Rhesus Rotavirus Entry into a Polarized Epithelium Is Endocytosis Dependent and Involves Sequential VP4 Conformational Changes

JOURNAL OF VIROLOGY, Mar. 2011, p. 2492 2503 Vol. 85, No. 6 0022-538X/11/$12.00 doi:10.1128/jvi.02082-10 Copyright 2011, American Society for Microbiology. All Rights Reserved. Rhesus Rotavirus Entry into a Polarized Epithelium Is Endocytosis Dependent and Involves Sequential VP4 Conformational Changes Marie Wolf,* Phuoc T. Vo, and Harry B. Greenberg Department of Medicine and Microbiology and Immunology, Stanford University School of Medicine, Stanford, California Received 30 September 2010/Accepted 22 December 2010 Rotavirus (RV) cell entry is an incompletely understood process, involving VP4 and VP7, the viral proteins composing the outermost layer of the nonenveloped RV triple-layered icosahedral particle (TLP), encasing VP6. VP4 can exist in three conformational states: soluble, cleaved spike, and folded back. In order to better understand the events leading to RV entry, we established a detection system to image input virus by monitoring the rhesus RV (RRV) antigens VP4, VP6, and VP7 at very early times postinfection. We provide evidence that decapsidation occurs directly after cell membrane penetration. We also demonstrate that several VP4 and VP7 conformational changes take place during entry. In particular, we detected, for the first time, the generation of folded-back VP5 in the context of the initiation of infection. Folded-back VP5 appears to be limited to the entry step. We furthermore demonstrate that RRV enters the cell cytoplasm through an endocytosis pathway. The endocytosis hypothesis is supported by the colocalization of RRV antigens with the early endosome markers Rab4 and Rab5. Finally, we provide evidence that the entry process is likely dependent on the endocytic Ca 2 concentration, as bafilomycin A1 treatment as well as an augmentation of the extracellular calcium reservoir using CaEGTA, which both lead to an elevated intraendosomal calcium concentration, resulted in the accumulation of intact virions in the actin network. Together, these findings suggest that internalization, decapsidation, and cell membrane penetration involve endocytosis, calcium-dependent uncoating, and VP4 conformational changes, including a fold-back. Rotaviruses (RVs) are the single most important cause of severe diarrhea requiring the hospitalization of infants and young children worldwide. Diarrheal disease caused by rotaviruses is associated with more than 500,000 deaths per year, predominantly in developing countries, and is a leading cause of pediatric hospitalizations. These viruses are also relatively common causes of disease in the elderly and the immunocompromised as well as a wide variety of animal species. Although much has been learned about various components of the viral replication cycle, the early RV entry pathway is still poorly understood. Unlike enveloped viruses that fuse to cell membranes, most nonenveloped viruses induce lysis or pore formation in the plasma or endocytic vesicle membranes in order to enter cells (43). Whether RV behaves like the other nonenveloped viruses during membrane penetration remains controversial. A direct entry of RV particles was initially proposed (26, 39), but more recent studies suggested an endocytosis step during RV entry (8, 33, 55). Most of the RV entry data were obtained by using the simian rhesus RV (RRV) strain and MA104 cells as a model, but different RV strains appear to use various endocytosis pathways (33). Although drugs affecting dynamin and cholesterol have been shown to impair RRV infection (33, 55), drugs and dominant negative mutants known to impair clathrin or caveolin-dependent endocytosis have no inhibitory effects on RRV replication (33, 55). Moreover, conflicting observations have been made about the effects of drugs that block endosomal acidification (8, 15, 33). There is * Corresponding author. Mailing address: Stanford University, VA- PAHCS, 3801 Miranda Avenue (154 C), Building 101, Room C4-181, Palo Alto, CA 94304. Phone: (650) 493-5000, ext. 63124. Fax: (650) 852-3259. E-mail: mmwolf@stanford.edu. Published ahead of print on 29 December 2010. thus no definitive model of the RV entry process at this time (2, 42, 54). The restricted tissue and host tropism displayed by RV during in vivo infection indicates