV-1 host cell migration triggered by gp120 via focal adhesion kinase (FAK) signaling. Finally, NHERF1 enhanced actin filament rearrangement in host cells, an important step in post-entry HIV-1 replication events. While postsynaptic density 95/disk-large/zonula occludens 2 (PDZ2) appears to be the major contributor in those events, other domains also participate in the regulation of gp120-induced signaling pathways. Altogether, our results suggest a very important role of the scaffold NHERF1 in the regulation of HIV-1 entry and replication.

Key words: CCR5 . gp120 . G protein-coupled receptor . Na1/H1 exchanger regulator factor 1 (NHERF1) . Signaling

Supporting Information available online

Introduction Chemokines are a family of chemotactic cytokines that bind G protein-coupled receptors (GPCRs) on the surface of target cells. Chemokines are involved in the recruitment and activation

Correspondence: Dr. Denis J. Dupre´ e-mail: [email protected]

& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

of a subset of leukocytes during immune and inflammatory responses [1], and play a role in the pathogenesis of many viralbased human diseases [2]. CXCR4 and CCR5 are used by human immunodeficiency virus type 1 (HIV-1) as co-receptors for viral entry, and their chemokine ligands are potent inhibitors of HIV-1 infection [3, 4]. Host cell entry of HIV-1 is mediated by sequential binding of the trimeric HIV envelope glycoprotein gp120 to CD4 and one of two primary chemokine co-receptors, CXCR4 or CCR5. This

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NHERF1 regulates gp120-induced internalization and signaling by CCR5, and HIV-1 production

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association triggers a series of conformational changes in the envelope that exposes the fusion peptide and induces the merger of the virus with the host cell. T lymphocyte-tropic, macrophagetropic (M-tropic) HIV-1 strains or the viral envelope gp120 interaction with CD4 and a co-receptor also stimulate various intracellular signaling events through heterotrimeric G proteins, such as phosphorylation of MAPK/ERK and focal adhesion kinase (FAK)/Pyk2, calcium mobilization, chemotaxis, Ras homolog gene family member A (RhoA)/Rho-associated protein kinase (ROCK) activation and filamentous actin cytoskeleton rearrangements [5–11]. These events are suggested to participate in efficient HIV-1 infection and replication. However, the CXCR4and CCR5-using HIV-1 envelope shows different signaling activation or gene expression profiles in HIV-1 host cells [12], implying that different internalization and intracellular trafficking pathways are used by CXCR4 and CCR5. It has been suggested that GPCR heteromerization could alter the properties of the receptors involved such that they respond differently to pharmacological agents, revealing a possible source of unexpected pharmacological diversity. Interestingly, Na1/H1 exchanger regulator factor 1 (NHERF1) was shown to interact only with CCR5 homodimers, and not with CXCR4 homodimers or CXCR4/CCR5 heterodimers [13]. It is thought that unique signaling partners within a particular pathway could account for specificity and diversity of signal transduction for the different receptor complexes expressed in one cell. The protein scaffold NHERF1, also known as ezrin-radixinmoesin (ERM) binding phosphoprotein 50 (EBP50), is a ubiquitous protein that contains three functional domains: two N-terminal tandem postsynaptic density 95/disk-large/zonula occludens (PDZ) domains and a C-terminal ERM domain [14]. NHERF1 was first identified as a cofactor essential for protein kinase A-mediated inhibition of Na1/H1 exchanger isoform 3 (NHERF3) [15]. Since then, it has been shown to be a crucial component for recycling and sorting of several receptors, ion channels and transporters [16–19]. Long-term epidemic studies have shown that most HIV-1 infections of the global pandemic use CCR5 as the HIV co-receptor to enter the host cells and CCR5 has been validated as a promising target for HIV prevention strategies, especially since a CCR5 antagonist, Maraviroc, was approved as the first in a new class of anti-HIV therapeutic drugs [20]. HIV-1 envelope gp120 binding to CCR5 activates cellular signaling and trafficking (internalization) pathways shown to be pivotal in the establishment of a successful infection and replication by HIV-1. Similarly, the cytoskeletal rearrangements following HIV-1 entry are required for viral uncoating, reverse transcription and nuclear importation [21]. We previously showed that NHERF1 can modulate both CCR5 internalization and cytoskeletal rearrangements upon receptor stimulation with RANTES [13]. Understanding how NHERF1 regulates CCR5 internalization and signaling following HIV-1 or gp120 binding to the receptor will reveal new aspects about HIV-1 entry into host cells. Our study shows that NHERF1 enhances M-tropic HIV-1 infection by upregulating the activation of M-tropic gp120-induced signaling cascades in host cells.


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Results NHERF1 effects on gp120-induced CCR5 internalization In a recent publication from our laboratory, we demonstrated that CCR5’s internalization could be modulated by NHERF1 following RANTES stimulation [13]. Here, we wanted to examine the effects of NHERF1 on gp120-induced CCR5 internalization. First, we studied the levels of CCR5 cell surface expression following CCR5/M-tropic HIV-1 (HIV-1CM strain) envelope gp120 stimulation. Our results show that gp120 stimulation of CCR5 promoted internalization, in comparison with unstimulated cells (Fig. 1A). When NHERF1 was overexpressed, CCR5 internalization was further promoted, with an approximately 40% decrease in CCR5 surface expression, in comparison with a 20% decrease in cells that were transfected with pcDNA3. In contrast, when fragments of NHERF1 corresponding to different domains were expressed, levels of CCR5 expression at the cell surface were comparable with those in the unstimulated cell. We wanted to determine the effects of the knockdown of endogenous NHERF1 and Fig. 1B shows the levels of inhibition by NHERF1 siRNAs. A complete knockdown was obtained with the combination of all three siRNAs, which were used in subsequent experiments. We evaluated the effect of NHERF1 knockdown on CCR5 internalization (Fig. 1C). NHERF1 knockdown resulted in a basal increase of cell surface expression, as measured by ELISA, while NHERF1 overexpression does not seem to alter the general internalization pattern, as both pcDNA3 and NHERF1 cells followed a similar internalization curve except at 60 min, where a slight trend towards an increase is observed.

