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1 Laboratory of Virology, Faculty of Veterinary Medicine, Ghent University, Belgium
2 Department of Clinical Chemistry, Microbiology and Immunology, Faculty of Medicine and Health Science, Ghent University, Belgium
Correspondence
Hans J. Nauwynck
hans.nauwynck{at}UGent.be
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Present address: Laboratoire de Vaccinologie et d'Immunologie Mucosale (LOVMI), Université Libre de Bruxelles, Belgium. 
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Cell-mediated immunity is impaired in FIP cats, as lymphocytes and cytotoxic T cells are depleted (Haagmans et al., 1996; Kipar et al., 2001; Paltrinieri et al., 2003). In previous work, we reported that the membrane-bound viral proteins, the spike and the membrane protein, are internalized upon antibody binding, which results in the loss of detectable viral proteins on the plasma membrane (Dewerchin et al., 2006). The fact that no surface-expressed viral antigens can be found on FCoV-infected monocytes isolated from naturally infected FIP cats, and that surface expression returns after isolation and in vitro cultivation of the monocytes, is the first indication that this immune-evasion strategy also exists in vivo (Cornelissen et al., 2007). If internalization could be blocked, then the humoral immune system would be able to contribute to the elimination of infected monocytes either by antibody-dependent complement-mediated or antibody-dependent cell-mediated lysis. This could open new perspectives for anti-feline infections peritonitis virus (FIPV) therapies. To elucidate how internalization can be blocked, the pathway needs to be identified.
Multiple pathways for ligand internalization have been described in literature and are represented in Fig. 1. There are four ‘classical’ pathways: phagocytosis, macropinocytosis, clathrin- and caveolae-mediated internalization. To our knowledge, four clathrin- and caveolae-independent internalization or ‘non-classical’ pathways have been described.
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Here we used biochemical, cell biological and genetic approaches to determine the internalization method of surface-expressed FIPV antigens and their destination.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Polyclonal anti-FCoV antibodies were kindly provided by P. Rottier (Utrecht University, The Netherlands). The antibodies were purified and biotinylated according to the manufacturer's instructions (Amersham Bioscience). Fluorescein isothiocyanate (FITC)-labelled polyclonal anti-FIPV antibodies were purchased from Veterinary Medical Research and Development (VMRD). The monoclonal antibody E22-2, recognizing the N-protein, was kindly provided by T. Hohdatsu (Kitasato University, Japan). The monocyte marker DH59B, recognizing CD172a, was purchased from VMRD. A rabbit polyclonal antibody against Rab 7 and goat polyclonal antibodies against early endosomal antigen 1 (EEA 1) and cathepsin D were purchased from Santa Cruz Biotechnology. Secondary antibodies and reagents [purchased from Molecular Probes (Invitrogen)] were goat anti-mouse–Texas red, goat anti-mouse–Alexa Fluor 350 (data not shown), streptavidin–Texas red, streptavidin–FITC and anti-rabbit– and anti-goat–Alexa Fluor 594 Zenon reagent.
Isolation and inoculation of blood monocytes.
Four FCoV-, feline leukemia virus- and feline immunodeficiency virus-negative cats were used as blood donors. Blood (10 ml) was collected from the vena jugularis in heparin (15 U ml–1) (Leo, Zaventem, Belgium) and mononuclear cells were separated on Ficoll-Paque (GE Healthcare). Cells were seeded on glass coverslips inserted into a 24-well dish, in RPMI 1640 medium containing 10 % fetal bovine serum (FBS), 0.3 mg glutamine ml–1, 100 U penicillin ml–1, 0.1 mg streptomycin ml–1, 0.1 mg kanamycin ml–1, 10 U heparin ml–1, 1 mM sodium pyruvate and 1 % non-essential amino-acids (100x). Non-adherent cells were removed by washing the dishes twice with RPMI 1640, 2 and 24 h post-seeding (hps). The adherent cells consisted of 86±7 % of monocytes (as assessed by fluorescent staining with the monocyte marker DH59B). Isolation efficiency fluctuated around 10 %. At 56 hps, monocytes were inoculated with FIPV at an m.o.i. of 5.