very specific host-virus interactions. These restricted interactions are likely influenced at least in part by a multistep process that begins with RV entry into epithelial cells. Several host cell RV receptors have been proposed. Terminal sialic acid (SA) augments the cell attachment of some RV strains, including RRV (12, 35), but is not essential for infection (66). Infection by the majority of rotaviruses, including human strains, might involve subterminal SA (17, 32). Several integrin family members ( 2 1, 4 1, x 2, and V 3 ) play a role in attachment and postattachment events in vitro (28, 30, 42) but may not all be essential for entry (36, 42). In addition, the heat shock protein hsc70 has also been implicated in late entry events and changes in the RV particle (31, 49). However, the cell surface receptors involved in viral entry in vivo, either in the gut or elsewhere, have not been well characterized. On the viral side, the outermost layer of the 100-nm triplelayered icosahedral particle (TLP) is composed of two proteins, VP4 and VP7, which are responsible for the initial virus-cell interaction. These 2 proteins fully encapsidate a double-layered particle (DLP), which is composed primarily of an assembly of VP6 trimers. VP7 is a calcium-binding trimeric glycoprotein located on the surface of the virion, and VP4 forms spikes protruding through the VP7 surface. Both VP4 and VP7 are necessary for early steps of infection (42). Recent studies using RRV have added a wealth of structural specificity to the understanding of both VP7 and VP4 (1, 9, 65). Both of these proteins contain motifs for binding members of the heterodimeric / integrin family (29, 67), and both induce neu- 2492

VOL. 85, 2011 VISUALIZATION OF ROTAVIRUS ENTRY 2493 tralizing antibodies (Abs) that, in part, block RV entry (52). In order to efficiently mediate productive infection, RV penetration of the epithelial cell membrane requires an initial trypsinmediated proteolytic cleavage of VP4 into two fragments, VP5* and VP8* (24). This cleavage is associated with the transition from a flexible form to a rigid spike that can be visualized by cryo-electron microscopy (EM) with approximate 2-fold symmetry in the portion that protrudes most from the virion, 3-fold symmetry of the part buried under the VP7 shell, and no symmetry between these regions. For the sialic acid binding strains of RV, the amino-terminal VP8* domain mediates initial cellular attachment via binding to a sialic acidcontaining moiety. Cell permeabilization properties have been localized to the carboxyl-terminal VP5* component of VP4 (22) and were recently suggested to depend on the hydrophobicity of the VP5 apex (40). VP5* also contains regions implicated in integrin binding (29). Based on the crystal structure, a head, body, stalk, and foot model was proposed, in which VP5* is part of the foot and the body and VP8* forms part of the body and head of the VP4 spike (20, 21). More recent structural studies identified three VP4 conformational states: soluble, cleaved spike (following trypsin activation), and folded back. While the folded-back form has been generated in vitro as a serial protease digestion product containing only the N- terminal half of VP5* in a folded-back state (VP5CT), it remains unclear as to whether and when it occurs during the infectious process (19, 63 65). The VP5 fold-back is hypothesized to happen during and to promote plasma membrane penetration at the time of initial viral entry into the cell (64). The VP5 fold-back depends on both membrane interactions and virus uncoating (63, 65), suggesting that this rearrangement is one of the final steps of RV entry. Interestingly, these VP4 sequential conformational changes resemble the conformational transitions of membrane fusion proteins of enveloped viruses (43). In this study, we have used an imaging approach to further interrogate the sequence of entry of RRV into a polarized epithelium. We established a detection system to image the input virus by monitoring RRV antigens VP4, VP6, and VP7 at very early times postinfection (p.i.), prior to the onset of detectable viral replication. We provide evidence that decapsidation occurs directly after cell membrane penetration, as assessed by the disappearance of trimeric VP7 staining. We also demonstrate that several VP4 conformational changes take place