NHERF1 effects on the M-tropic HIV-1 infection Since CCR5 cell surface expression levels affect HIV-1 entry, we investigated the effects of NHERF1 overexpression on HIV-1 production in infected host cells. We challenged human osteosarcoma cells coexpressing CD4 and CCR5 (HOS-CD4-CCR5) cells with the HIV-1Ada-M M-tropic strain at a multiplicity of infection (MOI) of 0.2. It is anticipated that HIV-1 infection will elevate p24 core antigen levels, which can be measured by ELISA. Our results show that when WT NHERF1 is overexpressed, HIV-1 p24 core antigen levels are increased, which suggests that NHERF1 increases HIV-1 infection and replication levels over control baseline (Fig. 2). Interestingly, overexpression of fragments of NHERF1 (ERM, PDZ2 or PDZ1-PDZ2) showed inhibition (about 25, 80 and 45%, respectively) of p24 core antigen level production. While NHERF1 ERM, PDZ2, PDZ1-PDZ2 fragments could effectively inhibit HIV-1Ada-M replication at low MOI titer (MOI 5 0.1) (data not shown), only PDZ2 could firmly inhibit HIV-1Ada-M replication by 80% at higher MOI titer (MOI 5 0.2) (Fig. 2). Inhibition of p24 core antigen level production by NHERF1 fragments was also observed with the HIV-1SF162 strain, although not to the same extent as with HIV-1Ada-M. www.eji-journal.eu

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Figure 2. NHERF1 domains affect HIV-1 production. HOS-CD4-CCR5 cells expressing WT NHERF1 or the indicated NHERF1 domains were challenged with M-tropic HIV-1Ada-M or HIV-1SF162 and, after 96 h of infection, HIV-1 core antigen p24 levels were measured in culture supernatant by an HIV Ag/Ab Elisa. Inhibition activity was determined by comparison with the HIV-1 challenging viral stock positive control (MOI 5 0.2). Results are shown using mean7SEM of p24 core antigen level of HIV-1-infected cell culture supernatant of three independent experiments. po0.05, po0.005, One-way analysis of variance (ANOVA) with Bonferroni’s post test.

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Figure 1. WT NHERF1 enhances CCR5 internalization. (A) HOS-CD4CCR5 cells expressing WT NHERF1 or the indicated NHERF1 domains were stimulated with 50 nM gp120 for 60 min at 371C. An anti-CCR5 antibody targeting an extracellular portion of the receptor was used to measure cell surface expression by ELISA. Results are shown as the relative intensity of receptor expression at the plasma membrane, where unstimulated pcDNA3 transfected cells were adjusted empirically to 100%. (B) 48 h post-transfection, the effect of NHERF1 siRNAs on NHERF1 expression were determined by Western blot using an NHERF1 Ab; a-tubulin serves as a loading control. (C) NHERF1 siRNA expression inhibits CCR5 internalization, as measured by ELISA with an anti- CCR5 Ab. Results show the mean7SEM of at least three independent experiments, and po0.05; po0.01 compared with control using Student’s t test. Error bars indicate standard error mean (SEM).

NHERF1 association with CCR5 Next we sought to confirm that CCR5 can interact with NHERF1, in HOS-CD4-CCR5 cells, as previously reported in HEK293 cells [13]. First, HA-tagged WT NHERF1, individual domain mutants PDZ1, PDZ2 and ERM, as well as PDZ1-PDZ2 and PDZ2-ERM constructs were expressed into HOS-CD4-CCR5 cells to ensure


their proper expression (Fig. 3A). To determine whether CCR5 interacts with NHERF1, HOS-CD4-CCR5 cell lysates expressing HA-tagged NHERF1 WT were immunoprecipitated with an antiCCR5 antibody, and then blotted with anti-HA or anti-NHERF1 antibodies. The immunoblot in Fig. 3B shows that NHERF1 has weak basal level interactions with the receptor, but is robustly recruited to CCR5 following gp120 stimulation. Both HEK293 and HOS cells express NHERF1 endogenously (Supporting Information Fig. 1). We wanted to determine whether this interaction was due only to overexpression of our proteins of interest or if the interaction occurs with endogenous levels of expression. Figure 3C shows the interaction of endogenous CCR5 with NHERF1, following stimulation with gp120.

