- Email: [email protected]
The minute virus of mice exploits different endocytic pathways for cellular uptake
Virology 482 (2015) 157–166
Contents lists available at ScienceDirect
Virology journal homepage: www.elsevier.com/locate/yviro
The minute virus of mice exploits different endocytic pathways for cellular uptake Pierre O. Garcin, Nelly Panté n Department of Zoology, University of British Columbia, Vancouver, British Columbia, Canada
art ic l e i nf o
a b s t r a c t
Article history: Received 11 January 2015 Returned to author for revisions 30 January 2015 Accepted 25 February 2015
The minute virus of mice, prototype strain (MVMp), is a non-enveloped, single-stranded DNA virus of the family Parvoviridae. Unlike other parvoviruses, the mechanism of cellular uptake of MVMp has not been studied in detail. We analyzed MVMp endocytosis in mouse LA9 fibroblasts and a tumor cell line derived from epithelial–mesenchymal transition through polyomavirus middle T antigen transformation in transgenic mice. By a combination of immunofluorescence and electron microscopy, we found that MVMp endocytosis occurs at the leading edge of migrating cells in proximity to focal adhesion sites. By using drug inhibitors of various endocytic pathways together with immunofluorescence microscopy and flow cytometry analysis, we discovered that MVMp can use a number of endocytic pathways, depending on the host cell type. At least three different mechanisms were identified: clathrin-, caveolin-, and clathrin-independent carrier-mediated endocytosis, with the latter occurring in transformed cells but not in LA9 fibroblasts. & 2015 Elsevier Inc. All rights reserved.
Keywords: Parvovirus Minute virus of mice MVM Cell entry Endocytosis Clathrin
Introduction The cellular entry mechanisms of many viruses are well known, as these mechanisms usually determine viral infectivity and offer potential anti-viral targets. However, the early infection events of the parvovirus minute virus of mice, prototype strain (MVMp), are only recently starting to be elucidated. MVMp is a small (about 26 nm in diameter), non-enveloped virus with a very small (5 kb) singlestranded DNA genome that carries only two open reading frames (ORFs). The 50 ORF encodes the structural proteins VP1 and VP2, while the 30 ORF encodes two non-structural proteins NS1 and NS2 (reviewed by Cotmore and Tattersall, 2006). VP1 and VP2 are produced by alternative splicing of the viral mRNA, and therefore VP1 contains the complete sequence of VP2, as well as a 143-residue unique N-terminal sequence. VP1 and VP2 compose the icosahedral capsid that protects the viral genome from environmental extremes and mediates viral interaction with cell surface receptor(s). NS1, the first viral protein to be expressed during infection, is a multifunctional nuclear phosphoprotein, with roles ranging from initiating viral DNA transcription to cell cycle arrest, DNA-damage response, and release of progeny virions (reviewed by Nuesh and Rommelaere, 2014). NS2 is essential for viral DNA amplification (Ruiz et al., 2011)
n Corresponding author. Fax: þ1 604 822 2416, Tel: 001 604 822 3369 (office) or 001 604 822 0664 (lab) E-mail address: [email protected] (N. Panté).
http://dx.doi.org/10.1016/j.virol.2015.02.054 0042-6822/& 2015 Elsevier Inc. All rights reserved.
and for the egress of progeny virions from the nucleus of infected cells (Eichwald et al., 2002). MVMp attaches to its target cells via sialic acid on an unknown glycoprotein receptor and subsequently enters the endosomal pathway. After endocytosis, MVMp escapes from endocytic compartments into the cytosol by means of the enzymatic action of a phospholipase A2 (PLA2) motif in the unique region of VP1 (Farr et al., 2005). Although the mechanism of endosome escape has been elucidated, the cellular internalization route of MVMp and the cellular factors involved in this process await further studies. Yet, the endocytic mechanisms used by other parvoviruses have been well characterized. For example, canine parvovirus and feline panleukopenia virus use the transferrin receptor to enter their host cells by clathrin-mediated endocytosis (CME) (Parker and Parrish, 2000; Parker et al., 2001); parvovirus B19 also enters via CME (Quattrocchi et al., 2012). Recent studies have shown that, in addition to CME, parvoviruses can use several other endocytic pathways. For example, adeno-associated virus type 2 (AAV-2) uses clathrin-independent carriers (CLICs) (Nonnenmacher and Weber, 2011), AAV-5 uses caveolae-dependent endocytosis (Bantel-Schaal et al., 2009), and porcine parvovirus uses both CME and macropinocytosis (Boisvert et al., 2010). Thus, although parvoviruses share some general features, the literature on their routes of cellular uptake does not allow for mechanistic generalizations. The cellular internalization mechanism used by MVMp has not been studied in detail. In principle, MVMp could use CME, as canine parvovirus does; caveolae-dependent endocytosis, as AAV-5 does; CLICs, as AAV-2 does; or any of the various novel endocytic
158
P.O. Garcin, N. Panté / Virology 482 (2015) 157–166
mechanisms that have been identified recently and are known to be used by several other viruses (Mercer et al., 2010; Schelhaas, 2010). The identification of the endocytic pathway used by MVMp is important to reconstitute its mechanism of infection completely and may help us to understand the oncotropic characteristics of this virus. We used electron microscopy (EM), immunofluorescence (IF) microscopy, and fluorescence-activated cell sorting (FACS) in combination with inhibitors of various endocytic pathways to elucidate the endocytic mechanisms used by MVMp. We found that MVMp is endocytosed from the base of filopodia and from cell-extracellular matrix (ECM) contact sites at the leading edge of migrating cells via at least three different endocytic pathways: clathrin-, caveolin-, and CLIC-mediated endocytosis.
Results Electron microscopy of MVMp cellular uptake To elucidate the MVMp uptake pathway, we performed EM analysis of two different cell lines infected with MVMp: LA9 mouse fibroblasts, a model for the study of MVMp infection, and mouse mammary cells transformed with polyomavirus middle T antigen (PyMT cells, Granovsky et al., 2000), highly migrating cells that are more susceptible to MVMp infection than non-motile cells (Garcin and Panté, 2014). To visualize a large number of virions in endocytic compartments close to the plasma membrane, we performed a short-term MVMp endocytosis assay by incubating the cells with MVMp at 4 1C for 15 min, and then switching the cells to 37 1C for 5 min (PyMT) or 10 min (LA9). After sample preparation for EM, ultrathin en-face cell sections (i.e., sections parallel to the cell monolayer) were positively stained and observed by transmission EM. As illustrated in Fig. 1, both LA9 and PyMT cells displayed MVMp-containing endocytic vesicles at the base of filopodia and near the plasma membrane. Control cells mock incubated with medium instead of MVMp lacked any virioncontaining vesicles (Fig. S1). Immunogold labeling using an antiMVMp capsid antibody confirmed that the round, electron-dense particles of about 26 nm in diameter observed in our electron micrographs were indeed MVMp particles (Fig. S2). The results shown in Fig. 1 are consistent with our recently published finding that MVMp particles cluster at the leading edge of migrating cells rapidly after binding to the cell surface (Garcin and Panté, 2014). These results indicate that MVMp could be endocytosed at the leading edge of migrating cells. Close inspection of the MVMp-containing vesicles by EM in both cell types revealed multiple MVMp particles internalized in coated vesicles, which exhibited all the hallmarks of clathrincoated vesicles (Fig. 2A, top panels). We also detected single MVMp particles in small vesicles and in flask-shaped invaginations typical of caveolae (Fig. 2A, bottom panels), and in elongated tubular compartments associated with CLICs (Fig. 2B). The latter structure was found only in PyMT cells. These results clearly indicate that MVMp uses several endocytic pathways. MVMp cellular uptake occurs in proximity to focal adhesions We previously reported that MVMp clusters at the leading edge of migrating cells (Garcin and Panté, 2014) and our EM results show that MVMp internalization occurs at the base of filopodia (Fig. 1). Thus, MVMp might be endocytosed in proximity to focal adhesion sites, which play a pivotal role during cell migration. We then investigated the proximity of MVMp clusters to focal adhesion sites by using the focal adhesion markers paxillin and α5-integrin. For these experiments, LA9 and PyMT cells were
assayed for MVMp uptake using our short-term MVMp endocytosis assay and prepared for IF microscopy using antibodies for MVMp and paxillin. In another experiment, the short-term MVMp endocytosis assay was performed in cells that were first transfected with α5-integrin-GFP. As shown in Fig. 3A, there was a clear proximity between MVMp clusters and both markers of focal adhesions, indicating that MVMp cellular uptake could indeed take place at contact sites between the cell and the ECM. To confirm the IF finding that MVMp cellular uptake occurs at cell-ECM contact sites, cells were assayed for MVMp uptake with our short-term MVMp endocytosis assay, prepared for EM, and sectioned vertically (i.e., perpendicular to the monolayer) to allow visualization of focal adhesion sites and ECM. Using this approach, vesicles containing MVMp particles forming at cell-ECM contact sites were visualized (Fig. 3B, right panel). EM analysis of vertically sectioned MVMp-infected cells also revealed vesicles containing MVMp particles at the base of filopodia (Fig. 3B, left panel). Thus, MVMp appears to be endocytosed by migrating cells during turnover of the plasma membrane or focal adhesions, and cellular uptake can occur immediately at the base of filopodia, or from focal adhesion sites. MVMp uses both clathrin- and lipid-raft mediated endocytosis Our EM analysis of MVMp-infected cells revealed virions internalized in clathrin-coated vesicles and non-coated pits (Figs. 1 and 2). To verify these observations, we performed our short-term MVMp endocytosis assay with LA9 and PyMT cells, and prepared the cells for IF microscopy using a clathrin-specific antibody. Because some clathrin-independent endocytic pathways are lipid raft-mediated, we also detected lipid rafts by using FITCconjugated cholera toxin B subunit in these experiments. As illustrated in Fig. 4, MVMp co-localized with both clathrin and cholera toxin B, indicating that MVMp uses CME and lipid-raft mediated endocytosis in both LA9 and PyMT cells. MVMp uses a variety of endocytic pathways Our EM results indicate that MVMp internalization occurs via clathrin- and caveolin-carriers, as well as CLICs (Fig. 2). To verify these findings, we investigated MVMp uptake in the presence of various drug inhibitors of these endocytic pathways. CME was inhibited by chlorpromazine, which induces the miss-assembly of clathrin-coated pits at the plasma membrane (Wang et al., 1993). The effect of chlorpromazine on cellular uptake of MVMp was studied using an IF MVMp endocytosis assay in the presence of drugs. For this assay LA9 and PyMT cells were first pretreated with chlorpromazine and bafilomycin A1 (bafA1) for 1 h, then incubated with MVMp at an MOI of 8 for 15 min at 4 1C, and subsequently maintained for 4 h at 37 1C (all in the presence of the drugs). BafA1 inhibits the vacuolar H þ -ATPase in the endosomal membrane that is responsible for acidification (Bayer et al., 1998), and thus bafA1 arrests MVMp in early endosomes, facilitating observation of the internalized virions. As a control, cells were treated with DMSO, instead of chlorpromazine. As documented in Fig. 5, chlorpromazine treatment reduced the MVMp uptake in both LA9 and PyMT cells, but some MVMp immunostaining was still detected in the cytoplasm. This observation supports our findings that cellular uptake of MVMp is via clathrin-dependent route, but also by other endocytic pathways. Similar MVMp uptake experiments were performed in cells treated with genistein, an inhibitor of caveolae-mediated uptake (Pelkmans et al., 2002). IF microscopy showed that genistein reduced MVMp uptake even though some virions were still found in the cells (Fig. 5). Consistent with our EM analysis (Fig. 2A),
P.O. Garcin, N. Panté / Virology 482 (2015) 157–166
159
Fig. 1. Electron micrographs of MVMp endocytosis at the leading edge of migrating cells. LA9 (A) and PyMT (B) cells were assayed for short-term MVMp endocytosis at an MOI of 32 and prepared for EM. Images were acquired using a TEM after en-face ultrathin sectioning (i.e., sectioning parallel to the cell monolayer), and positive staining. Arrowheads point to areas that are enlarged in panels Z1 and Z2.
