doi: 10.1128/JVI.02255-16. Print 2017 Apr 1.
The Measles Virus Receptor SLAMF1 Can Mediate Particle Endocytosis
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- PMID:28100610
- PMCID: PMC5355598
- DOI: 10.1128/JVI.02255-16
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The Measles Virus Receptor SLAMF1 Can Mediate Particle Endocytosis
Daniel Gonçalves-Carneiro et al. J Virol..
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Abstract
The signaling lymphocyte activation molecule F1 (SLAMF1) is both a microbial sensor and entry receptor for measles virus (MeV). Herein, we describe a new role for SLAMF1 to mediate MeV endocytosis that is in contrast with the alternative, and generally accepted, model that MeV genome enters cells only after fusion at the cell surface. We demonstrated that MeV engagement of SLAMF1 induces dramatic but transient morphological changes, most prominently in the formation of membrane blebs, which were shown to colocalize with incoming viral particles, and rearrangement of the actin cytoskeleton in infected cells. MeV infection was dependent on these dynamic cytoskeletal changes as well as fluid uptake through a macropinocytosis-like pathway as chemical inhibition of these processes inhibited entry. Moreover, we identified a role for the RhoA-ROCK-myosin II signaling axis in this MeV internalization process, highlighting a novel role for this recently characterized pathway in virus entry. Our study shows that MeV can hijack a microbial sensor normally involved in bacterial phagocytosis to drive endocytosis using a complex pathway that shares features with canonical viral macropinocytosis, phagocytosis, and mechanotransduction. This uptake pathway is specific to SLAMF1-positive cells and occurs within 60 min of viral attachment. Measles virus remains a significant cause of mortality in human populations, and this research sheds new light on the very first steps of infection of this important pathogen.IMPORTANCE Measles is a significant disease in humans and is estimated to have killed over 200 million people since records began. According to current World Health Organization statistics, it still kills over 100,000 people a year, mostly children in the developing world. The causative agent, measles virus, is a small enveloped RNA virus that infects a broad range of cells during infection. In particular, immune cells are infected via interactions between glycoproteins found on the surface of the virus and SLAMF1, the immune cell receptor. In this study, we have investigated the steps governing entry of measles virus into SLAMF1-positive cells and identified endocytic uptake of viral particles. This research will impact our understanding of morbillivirus-related immunosuppression as well as the application of measles virus as an oncolytic therapeutic.
Keywords: SLAMF1; endocytosis; fusion; macropinocytosis; measles; morbillivirus; virus attachment; virus entry.
Copyright © 2017 Gonçalves-Carneiro et al.
Figures
MeV induces the formation of transient membrane structures in SLAMF1-positive lymphocytes. (A) B-lymphoblastoid cells were inoculated with recombinant MeV (MOI, ∼0.1) engineered to express EGFP and incubated for 72 h prior to visualization by UV phase-contrast microscopy at high and low magnifications (mag'). (B) B-lymphoblastoid cells were infected with MeV (MOI, ∼0.01) and incubated at 37°C prior to quantification of viral progeny, by 50% tissue culture infective dose (TCID50), at the indicated times. HPI, hours postinfection. (C) MeV was purified by ultracentrifugation using a stepped sucrose gradient. The opalescent band located at 30-45% sucrose cushion interface was extracted and dialyzed. Protein lysates from these purified virus preparations were analyzed by silver staining and Western blot analysis. Mk, protein marker. (D) B-lymphoblastoid cells were synchronously infected with MeV (MOI, ∼20) or mock infected with 20% FBS-containing DMEM before fixation, preparation, and visualization by SEM. (E) Images were blinded and representative micrographs quantified for membrane blebs and membrane ruffles. Statistical analysis was performed using the Studentt test. ***,P < 0.001. n.s., nonsignificant.