during entry: VP8* staining is lost at the time of cell penetration and is not found within the cytoplasm. On the other hand, VP5* is detected in the cell cytoplasm within an hour of infection. We detected, for the first time, the generation of the folded-back conformation of VP5 in the context of the initiation of infection. This folded-back conformation appears to be limited to the entry step. Based on the imaging analysis and the use of pharmacological intervention, we furthermore demonstrate that RV enters the cell cytoplasm through an endocytosis pathway. The endocytosis hypothesis is supported by the colocalization of RRV antigens with the early endosome markers Rab4 and Rab5. Taken together, our results give new and additional insights into RV entry. Finally, we provide evidence that the entry process is likely dependent on the endocytic Ca 2 concentration, as the inhibition of the vacuolar H -ATPase (V-ATPase), which leads to an elevation of the intraendosomal Ca 2 concentration, blocked viral entry. Additionally, an augmentation of the extracellular calcium reservoir, which leads to an elevated intraendosomal calcium concentration and to a delay in the dissipation of the Ca 2 gradient, resulted in the accumulation of TLP-associated VP7 in the actin network. MATERIALS AND METHODS Cells, virus, and bacteria. MDCK cells (a kind gift from Manuel Amieva, Stanford University) were grown in Dulbecco s modified Eagle s medium (DMEM) supplemented with 10% fetal calf serum (FCS) and 100 IU penicillin and streptomycin/ml. MDCK cells were seeded onto 12-mm polycarbonate tissue culture inserts with 0.4- m pores (Transwell filters, Costar; Corning Inc., Corning, NY) at a density of 10 6 cells/cm 2 and supplemented with fresh medium every day for 5 days. The integrity of the cellular monolayer was assessed by measuring the transepithelial resistance at day 5 (Millicell; Millipore, Bedford, MA), as described elsewhere previously (61). Rotavirus strain RRV was grown as previously described (16). Concentrated virus preparations were obtained by centrifuging bulk RRV preparations at 1,000 rpm for 5 min to remove cell debris, and the supernatants were centrifuged at 110,000 g for 2 h. Pellets were resuspended in small volumes, and titers were determined with MA104 cells. The titers of the concentrated RRV preparation used ranged from 2 10 9 to 7 10 9 PFU per ml (37). RRV TLPs and DLPs, produced as previously described, were purified from MA104 cell lysates by cesium chloride density gradient centrifugation following Genetron extraction and pelleting through a sucrose cushion (6, 13). DLPs were created by treating TLPs with 20 nm EDTA as previously described (17). Viral preparations were trypsin activated (5 g/ml) at 37 C for 20 to 30 min prior to infection. Virus, as indicated, was added to the upper chamber of the transwell on the cell monolayer at room temperature, and infected cells were immediately put in 5% CO 2 at 37 C. After infection (10 min or 1 h postinfection [p.i.] at 37 C), cells were washed 3 times with phosphate-buffered saline (PBS), fixed with 3% paraformaldehyde in PBS for 10 min, and immunostained as described below. Green fluorescent protein (GFP)-expressing Listeria monocytogenes strain LM 124 (47), derived from wild-type strain 10403S, was a kind gift from Manuel Amieva and was grown as previously described (47, 48). Antibodies. The monoclonal antibodies (MAbs) used in this study were all produced as previously described (25). MAbs recognized the following RV antigens: VP6 (1E11); VP4 and VP8* (7A12); VP5* (2G4); soluble, Western blot reactive, monomeric VP4 (VP5 region) (HS-2); folded-back-specific VP5 (1E2); and VP7 in the trimeric and virion-associated (159) or soluble, not associated with the virion, (60) conformation (52, 58, 65). Commercial rabbit polyclonal antibodies directed against the endosomal markers Rab4, Rab5, and Rab7 were purchased from Abcam. GFP-expressing L. monocytogenes was detected by the incubation of samples with biotin-conjugated rabbit anti-l. monocytogenes antibody for all antigens (YVS4207; Accurate Chemical & Scientific Corp., Westbury, NY). Immunofluorescence and confocal imaging. Polarized MDCK cells were infected with trypsin-activated RRV at a multiplicity of infection (MOI) of 500 for 10 min, 1 h, or 16 h; washed 3 times with PBS; and fixed with 