NHERF1 effects on MAPK/ERK activation Chemokine receptor signaling pathways were previously reported to be involved in HIV-1 infection and replication through the CD4-CCR5/CXCR4 axis in host cells. For example, activation of the MAPK/ERK signaling pathway was reported during HIV-1 replication events like reverse transcription [11]. NHERF1 was previously shown to promote MAPK/ERK phosphorylation following CCR5 stimulation with RANTES [13]. MAPK/ERK phosphorylation levels were probed in HOS-CD4-CCR5 cells expressing NHERF1 or domain fragments in presence or absence of gp120. Here, we show that WT NHERF1 can increase ERK-1/2 phosphorylation levels following gp120 stimulation (Fig. 4A, quantified in Fig. 4B). In order to confirm that the effects we observed were due to the overexpression of NHERF1, and not by a general effect on the signaling pathway induced by overexpression of a protein, NHERF1 levels were measured basally and in

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Figure 3. Association of NHERF1 with CCR5. (A) Left panel: schematic representation of the different constructs used in the study. Right panel: HAtagged WT NHERF1, individual NHERF1 domain mutants PDZ1, PDZ2 and ERM, and combined NHERF1 domain mutants PDZ1-PDZ2 and PDZ2-ERM were transfected into HOS-CD4-CCR5 cells and expression determined by Western blot of cell lysates with an anti-HA Ab. (B) Binding of NHERF1 to CCR5. Cell lysates from NHERF1-HA-expressing HOS-CD4-CCR5 cells incubated in the presence or absence of gp120 (50 nM, 15 min) were immunoprecipitated with an anti-CCR5 antibody. The immunoprecipitate or lysate prior to immunoprecipitation were analyzed by Western blot with anti-HA or anti-NHERF1 followed by a horseradish peroxidase-conjugated secondary antibody to show co-immunoprecipitation and expression of each protein respectively. (C) Co-immunoprecipitation results show the interaction between endogenous CCR5 and NHERF1, following a 15 min stimulation with 50 nM gp120. Results shown are representative of three independent experiments.

presence of NHERF1 siRNA, showing our capacity to inhibit NHERF1 expression in HOS cells (Fig. 4C). Figure 4D shows the effects of NHERF1 siRNA expression on ERK-1/2 phosphorylation. Our results clearly demonstrate that the ERK-1/2 phosphorylation is completely abolished in absence of NHERF1. Several NHERF1 domains (PDZ1, PDZ2 and ERM) can also contribute to the increase of phosphorylation as seen in Fig. 4A, but only ERM’s effect was significant (Fig. 4B), compared to unstimulated cells. The relative intensity analysis of several immunoblots showed upregulated phosphorylation of ERK-1/2 in presence of NHERF1 WT and ERM, indicating a potentially greater role for ERM in the activation of this signaling pathway than for the other domains.

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Figure 4. Regulation of HIV-1 gp120-induced MAPK/ERK activation by NHERF1. (A) HOS-CD4-CCR5 expressing the indicated NHERF1 constructs and stimulated with 50 nM gp120 for 15 min at 371C where indicated and were analyzed by Western blot to detect ERK-1/2 and pERK-1/2. (B) Results from three independent experiments were quantified for pERK levels, with results using pcDNA3/vehicle being set empirically at 100%. (C) 48 h post-transfection, the effect of NHERF1 siRNAs on NHERF1 expression were determined by Western blot using an NHERF1 Ab; a-tubulin serves as a loading control. (D) 48 h posttransfection, the effect of NHERF1 siRNA on ERK-1/2 phosphorylation was determined by western blot using an p-ERK1/2 Ab; ERK1/2 serves as a loading control. Western blots are representative of at least three experiments. po0.05, using Two-way ANOVA with Bonferroni’s post test. Error bars indicate SEM.


FAK activation coordinates various cellular responses including cell adhesion, polarization, migration, survival and death [22]. Both FAK and NHERF1 are involved in several of these events, so the role of NHERF1 in FAK signal transduction events was evaluated. To determine the duration of stimulation required for gp120-induced FAK phosphorylation, cells were stimulated with 50 nM of gp120 for a time course between 0 and 30 min. When stimulated for 15 min at 371C, FAK phosphorylation reached its maximal level (Fig. 5A). We then co-expressed NHERF1 constructs in HOS-CD4-CCR5 cells and stimulated them with 50 nM gp120 for 15 min at 371C to analyze the effect of NHERF1 on FAK phosphorylation. Our results show that gp120-stimulated cells overexpressing NHERF1 WT could upregulate FAK phosphorylation (Fig. 5B), by approximately fourfold (Fig. 5C). PDZ2ERM and ERM domain also displayed an increase of phospho-FAK while all other constructs could not significantly alter FAK phosphorylation levels (Fig. 5B and C). Figure 5D shows that the effects on FAK phosphorylation can be observed at the endogenous levels of expression, and Fig. 5E shows that NHERF1 siRNA can inhibit those events. We next determined whether FAK or phosphorylated FAK were recruited to the activated CCR5 receptor, and the effect of

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Figure 5. Regulation of FAK signaling pathway activation by NHERF1. (A) Cells expressing CD4 and CCR5 were stimulated with 50 nM gp120 for 0, 5, 10, 15 and 30 min at 371C and immunoblots of cell lysates were performed using an anti-phospho-FAK (Y397) or an anti-FAK monoclonal antibody. (B) Phosphorylated (Y397) or total FAK levels were measured in HOS-CD4-CCR5 cells transfected with WT NHERF1 or the indicated NHERF1 domain fragments by Western blotting. (C) Histogram representing the relative intensity of phospho-FAK of three independent experiments, compared to pcDNA3, empirically set to 100%. (D) PcDNA3 or NHERF1 transfected HOS-CD4-CCR5 cell lysates stimulated or not with gp120 were immunoprecipitated with an anti-CCR5 antibody. The immunoprecipitate or lysate prior to immunoprecipitation were analyzed by western blot for detection of phospho-FAK or total FAK respectively. (E) 48 h post-transfection, the effect of NHERF1 siRNA on FAK phosphorylation were determined by Western blot using an p-FAK Ab; FAK serves as a loading control. Western blots are representative of four experiments. po0.05, po0.005, using Two-way ANOVA with Bonferroni’s post test. Error bars indicate SEM of four experiments.