Internalization inhibition assays.
Twelve hours after inoculation, monocytes seeded on glass coverslips were pre-incubated for 30 min at 37 °C with 5 % CO2 with one of the following agents dissolved in RPMI (all from Sigma-Aldrich unless stated otherwise): 500 µM amantadine, 0.1 µM wortmannin, 0.74 nM Clostridium difficile toxin B, 20 µM latrunculin B (ICN Biochemicals), 10 mM methyl-β-cyclodextrin, 50 µg nystatin ml–1, 40 µM dynamin inhibitory peptide (Tocris Cookson), 300 nM staurosporine, 10 mM sodium fluoride, 50 µg genistein ml–1 or 50 µM dephostatin (Calbiochem). The working concentration of each reagent was based on published values and was optimized qualitatively in internalization assays with control ligands (data not shown; see Supplementary Table S1, available in JGV Online). Viability of the cells during the inhibition assay was tested for each inhibitor using ethidium bromide monoazide (Molecular Probes) and was always >99 %.
After pre-treatment, the cells were incubated with polyclonal biotinylated anti-FIPV antibodies in the presence of one of the given inhibitors for 30 min. Then, cells were fixed with 1 % formaldehyde, permeabilized with 0.1 % Triton X-100 and incubated with streptavidin–Texas red. Infected cells were visualized with polyclonal anti-FIPV–FITC. The glass coverslips were mounted on microscope slides using glycerin/PBS (0.9 : 0.1, v/v) with 2.5 % 1,4-diazabicyclo(2,2,2)octane and analysed using confocal microscopy. Monocytes were counted as cells with fully internalized antigen–antibody complexes when no labelling could be observed at the plasma membrane. Percentages of cells with fully internalized complexes were calculated relative to the total amount of monocytes that showed antibody binding, and thus had membrane expression before antibodies were added (about 50 % of the infected cells) (Dewerchin et al., 2005). Because of the variability in the amount of cells with membrane expression, visualization of the complexes remaining at the plasma membrane was required. Therefore, an acid washing step to remove the extracellular antibodies was not performed.
To test the effectiveness of the reagents, a suitable control was used in each experiment. Monocytes seeded on glass coverslips were pre-incubated for 30 min with one of the inhibitors. After treatment, the cells were incubated with biotinylated human transferrin (Sigma-Aldrich) or fluorescent 1 µm polystyrene microspheres (FluoSpheres; Molecular Probes) in the presence of the inhibitor. Then, cells were fixed with formaldehyde and permeabilized with Triton X-100. The biotinylated transferrin was visualized with streptavidin–FITC; in cells incubated with fluorescent beads, actin was visualized with phalloidin–Texas red (Molecular Probes). The glass coverslips were mounted on microscope slides and analysed by confocal microscopy. For the controls, the monocytes were counted analogously to the FIPV-infected cells: ligands were considered ‘fully internalized’ when they were only observed inside the cell. Fluorescent beads were considered internalized when they were found inside the cortical actin labelling.
Transfer plasmid construction.
The TRIP
U3-CMV-WPRE vector [TRIPU3-CMV–GFP-WPRE in which green fluorescent protein (GFP) was deleted by BamHI–SalI digestion] was used as transfer vector, pMD.G was used as the envelope plasmid and p8.91 as the packaging plasmid as described by Stove et al. (2005).
Eps15 is a protein that is essential for the docking of adaptor protein-2 (AP-2) to the plasma membrane during the assembly of clathrin-coated pits (Benmerah et al., 1998, 1999). The dominant-negative (DN) mutant of Eps15, named DIII, has a deletion at the Eps15 homology (EH) and coiled-coil domains and a C-terminal enhanced GFP (EGFP) tag. The construct D32, with an additional deletion of the AP-2-binding site, was used as a control. Both constructs were kindly provided by A. Benmerah and excised from pEGFP-C2 by BclI and Eco47III digestion and double-blunt cloned into TRIPU3-CMV-WPRE vector.