3% paraformaldehyde in 100 mm phosphate buffer (ph 7.4) for 10 min. Permeabilization conditions. In some experiments cells were immunostained directly under permeabilizing conditions. When needed, cells were first immunostained under nonpermeabilizing conditions (PBS plus 3% bovine serum albumin) to detect viral antigens present at the cell surface. The cells were subsequently immunostained by using permeabilizing buffers (PBS, 1% saponin, 1% Triton X-100, and 3% bovine serum albumin) with different secondary antibodies to detect total viral antigens present both on the cell surface and within the cell. VP6 (1E11), trimeric (159) and monomeric (60) VP7, VP5* (2G4), and VP8* (7A12) immunostaining. Cells were either immunostained under permeabilizing conditions only or first incubated with anti-rv MAb and Alexa Fluor (AF) 647 anti-mouse secondary antibody (Invitrogen) under nonpermeabilizing conditions, as described above. Cells were subsequently stained in a second step with anti-rv MAb and AF 488 anti-mouse secondary antibody under permeabilizing conditions (Invitrogen) and AF 594 phalloidin (Invitrogen). Folded-back VP5 (1E2) immunostaining. Cells in the first step were incubated with VP5 trimer-specific MAb 1E2 and goat anti-mouse AF 647 antibody (Invitrogen) followed by donkey anti-goat AF secondary antibody (Invitrogen) under nonpermeabilizing conditions. In a second step, the cells were incubated with 1E2 and goat anti-mouse AF 488 antibody (Invitrogen) followed by donkey

2494 WOLF ET AL. J. VIROL. anti-goat AF 488 secondary antibody (Invitrogen) under permeabilizing conditions and AF 594 phalloidin. L. monocytogenes immunofluorescence. An immunofluorescence inside/outside staining that distinguishes extracellular from intracellular GFP-tagged L. monocytogenes was performed as previously described (47). Briefly, MDCK cells were infected with L. monocytogenes at an MOI of 5 for 1 h and fixed. The total amount of L. monocytogenes bacteria was visualized due to its GFP expression, whereas cell surface-attached L. monocytogenes bacteria were detected by using nonpermeabilizing conditions, anti-l. monocytogenes polyclonal antibody, and the appropriate AF 647 secondary antibody. Colocalization experiments. Cells were immunostained using only permeabilizing conditions to detect the total numbers of viral antigens present on or in the cell. In addition, cells were incubated with a 1/100 dilution of polyclonal antibodies to Rab4, Rab5, or Rab7 (Abcam) and AF 488 anti-rabbit secondary antibodies (Invitrogen). All samples were then mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, CA) and imaged with a confocal microscope (LSM510; Zeiss, Jena, Germany) using 0.2- m sections. z stacks were reconstructed into three dimensions by using Volocity software (Improvision; Perkin- Elmer Company, Coventry, England). Staining quantification. Using Volocity software, immunostained dots were identified and quantified in an automated way based on the intensity and size of each dot ( 1 m and 20 m). Cell surface antigens were quantified based on the staining obtained under nonpermeabilizing conditions (AF 647 staining), whereas total antigen was quantified by using staining under permeabilizing conditions (AF 488 staining). In order to distinguish antigens present in the actin network located just below the cell surface from fully cytoplasmic viral antigens, sections of a 1- m thickness were cropped in the z stacks, and staining was quantified for each section. Actin-positive sections were defined in the apical sections of the epithelium by a total surface at least 80% positive for phalloidin. Dots were counted in at least 3 separate experiments, and each experiment consisted of a quantification of 3 representative fields for each condition. Pharmacological interventions. Cells were pretreated with each of the following drugs at the indicated concentrations at 37 C for 30 min to 1 h prior to and/or during infection: 100 nm bafilomycin A1 (BafA1) (Sigma), 20 nm concanamycin A (Sigma), 10 M chloroquine (Sigma), 10 mm ammonium chloride (Sigma), 10 mm methyl- -cyclodextrin (Sigma), 100 M dynasore (Sigma), 50 M leupeptin (EMD), or 5 M cathepsin inhibitor III (Fisher). Since some of these drugs are soluble only in dimethyl sulfoxide (DMSO) or ethanol, the effect of similar volumes of pure DMSO or ethanol on infectivity was tested as