NHERF1 overexpression on the interaction of FAK/phospho-FAK with CCR5. NHERF1- or pcDNA3-transfected HOS-CD4-CCR5 cells were lysed, and lysates were immunoprecipitated with an anti-CCR5 antibody. The precipitated proteins were blotted with anti-FAK or phospho-FAK antibody. The blots indicate that FAK but not phosphorylated FAK interacts with CCR5 following a 15 min stimulation with 50 nM gp120 (Fig. 5D). Our results suggest a modest increase in FAK recruitment in presence of NHERF1, in comparison with pcDNA3 transfected cells. Moreover, we show that NHERF1 also gets recruited to CCR5 following gp120 stimulation, suggesting that CCR5, FAK and NHERF1 could be part of a signaling complex together.

tested via a GST-Rhotekin-RhoA binding domain pulldown assay. Activated RhoA from a cell lysate is incubated with the GSTRhotekin RhoA binding domain. Only activated RhoA will bind to the domain, which can then be immunoblotted for RhoA presence. Our data show that upon CCR5 stimulation with gp120, NHERF1 potentiated RhoA activation (about 7.5-fold), when compared to pcDNA3-transfected HOS-CD4-CCR5 cells (Fig. 6A). NHERF1 siRNA expression resulted in a significant decrease in RhoA activation (Fig. 6B). Analysis of the effect of the different NHERF1 domains suggests a contribution of various domains to RhoA activation (Fig. 6C). Our data also indicate that the PDZ2-ERM domain increases RhoA activation, although not as strongly as WT NHERF1.

Effects of NHERF1 on RhoA activation

Effects of NHERF1 on filamentous actin rearrangement

The RhoA GTPase is involved in regulating actin cytoskeletal organization, gene expression, cell proliferation and survival. The RhoA signal transduction pathway also regulates HIV-1 postentry viral replication. Both NHERF1 and RhoA are involved in the cytoskeleton remodeling [23]. Since both NHERF1 and RhoA participate in cytoskeleton remodeling, we wanted to determine whether NHERF1 was upstream in the RhoA signaling cascade (e.g. can NHERF1 activate RhoA) or if they worked independently. Activation of RhoA level (RhoA-guanosine triphosphate (GTP)) was

CCR5 internalization was previously shown to be dependent on actin rearrangements and activation of small G proteins in a Rho-dependent manner [24]. We wanted to determine whether gp120-induced CCR5 stimulation would be able to trigger HOS cells cytoskeletal actin remodeling. HOS-CD4-CCR5 cells expressing NHERF1 WT or NHERF1 domain fragments were stimulated with gp120 to detect phalloidin-labeled actin via immunofluorescence microscopy. In NHERF1 WT expressing cells (Fig. 7B), we observed stronger formation of filamentous actin (indicated


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migration. Our results demonstrate that HIV-1CM gp120 can trigger HOS cells migration (Fig. 8A); and the FAK-specific inhibitor (FAK inhibitor 14, FI14) was able to inhibit cell migration of gp120 stimulated cells. Interestingly, NHERF1 coexpression potentiated FAK-mediated cell migration (about tenfold increase, Fig. 8A), compared to pcDNA3 transfected cells. In presence of FAK inhibitor 14, NHERF1 potentiation of cell migration was completely abolished. When HOS-CD4-CCR5 cells are co-expressing the domain fragments, none of the fragments could induce cell migration any differently than pcDNA3-expressing cells (Fig. 8B). Similar results were obtained in another cell type, Jurkat T cells, expressing the same constructs (Fig. 8C). In HOS cells, the PDZ1 domain and to a smaller extent the PDZ1-PDZ2 construct appeared to block cell migration. Our results suggest that NHERF1 fragments could potentially block gp120-induced, FAK-dependent cell migration.

Discussion

Figure 6. Effect of NHERF1 on RhoA activation. (A) RhoA activation levels (RhoA-GTP) were determined by GST-Rhotekin-RBD pulldown assays with WT NHERF1; cell lysates from serum starved normal (untransfected) HOS-CD4-CCR5 cells loaded with GDP (GDP lane) or GTPgS (GTPgS lane) served as controls. Stimulations were performed with 50 nM gp120 for 15 min. All transfected cell extracts and GDP- or GTPgS-treated lysates were incubated with 50 mg of Rhotekin-RBD beads. Aliquots of 20 ng of His-RhoA were used as the control protein (first lane). PVDF membranes were probed with an anti-RhoA antibody. (B) Immunoblots showing the effect of NHERF1 siRNA knockdown on RhoA activation as described in (A). (C) Immunoblots showing the effect of NHERF1 domains on RhoA activation as described in (A). Western blots are representative of three experiments.

by arrows), compared to unstimulated or gp120-stimulated (Fig. 7A) pcDNA3-transfected cells. Transfection with NHERF1 domains prevented actin rearrangements in all cases, except for the PDZ1-PDZ2 and PDZ2-ERM fragments, which also displayed filamentous actin rearrangements (Fig. 7F and G).