The EGFP-tagged wild-type (WT) and DN caveolin-1 constructs were a kind gift from A. Helenius (Kurzchalia et al., 1992; Pelkmans et al., 2001). The WT caveolin-1–GFP construct was amplified by PCR from the pEGFP-N1 vector and cloned into the TRIPU3-CMV-WPRE vector. The DN caveolin-1–GFP construct was excised from pEGFP-C1 by Eco47III and BamHI digestion and double-blunt cloned into the TRIPU3-CMV-WPRE vector.
The EGFP-tagged WT and DN dynamin 2(aa) were kindly provided by M. McNiven (Cao et al., 1998, 2000). Both constructs were excised from pEGFP-N1 by HindIII and XbaI digestion and double-blunt cloned into the TRIPDU3-CMV-WPRE vector.
Biological activity of all constructs was tested on CrFK cells before and after transfer to pTRIPU3-CMV-WPRE.
Production of lentiviral supernatant.
293FT cells (Invitrogen) were seeded in a 25 cm2 culture dish in Iscove's modified Dulbecco's medium supplemented with 100 U penicillin ml–1, 0.1 mg streptomycin ml–1 and 10 % heat-inactivated FBS. At 70 % confluency, cells were co-transfected with 1.66 µg packaging plasmids, 3.33 µg envelope plasmids and 3.33 µg transfer plasmids using a Calcium Phosphate Transfection kit (Invitrogen). Viral supernatant was harvested 40 h later.
Viral gene transfer, superinfection and internalization assays.
Feline monocytes were isolated as described previously (Dewerchin et al., 2005). After 3 h, the cells were washed and the medium was replaced with lentiviral supernatant. At 24 hps, cells were washed and fresh medium was added. At 56 hps, cells were inoculated with FIPV. Twelve hours later, internalization assays were performed by adding biotinylated anti-FIPV antibodies for 30 min. Then, cells were fixed with formaldehyde, permeabilized with Triton X-100 and incubated with streptavidin–Texas red to visualize the antigen–antibody complexes. Next, infected cells were visualized with anti-N monoclonal antibodies and goat anti-mouse–Alexa Fluor 350. Transduction efficiencies varied between 10 and 50 %.
For the controls, transduced cells were incubated with biotinylated transferrin or biotinylated cholera toxin B (Sigma-Aldrich). Then, cells were fixed and permeabilized, and ligands were visualized with streptavidin–Texas red.
Co-localization studies with endosomal markers.
Twelve hours after inoculation, monocytes were incubated with biotinylated anti-FIPV polyclonal antibodies. At different times after antibody addition, cells were fixed and permeabilized, and antigen–antibody complexes were visualized with streptavidin–FITC followed by a blocking step with 10 % negative goat serum. To visualize early endosomes, goat polyclonal antibodies against EEA 1 were tagged with anti-goat–Alexa Fluor 594 Zenon reagent. To visualize late endosomes and lysosomes, rabbit polyclonal antibodies against Rab 7 and cathepsin D, respectively, were tagged with anti-rabbit–Alexa Fluor 594 Zenon reagent. After 45 min of incubation with the tagged antibodies, cells were fixed to stabilize the Zenon reagent. Finally, infected cells were visualized with anti-N monoclonal antibodies and goat anti-mouse–Alexa Fluor 350.
Confocal laser scanning microscopy.
Samples were examined with a Leica TCS SP2 laser scanning spectral confocal system (Leica Microsystems) linked to a DM IRB inverted microscope (Leica Microsystems). Argon and helium–neon laser lights were used to excite FITC (488 nm line) and Texas red or Alexa Fluor 594 (543 nm line) fluorochromes. The images were obtained with Leica confocal software and processed with the GNU image manipulation program.
Statistical analysis.
Triplicate assays were compared using a Mann–Whitney U test with SPSS 11.0. P values <0.05 were considered significantly different. For each assay, between 20 and 60 cells were counted.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Binding of specific antibodies to these proteins leads to rapid internalization of the antigen–antibody complexes (Dewerchin et al., 2006). In this paper we aimed to investigate the endocytic route through which the antigen–antibody complexes were internalized.