a control. In order to evaluate the specificity of the drug for RV cell entry, cells were also treated starting 1 h after infection as a control. Furthermore, none of the drugs tested had an effect on RV infectivity when assessed by direct in vitro treatment of virus stocks for 1 h with each drug followed by serial dilution and a titration-based focus assay. CaEGTA treatment. Cells were treated with 10 mm ethylene glycol-bis( aminoethyl ether)-n,n,n,n -tetraacetic acid (CaEGTA) at the time of infection and infected for 15 min at 37 C (8). Low-pH pulses. Cells were pretreated with BafA1 for 30 min at 37 C, infected for1hat37 C, washed 3 times with PBS, incubated for 3 min with citrate buffers ranging from ph 3 to 7, washed again 3 times with PBS, incubated for2hat37 C in DMEM, fixed with 3% paraformaldehyde in PBS for 15 min, and immunostained as described above. Statistical analysis. The statistical significance of differences between means was evaluated by using a one-way analysis of variance (ANOVA) test followed by Tukey s multiple-comparison posttest. P values of less than 0.05 were considered to be significant. RESULTS Input RV can be visualized during cell entry and infection. In order to examine RV entry microscopically, we needed a model system to efficiently visualize the very early steps of infection. To enable these experiments, we took advantage of a panel of MAbs directed at selected structural proteins of the virion which would be expected to be present on input virus. These MAbs specifically bound to various VP4 or VP7 conformations or regions as well as to the trimeric form of VP6, the major structural protein of the virus and the primary component of the DLP. Our working hypothesis was that we could identify RV MAbs that would be specific and sensitive enough to visualize and monitor the fate of input infectious RV during the entry process. We selected the simian RRV strain for our studies for three reasons. First, the RRV entry pathway is in part sialic acid dependent, which simplifies the model (35). Second, RRV is highly infectious to a large number of cell lines of different host origins and grows to high titers, making it perhaps one of the easiest rotaviruses to study with this model system. Finally, and most importantly, several of our VP7 and VP4 MAbs are RRV specific. We established a classic model polarized epithelium system using the MDCK canine epithelial cell line. MDCK cells form a very well-characterized, highly polarized epithelium when grown on permeable filter supports (48, 59). Interestingly, MDCK cells express all integrins described previously to play a role in RV entry (44, 56). As MDCK cells have seldom been used to study RV infection (11, 51, 61), we first established that they were permissive for RRV infection. While the yield from MDCK cells infected with RRV at a multiplicity of infection (MOI) of 1 was approximately 6- to 10-fold lower than the yield from MA104 cells infected at the same MOI, the resulting titer was 10,000-fold above the input background titer (data not shown). This indicated that MDCK cells represent a permissive host for efficient RRV replication. Of note, the MOIs used in this study were calculated based on virus titration on MA104 cells. Thus, an MOI of 500 actually corresponds to an MOI of 50 to 80 in MDCK cells. We next examined whether this cell culture system could be employed to examine the very earliest events in infection. In doing so, we utilized a confocal microscopic approach that was previously used to study early events in bacterium-host cell interactions (48). Cells were infected for short times (10 s to 1 h) at high MOIs, conditions chosen to enhance our ability to visualize input virus binding to and entering cells very early in the infection cycle. Of note, the nonstructural proteins NSP2 and NSP4 cannot be visualized by the immunostaining of infected cells earlier than 2 to 3 h postinfection, nor can radiolabeled viral proteins be detected at these early time points (10), supporting the conclusion that the immunostaining observed is reflective of input virus and not viral replication. Indeed, we showed that at 1 h postinfection, NSP2 was not visualized in the cell (Fig. 1A). In addition, mock-infected cells were stained for all MAbs used in this study, and the micrographs obtained were all identical to those in Fig. 1A. A