FAK-mediated cell migration assay Cell migration is associated with cytoskeletal reorganization. The FAK signaling pathway is involved in cell migration and adhesion processes of several cell types [22]. Here, we asked whether FAK signaling following HIV-1CM gp120 stimulation contributed to cell migration, and how NHERF1 would then affect cell


Binding of HIV-1 to CD4 and CCR5/CXCR4 initiates a cascade of cellular events, including receptor internalization, activation of various kinases, calcium mobilization, actin rearrangements and cell migration. NHERF1, a molecular scaffold, is an important regulator the signaling and intracellular trafficking of several GPCRs, including CCR5 [25–27]. CCR5 interacts with NHERF1 via a PDZ-interacting sequence at its C-terminus [13]. We previously demonstrated a role of NHERF1 in the regulation of RANTES-activated CCR5. However, the role of NHERF1 in the regulation of CCR5 signal transduction during HIV-1 or viral envelope gp120 simulation is not known. Here, we demonstrate the influence of NHERF1 on the internalization of CCR5 induced by gp120, associated with an increase in HIV-1 production in host cells. Furthermore, NHERF1 is able to modulate several key signaling pathways involved in HIV-1 replication as well as cellular rearrangements and migration. To our knowledge, these results represent the first evidence of NHERF1 involvement in the regulation of HIV-1 signaling cascade. We demonstrate that the interaction between CCR5 and NHERF1 can be modulated by gp120 stimulation (Fig. 3C). As opposed to the strong signal reported in HEK293 cells, very little interaction with CCR5 is observed basally in HOS cells. It is worth mentioning that disruption of the c-terminal PDZ ligand of NHERF1 by the addition of a tag may have significant consequences on the affinity of NHERF1 for its targets and on the subcellular distribution of the protein in some cell types. Yet, this does not appear to be the case for the interaction of CCR5 and NHERF1 in HOS cells. The recruitment of NHERF1 to an activated receptor is consistent with several reports [28, 29]. For example, the b2-adrenergic and platelet-derived growth factor receptors were both shown to recruit NHERF1 following stimulation. Given the important role of NHERF1 in trafficking regulation, we evaluated NHERF1’s effect on CCR5 internalization. NHERF1 recruitment to CCR5 is associated with an increase in receptor internalization levels. Interestingly, fragments of

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HIGHLIGHTS

Figure 7. Effects of NHERF1 on gp120-induced actin rearrangement. HOS-CD4-CCR5 cells were transfected with HA-tagged WT NHERF1 or HA-tagged NHERF1 domain constructs in the presence or absence of gp120 and stained with either anti-HA Alexa Fluor 488-labeled Abs or Alexa Fluor 647-conjugated phalloidin. (A–G) pcDNA3, NHERF1-HA WT, PDZ1-HA, PDZ2-HA, ERM-HA, PDZ1-PDZ2-HA and PDZ2-ERM-HA, respectively. Images were acquired by fluorescence microscopy. Arrows indicate actin filaments. Scale bars represent 10 mm.

NHERF1 abolish CCR5 internalization, likely via competition of the domains with the receptor and other protein partners in the internalization process. gp120-induced CCR5 activation also upregulated a number of signal cascade pathways, such as MAPK/ ERK, FAK and RhoA, all of which play crucial roles in HIV-1 pathogenesis. The gp120-triggered MAPK/ERK activation plays a critical role in the viral reverse transcription, and host immunopathogenesis, and AIDS neurotoxicity [11, 30, 31]. NHERF1enhanced MAPK/ERK activation may contribute to the accelerated viral replication and cell proliferation required for viral production as previously suggested. [32]. It was also reported that phosphorylation of the MAPK/ERK was increased in a CD41


CCR51 U87 cell line treated with R5 HIV-1 isolate. MAPK/ERK appears, in this case, to phosphorylate the HIV-1 pre-integration complex, a step necessary for nuclear translocation and successful integration [33]. Therefore, MAPK/ERK plays diverse roles under different cells and tissue contexts during HIV-1 infection. Interestingly, WT NHERF1 can promote ERK phosphorylation, while different domains of NHERF1 can inhibit this increased phosphorylation. The ERM domain also showed an increase in activation of MAPK/ERK. It was previously reported that ERM proteins are linked to the regulation of MAPK signaling pathways activation [34]. FAK activation is important for HIV-1 entry, post-entry replication events, cell survival and chemotaxis [35]. FAK phosphor-

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Jurkat cells Figure 8. NHERF1 effect on cell migration. Data from representative photomicrographs (20 ) for the various conditions for migrated cells are shown in the histogram. FI14 indicates FAK inhibitor 14. Migration was measured via a transwell assay, where cells were incubated for 3 h in a well separated by a membrane from 10 nM gp120. Cells that crossed the membrane were then stained with crystal violet and counted. (A) Migration of HOS-CD4-CCR5 cells transfected with the indicated constructs in the presence/absence of FAK inhibitor 14 (FI14) and/or gp120. (B) Effect of NHERF1 domains on HOS-CD4-CCR5 cells migration in the presence or absence of FI14. (C) Effect of NHERF1 domains on the migration of Jurkat cells in the presence or absence of FI14. Results are representative of three independent experiments. po0.05, using One-way ANOVA with Bonferroni’s post test. Error bars indicate 1SEM of all replicates.