IF microscopy showed that MVMp uses caveolar endocytosis and other endocytic pathways. Dynamin is involved in several endocytic pathways, including CME, caveolae-mediated endocytosis, phagocytosis, and flotillindependent endocytosis (Doherty and McMahon, 2009). To study the role of dynamin in MVMp cellular uptake, the MVMp endocytosis assay was performed in cells incubated with the dynamin inhibitor dynasore (Macia et al., 2006). Dynasore prevented MVMp uptake into LA9 cells; instead of the typical accumulation of MVMp at the nuclear periphery (Fig. 5, Control panel), the virus accumulated at the cell surface (Fig. 5, Dyna panel). However, dynasore only partially inhibited MVMp uptake by PyMT cells (Fig. 5). These results indicate that MVMp is endocytosed in a dynamin-dependent manner in LA9 cells, but uses a dynaminindependent pathway in PyMT cells. To quantify MVMp uptake in the presence of drugs that inhibit several endocytic pathways, we used FACS analysis after labeling
the cells with a monoclonal antibody against MVMp. Because treatment with 0.1–0.5 unit/ml of neuraminidase (which cleaves terminal sialic acid residues from plasma membrane glycans) in our endocytosis assays removed some but not all the virus from the cell membrane, we developed an indirect MVMp uptake assay in which cells were not permeabilized and, therefore, only viruses attached to the plasma membrane were detected by flow cytometry. In this assay after pretreatment of the cells with drugs for 1 h and an initial incubation of cells with MVMp at an MOI of 8 for 15 min at 4 1C, the cells were further incubated at 37 1C for 4 h in the presence of the drugs inhibitors of endocytic pathways or DMSO (uptake control). As a control binding assay, cells were incubated for 4 h at 4 1C instead of 37 1C. The cells were then labeled with an anti-MVMp antibody and corresponding secondary antibody and analyzed by flow cytometry. As illustrated in Fig. 6, FACS analysis of cells treated with chlorpromazine, genistein, or dynasore confirmed our IF microscopy finding that MVMp
160
P.O. Garcin, N. Panté / Virology 482 (2015) 157–166
Fig. 2. Electron micrographs of MVMp-containing vesicles. (A) LA9 and PyMT cells were assayed for short-term MVMp endocytosis at an MOI of 32 and prepared for EM analysis. Images were acquired using a TEM after en-face ultrathin sectioning (i.e., section parallel to the cell monolayer), and positive staining. Arrowheads point to MVMp particles. Shown are electron micrographs illustrating the presence of MVMp particles in clathrin-coated vesicles (A, upper panels), in flask-shaped vesicles (A, lower panels), and in elongated tubular compartments in PyMT cells (B).
can use several endocytic pathways (Fig. 5). We found that chlorpromazine and genistein partially inhibited cellular uptake of MVMp in both LA9 and PyMT cells (Fig. 6). In contrast, dynasore completely blocked MVMp cellular uptake in LA9 cells, but did not eliminate cellular uptake in PyMT cells, although it was more efficient than chlorpromazine or genistein (Fig. 6). Thus, MVMp is endocytosed in a dynamin-dependent manner via clathrindependent and -independent endocytosis in LA9 cells, but in PyMT cells, MVMp uses an additional dynamin-independent endocytic pathway. This is consistent with our EM observation of MVMp in tubular compartments typical of CLICs in PyMT cells (Fig. 2B). Taken together, our IF and FACS data indicate that MVMp uses various endocytic pathways that may vary depending on target cell type. MVMp is internalized by CLICs in PyMT cells, but not in LA9 cells The CLIC endocytic pathway involves glycosylphosphatidylinositol (GPI) (Howes et al., 2010). Since we observed MVMp in CLIC compartments in PyMT cells by EM (Fig. 2B), we hypothesized that MVMp internalization in these cells occurs through the CLIC pathway. To test this hypothesis, we performed our short-term MVMp endocytosis assay in LA9 and PyMT cells transfected with GPI–GFP. As
documented in Fig. 7, only PyMT cells showed co-localization of MVMp clusters with GPI–GFP at the leading edge. This result supports our conclusion that MVMp can exploit CLICs for internalization in PyMT cells, but not in LA9 cells.
Discussion Many studies have characterized the DNA replication mechanism of MVMp and other parvoviruses, while less is known about the molecular mechanism of early MVMp infection. Understanding the mechanism of MVMp endocytosis might shed light on the oncotropic properties of this virus. Here we report the characterization of MVMp uptake in LA9 mouse fibroblast and PyMT mouse epithelial mammary tumor cells. We found that MVMp cellular uptake occurs at the leading edge of migrating cells via several distinct endocytic pathways. MVMp particles clustered at the leading edge of migrating LA9 and PyMT cells and were endocytosed in these areas. MVMp clusters co-localized with markers of focal adhesions (Fig. 3A), indicating the potential involvement of these structures in MVMp internalization. This finding was supported by EM analysis of vertically sectioned cells assayed for MVMp uptake, in which MVMp-containing endocytic vesicles formed at the base of
P.O. Garcin, N. Panté / Virology 482 (2015) 157–166
161
Fig. 3. MVMp cellular uptake occurs in proximity to focal adhesions. (A) Cells were transfected (α5-GFP panel) or not (Pax panel) with an α5-integrin-GFP (green) construct for 24 h, assayed for short-term MVMp endocytosis, and prepared for IF microscopy. MVMp (red) and paxillin (green) were detected using specific antibodies. Zoom panel shows high magnification images of areas marked with “Z”. (B) Electron micrographs of MVMp endocytosis at the base of a filopodia and from cell-ECM contact sites. Cells were assayed for short-term MVMp endocytosis and prepared for EM. Images were acquired using a TEM after ultrathin cross sectioning (i.e., section perpendicular to the cell monolayer) and positive staining. Arrowheads point to endocytic vesicles containing MVMp. FP: filopodia. ECM: extracellular matrix.
filopodia, but also at cell-ECM contact sites (Fig. 3B). This is consistent with an earlier EM study of MVMp uptake in LA9 cells, which showed massive amounts of MVMp particles internalized at cell-ECM contact sites (Linser et al., 1979). Early EM studies visualized MVMp in clathrin-coated vesicles (Linser et al., 1977), and this endocytic pathway was thought to be the uptake mechanism for MVMp and others parvoviruses.
Nevertheless, recent publications have shown that in addition to clathrin-mediated endocytosis, some parvoviruses could use internalization by CLICs, caveolae-dependent internalization, and macropinocytosis (Nonnenmacher and Weber, 2011; Boisvert et al., 2010; Bantel-Schaal et al., 2009). Thus, MVMp could use any or several of the internalization mechanisms described for other parvoviruses. We confirmed that MVMp co-localizes with clathrin by IF microscopy
162
P.O. Garcin, N. Panté / Virology 482 (2015) 157–166
Fig. 4. IF microscopy analysis of MVMp endocytosis. Cells were assayed for short-term MVMp endocytosis at an MOI of 8 in presence of FITC-conjugated cholera toxin B (CtxB panel, green) or not (Clath panel), and prepared for IF microscopy. MVMp (red) and Clathrin (green) were detected using specific antibodies. Zoom panel shows high magnification images of areas marked with “Z”.