First, phagocytosis and macropinocytosis were tested as possible internalization routes using the inhibitors wortmannin and latrunculin B. Activity of the inhibitors was validated by determining their effect on the phagocytosis of fluorescent beads. Fig. 2(a) shows cells after the internalization assay in the presence of the inhibitors. The internalization of the control beads was reduced to 49±11 and 18±6 % of untreated controls for wortmannin and latrunculin B, respectively, while the antibody-induced internalization of viral proteins remained unaffected (Fig. 2b). These data suggest that viral proteins were not internalized via phagocytosis nor macropinocytosis and that internalization did not require dynamic actin.
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Possible independence of clathrin-mediated internalization was confirmed using a DN mutant of Eps15, named DIII. The construct D32 was used as a control. To enable expression of the DN protein in primary monocytes, the constructs were cloned into a lentiviral expression system. In monocytes transduced with DIII, the uptake of the control ligand transferrin was reduced to 40±9 %, while the internalization of viral proteins in DIII-transduced FIPV-infected monocytes remained unaffected (Fig. 3). The image of transferrin uptake in a monocyte transduced with DIII shows very little staining at the plasma membrane (Fig. 3). This is because internalization assays are performed when the DN proteins have been expressed for about 12 h. By that time, the transferrin receptors that are locked at the plasma membrane are mostly saturated by the unlabelled transferrin that is present in the culture medium. In monocytes transduced with the control construct D32, internalization of both transferrin and viral proteins was not significantly different from the untransduced controls. Together, these results indicate that antibody-induced internalization of viral antigens in FIPV-infected cells occurs independently of clathrin.
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). Fig. 2(a) shows accumulation of albumin in the preformed caveolae that are locked underneath the plasma membrane, when monocytes are treated with nystatin or methyl-β-cyclodextrin. As illustrated in Fig. 2(a), the inhibitors were unable to completely block the internalization of caveolae in most monocytes.
The independence of the internalization process from caveolae was verified using a GFP-tagged DN mutant of caveolin-1, which was cloned into the lentiviral expression system (Kurzchalia et al., 1992; Pelkmans et al., 2001). GFP-tagged WT caveolin-1 was used as a control . In monocytes transduced with DN caveolin-1, there was no reduction in the internalization of viral antigens, whereas the uptake of the control ligand cholera toxin B was reduced to 40±5 % of the untransduced monocytes (Fig. 4). Transduction with WT caveolin did not have an effect on the internalization of viral proteins or cholera toxin B. Taken together, these results show that the internalization of viral surface-expressed proteins is not mediated by caveolae.
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). Independence from rafts was also reinforced by the results with the inhibitor nystatin (Fig. 2).
To confirm the independence of dynamin, the only structural protein that characterizes two of the clathrin- and caveolae-independent pathways, a GFP-tagged DN mutant from dynamin (2aa) (K44A) was used (Cao et al., 1998). GFP-tagged WT dynamin (2aa) was used as a control (Cao et al., 2000). The constructs were cloned into the lentiviral expression system to allow expression in primary monocytes. Transduction of monocytes with the DN dynamin construct led to a decrease in uptake of the control ligand transferrin to 32±13 % of the untransduced control, while transduction with the WT dynamin construct lead to an increase to 161±21 %. In contrast, internalization of the viral antigen–antibody complexes remained unaffected by either construct (Fig. 5). These results indicate that dynamin, rafts and Rho-GTPases are not required for the internalization of viral antigens.
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). Furthermore, the inhibitors methyl-β-cyclodextrin, dynamin inhibitory peptide and the DN dynamin construct confirmed these results, since they can block clathrin- and caveolae-mediated endocytosis (and two independent pathways) simultaneously.
Internalization of viral antigens is regulated by a serine/threonine kinase
These data show that the proteins known to mediate internalization processes (actin, dynamin, clathrin and caveolin) were not required for internalization. To obtain an insight into the regulation of the internalization process presented here, the importance of phosphorylation and/or dephosphorylation was tested with chemical inhibitors. Representative confocal images of the internalization assays in the presence of inhibitors, and quantification of the results are given in Fig. 6.