MeV infection of SLAMF1-recombinant A549 cells induces blebbing, filopodium formation, and cellular retraction. (A) A549 or A549-SLAMF1 cells were infected with MeV (MOI, ∼1) and incubated for 48 h prior to cell lysis, clarification of supernatant, and quantification of viral progeny via TCID50 (n.d., not detected). (B) Similarly, A549 and A549-SLAMF1 cells were infected with MeV-PP reporter virus and incubated for 72 h prior to lysis and measurement of luciferase activity. (C and D) A549-SLAM cells were serum starved overnight and inoculated with MeV (MOI = 45) for 1 h at 4°C. Cells were washed with PBS and incubated at 37°C for 30 min before fixation with 4% PFA and visualization by phase-contrast microscopy. Representative micrographs of infected and uninfected cells are provided at a magnification of ×40. (C) The percentage of cells that presented membrane blebs per field of view was calculated for both samples (D). (E and F) Similarly, serum-starved A549-SLAMF1 cells were synchronously infected with MeV (MOI = 30) and incubated at 37°C for 0, 10, 30 or 60 min postinfection (mpi) before fixation and imaging by SEM. The cell surface of uninfected and infected cells presented small filopodium-like structures at the surface (orange arrowheads), but only infected cells presented membrane blebs (white arrowheads) (E). Infected A549-SLAMF1 cells retracted after infection, in association with these membrane blebs; however, this was transient and lasted only 60 min (F). All statistical analysis was performed using the Studentt test. **,P < 0.005; error bars indicate SDs.
MeV particles are endocytosed. (A) Diagram of experimental setup. Purified MeV was attached to A549-SLAMF1 cells (MOI = 30) at 4°C for 1 h, washed with PBS to remove unbound virus, and incubated for 0, 15, or 30 min at 37°C prior to trypsin treatment. (B) MeV particles were incubated with trypsin at 37°C for 7 min prior to addition of an equal volume of complete fetal calf serum (FCS)-containing cell culture medium. Lysates were generated with Laemmli buffer and resolved on a 15% SDS-PAGE gel, followed by Western blotting with anti-MeV H antibody. (C) Total cell lysates were generated and resolved on a 15% SDS-PAGE gel prior to Western blotting using a polyclonal antibody raised against the cytoplasmic tail of MeV H. Three H-specific bands were detected with approximate sizes of 75 kDa (black arrowhead; full length), 24 kDa (blue arrowhead), and 20 kDa (white arrowhead), the latter two detected only after trypsin treatment. The data are representative of those from four individual experiments. Of note, the specificity of this assay was preserved using an antibody specific to the intravirion tail of MeV H that allowed detection of tryptically digested variants. (D) A549-SLAMF1 cells were synchronously infected with MeV (MOI, ∼1), incubated for the indicated times (minutes postinfection), and trypsinized for 7 min at 37°C. Cells were pelleted, resuspended in complete medium, and incubated for 48 h, and virus infectivity was determined (error bars indicate SDs). (E and F) HEK293T-SLAMF1 cells carrying a split form of Renilla luciferase (RLuc1) were synchronously infected with purified MeV (MOI = 30) for 60 min at 4°C, washed with PBS, and incubated for 0, 15, 30 or 60 min postinfection (mpi), with or without 5 μg/ml of chlorpromazine (CPZ), an inhibitor of clathrin pit formation and membrane protein sequestration, at 37°C prior to the addition of HEK293T-SLAMF1 cells expressing the complementary part of the luciferase (RLuc2). Cells were cocultured for 90 min before addition of the luciferase substrate coelenterazine and measurement of light in a luminometer. Values were normalized by subtracting the values from uninfected cells and plotted as relative light units (error bars indicate standard errors). Statistical analysis was performed using the Studentt test. **,P < 0.005; ***,P < 0.001. n.s., nonsignificant.
MeV particles are internalized in a clathrin-, caveolin-, and dynamin-2-independent manner. (A) A549-SLAMF1 cells were transfected with plasmids expressing dominant negative (DN) mutants of EPS15, cav-1, and Dyn2 and 48 h posttransfection were transduced with MeV- or VSV-PPs. As a control, cells were transfected with an empty plasmid (Vector). After 72 h, cells were lysed and luciferase activity was measured. (B) A549-SLAMF1 cells were pretreated with chloroquine (CQ; 50 μg/ml), bafilomycin A1 (BAF; 50 μg/ml), dynasore (DYN; 100 μM), or solvent for 30 min at 37°C. MeV- or VSV-PP encoding the firefly luciferase reporter was added to cells and incubated for 3 h. Medium was replaced and cells were incubated for 72 h before lysis, addition of the luciferase substrate luciferin, and measurement of produced light. Statistical analysis was performed using the Studentt test. *,P < 0.05; **,P < 0.005. n.s., nonsignificant (error bars indicate standard errors).