(Fig. 4), and used EM to demonstrate MVMp in clathrin-coated vesicles (Fig. 2A). However, our EM analysis also revealed MVMp particles in flask-shaped vesicles characteristic of caveolaedependent endocytosis (Fig. 2A), and in elongated tubular compartments with the appearance of CLICs (Fig. 2B). These results clearly indicate that MVMp can use various endocytic mechanisms. This conclusion was also supported by our IF co-localization of MVMp with cholera toxin B subunit, indicating that MVMp uses both clathrin- and lipid-raft-mediated endocytosis. To further document the involvement of clathrin, we performed IF and FACS analyses of MVMp uptake in the presence of chlorpromazine, an inhibitor of clathrin-mediated endocytosis, and found that MVMp uptake was partially reduced in the presence of this inhibitor (Figs. 5 and 6). Thus, MVMp enters target cells by clathrin-dependent and other routes of endocytosis. Genistein (an inhibitor of caveolar endocytosis) also reduced, but did not completely inhibit, cellular uptake of MVMp (Figs. 5 and 6), indicating that in addition to clathrin, caveolin could drive endocytosis of MVMp. Our EM analysis confirmed these results (Fig. 4). We also used dynasore, an inhibitor of dynamins, and found slightly different results. While dynasore
completely blocked MVMp uptake in LA9 cells, its inhibitory effect was not complete in PyMT cells (Figs. 5 and 6). These results indicate that both clathrin and caveolin drive MVMp endocytosis in a dynamin-dependent manner in LA9 cells, but MVMp can use an additional dynamin-independent endocytic route in PyMT cells. Based on our EM (Fig. 4) and IF analysis of MVMp co-localization with GPI–GFP (Fig. 7), we suggest that CLIC-mediated endocytosis, which is dynamin-independent (Howes et al., 2010), is also used by MVMp to enter PyMT cells. It is well accepted that the leading edge of migrating cells features high levels of endocytosis to allow for recycling of the plasma membrane or degradation of mature focal adhesion complexes (reviewed in Caswell et al., 2009; Nagano et al., 2012); these mechanisms can occur via clathrin-, caveolin-, and CLIC-mediated endocytosis (Howes et al., 2010; Ezratty et al., 2009; Shi and Sottile, 2008). Our findings demonstrate that MVMp uses all of these internalization mechanisms. Thus, we propose that for MVMp uptake, the virus takes advantage of the various endocytic pathways available for turnover of the plasma membrane or disassembly of focal adhesions during cell migration (summarized in Fig. 8). Given the crucial implication of focal adhesion turnover during cancer cell
P.O. Garcin, N. Panté / Virology 482 (2015) 157–166
163
Fig. 5. MVMp uses various endocytic pathways as shown by IF microscopy. LA9 and PyMT cells were grown onto glass coverslips, assayed for MVMp uptake at an MOI of 8 in the presence of various drug inhibitors of endocytosis, and prepared for IF microscopy. MVMp (red) was detected using a specific antibody, and DAPI was used to observe the nucleus. Zoom panel shows high magnification images of areas marked with “Z”. CPZ: chlorpromazine; Genist: genistein; Dyna: dynasore.
migration and invasion, this could also explain why MVMp preferentially infects some cancer cells. In summary, our results demonstrate that MVMp particles cluster at the base of filopodia and use multiple endocytic pathways that involve clathrin-coated vesicles as well as caveolin- and CLICmediated endocytosis. The endocytic pathway used by MVMp may depend on the cell type and whether or not the cell is engaged in migration.
Materials and methods Cell culture and virus production LA9 and PyMT cells were maintained at 5% CO2 and 37 1C in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% glutamine, and penicillin– streptomycin. MVMp was produced and purified based on the
procedures described by Tattersall et al. (1976) and Williams et al. (2004) with an extended step of dialysis to completely remove cesium chloride. The yield was assessed by plaque assay to be 2.1 109 plaque-forming units per milliliter (pfu/ml). Antibodies and reagents PyMT-transformed mouse epithelial mammary tumor cell line was a generous gift from Drs. J.W. Dennis (University of Toronto) and I.R. Nabi (University of British Columbia). Mouse anti MVMp capsid (MAb B7, Kaufmann et al., 2007) and mouse NS1 (Yeung et al., 1991) antibodies were a generous gift from Dr. P. Tattersall (Yale School of Medicine). The α5-integrin-GFP construct was a generous gift from Dr. A.F. Horwitz (University of Virginia). The GPI– GFP construct was a generous gift from Dr. J. Lippincott-Schwartz (NIH). Rabbit clathrin (ab59710) antibody was purchased from Abcam. Cholera toxin B subunit conjugated with FITC (FITC-CTxB), bafilomycin A1, dynasore hydrate, genistein, and chlorpromazine
164
P.O. Garcin, N. Panté / Virology 482 (2015) 157–166
Fig. 6. MVMp uses various endocytic pathways as shown by FACS. (A) LA9 and PyMT cells were grown onto plastic dishes, assayed for MVMp uptake at an MOI of 8 in presence of various drug inhibitors of endocytosis, and prepared for FACS analysis. MVMp was detected using a specific antibody. The cells were not permeabilized, and the decrease in viral fluorescence reflects the removal of the virus from the cells surface by endocytosis. (B) Quantification of the percentage of MVMp uptake inhibition from three experiments performed as described in A (data are mean7 standard error of the mean, *p o0,05; **p o 0,01). CPZ: chlorpromazine; Genist: genistein; Dyna: dynasore.
Fig. 7. MVMp co-localization with GPI–GFP in PyMT but not LA9 cells. LA9 and PyMT cells were transfected for 24 h with a GPI–GFP (green) construct, assayed for short-term MVMp endocytosis, and prepared for IF microscopy. MVMp (red) was detected using a specific antibody. Zoom panel shows high magnification images of areas marked with “Z”.
hydrochloride were purchased from Sigma. AlexaFluor 647- and 549-conjugated secondary antibodies were purchased from Jackson ImmunoResearch laboratories. The Phycoerythrin (PE)-conjugated secondary antibody for flow cytometry was purchased from IMgenex. Short-term MVMp endocytosis assay Cells grown as monolayers at about 50% confluence were incubated with MVMp (MOI of 8) in infection medium (IM: DMEM þ1% glutamine) for 15 min at 4 1C. The virus-containing
medium was then replaced with fresh IM, the cells were incubated at 37 1C for 5 min (PyMT cells) or 10 min (LA9 cells) and prepared for IF microscopy as indicated below. MVMp was detected with a specific anti-capsid antibody (MAb B7). For analysis of MVMp co-localization with α5-integrin, the cells were transfected with α5-integrin-GFP 24 h before infection using lipofectamin 2000 reagent (Invitrogen) according to the manufacturer's indications. For analysis of MVMp colocalization with cholera toxin B subunit, 2 mg/ml FITC-CtxB was added with MVMp during the initial 15 min incubation period at 4 1C.
P.O. Garcin, N. Panté / Virology 482 (2015) 157–166
165
Fig. 8. Model for MVMp early infection steps. Five steps are illustrated labeled 1–5. (1) In the first step, MVMp binds to its receptor at the plasma membrane; (2) in a second step, MVMp particles use filopodia to accumulate at the leading edge of the cell. This is followed by (3) MVMp endocytosis during plasma membrane turnover and focal adhesion disassembly, (4) trafficking of MVMp-containing endosomes along microtubules, and (5) MVMp escape from late endosomes to the cytoplasm using the PLA2 motif in the unique VP1 region.
MVMp endocytosis assay in the presence of drugs For IF analysis of MVMp uptake in presence of drug inhibitors of endocytosis, cells grown as monolayers at about 50% confluence were treated for 1 h at 37 1C with PBS containing 100 nM bafA1 in addition to 25 mM chlorpromazine (an inhibitor of clathrin-mediated endocytosis), 100 mM dynasore (an inhibitor of dynamins), or 50 mg/ ml genistein (an inhibitor of caveolar endocytosis). As control, cells were incubated with DMSO (diluted 1:1000). Cells were subsequently incubated with MVMp (MOI of 8) for 15 min at 4 1C in IM. The virus-containing medium was then replaced with PBS containing 100 nM bafA1, and the cells were incubated for 4 h at 37 1C and 5% CO2, before preparation for IF microscopy. MVMp was detected with a specific anti-capsid antibody. Optimal concentrations of the drugs used were determined by dose-dependent control experiments testing their efficiency of inhibition during uptake experiments of FITC-conjugated transferrin or cholera toxin B subunit. Immunofluorescence microscopy Cells grown as monolayers on glass coverslips and assayed as indicated above were rinsed once with PBS, fixed with 4% paraformaldehyde in water for 10 min at 4 1C, permeabilized with 0.2% Triton X-100 in PBS containing 2.5% BSA for 2 min at room temperature (RT), and blocked with PBS containing 2.5% BSA for 30 min at RT. Cells were incubated with primary antibodies for 1 h at RT, washed with PBS containing 2.5% BSA, and then incubated with fluorescently-labeled secondary antibodies for 20 min at RT. Cells were rinsed several times with PBS containing 2.5% BSA and mounted in Prolong Gold Antifade with DAPI. Confocal microscopy and image analysis All IF images shown here were acquired using a Fluoview 1000 confocal laser-scanning microscope (Olympus). Images of single cells were obtained with a 100 plan apochromatic objective (pinhole 185 mm), and the lower magnification images with a 60 plan apochromatic (pinhole 150 mm). For the short-term endocytosis assays, images were acquired in the plane of focal contacts.