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Phosphorylation.
Next, a role for kinases was investigated. Treatment with the broad range serine/threonine kinase inhibitor staurosporine resulted in a reduction in viral protein internalization (21±2 % of control) to a similar extent as the control ligand, beads (23±4 % of control). Fig. 6(a) shows an FIPV-infected monocyte without internalized viral proteins and a monocyte with partial internalization. Using the tyrosine kinase inhibitor genistein, the internalization of viral antigens amounted to 81±4 % of that of the untreated control, while the internalization of the control ligand albumin was 3±5 % of that of the untreated control. The reduction in internalization caused by genistein was significant but small compared with the control ligand albumin. This minor effect could be due to a non-specific action of the drug or it could be that tyrosine kinases play a role in the later stages of the internalization process, such as intracellular transportation. Taken together, these data indicate an important role for a serine/threonine kinase(s) in the antibody-induced internalization of surface-expressed viral proteins in FIPV-infected monocytes.
Co-localization of viral antigen–antibody complexes and endosomal compartments
After internalization, ligands are transported in endocytic compartments. To visualize this intracellular trafficking, co-localization stainings were performed with markers for early endosomes, late endosomes and lysosomes. First, passage through early endosomes was checked by staining EEA 1. The upper row of images in Fig. 7 shows that co-localization was only observed at 0.5 min. In most monocytes, few internalized antigen–antibody complexes were found to co-localize with EEA 1. This indicates that the antigen–antibody complexes resided in early endosomes only briefly before they were transported further into the cell. Co-localization staining with Rab 7 (a late endosome marker) confirmed that the antigen–antibody complexes moved quickly to the late endosomes, in which complexes were observed as soon as 1 min after internalization (Fig. 7, middle row). The antigen–antibody complexes accumulated in the late endosomes until 30 min after antibody addition. By 60 min, all complexes had left the late endosomes. Staining with the lysosome marker cathepsin D showed no co-localization at any time point (Fig. 7, lower row).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). In 1931, membrane ruffles were seen on macrophages, a phenomenon that was later named macropinocytosis (Lewis, 1931). In the 1950s, electron microscopists described cave-like invaginations at the plasma membrane, which were called ‘caveolae intracellulare’. However, it took until 1992 for the major structural protein, caveolin, to be identified (Rothberg et al., 1992). In 1964, internalization through vesicles covered with a dense protein coat was first described (Roth & Porter, 1964). It took another 10 years for the major coat protein, clathrin, to be characterized (Pearse, 1975). Since then, knowledge about these four ‘classical’ internalization pathways has been gathered at increasing speed. Around the turn of the century, the first clathrin- and caveolae-independent pathways were reported (Gilbert & Benjamin, 2000; Lamaze et al., 2001). To date, four independent pathways have been described, two of which involve dynamin for pinching off the vesicle from the plasma membrane. For the other two, no structural protein has been identified so far. They are distinguished from each other solely by the finding that they are mediated by different Rho-GTPases. There are several reports of viruses using an independent pathway for entry into the host cell, although most pathways have not been characterized fully (Gilbert & Benjamin, 2000; Gilbert et al., 2003; Sanchez-San Martin et al., 2004; Damm et al., 2005; Van Hamme et al., 2008).
In this work, we sought to identify the pathway through which the surface-expressed viral proteins in FIPV-infected monocytes were internalized after binding of antibodies. The techniques that were used were based on immunofluorescence and confocal microscopy. Considering the fact that the in vitro infection rate of monocytes varies between 0.1 and 1 %, of which around 50 % have membrane expression, experiments that require a lot of starting material (e.g. flowcytometry or Western blots) could not be performed. In addition, we were restricted to monocytes to perform our experiments since internalization cannot be induced in CrFK or fcwf cell lines (Dewerchin et al., 2006).