ylation, in the presence of NHERF1, is also increased following gp120 stimulation. Interestingly, the entire NHERF1 protein is not required to obtain FAK activation. PDZ2-ERM and ERM domains also displayed an increase in FAK phosphorylation. The amino-terminal region of FAK contains a region of sequence homology with band 4.1 and ERM proteins termed a FERM domain. FAK was previously reported to associate with CD4 when stimulated by HIV-1 gp120 [36]. Activation of the FAK signaling pathway by HIV-1 envelope is an important pathogenic mechanism of dysregulated cellular activation and trafficking during HIV infection [8, 37]. The exact role of NHERF1 in the increased phosphorylation of FAK is still unknown, but NHERF1 could potentially serve as a scaffold, eliciting the recruitment of FAK to CCR5 and the downstream activation (phosphorylation) of FAK to activate the signaling pathway. NHERF1-enhanced FAK activation also promotes HOS and Jurkat cells migration. Migration of cells requires important cellular morphological changes, which involve actin rearrangements. As previously reported, gp120 induces cofilin phosphorylation/inactivation and RhoA activation through a RhoA-ROCK-dependent pathway [10]. Upregulated RhoA activation by NHERF1 was observed as well as actin rearrangement, which is employed by viruses like HIV-1 to establish infection. Recently, a RhoA effector, citron kinase, was reported to preferentially enhance HIV-1 virion production by stimulating the endosomal compartments and exocytosis [38]. NHERF1 has also been involved in the regulation of endosomal sorting for several GPCRs [39]. NHERF1, via its ERM domain, could regulate signaling events related to actin rearrangements. It appears that RhoA and NHERF1 could have some overlapping and yet complementary roles that could likely finetune the levels of actin rearrangements in a cell. Taken together, our results show the effects of NHERF1 and its individual domains on the regulation of MAPK/ERK, FAK and RhoA signaling pathways. The PDZ2 domain, shown to be involved in the interaction with CCR5, also modulates the biological function of NHERF1. There is increasing evidence suggesting that FAK activation can promote RhoA-mediated signaling activation, and that the FAK-RhoA cross-talk might be involved in the CCR5-induced cellular signal cascades. Considering the similarities in the activation levels of FAK and RhoA signaling pathways by PDZ2-ERM, and the involvement of both pathways in HIV infection, we hypothesize that a cross-talk between the two pathways or a FAK-RhoA signal transduction pathway exists upon HIV-1 or gp120 association with receptors that could be regulated by NHERF1. In this case, NHERF1 could potentially be involved in a HIV-1 viral strategy to establish a successful infection and post-entry replication in host cells. In the case of HIV-1-induced membrane fusion, activation of RhoA and subsequent actin cytoskeletal reorganizations are required for efficient virus entry and infection [40]. NHERF1 could likely direct RhoA to CCR5-bound HIV-1 complexes, and promote localized membrane fusion. Therefore, the role of NHERF1 in regulating the HIV-1 infection and replication might be extremely important. NHERF1 might not be the only PDZ-containing scaffold important for viral infections. Recently, a PDZ domain-

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containing protein PDZD8 was identified to promote retroviral infection [41], which displayed the involvement of PDZ-domain family members in HIV-1 replication. Our study illustrates the potential role of a molecular scaffold, NHERF1, in the regulation of HIV-1 gp120-induced signal transduction. The development of strategies targeting intracellular targets such as protein scaffolds could likely be included in the design and development of novel anti-HIV drug therapies.

Materials and methods Cell culture HEK293 and HOS-CD4-CCR5 (obtained from Dr. Nathaniel Landau through the NIH AIDS Research and Reference Reagents Program, Division of AIDS, NIAID, NIH) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) high glucose supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 mg/mL streptomycin. Jurkat cells were maintained in RPMI 1640 supplemented with 10% FBS and 100 U/mL penicillin and 100 mg/mL streptomycin.

Cell-surface expression assay HEK293 cells were co-transfected with the receptor constructs, NHERF1 WT or domain fragments of NHERF1 (PDZ domain1 (PDZ1), PDZ domain 2 (PDZ2), ERM, PDZ1-PDZ2, PDZ2-ERM). An aliquot of 1 mg of each cDNA was transfected into each well of a six-well plate, and total DNA/dish was kept constant by adding pcDNA3 vector as required. Further, 48 h post-transfection, cells were stimulated with CCR5-tropic gp120 for up to 60 min. Cells were then washed with phosphate-buffered saline (PBS), fixed with 3.7% formaldehyde in TBS for 5 min. After three washes with TBS, cells were incubated for 45 min in 1% BSA-TBS, and then for 1 h in 1% BSA-TBS with relevant primary antibody. Cells were gently washed twice with TBS, incubated, blocked again in 1% BSA-TBS for 15 min, and then with 1% BSATBS containing relevant horseradish peroxidase-coupled secondary antibody for 60 min. Cells were washed again twice with TBS. The o-Phenylenediamine dihydrochloride substrate in a citrate buffer was then prepared and added to the cells to induce the colorimetric reaction. The reaction was stopped with 3N HCl when color appeared. The colorimetric assay was then read on a plate reader (Perkin Elmer Envision) at 492 nm.