For endocytosis assays in the presence of drugs, images were acquired in the plane of the nucleus. Electron microscopy Cells grown as monolayers at about 60% confluence on Aclar films (Pelco) were assayed for short-term MVMp endocytosis at an MOI of 32 as indicated above. The cells were then washed in PBS, fixed with 4% glutharaldehyde in 0.1 M sodium cacodylate for 1 h at 4 1C, post-fixed with 1% OsO4 in 0.1 M sodium cacodylate for 1 h at 4 1C, and stained en bloc with 1% uranyl acetate. Cells were then dehydrated and embedded in Epon. Ultrathin (50 nm) sections resulting from en-face or cross sectioning (i.e., sections parallel or perpendicular to the cell monolayer) were cut on a Leica Ultracut Ultramicrotome (Leica Microsystems) using a diamond knife (Diatome), and positively stained with 2% uranyl acetate and 2% lead citrate. Images were acquired using a FEI Tecnai G2 transmission electron microscope operated at an acceleration voltage of 120 kV. Micrographs were digitally recorded using an Eagle 4k CCD camera (FEI). All the EM images showed are representative for at least 3 independent experiments. MVMp uptake assay for FACS analysis Cells grown as monolayers at about 60% confluence were grown on tissue culture dishes and treated for 1 h at 37 1C with PBS containing 25 mM chlorpromazine (an inhibitor of clathrinmediated endocytosis), 100 mM dynasore (an inhibitor of dynamins), or 50 mg/ml genistein (an inhibitor of caveolar endocytosis). As control, cells were incubated with DMSO (diluted 1:1000). Cells were subsequently incubated with MVMp (MOI of 8) in IM containing the drugs for 15 min at 4 1C. The virus-containing medium was then replaced with fresh PBS containing the various drugs indicated above and the cells were incubated for 4 h at 4 1C (binding control) or 37 1C (uptake control). After a wash in ice cold PBS, the cells were detached with trypsin–EDTA for 30 min at 4 1C, fixed with 2% paraformaldehyde in water for 15 min at 4 1C, and blocked with PBS containing 2.5% BSA for 20 min at 4 1C. Cells were then incubated with MVMp anti-capsid antibody (dilution 1:200) for 30 min at 4 1C, washed with PBS containing 2.5% BSA, and incubated with PE-conjugated
166
P.O. Garcin, N. Panté / Virology 482 (2015) 157–166
secondary antibody (dilution 1:500) for 30 min at 4 1C. After several washes in PBS containing 2.5% BSA, the cells were analyzed with a FACS LSRII flow cytometer (BD Biosciences). Optimal concentrations of the drugs used were determined by dose-dependent control experiments testing their efficiency of inhibition during uptake experiments of FITC-conjugated transferrin or cholera toxin B subunit. Acknowledgments We thank Drs. James W. Dennis (University of Toronto) and Ivan R. Nabi (University of British Columbia) for the PyMT-transformed mouse epithelial mammary tumor cell line. We thank Dr. Peter Tattersall (Yale School of Medicine) for the anti-MVMp capsid and NS1 antibodies. We thank Dr. Alan F. Horwitz (University of Virginia) for the α5-integrin-GFP construct, and Dr. Jennifer Lippincott-Schwartz (NIH) for the GPI–GFP construct. We would like to thank Dr. Pascal St. Pierre (Zeiss) for help with confocal microscopy analysis, and Dr. Wayne Vogl (University of British Columbia) for helpful advice in TEM analysis. We thank Drs. Andy Johnson and Lixin Zhou (University of British Columbia) for helpful advice with flow cytometry analysis. This work was supported by Grants from the Canada Foundation for Innovation (CFI 22559), Canadian Institutes of Health Research (CIHR MOP 111270), and Natural Sciences and Engineering Research Council of Canada (NSERC RGPAS 412254-11 and RGPIN 227926-11). NP is a scholar from the Peter Wall Institute for Advanced Studies at UBC. Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.virol.2015.02.054. References Bantel-Schaal, U., Braspenning-Wesch, I., Kartenbeck, J., 2009. Adeno-associated virus type 5 exploits two different entry pathways in human embryo fibroblasts. J. Gen. Virol. 90, 317–322. Bayer, N., Schober, D., Prchla, E., Murphy, R.F., Blaas, D., Fuchs, R., 1998. Effect of bafilomycin A1 and nocodazole on endocytic transport in HeLa cells: implications for viral uncoating and infection. J. Virol. 72, 9645–9655. Boisvert, M., Fernandes, S., Tijssen, P., 2010. Multiple pathways involved in porcine parvovirus cellular entry and trafficking toward the nucleus. J. Virol. 84, 7782–7792. Caswell, P.T., Vadrevu, S., Norman, J.C., 2009. Integrins: masters and slaves of endocytic transport. Nat. Rev. Mol. Cell Biol. 10, 843–853. Cotmore, S.F., Tattersall, P., 2006. A rolling-hairpin strategy: basic mechanisms of DNA replication in the parvoviruses. In: Kerr, JR, Cotmore, SF, Bloom, ME, Linden, CR., Parrish, CR. (Eds.), Parvoviruses. Hodder Arnold, London, UK, pp. 171–188. Doherty, G.J., McMahon, H.T., 2009. Mechanisms of endocytosis. Annu. Rev. Biochem. 78, 857–902. Eichwald, V., Daeffler, L., Klein, M., Rommelaere, J., Salome, N., 2002. The NS2 proteins of parvovirus minute virus of mice are required for efficient nuclear egress of progeny virions in mouse cells. J. Virol. 76, 10307–10319. Ezratty, E.J., Bertaux, C., Marcantonio, E.E., Gundersen, G.G., 2009. Clathrin mediates integrin endocytosis for focal adhesion disassembly in migrating cells. J. Cell Biol. 187, 733–747.
Farr, G.A., Zhang, L.G., Tattersall, P., 2005. Parvoviral virions deploy a capsidtethered lipolytic enzyme to breach the endosomal membrane during cell entry. Proc. Natl. Acad. Sci. USA 102, 17148–17153. Garcin, P.O., Panté, N., 2014. Cell migration is another player of the minute virus of mice infection. Virology 468-470, 150–159. Granovsky, M., Fata, J., Pawling, J., Muller, W.J., Khokha, R., Dennis, J.W., 2000. Suppression of tumor growth and metastasis in Mgat5-deficient mice. Nat. Med. 6, 306–312. Howes, M.T., Kirkham, M., Riches, J., Cortese, K., Walser, P.J., Simpson, F., Hill, M.M., Jones, A., Lundmark, R., Lindsay, M.R., Hernandez-Deviez, D.J., Hadzic, G., McCluskey, A., Bashir, R., Liu, L., Pilch, P., McMahon, H., Robinson, P.J., Hancock, J.F., Mayor, S., Parton, R.G., 2010. Clathrin-independent carriers form a high capacity endocytic sorting system at the leading edge of migrating cells. J. Cell Biol. 190, 675–691. Kaufmann, B., Lopez-Bueno, A., Mateu, M.G., Chipman, P.R., Nelson, C.D., Parrish, C.R., Almendral, J.M., Rossmann, M.G., 2007. Minute virus of mice, a parvovirus, in complex with the Fab fragment of a neutralizing monoclonal antibody. J. Virol. 81, 9851–9858. Linser, P., Bruning, H., Armentrout, R.W., 1977. Specific binding sites for a parvovirus, minute virus of mice, on cultured mouse cells. J. Virol. 24, 211–221. Linser, P., Bruning, H., Armentrout, R.W., 1979. Uptake of minute virus of mice into cultured rodent cells. J. Virol. 31, 537–545. Macia, E., Ehrlich, M., Massol, R., Boucrot, E., Brunner, C., Kirchhausen, T., 2006. Dynasore, a cell-permeable inhibitor of dynamin. Dev. Cell 10, 839–850. Mercer, J., Schelhaas, M., Helenius., A., 2010. Virus entry by endocytosis. Annu. Rev. Biochem. 79, 803–833. Nagano, M., Hoshino, D., Koshikawa, N., Akizawa, T., Seiki, M., 2012. Turnover of focal adhesions and cancer cell migration. Int. J. Cell Biol. 2012, 310616. Nonnenmacher, M., Weber, T., 2011. Adeno-associated virus 2 infection requires endocytosis through the CLIC/GEEC pathway. Cell Host Microbe 10, 563–576. Nuesh, J.P., Rommelaere, J., 2014. Tumor suppressing properties of rodent parvoviruses NS1 proteins and their derivatives. Adv. Exp. Med. Biol. 818, 99–124. Quattrocchi, S., Ruprecht, N., Bonsch, C., Bieli, S., Zurcher, C., Boller, K., Kempf, C., Ros, C., 2012. Characterization of the early steps of human parvovirus B19 infection. J. Virol. 86, 9274–9284. Parker, J.S., William, J.M., Wang, D., O’Brien, J.S., Parrish, C.R., 2001. Canine and Feline parvoviruses can use human transferrin receptors to bind, enter, and infect cells. J. Virol. 75, 3896–3902. Parker, J.S., Parrish, C.R., 2000. Cellular uptake and infection by canine parvovirus involves rapid dynamin-regulated clathrin-mediated endocytosis, followed by slower intracellular trafficking. J. Virol. 74, 1919–1930. Pelkmans, L., Puntener, D., Helenius, A., 2002. Local actin polymerization and dynamin recruitment in SV40-induced internalization of caveolae. Science 296, 535–539. Ruiz, L., Mihaylov, L.S., Cotmore, S.F., Tattersall, P., 2011. Recruitment of DNA replication and damage response proteins to viral replication centers during infection with NS2 mutants of minute virus of mice (MVM). Virology 410, 375–384. Schelhaas, M., 2010. Come in and take your coat off – how host cells provide endocytosis for virus entry. Cell. Microbiol. 12, 1378–1388. Shi, F., Sottile, J., 2008. Caveolin-1-dependent beta1 integrin endocytosis is a critical regulator of fibronectin turnover. J. Cell Sci. 121, 2360–2371. Tattersall, P., Cawte, P.J., Shatkin, A.J., Ward, D.C., 1976. Three structural polypeptides coded for by minute virus of mice, a parvovirus. J. Virol. 20, 273–289. Wang, L.H., Rothberg, K.G., Anderson, R.G., 1993. Mis-assembly of clathrin lattices on endosomes reveals a regulatory switch for coated pit formation. J. Cell Biol. 123, 1107–1117. Williams, W.P., Tamburic, L., Astell, C.R., 2004. Increased levels of B1 and B2 SINE transcripts in mouse fibroblast cells due to minute virus of mice infection. Virology 327, 233–241. Yeung, D.E., Brown, G.W., Tam, P., Russnak, R.H., Wilson, G., Clark-Lewis, I., Astell, C.R., 1991. Monoclonal antibodies to the major nonstructural nuclear protein of minute virus of mice. Virology 181, 35–45.