The surface-expressed viral proteins in a FIPV-infected monocyte are the spike protein and the membrane protein (Dewerchin et al., 2006). The experiments presented here showed that internalization of these proteins was not clathrin-mediated, since it could not be blocked by any of the six inhibitors (amantadine, latrunculin B, methyl-β-cyclodextrin, dynamin inhibitory peptide, C. difficile toxin B and sodium fluoride) or two DN constructs (eps15 and dynamin) that are known to inhibit clathrin-mediated internalization. Independence from caveolae was also confirmed by using the six inhibitors and two DN constructs. The experiments further indicated that the antigen–antibody complexes were internalized via a new pathway regulated by a serine/threonine kinase(s) but not by phosphatases or tyrosine kinases. Future research will focus on identifying which serine/threonine kinase(s) is of importance.
After internalization, the viral antigen–antibody complexes were further transported into the cell. The main pinocytic trafficking routes are the recycling and the degradative pathway (Mellman, 1996). In the internalization process studied here, internalized antigen–antibody complexes could be observed inside the cell for more than 1 h, making the recycling pathway a highly unlikely route for trafficking. Therefore, co-localization stainings with markers for the degradative endocytic pathway were performed. The stainings revealed that the antigen–antibody complexes resided in the early endosomes only briefly, followed by accumulation of the antigen–antibody complexes in the late endosomes. The complexes left the late endosome between 30 and 60 min but were not observed in the lysosomes, indicating that they did not follow the degradative pathway. The fact that the antigen–antibody complexes could still be observed 90 min after antibody addition confirmed that the complexes were not degraded. It remains to be elucidated whether these antigen–antibody complexes might be of importance during the infection cycle. A model for the internalization pathway combining all the results is given in Fig. 8.
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). This implies that specific cellular components are required that are not ubiquitously expressed, adding to the likelihood of finding a compound that will specifically block this internalization route while leaving the more conventional pathways undisturbed.
In previous reports, we described three patterns of FIPV infection kinetics in monocytes: (i) sustained infection, (ii) no sustained infection and (iii) no infection (Dewerchin et al., 2005). For the experiments in the current paper, monocytes from cats belonging to the first and second pattern were used (at 12 h post-inoculation). No differences were observed in the amount of cells with membrane expression or in the percentage of cells that showed internalization both in the presence or absence of inhibitors. This indicates that internalization is not cat-dependent and that all FCoV-infected monocytes internalize their viral proteins via the same pathway. Even though only about 1 % of the monocytes was infected with FIPV, the fact that every infected monocyte was able to internalize its viral proteins adds to the likelihood that internalization occurs in vivo as well. In addition, we have found that FCoV-infected monocytes in naturally infected FIP cats show no membrane expression (Cornelissen et al., 2007). However, membrane expression returns after in vitro cultivation, which indicates that internalization might occur in vivo. By further characterizing this internalization pathway in vitro and determining which cellular proteins are of importance, we will be able to verify whether viral proteins in naturally infected monocytes are internalized via the same pathway.
In conclusion, surface-expressed viral proteins in FIPV-infected monocytes are internalized through a clathrin- and caveolae-independent internalization pathway that is independent of actin, rafts, dynamin, Rho-GTPases, phosphatases and tyrosine kinases, but dependent on a serine/threonine kinase(s). Internalization of viral antigens through this pathway led to trafficking via the early endosomes to the late endosomes but not to the lysosomes. Despite the growing number of independent internalization pathways, very little is known about the molecular mechanisms underlying these pathways. But, as the pathways are better characterized, it is becoming apparent that they are very well regulated by the cell itself and by the ligand that is being internalized. Undoubtedly, more internalization pathways await discovery and characterization, which might lead to a better understanding of the complex network of all internalization processes.
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ACKNOWLEDGEMENTS |
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2 constructs, to A. Helenius for the WT and DN caveolin-1 constructs, to M. McNiven for the WT and DN dynamin 2 constructs and to J. Vicca for supplying fluorescent beads. We thank E. Naessens for technical assistance and J. Vandekerckhove and C. Ampe for critical reading. |
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Received 18 March 2008;
accepted 29 June 2008.
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