MeV induces fluid uptake in a SLAMF1-dependent manner. (A) A549-SLAMF1 cells were synchronously infected with MeV (MOI = 10) for 1 h at 4°C, washed in cold PBS, and incubated at 37°C for 20 min with DMEM containing 0.25 mg/ml of dextran-Alexa Fluor 488 conjugate. Alternatively, cells were treated with a 200 nM concentration of the macropinocytosis inducer phorbol 12-myristate 13-acetate (PMA). Cells were moved to ice, washed, bleached, and fixed in 4% PFA. Samples were analyzed by CSLM. Representative micrographs of uninfected, MeV-infected, and PMA-treated cells were recorded. The bar represents 15 μm. (B) Quantification of internalized dextran (pseudocolored green) was performed by calculating corrected total cell fluorescence based on mean fluorescence intensity (MFI). Statistical analysis was performed using the Studentt test. *,P < 0.05 (error bars indicate standard errors). (C) To assess the role of SLAMF1 in fluid uptake during MeV infection, A549 and A549-SLAMF1 cells were synchronously mock or MeV infected (MOI = 10), incubated with dextran-containing PBS for 30 min, fixed, and analyzed by CSLM (left) with the mean fluorescent intensity quantified and expressed relative to the background (right). (D) B-lymphoblastoid cells (LCLs) were attached to a glass coverslip, synchronously infected with MeV (MOI, ∼20), and incubated with DMEM containing 0.25 mg/ml of dextran-Alexa Fluor 488 conjugate. Cells were moved to ice, washed, bleached, and fixed before analysis by CSLM. The bar represents 5 μm. (E) A549-SLAMF1 cells were synchronously infected with MeV (MOI = 10), washed and incubated at 37°C in dextran-containing PBS for the indicated times, fixed, prepared, and analyzed by CSLM. Dextran is showed in green, while MeV N is pseudocolored in red; arrowheads indicate colocalization of N and dextran-positive vesicles. (F) Using a similar approach and CSLM analysis, the colocalization of MeV N puncta with dextran-containing endosomes was quantified at 0, 15, 30, 45, and 60 min postinfection. Statistical analysis was performed using analysis of variance (ANOVA);F (, ; ratio variables obtained with a Brown-Forsythe test) = 6.076;P = 0.0005 (error bars indicate standard errors). (G) Intensity analysis was performed using Leiss ZEN software, by profiling of red and green channels along the indicated line.
MeV infection is inhibited by amiloride. (A) A549-SLAMF1 cells were treated with 25 μM EIPA prior to infection with MeV- or VSV-PPs for 3 h SLAMF1 and incubation in complete medium. Luciferase activity was measured 72 h later. (B) A549-SLAMF1 cells were pretreated with EIPA and infected with MeV (MOI = 1) at the indicated concentrations, before trypsinization and culture at 37°C for an additional 6.5 h. Cells were detached with 2 mM EDTA solution, and GFP fluorescence was analyzed by flow cytometry. (C) A549-SLAM cells were incubated with EIPA at the indicated concentration for 30 min, washed, and incubated with 200 nM PMA in dextran-containing PBS for 30 min. Cells were then bleached, washed, and prepared for CSLM. (D) A549-SLAMF1 cells were transfected with constructs encoding myc-tagged dominant negative (DN) mutants (Rac1 N17 and Cdc42 N17) or GFP-tagged constitutively active (CA) forms (Rac1 L61 and Cdc42 L61) of the indicated RhoGTPases. Cells were then infected with MeV-PP and luciferase activity was measured 72 h later. (E) Similarly, cells were transfected with the same constructs, infected with MeV (MOI = 1), and incubated for 24 h. Infected cells were quantified by UV microscopy. (F) Cells were transfected with plasmid constructs encoding wild-type (WT) or a CA mutant of PAK-1 and infected with MeV-PP before quantification by luciferase assay. (G) A549-SLAMF1 cells were transfected with either random siRNA (control), siRNA targeting PAK-1, or a carrier. Seventy-two hours later, cells were infected with MeV (MOI = 1), incubated for 24 h, and lysed for Western blot analysis. In all instances the expression of DN, WT, or CA proteins was confirmed by Western blotting or fluorescence microscopy (not shown). Statistical analysis was performed using the Studentt test. *,P < 0.05; **,P < 0.005. n.s., nonsignificant (error bars indicate SDs).