Fluorescence microscopy HIV-1 strains and infection assay The CCR5/M-tropic HIV-1Ada-M and HIV-1SF162 strains were obtained from Dr. Howard Gendelman and Dr. Jay Levy through the AIDS Research and Reference Reagent Program. HOS-CD4CCR5 cells transfected with WT NHERF1 or domain fragments were seeded in 24-well plates 48 h post-transfection, and then challenged with the HIV-1 strains. After 96 h of infection, cell-free supernatants were collected and inactivated by 0.5% Triton X-100. The level of virus antigen was then measured by an HIVAg/Ab ELISA Kit (WANTAI Bio-Pharm). The inhibition of virus replication was calculated by the Reed and Muench method as previously described [42].

DNA construction and transfection The HA-tagged NHERF1 WT and fragment domains (PDZ1-HA, PDZ2-HA, ERM-HA, PDZ1-PDZ2-HA, PDZ2-ERM-HA) were kind gifts from Dr. Jean-Luc Parent (Universite´ de Sherbrooke, Canada). The pGFP2-N2-CD4 and pRluc-CCR5 recombinant DNA plasmids TM were constructed in our lab. NHERF1 Stealth Select RNAi siRNA (set of three; catalog numbers HSS145123, HSS145124 and HSS145125) were purchased from Invitrogen. Transfections with NHERF1 constructs were carried out using Lipofectamine 2000 manufacturer’s instructions (Invitrogen) (Supporting Information Fig. 2). Jurkat cells were plated in six-well plates and transfected with Lipofectamine LTX and PLUS reagents (Invitrogen), as prescribed in the Jurkat cell optimized transfection protocol (approximatively 20% transfection efficiency).


HOS-CD4-CCR5 cells transfected with NHERF1 constructs were grown overnight on laminin-coated glass coverslips in the presence of serum. Cells were washed with media and then incubated in serum-free media containing either DMEM vehicle or 50 nM HIV-1CM gp120 (Cedarlane) for 15 min at 371C. Cells were then fixed for 15 min in 3.7% w/v paraformaldehyde in PBS and permeabilized for 5 min in 0.1% Triton X-100 in PBS at room temperature. Filamentous actin was labeled with Alexa Fluor 647-conjugated phalloidin (Invitrogen) for 25 min at room temperature. The coverslips were washed with PBS, drained and mounted onto glass slides using a drop of VectaMount permanent mounting medium (Vector laboratories). Fluorescence microscopy was performed under 60 oil immersion on an Olympus IX81 microscope (Olympus) equipped with a Photometrics coolSNAP HQ2 camera and excite series 120Q light source, and Images were obtained with Metamorph Image software (Universal Imaging).

Cell migration assay HOS-CD4-CCR5 cells expressing NHERF1 WT or fragments were collected 48 h post-transfection by centrifugation and resuspended to a density of 0.5 105 cells/mL in 100 mL DMEM medium with 0.01% BSA. Jurkat cells cotransfected with CCR5 and pcDNA3, NHERF1 WT, or NHERF1 domain mutants were collected by centrifugation and resuspended to a density of 1 105 cells/mL in 100 mL RPMI 1640 with 0.01% BSA without serum. The cells were added to the upper chamber of a 24-well

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Transwell plate (Corning; 5 mm pore size polycarbonate membranes) and the lower chamber received the same medium supplemented with 10 nM HIV-1CM gp120. The chambers were incubated for 3 h in a 371C, 5% CO2 chamber. For inhibitor studies, cells were pretreated with 10 mM FAK inhibitor 14 (Santa Cruz Biotechnology) for 30 min at 371C prior to addition to the Transwell plate. The inhibitor remained in both chambers of each well for the duration of the cell migration. The cells were allowed to migrate for 5 h at 371C. Non-migrated cells on the upper side of the membrane were removed with a cotton swab while the cells that had migrated to the underside of the membrane were fixed with chilled methanol for 20 min at room temperature, washed twice with PBS and stained with 0.5% crystal violet. Migration was assessed by counting three 20 fields using an inverted fluorescence microscope. The computer generated cells counts using with ImageJ 4.3 (NIH) were confirmed by manual counting of the migrated cells in representative experiments.

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was immediately flash-frozen in liquid nitrogen, and stored at 701C for later use. The remaining 10% lysate in each sample was for used for total protein concentration measurements using the ready-to-use Bradford reagent (Fermentas) and saved for Western immunoblotting loading controls to assess total RhoA expression levels in the cell lysate. For activated RhoA pulldown assays, 800 mg of total cell lysate was incubated with 15 mg of GST-Rhotekin-RBD beads for 1 h at 41C. The beads were then centrifugated for 1 min at 5000 g and washed once with icecold Wash Buffer, resuspended in Laemmli Buffer and boiled for 2 min prior to running on a 12% SDS-PAGE gel, and then transferred to a PVDF membrane (BioRad). Mouse anti-RhoA monoclonal antibody at a dilution ratio of 1:500 in TBS-T was used for Western blotting of GST-Rhotekin-RBD pull-down or whole-cell lysate input.