Contents lists available at ScienceDirect
Virology journal homepage: www.elsevier.com/locate/yviro
The minute virus of mice exploits different endocytic pathways for cellular uptake Pierre O. Garcin, Nelly Panté n Department of Zoology, University of British Columbia, Vancouver, British Columbia, Canada
art ic l e i nf o
a b s t r a c t
Article history: Received 11 January 2015 Returned to author for revisions 30 January 2015 Accepted 25 February 2015
The minute virus of mice, prototype strain (MVMp), is a non-enveloped, single-stranded DNA virus of the family Parvoviridae. Unlike other parvoviruses, the mechanism of cellular uptake of MVMp has not been studied in detail. We analyzed MVMp endocytosis in mouse LA9 fibroblasts and a tumor cell line derived from epithelial–mesenchymal transition through polyomavirus middle T antigen transformation in transgenic mice. By a combination of immunofluorescence and electron microscopy, we found that MVMp endocytosis occurs at the leading edge of migrating cells in proximity to focal adhesion sites. By using drug inhibitors of various endocytic pathways together with immunofluorescence microscopy and flow cytometry analysis, we discovered that MVMp can use a number of endocytic pathways, depending on the host cell type. At least three different mechanisms were identified: clathrin-, caveolin-, and clathrin-independent carrier-mediated endocytosis, with the latter occurring in transformed cells but not in LA9 fibroblasts. & 2015 Elsevier Inc. All rights reserved.
Keywords: Parvovirus Minute virus of mice MVM Cell entry Endocytosis Clathrin
Introduction The cellular entry mechanisms of many viruses are well known, as these mechanisms usually determine viral infectivity and offer potential anti-viral targets. However, the early infection events of the parvovirus minute virus of mice, prototype strain (MVMp), are only recently starting to be elucidated. MVMp is a small (about 26 nm in diameter), non-enveloped virus with a very small (5 kb) singlestranded DNA genome that carries only two open reading frames (ORFs). The 50 ORF encodes the structural proteins VP1 and VP2, while the 30 ORF encodes two non-structural proteins NS1 and NS2 (reviewed by Cotmore and Tattersall, 2006). VP1 and VP2 are produced by alternative splicing of the viral mRNA, and therefore VP1 contains the complete sequence of VP2, as well as a 143-residue unique N-terminal sequence. VP1 and VP2 compose the icosahedral capsid that protects the viral genome from environmental extremes and mediates viral interaction with cell surface receptor(s). NS1, the first viral protein to be expressed during infection, is a multifunctional nuclear phosphoprotein, with roles ranging from initiating viral DNA transcription to cell cycle arrest, DNA-damage response, and release of progeny virions (reviewed by Nuesh and Rommelaere, 2014). NS2 is essential for viral DNA amplification (Ruiz et al., 2011)
n Corresponding author. Fax: þ1 604 822 2416, Tel: 001 604 822 3369 (office) or 001 604 822 0664 (lab) E-mail address: [email protected] (N. Panté).
http://dx.doi.org/10.1016/j.virol.2015.02.054 0042-6822/& 2015 Elsevier Inc. All rights reserved.
and for the egress of progeny virions from the nucleus of infected cells (Eichwald et al., 2002). MVMp attaches to its target cells via sialic acid on an unknown glycoprotein receptor and subsequently enters the endosomal pathway. After endocytosis, MVMp escapes from endocytic compartments into the cytosol by means of the enzymatic action of a phospholipase A2 (PLA2) motif in the unique region of VP1 (Farr et al., 2005). Although the mechanism of endosome escape has been elucidated, the cellular internalization route of MVMp and the cellular factors involved in this process await further studies. Yet, the endocytic mechanisms used by other parvoviruses have been well characterized. For example, canine parvovirus and feline panleukopenia virus use the transferrin receptor to enter their host cells by clathrin-mediated endocytosis (CME) (Parker and Parrish, 2000; Parker et al., 2001); parvovirus B19 also enters via CME (Quattrocchi et al., 2012). Recent studies have shown that, in addition to CME, parvoviruses can use several other endocytic pathways. For example, adeno-associated virus type 2 (AAV-2) uses clathrin-independent carriers (CLICs) (Nonnenmacher and Weber, 2011), AAV-5 uses caveolae-dependent endocytosis (Bantel-Schaal et al., 2009), and porcine parvovirus uses both CME and macropinocytosis (Boisvert et al., 2010). Thus, although parvoviruses share some general features, the literature on their routes of cellular uptake does not allow for mechanistic generalizations. The cellular internalization mechanism used by MVMp has not been studied in detail. In principle, MVMp could use CME, as canine parvovirus does; caveolae-dependent endocytosis, as AAV-5 does; CLICs, as AAV-2 does; or any of the various novel endocytic
158
P.O. Garcin, N. Panté / Virology 482 (2015) 157–166
mechanisms that have been identified recently and are known to be used by several other viruses (Mercer et al., 2010; Schelhaas, 2010). The identification of the endocytic pathway used by MVMp is important to reconstitute its mechanism of infection completely and may help us to understand the oncotropic characteristics of this virus. We used electron microscopy (EM), immunofluorescence (IF) microscopy, and fluorescence-activated cell sorting (FACS) in combination with inhibitors of various endocytic pathways to elucidate the endocytic mechanisms used by MVMp. We found that MVMp is endocytosed from the base of filopodia and from cell-extracellular matrix (ECM) contact sites at the leading edge of migrating cells via at least three different endocytic pathways: clathrin-, caveolin-, and CLIC-mediated endocytosis.
Results Electron microscopy of MVMp cellular uptake To elucidate the MVMp uptake pathway, we performed EM analysis of two different cell lines infected with MVMp: LA9 mouse fibroblasts, a model for the study of MVMp infection, and mouse mammary cells transformed with polyomavirus middle T antigen (PyMT cells, Granovsky et al., 2000), highly migrating cells that are more susceptible to MVMp infection than non-motile cells (Garcin and Panté, 2014). To visualize a large number of virions in endocytic compartments close to the plasma membrane, we performed a short-term MVMp endocytosis assay by incubating the cells with MVMp at 4 1C for 15 min, and then switching the cells to 37 1C for 5 min (PyMT) or 10 min (LA9). After sample preparation for EM, ultrathin en-face cell sections (i.e., sections parallel to the cell monolayer) were positively stained and observed by transmission EM. As illustrated in Fig. 1, both LA9 and PyMT cells displayed MVMp-containing endocytic vesicles at the base of filopodia and near the plasma membrane. Control cells mock incubated with medium instead of MVMp lacked any virioncontaining vesicles (Fig. S1). Immunogold labeling using an antiMVMp capsid antibody confirmed that the round, electron-dense particles of about 26 nm in diameter observed in our electron micrographs were indeed MVMp particles (Fig. S2). The results shown in Fig. 1 are consistent with our recently published finding that MVMp particles cluster at the leading edge of migrating cells rapidly after binding to the cell surface (Garcin and Panté, 2014). These results indicate that MVMp could be endocytosed at the leading edge of migrating cells. Close inspection of the MVMp-containing vesicles by EM in both cell types revealed multiple MVMp particles internalized in coated vesicles, which exhibited all the hallmarks of clathrincoated vesicles (Fig. 2A, top panels). We also detected single MVMp particles in small vesicles and in flask-shaped invaginations typical of caveolae (Fig. 2A, bottom panels), and in elongated tubular compartments associated with CLICs (Fig. 2B). The latter structure was found only in PyMT cells. These results clearly indicate that MVMp uses several endocytic pathways. MVMp cellular uptake occurs in proximity to focal adhesions We previously reported that MVMp clusters at the leading edge of migrating cells (Garcin and Panté, 2014) and our EM results show that MVMp internalization occurs at the base of filopodia (Fig. 1). Thus, MVMp might be endocytosed in proximity to focal adhesion sites, which play a pivotal role during cell migration. We then investigated the proximity of MVMp clusters to focal adhesion sites by using the focal adhesion markers paxillin and α5-integrin. For these experiments, LA9 and PyMT cells were
assayed for MVMp uptake using our short-term MVMp endocytosis assay and prepared for IF microscopy using antibodies for MVMp and paxillin. In another experiment, the short-term MVMp endocytosis assay was performed in cells that were first transfected with α5-integrin-GFP. As shown in Fig. 3A, there was a clear proximity between MVMp clusters and both markers of focal adhesions, indicating that MVMp cellular uptake could indeed take place at contact sites between the cell and the ECM. To confirm the IF finding that MVMp cellular uptake occurs at cell-ECM contact sites, cells were assayed for MVMp uptake with our short-term MVMp endocytosis assay, prepared for EM, and sectioned vertically (i.e., perpendicular to the monolayer) to allow visualization of focal adhesion sites and ECM. Using this approach, vesicles containing MVMp particles forming at cell-ECM contact sites were visualized (Fig. 3B, right panel). EM analysis of vertically sectioned MVMp-infected cells also revealed vesicles containing MVMp particles at the base of filopodia (Fig. 3B, left panel). Thus, MVMp appears to be endocytosed by migrating cells during turnover of the plasma membrane or focal adhesions, and cellular uptake can occur immediately at the base of filopodia, or from focal adhesion sites. MVMp uses both clathrin- and lipid-raft mediated endocytosis Our EM analysis of MVMp-infected cells revealed virions internalized in clathrin-coated vesicles and non-coated pits (Figs. 1 and 2). To verify these observations, we performed our short-term MVMp endocytosis assay with LA9 and PyMT cells, and prepared the cells for IF microscopy using a clathrin-specific antibody. Because some clathrin-independent endocytic pathways are lipid raft-mediated, we also detected lipid rafts by using FITCconjugated cholera toxin B subunit in these experiments. As illustrated in Fig. 4, MVMp co-localized with both clathrin and cholera toxin B, indicating that MVMp uses CME and lipid-raft mediated endocytosis in both LA9 and PyMT cells. MVMp uses a variety of endocytic pathways Our EM results indicate that MVMp internalization occurs via clathrin- and caveolin-carriers, as well as CLICs (Fig. 2). To verify these findings, we investigated MVMp uptake in the presence of various drug inhibitors of these endocytic pathways. CME was inhibited by chlorpromazine, which induces the miss-assembly of clathrin-coated pits at the plasma membrane (Wang et al., 1993). The effect of chlorpromazine on cellular uptake of MVMp was studied using an IF MVMp endocytosis assay in the presence of drugs. For this assay LA9 and PyMT cells were first pretreated with chlorpromazine and bafilomycin A1 (bafA1) for 1 h, then incubated with MVMp at an MOI of 8 for 15 min at 4 1C, and subsequently maintained for 4 h at 37 1C (all in the presence of the drugs). BafA1 inhibits the vacuolar H þ -ATPase in the endosomal membrane that is responsible for acidification (Bayer et al., 1998), and thus bafA1 arrests MVMp in early endosomes, facilitating observation of the internalized virions. As a control, cells were treated with DMSO, instead of chlorpromazine. As documented in Fig. 5, chlorpromazine treatment reduced the MVMp uptake in both LA9 and PyMT cells, but some MVMp immunostaining was still detected in the cytoplasm. This observation supports our findings that cellular uptake of MVMp is via clathrin-dependent route, but also by other endocytic pathways. Similar MVMp uptake experiments were performed in cells treated with genistein, an inhibitor of caveolae-mediated uptake (Pelkmans et al., 2002). IF microscopy showed that genistein reduced MVMp uptake even though some virions were still found in the cells (Fig. 5). Consistent with our EM analysis (Fig. 2A),
P.O. Garcin, N. Panté / Virology 482 (2015) 157–166
159
Fig. 1. Electron micrographs of MVMp endocytosis at the leading edge of migrating cells. LA9 (A) and PyMT (B) cells were assayed for short-term MVMp endocytosis at an MOI of 32 and prepared for EM. Images were acquired using a TEM after en-face ultrathin sectioning (i.e., sectioning parallel to the cell monolayer), and positive staining. Arrowheads point to areas that are enlarged in panels Z1 and Z2.