The RhoA-ROCK-myosin II axis is important for virus infection. (A) A549-SLAMF1 cells were transfected with constructs encoding myc-tagged DN (Rho N19) or GFP-tagged CA (Rho L63) of RhoA. Cells were infected with MeV-PP, and luciferase activity was measured 72 h later. (B) Similarly, cells were transfected with the same constructs, infected with MeV (MOI = 1), and incubated for 24 h; infected cells were quantified under UV microscopy. (C) A549-SLAMF1 cells were pretreated with the ROCKI inhibitor H-1152 at the indicated concentrations for 5 h before infection with MeV- or VSV-PP, and luciferase activity was measured 72 h later. (D) A549-SLAMF1 cells were pretreated with blebbistatin (BLEB) or solvent for 30 min before infection with MeV- or VSV-PP for 3 h, unbound virus was removed by washing, and cells were incubated for 72 h in complete medium prior to measurement of luciferase activity. (E) In a similar experiment, cells were pretreated with 100 μM BLEB for 30 min and infected with MeV (MOI = 1) for 1 h. Cells were trypsinized and incubated for 24 h before quantification of the frequency of GFP+ cells and total yield of virus. (F) A549-SLAM cells were incubated with blebbistatin at the indicated concentration for 30 min, washed, and incubated with 200 nM PMA in dextran-containing PBS for 30 min. Cells were then bleached, washed, and prepared for CSLM. Please refer to Fig. 6C for relevant control image. (G) To address if blebbistatin had a dose-dependent effect on MeV entry, A549-SLAMF1 cells were either pre- or posttreated with the drug at the indicated concentrations, trypsinized, and incubated at 37°C for 24 h. Total cell lysates were generated and resolved on a 15% SDS-PAGE gel prior to Western blotting using a polyclonal antibody raised specifically for MeV H cytoplasmic tail. Statistical analysis was performed using the Studentt test. *,P < 0.05; **,P < 0.005. n.s., nonsignificant (error bars indicate SDs).
MeV induces cytoskeleton reorganization in a SLAMF1-dependent manner. (A and C) A549-SLAMF1 cells were synchronously infected with MeV (MOI, ∼15) for 1 h at 4°C (A) or incubated with 20% FBS-DMEM under the same conditions (No virus) (C). Cells were washed in cold PBS and incubated at 37°C for 0, 10, 20, or 60 min prior to fixation with 4% PFA, staining with phalloidin-TRITC (pseudocolored in white), and analysis by CSLM. Orange arrowheads indicate actin-enriched domains. (B) The average cell area (in pixel units) of individual cells was calculated based on representative micrographs of each condition using ImageJ. Statistical analysis was performed using the Studentt test. *,P < 0.05 (error bars indicate SDs). (D) To assess the role of SLAMF1 in inducing the contraction of the cytoskeleton during MeV infection, A549 and A549-SLAMF1 cells were synchronously mock or MeV infected (MOI = 10), incubated at 37°C for 30 min, fixed, and analyzed by CSLM. The bar represents 30 μm. (E) Six representative micrographs of each condition were analyzed, and the average cell area was calculated. Statistical analysis was performed using a one-tailed Studentt test. *,P < 0.05 (error bars indicate SDs). (F) A549-SLAMF1 cells were synchronously infected with MeV (MOI = 15), incubated at 37°C for 0, 15, 30, or 60 min, fixed, and prepared for CSLM. Blue arrowheads represent MeV N (in red) and actin (white) colocalization. (G) A549-SLAMF1 cells were synchronously infected with MeV (MOI, ∼15), washed, and incubated at 37°C for 0, 10, 20, 30, or 45 min prior to lysis and ERM phosphorylation analysis by SDS-PAGE and Western blotting. (H) A549-SLAMF1 cells were pre- or posttreated with the indicated concentrations of cytochalasin D (CytoD) or jasplakinolide (Jasp) and infected with MeV (MOI = 1). Total cell lysates were generated 24 h postinfection and resolved on a 15% SDS-PAGE gel prior to Western blotting using a polyclonal antibody raised against the cytoplasmic tail of MeV H.
Model of endocytic MeV entry into SLAMF1-positive cells. (A) MeV binds to human SLAMF1 (CD150) via a specific protein-protein interaction between the viral hemagglutinin and SLAMF1. (B) Within 10 min, the receptor interaction induces the formation of membrane blebs, governed in part by the RhoA-ROCK-myosin II axis. Inhibition of this axis (with chemical inhibitors or dominant negative mutants) reduces MeV entry. (C) The formation of membranous blebs is followed by an acute retraction of the cell, orchestrated by the cortical cytoskeleton. Perturbation of this process with chemical inhibitors of actin modulation also reduces MeV entry. (D) The internalization of MeV particles is concurrent with fluid uptake via a macropinocytosis-like pathway, a process that is sensitive to chemical inhibition with EIPA. Within 45 min, infected cells begin to reestablish their characteristic morphology through specific phosphorylation of ERM proteins.
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