Western immunoblotting Immunoprecipitation assay Stimulation of transfected HOS-CD4-CCR5 cells and immunoprecipitation were carried out as previously described [13]. Briefly, 48 h post-transfection, cells were harvested and then treated for 15 min at 371C with 50 nM recombinant HIV-1CM envelope gp120 or medium vehicle treatment as control. After treatment, cells were pelleted and lysed with RIPA lysis buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 150 mM NaCl, 0.5% sodium deoxycholate, 1% Nonidet P-40) plus Complete EDTA-free Protease Inhibitor Cocktail (Roche). Cell lysates were pre-cleared by incubation for 30 min at 41C with Protein A-sepharose (Sigma-Aldrich) with BSA. Immunoprecipitations were performed using a goat anti-CCR5 antibody (Santa Cruz Biotechnology) followed by incubation with Protein A-sepharose at 41C. Immunoprecipitated proteins were resolved by SDS-PAGE and were sequentially immunoblotted with anti-phospho-FAK (Tyr379, Santa Cruz Biotechnology), or anti-FAK (BioVision). To detect NHERF1-HA interaction with FAK, we employed anti-HA monoclonal antibody to immunoprecipitate NHERF1-HA, and then immunoblotted with using the anti-FAK antibody.

RhoA activation assay Cellular levels of activated RhoA (associated with GTP) were determined by a pull-down assay using the Rho Activation Assay Biochem Kit manufacturer’s manual (Cytoskeleton). HOSCD4-CCR5 cells were seeded at the density of 1.4 105 cells per 10-cm culture dish in DMEM medium with 10% FBS overnight. Then, cells were transfected with 8 mg of the NHERF1 or domain fragments. And 24 h post-transfection, HOS-CD4-CCR5 cells were cultured in DMEM without FBS; 48 h post-transfection, cells were stimulated with 50 nM gp120, then washed and lysed in ice-cold Cell Lysis Buffer and spun at 10 000 rpm in a microcentrifuge at 41C for 2 min. About 90% of cleared cell lysate


HEK293 or HOS cells stably expressing CD4 and CCR5 were serum starved 24 h post-transfection and then treated with 50 nM of HIV-1CM gp120 for 15 min at 371C. And 48 h posttransfection, cells were lysed in RIPA buffer, sonicated and boiled at 95–1001C for 5 min in Laemmli sample buffer (BioRad). Aliquots of proteins were resolved by SDS-PAGE on 8–12% polyacrylamide gels and transferred to nitrocellulose membranes (BioRad). Immunoblots were then probed with anti-phosphoERK or anti-phospho-FAK primary antibodies followed by HRPconjugated goat anti-rabbit or anti-mouse IgG (1:10 000) secondary antibodies. Immunoblots were visualized using Western Lightning Plus-ECL Enhanced Chemiluminescence Substrate (Perkin Elmer). Protein loading was controlled by stripping the membrane and reprobing with anti-MAPK/ERK, anti-FAK antibodies. ImageJ 4.3 (NIH) was used to analyze immunoblot band densities.

Statistical analysis The data are expressed as a mean value7SEM for at least three independent experiments. Statistical analysis was performed using a Student’s t test or analysis of variance (ANOVA) with Bonferroni’s post test in Prism software package (GraphPad). The significance level was set at po0.05.

Acknowledgements: This work was partially supported by grants from the Canadian Institutes of Health Research (No. HOP-86863) and NSERC (No. RGPIN/355310-2008) to D.J.D. D.J.D. is the recipient of a CIHR New Investigator Award and Dalhousie Medical Research Foundation New Investigator Award. This work was also partially supported by grants from National Basical Research Program of China (No.2009CB522306),

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Eleven Five-Year Key Scientific and Technological Program of China (No. 2009ZX09501-029, 2008ZX10001-015, 2008ZX10005-002), and National Natural Science Fundation of China (No. U0832601) to Y.T.Z. We would like to thank Nicholle Charette and Patrick Holland for their valuable comments on this manuscript.

13 Hammad, M. M., Kuang, Y. Q., Yan, R., Allen, H. and Dupre´, D. J., Na1/ H1 exchanger regulatory factor-1 is involved in chemokine receptor homodimer CCR5 internalization and signal transduction but does not affect CXCR4 homodimer or CXCR4-CCR5 heterodimer. J. Biol. Chem. 2010. 285: 34653–34664. 14 Hung, A. Y. and Sheng, M., PDZ domains: structural modules for protein complex assembly. J. Biol. Chem. 2002. 277: 5699–5702.

Conflict of interest: The authors declare no financial or commercial conflict of interest.

15 Weinman, E. J., Steplock, D., Wang, Y. and Shenolikar, S., Characterization of a protein cofactor that mediates protein kinase A regulation of the renal brush border membrane Na(1)-H1 exchanger. J. Clin. Invest. 1995. 95: 2143–2149. 16 Cao, T. T., Deacon, H. W., Reczek, D., Bretscher, A. and von Zastrow, M.,

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Abbreviations: ERM: ezrin-radixin-moesin FAK: focal adhesion kinase GPCR: G protein-coupled receptor HOS-CD4-CCR5: human osteosarcoma cells coexpressing CD4 and CCR5 M-tropic: macrophage-tropic NHERF1: Na1/H1 exchanger regulator factor 1 PDZ: postsynaptic density 95/disk-large/zonula occludens RhoA: Ras homolog gene family member A ROCK: Rho-associete protein kinase Full correspondence: Dr. Denis J. Dupre´, Department of Pharmacology, Faculty of Medicine, Room 6C, Sir Charles Tupper Medical Building, Dalhousie University, 5850 College St. PO BOX 15 000, Halifax, NS, B3H 4R2 Canada Fax: 11-902-494-1388 e-mail: [email protected]

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Received: 27/5/2011 Revised: 15/9/2011 Accepted: 21/10/2011 Accepted article onlline: 26/10/2011

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