IF microscopy showed that MVMp uses caveolar endocytosis and other endocytic pathways. Dynamin is involved in several endocytic pathways, including CME, caveolae-mediated endocytosis, phagocytosis, and flotillindependent endocytosis (Doherty and McMahon, 2009). To study the role of dynamin in MVMp cellular uptake, the MVMp endocytosis assay was performed in cells incubated with the dynamin inhibitor dynasore (Macia et al., 2006). Dynasore prevented MVMp uptake into LA9 cells; instead of the typical accumulation of MVMp at the nuclear periphery (Fig. 5, Control panel), the virus accumulated at the cell surface (Fig. 5, Dyna panel). However, dynasore only partially inhibited MVMp uptake by PyMT cells (Fig. 5). These results indicate that MVMp is endocytosed in a dynamin-dependent manner in LA9 cells, but uses a dynaminindependent pathway in PyMT cells. To quantify MVMp uptake in the presence of drugs that inhibit several endocytic pathways, we used FACS analysis after labeling
the cells with a monoclonal antibody against MVMp. Because treatment with 0.1–0.5 unit/ml of neuraminidase (which cleaves terminal sialic acid residues from plasma membrane glycans) in our endocytosis assays removed some but not all the virus from the cell membrane, we developed an indirect MVMp uptake assay in which cells were not permeabilized and, therefore, only viruses attached to the plasma membrane were detected by flow cytometry. In this assay after pretreatment of the cells with drugs for 1 h and an initial incubation of cells with MVMp at an MOI of 8 for 15 min at 4 1C, the cells were further incubated at 37 1C for 4 h in the presence of the drugs inhibitors of endocytic pathways or DMSO (uptake control). As a control binding assay, cells were incubated for 4 h at 4 1C instead of 37 1C. The cells were then labeled with an anti-MVMp antibody and corresponding secondary antibody and analyzed by flow cytometry. As illustrated in Fig. 6, FACS analysis of cells treated with chlorpromazine, genistein, or dynasore confirmed our IF microscopy finding that MVMp
160
P.O. Garcin, N. Panté / Virology 482 (2015) 157–166
Fig. 2. Electron micrographs of MVMp-containing vesicles. (A) LA9 and PyMT cells were assayed for short-term MVMp endocytosis at an MOI of 32 and prepared for EM analysis. Images were acquired using a TEM after en-face ultrathin sectioning (i.e., section parallel to the cell monolayer), and positive staining. Arrowheads point to MVMp particles. Shown are electron micrographs illustrating the presence of MVMp particles in clathrin-coated vesicles (A, upper panels), in flask-shaped vesicles (A, lower panels), and in elongated tubular compartments in PyMT cells (B).
can use several endocytic pathways (Fig. 5). We found that chlorpromazine and genistein partially inhibited cellular uptake of MVMp in both LA9 and PyMT cells (Fig. 6). In contrast, dynasore completely blocked MVMp cellular uptake in LA9 cells, but did not eliminate cellular uptake in PyMT cells, although it was more efficient than chlorpromazine or genistein (Fig. 6). Thus, MVMp is endocytosed in a dynamin-dependent manner via clathrindependent and -independent endocytosis in LA9 cells, but in PyMT cells, MVMp uses an additional dynamin-independent endocytic pathway. This is consistent with our EM observation of MVMp in tubular compartments typical of CLICs in PyMT cells (Fig. 2B). Taken together, our IF and FACS data indicate that MVMp uses various endocytic pathways that may vary depending on target cell type. MVMp is internalized by CLICs in PyMT cells, but not in LA9 cells The CLIC endocytic pathway involves glycosylphosphatidylinositol (GPI) (Howes et al., 2010). Since we observed MVMp in CLIC compartments in PyMT cells by EM (Fig. 2B), we hypothesized that MVMp internalization in these cells occurs through the CLIC pathway. To test this hypothesis, we performed our short-term MVMp endocytosis assay in LA9 and PyMT cells transfected with GPI–GFP. As
documented in Fig. 7, only PyMT cells showed co-localization of MVMp clusters with GPI–GFP at the leading edge. This result supports our conclusion that MVMp can exploit CLICs for internalization in PyMT cells, but not in LA9 cells.
Discussion Many studies have characterized the DNA replication mechanism of MVMp and other parvoviruses, while less is known about the molecular mechanism of early MVMp infection. Understanding the mechanism of MVMp endocytosis might shed light on the oncotropic properties of this virus. Here we report the characterization of MVMp uptake in LA9 mouse fibroblast and PyMT mouse epithelial mammary tumor cells. We found that MVMp cellular uptake occurs at the leading edge of migrating cells via several distinct endocytic pathways. MVMp particles clustered at the leading edge of migrating LA9 and PyMT cells and were endocytosed in these areas. MVMp clusters co-localized with markers of focal adhesions (Fig. 3A), indicating the potential involvement of these structures in MVMp internalization. This finding was supported by EM analysis of vertically sectioned cells assayed for MVMp uptake, in which MVMp-containing endocytic vesicles formed at the base of
P.O. Garcin, N. Panté / Virology 482 (2015) 157–166
161
Fig. 3. MVMp cellular uptake occurs in proximity to focal adhesions. (A) Cells were transfected (α5-GFP panel) or not (Pax panel) with an α5-integrin-GFP (green) construct for 24 h, assayed for short-term MVMp endocytosis, and prepared for IF microscopy. MVMp (red) and paxillin (green) were detected using specific antibodies. Zoom panel shows high magnification images of areas marked with “Z”. (B) Electron micrographs of MVMp endocytosis at the base of a filopodia and from cell-ECM contact sites. Cells were assayed for short-term MVMp endocytosis and prepared for EM. Images were acquired using a TEM after ultrathin cross sectioning (i.e., section perpendicular to the cell monolayer) and positive staining. Arrowheads point to endocytic vesicles containing MVMp. FP: filopodia. ECM: extracellular matrix.
filopodia, but also at cell-ECM contact sites (Fig. 3B). This is consistent with an earlier EM study of MVMp uptake in LA9 cells, which showed massive amounts of MVMp particles internalized at cell-ECM contact sites (Linser et al., 1979). Early EM studies visualized MVMp in clathrin-coated vesicles (Linser et al., 1977), and this endocytic pathway was thought to be the uptake mechanism for MVMp and others parvoviruses.
Nevertheless, recent publications have shown that in addition to clathrin-mediated endocytosis, some parvoviruses could use internalization by CLICs, caveolae-dependent internalization, and macropinocytosis (Nonnenmacher and Weber, 2011; Boisvert et al., 2010; Bantel-Schaal et al., 2009). Thus, MVMp could use any or several of the internalization mechanisms described for other parvoviruses. We confirmed that MVMp co-localizes with clathrin by IF microscopy
162
P.O. Garcin, N. Panté / Virology 482 (2015) 157–166
Fig. 4. IF microscopy analysis of MVMp endocytosis. Cells were assayed for short-term MVMp endocytosis at an MOI of 8 in presence of FITC-conjugated cholera toxin B (CtxB panel, green) or not (Clath panel), and prepared for IF microscopy. MVMp (red) and Clathrin (green) were detected using specific antibodies. Zoom panel shows high magnification images of areas marked with “Z”.
(Fig. 4), and used EM to demonstrate MVMp in clathrin-coated vesicles (Fig. 2A). However, our EM analysis also revealed MVMp particles in flask-shaped vesicles characteristic of caveolaedependent endocytosis (Fig. 2A), and in elongated tubular compartments with the appearance of CLICs (Fig. 2B). These results clearly indicate that MVMp can use various endocytic mechanisms. This conclusion was also supported by our IF co-localization of MVMp with cholera toxin B subunit, indicating that MVMp uses both clathrin- and lipid-raft-mediated endocytosis. To further document the involvement of clathrin, we performed IF and FACS analyses of MVMp uptake in the presence of chlorpromazine, an inhibitor of clathrin-mediated endocytosis, and found that MVMp uptake was partially reduced in the presence of this inhibitor (Figs. 5 and 6). Thus, MVMp enters target cells by clathrin-dependent and other routes of endocytosis. Genistein (an inhibitor of caveolar endocytosis) also reduced, but did not completely inhibit, cellular uptake of MVMp (Figs. 5 and 6), indicating that in addition to clathrin, caveolin could drive endocytosis of MVMp. Our EM analysis confirmed these results (Fig. 4). We also used dynasore, an inhibitor of dynamins, and found slightly different results. While dynasore
completely blocked MVMp uptake in LA9 cells, its inhibitory effect was not complete in PyMT cells (Figs. 5 and 6). These results indicate that both clathrin and caveolin drive MVMp endocytosis in a dynamin-dependent manner in LA9 cells, but MVMp can use an additional dynamin-independent endocytic route in PyMT cells. Based on our EM (Fig. 4) and IF analysis of MVMp co-localization with GPI–GFP (Fig. 7), we suggest that CLIC-mediated endocytosis, which is dynamin-independent (Howes et al., 2010), is also used by MVMp to enter PyMT cells. It is well accepted that the leading edge of migrating cells features high levels of endocytosis to allow for recycling of the plasma membrane or degradation of mature focal adhesion complexes (reviewed in Caswell et al., 2009; Nagano et al., 2012); these mechanisms can occur via clathrin-, caveolin-, and CLIC-mediated endocytosis (Howes et al., 2010; Ezratty et al., 2009; Shi and Sottile, 2008). Our findings demonstrate that MVMp uses all of these internalization mechanisms. Thus, we propose that for MVMp uptake, the virus takes advantage of the various endocytic pathways available for turnover of the plasma membrane or disassembly of focal adhesions during cell migration (summarized in Fig. 8). Given the crucial implication of focal adhesion turnover during cancer cell
P.O. Garcin, N. Panté / Virology 482 (2015) 157–166
163
Fig. 5. MVMp uses various endocytic pathways as shown by IF microscopy. LA9 and PyMT cells were grown onto glass coverslips, assayed for MVMp uptake at an MOI of 8 in the presence of various drug inhibitors of endocytosis, and prepared for IF microscopy. MVMp (red) was detected using a specific antibody, and DAPI was used to observe the nucleus. Zoom panel shows high magnification images of areas marked with “Z”. CPZ: chlorpromazine; Genist: genistein; Dyna: dynasore.
migration and invasion, this could also explain why MVMp preferentially infects some cancer cells. In summary, our results demonstrate that MVMp particles cluster at the base of filopodia and use multiple endocytic pathways that involve clathrin-coated vesicles as well as caveolin- and CLICmediated endocytosis. The endocytic pathway used by MVMp may depend on the cell type and whether or not the cell is engaged in migration.
Materials and methods Cell culture and virus production LA9 and PyMT cells were maintained at 5% CO2 and 37 1C in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% glutamine, and penicillin– streptomycin. MVMp was produced and purified based on the
procedures described by Tattersall et al. (1976) and Williams et al. (2004) with an extended step of dialysis to completely remove cesium chloride. The yield was assessed by plaque assay to be 2.1 109 plaque-forming units per milliliter (pfu/ml). Antibodies and reagents PyMT-transformed mouse epithelial mammary tumor cell line was a generous gift from Drs. J.W. Dennis (University of Toronto) and I.R. Nabi (University of British Columbia). Mouse anti MVMp capsid (MAb B7, Kaufmann et al., 2007) and mouse NS1 (Yeung et al., 1991) antibodies were a generous gift from Dr. P. Tattersall (Yale School of Medicine). The α5-integrin-GFP construct was a generous gift from Dr. A.F. Horwitz (University of Virginia). The GPI– GFP construct was a generous gift from Dr. J. Lippincott-Schwartz (NIH). Rabbit clathrin (ab59710) antibody was purchased from Abcam. Cholera toxin B subunit conjugated with FITC (FITC-CTxB), bafilomycin A1, dynasore hydrate, genistein, and chlorpromazine
164
P.O. Garcin, N. Panté / Virology 482 (2015) 157–166
Fig. 6. MVMp uses various endocytic pathways as shown by FACS. (A) LA9 and PyMT cells were grown onto plastic dishes, assayed for MVMp uptake at an MOI of 8 in presence of various drug inhibitors of endocytosis, and prepared for FACS analysis. MVMp was detected using a specific antibody. The cells were not permeabilized, and the decrease in viral fluorescence reflects the removal of the virus from the cells surface by endocytosis. (B) Quantification of the percentage of MVMp uptake inhibition from three experiments performed as described in A (data are mean7 standard error of the mean, *p o0,05; **p o 0,01). CPZ: chlorpromazine; Genist: genistein; Dyna: dynasore.
Fig. 7. MVMp co-localization with GPI–GFP in PyMT but not LA9 cells. LA9 and PyMT cells were transfected for 24 h with a GPI–GFP (green) construct, assayed for short-term MVMp endocytosis, and prepared for IF microscopy. MVMp (red) was detected using a specific antibody. Zoom panel shows high magnification images of areas marked with “Z”.
hydrochloride were purchased from Sigma. AlexaFluor 647- and 549-conjugated secondary antibodies were purchased from Jackson ImmunoResearch laboratories. The Phycoerythrin (PE)-conjugated secondary antibody for flow cytometry was purchased from IMgenex. Short-term MVMp endocytosis assay Cells grown as monolayers at about 50% confluence were incubated with MVMp (MOI of 8) in infection medium (IM: DMEM þ1% glutamine) for 15 min at 4 1C. The virus-containing
medium was then replaced with fresh IM, the cells were incubated at 37 1C for 5 min (PyMT cells) or 10 min (LA9 cells) and prepared for IF microscopy as indicated below. MVMp was detected with a specific anti-capsid antibody (MAb B7). For analysis of MVMp co-localization with α5-integrin, the cells were transfected with α5-integrin-GFP 24 h before infection using lipofectamin 2000 reagent (Invitrogen) according to the manufacturer's indications. For analysis of MVMp colocalization with cholera toxin B subunit, 2 mg/ml FITC-CtxB was added with MVMp during the initial 15 min incubation period at 4 1C.
P.O. Garcin, N. Panté / Virology 482 (2015) 157–166
165
Fig. 8. Model for MVMp early infection steps. Five steps are illustrated labeled 1–5. (1) In the first step, MVMp binds to its receptor at the plasma membrane; (2) in a second step, MVMp particles use filopodia to accumulate at the leading edge of the cell. This is followed by (3) MVMp endocytosis during plasma membrane turnover and focal adhesion disassembly, (4) trafficking of MVMp-containing endosomes along microtubules, and (5) MVMp escape from late endosomes to the cytoplasm using the PLA2 motif in the unique VP1 region.
MVMp endocytosis assay in the presence of drugs For IF analysis of MVMp uptake in presence of drug inhibitors of endocytosis, cells grown as monolayers at about 50% confluence were treated for 1 h at 37 1C with PBS containing 100 nM bafA1 in addition to 25 mM chlorpromazine (an inhibitor of clathrin-mediated endocytosis), 100 mM dynasore (an inhibitor of dynamins), or 50 mg/ ml genistein (an inhibitor of caveolar endocytosis). As control, cells were incubated with DMSO (diluted 1:1000). Cells were subsequently incubated with MVMp (MOI of 8) for 15 min at 4 1C in IM. The virus-containing medium was then replaced with PBS containing 100 nM bafA1, and the cells were incubated for 4 h at 37 1C and 5% CO2, before preparation for IF microscopy. MVMp was detected with a specific anti-capsid antibody. Optimal concentrations of the drugs used were determined by dose-dependent control experiments testing their efficiency of inhibition during uptake experiments of FITC-conjugated transferrin or cholera toxin B subunit. Immunofluorescence microscopy Cells grown as monolayers on glass coverslips and assayed as indicated above were rinsed once with PBS, fixed with 4% paraformaldehyde in water for 10 min at 4 1C, permeabilized with 0.2% Triton X-100 in PBS containing 2.5% BSA for 2 min at room temperature (RT), and blocked with PBS containing 2.5% BSA for 30 min at RT. Cells were incubated with primary antibodies for 1 h at RT, washed with PBS containing 2.5% BSA, and then incubated with fluorescently-labeled secondary antibodies for 20 min at RT. Cells were rinsed several times with PBS containing 2.5% BSA and mounted in Prolong Gold Antifade with DAPI. Confocal microscopy and image analysis All IF images shown here were acquired using a Fluoview 1000 confocal laser-scanning microscope (Olympus). Images of single cells were obtained with a 100 plan apochromatic objective (pinhole 185 mm), and the lower magnification images with a 60 plan apochromatic (pinhole 150 mm). For the short-term endocytosis assays, images were acquired in the plane of focal contacts.
For endocytosis assays in the presence of drugs, images were acquired in the plane of the nucleus. Electron microscopy Cells grown as monolayers at about 60% confluence on Aclar films (Pelco) were assayed for short-term MVMp endocytosis at an MOI of 32 as indicated above. The cells were then washed in PBS, fixed with 4% glutharaldehyde in 0.1 M sodium cacodylate for 1 h at 4 1C, post-fixed with 1% OsO4 in 0.1 M sodium cacodylate for 1 h at 4 1C, and stained en bloc with 1% uranyl acetate. Cells were then dehydrated and embedded in Epon. Ultrathin (50 nm) sections resulting from en-face or cross sectioning (i.e., sections parallel or perpendicular to the cell monolayer) were cut on a Leica Ultracut Ultramicrotome (Leica Microsystems) using a diamond knife (Diatome), and positively stained with 2% uranyl acetate and 2% lead citrate. Images were acquired using a FEI Tecnai G2 transmission electron microscope operated at an acceleration voltage of 120 kV. Micrographs were digitally recorded using an Eagle 4k CCD camera (FEI). All the EM images showed are representative for at least 3 independent experiments. MVMp uptake assay for FACS analysis Cells grown as monolayers at about 60% confluence were grown on tissue culture dishes and treated for 1 h at 37 1C with PBS containing 25 mM chlorpromazine (an inhibitor of clathrinmediated endocytosis), 100 mM dynasore (an inhibitor of dynamins), or 50 mg/ml genistein (an inhibitor of caveolar endocytosis). As control, cells were incubated with DMSO (diluted 1:1000). Cells were subsequently incubated with MVMp (MOI of 8) in IM containing the drugs for 15 min at 4 1C. The virus-containing medium was then replaced with fresh PBS containing the various drugs indicated above and the cells were incubated for 4 h at 4 1C (binding control) or 37 1C (uptake control). After a wash in ice cold PBS, the cells were detached with trypsin–EDTA for 30 min at 4 1C, fixed with 2% paraformaldehyde in water for 15 min at 4 1C, and blocked with PBS containing 2.5% BSA for 20 min at 4 1C. Cells were then incubated with MVMp anti-capsid antibody (dilution 1:200) for 30 min at 4 1C, washed with PBS containing 2.5% BSA, and incubated with PE-conjugated
166
P.O. Garcin, N. Panté / Virology 482 (2015) 157–166
secondary antibody (dilution 1:500) for 30 min at 4 1C. After several washes in PBS containing 2.5% BSA, the cells were analyzed with a FACS LSRII flow cytometer (BD Biosciences). Optimal concentrations of the drugs used were determined by dose-dependent control experiments testing their efficiency of inhibition during uptake experiments of FITC-conjugated transferrin or cholera toxin B subunit. Acknowledgments We thank Drs. James W. Dennis (University of Toronto) and Ivan R. Nabi (University of British Columbia) for the PyMT-transformed mouse epithelial mammary tumor cell line. We thank Dr. Peter Tattersall (Yale School of Medicine) for the anti-MVMp capsid and NS1 antibodies. We thank Dr. Alan F. Horwitz (University of Virginia) for the α5-integrin-GFP construct, and Dr. Jennifer Lippincott-Schwartz (NIH) for the GPI–GFP construct. We would like to thank Dr. Pascal St. Pierre (Zeiss) for help with confocal microscopy analysis, and Dr. Wayne Vogl (University of British Columbia) for helpful advice in TEM analysis. We thank Drs. Andy Johnson and Lixin Zhou (University of British Columbia) for helpful advice with flow cytometry analysis. This work was supported by Grants from the Canada Foundation for Innovation (CFI 22559), Canadian Institutes of Health Research (CIHR MOP 111270), and Natural Sciences and Engineering Research Council of Canada (NSERC RGPAS 412254-11 and RGPIN 227926-11). NP is a scholar from the Peter Wall Institute for Advanced Studies at UBC. Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.virol.2015.02.054. References Bantel-Schaal, U., Braspenning-Wesch, I., Kartenbeck, J., 2009. Adeno-associated virus type 5 exploits two different entry pathways in human embryo fibroblasts. J. Gen. Virol. 90, 317–322. Bayer, N., Schober, D., Prchla, E., Murphy, R.F., Blaas, D., Fuchs, R., 1998. Effect of bafilomycin A1 and nocodazole on endocytic transport in HeLa cells: implications for viral uncoating and infection. J. Virol. 72, 9645–9655. Boisvert, M., Fernandes, S., Tijssen, P., 2010. Multiple pathways involved in porcine parvovirus cellular entry and trafficking toward the nucleus. J. Virol. 84, 7782–7792. Caswell, P.T., Vadrevu, S., Norman, J.C., 2009. Integrins: masters and slaves of endocytic transport. Nat. Rev. Mol. Cell Biol. 10, 843–853. Cotmore, S.F., Tattersall, P., 2006. A rolling-hairpin strategy: basic mechanisms of DNA replication in the parvoviruses. In: Kerr, JR, Cotmore, SF, Bloom, ME, Linden, CR., Parrish, CR. (Eds.), Parvoviruses. Hodder Arnold, London, UK, pp. 171–188. Doherty, G.J., McMahon, H.T., 2009. Mechanisms of endocytosis. Annu. Rev. Biochem. 78, 857–902. Eichwald, V., Daeffler, L., Klein, M., Rommelaere, J., Salome, N., 2002. The NS2 proteins of parvovirus minute virus of mice are required for efficient nuclear egress of progeny virions in mouse cells. J. Virol. 76, 10307–10319. Ezratty, E.J., Bertaux, C., Marcantonio, E.E., Gundersen, G.G., 2009. Clathrin mediates integrin endocytosis for focal adhesion disassembly in migrating cells. J. Cell Biol. 187, 733–747.
Farr, G.A., Zhang, L.G., Tattersall, P., 2005. Parvoviral virions deploy a capsidtethered lipolytic enzyme to breach the endosomal membrane during cell entry. Proc. Natl. Acad. Sci. USA 102, 17148–17153. Garcin, P.O., Panté, N., 2014. Cell migration is another player of the minute virus of mice infection. Virology 468-470, 150–159. Granovsky, M., Fata, J., Pawling, J., Muller, W.J., Khokha, R., Dennis, J.W., 2000. Suppression of tumor growth and metastasis in Mgat5-deficient mice. Nat. Med. 6, 306–312. Howes, M.T., Kirkham, M., Riches, J., Cortese, K., Walser, P.J., Simpson, F., Hill, M.M., Jones, A., Lundmark, R., Lindsay, M.R., Hernandez-Deviez, D.J., Hadzic, G., McCluskey, A., Bashir, R., Liu, L., Pilch, P., McMahon, H., Robinson, P.J., Hancock, J.F., Mayor, S., Parton, R.G., 2010. Clathrin-independent carriers form a high capacity endocytic sorting system at the leading edge of migrating cells. J. Cell Biol. 190, 675–691. Kaufmann, B., Lopez-Bueno, A., Mateu, M.G., Chipman, P.R., Nelson, C.D., Parrish, C.R., Almendral, J.M., Rossmann, M.G., 2007. Minute virus of mice, a parvovirus, in complex with the Fab fragment of a neutralizing monoclonal antibody. J. Virol. 81, 9851–9858. Linser, P., Bruning, H., Armentrout, R.W., 1977. Specific binding sites for a parvovirus, minute virus of mice, on cultured mouse cells. J. Virol. 24, 211–221. Linser, P., Bruning, H., Armentrout, R.W., 1979. Uptake of minute virus of mice into cultured rodent cells. J. Virol. 31, 537–545. Macia, E., Ehrlich, M., Massol, R., Boucrot, E., Brunner, C., Kirchhausen, T., 2006. Dynasore, a cell-permeable inhibitor of dynamin. Dev. Cell 10, 839–850. Mercer, J., Schelhaas, M., Helenius., A., 2010. Virus entry by endocytosis. Annu. Rev. Biochem. 79, 803–833. Nagano, M., Hoshino, D., Koshikawa, N., Akizawa, T., Seiki, M., 2012. Turnover of focal adhesions and cancer cell migration. Int. J. Cell Biol. 2012, 310616. Nonnenmacher, M., Weber, T., 2011. Adeno-associated virus 2 infection requires endocytosis through the CLIC/GEEC pathway. Cell Host Microbe 10, 563–576. Nuesh, J.P., Rommelaere, J., 2014. Tumor suppressing properties of rodent parvoviruses NS1 proteins and their derivatives. Adv. Exp. Med. Biol. 818, 99–124. Quattrocchi, S., Ruprecht, N., Bonsch, C., Bieli, S., Zurcher, C., Boller, K., Kempf, C., Ros, C., 2012. Characterization of the early steps of human parvovirus B19 infection. J. Virol. 86, 9274–9284. Parker, J.S., William, J.M., Wang, D., O’Brien, J.S., Parrish, C.R., 2001. Canine and Feline parvoviruses can use human transferrin receptors to bind, enter, and infect cells. J. Virol. 75, 3896–3902. Parker, J.S., Parrish, C.R., 2000. Cellular uptake and infection by canine parvovirus involves rapid dynamin-regulated clathrin-mediated endocytosis, followed by slower intracellular trafficking. J. Virol. 74, 1919–1930. Pelkmans, L., Puntener, D., Helenius, A., 2002. Local actin polymerization and dynamin recruitment in SV40-induced internalization of caveolae. Science 296, 535–539. Ruiz, L., Mihaylov, L.S., Cotmore, S.F., Tattersall, P., 2011. Recruitment of DNA replication and damage response proteins to viral replication centers during infection with NS2 mutants of minute virus of mice (MVM). Virology 410, 375–384. Schelhaas, M., 2010. Come in and take your coat off – how host cells provide endocytosis for virus entry. Cell. Microbiol. 12, 1378–1388. Shi, F., Sottile, J., 2008. Caveolin-1-dependent beta1 integrin endocytosis is a critical regulator of fibronectin turnover. J. Cell Sci. 121, 2360–2371. Tattersall, P., Cawte, P.J., Shatkin, A.J., Ward, D.C., 1976. Three structural polypeptides coded for by minute virus of mice, a parvovirus. J. Virol. 20, 273–289. Wang, L.H., Rothberg, K.G., Anderson, R.G., 1993. Mis-assembly of clathrin lattices on endosomes reveals a regulatory switch for coated pit formation. J. Cell Biol. 123, 1107–1117. Williams, W.P., Tamburic, L., Astell, C.R., 2004. Increased levels of B1 and B2 SINE transcripts in mouse fibroblast cells due to minute virus of mice infection. Virology 327, 233–241. Yeung, D.E., Brown, G.W., Tam, P., Russnak, R.H., Wilson, G., Clark-Lewis, I., Astell, C.R., 1991. Monoclonal antibodies to the major nonstructural nuclear protein of minute virus of mice. Virology 181, 35–45.