trimer-specific VP6 MAb (1E11) was initially used to monitor RRV entry. The epithelium was imaged by confocal microscopy, and cell edges were visualized indirectly by using phalloidin staining of the F-actin cytoskeleton located just below the cell membrane (60). In these studies, VP6 staining was observed to increase over the 1-h time period. VP6 was visualized initially only at discrete locations in intimate contact with the actin network at 10 min postinfection (Fig. 1B). Conversely, numerous discrete foci were detected both close to the cell membrane and deep within the cell cytoplasm at 1 h p.i. (Fig. 1C). Since VP6 is not present on the surface of intact TLPs and is not exposed until TLPs initiate uncoating to become DLPs, the localization of VP6 in the actin network, very close to the cell membrane (Fig. 1B, white arrows), supports the conclusion that RRV uncoating initiates in a compartment intimately linked to the cell membrane. On the other hand, by

VOL. 85, 2011 VISUALIZATION OF ROTAVIRUS ENTRY 2495 FIG. 1. Detection of input RV antigens in a polarized epithelium. Polarized MDCK cells were infected with tissue culture-derived RRV (A to C and E to I) or monodispersed density gradient-purified RRV TLPs (D) at a high MOI (MOI of 500) and stained with monoclonal antibodies (MAbs) specific to NSP2 (MAb 191) (A), trimeric VP6 (MAb 1E11) for 10 min (B) or 1 h postinfection (C and D), VP8* (MAb 7A12) (E), VP5* (MAb 2G4) (F), monomeric VP4 (MAb HS2) (G), trimeric VP7 (MAb 159) (H), or soluble monomeric VP7 (MAb 60) (I) at the indicated time intervals postinoculation. Optical sections were taken at a 0.2- m resolution through the cell monolayers, and z stacks were reconstructed into three dimensions. RV antigens are visualized in green, while red staining corresponds to the actin network visualized using labeled phalloidin. White and black arrows indicate actin-associated and intracellular staining, respectively, as described in Materials and Methods. Mouse anti-rv MAbs were detected by using a donkey anti-mouse Alexa 488 antibody. These micrographs are representative of at least 3 separate experiments, and for each experiment, at least 3 fields were quantified. Scale bar, 10 m. 1 h postinfection, large amounts of VP6 staining did not localize to the actin network and were instead clearly present in the cytoplasm (Fig. 1C, black arrows). In order to determine if the individual focus staining corresponded to individual RRV particles that had associated with or entered the cell, experiments were performed by using density gradient-purified and monodispersed (via 0.22- m ultrafiltration) preparations of TLPs (as monitored by negativestained electron microscopy [EM] [data not shown]). The same staining pattern was observed by using the purified and ultrafiltered TLPs as that observed with the crude RV supernatant (Fig. 1D versus C), suggesting that, in this panel, each dot corresponds to an individual or a small aggregate of viral particles. While the fluorescent halo observed in the confocal images is not directly proportional to the size of a detected object (34), the difference in dot sizes between Fig. 1C and D suggests that very small aggregates, but not individual particles, are likely being detected using the nonpurified and nonfiltered virus preparations. Since staining patterns were highly similar under both conditions and dots were easier to see using nonpurified preparations, we decided to use nonpurified virus preparations for further studies. Nevertheless, we made sure that individual particles and small aggregates do use the same entry pathway by reproducing all major findings of this paper using purified TLPs (data not shown). Of note, very similar confocal immunostaining photomicrographs were observed previously in studies of cell binding to and entry into MA104 cells of purified baculovirus-expressed recombinant TLP viruslike particles (VLPs) (7). That same study observed baculovirus-expressed recombinant DLPs (lacking VP4 and VP7) also attached to the MA104 cell surface. However, in our studies, MDCK cells exposed to purified DLPs did not reveal any cell surface or intracellular VP6 staining (identical to Fig. 1A). Finally, cells exposed to entry-incompetent, non-trypsin-activated, nonpurified RRV (titer 100-fold lower than that of trypsin-activated virus) exhibited only rare VP6 cell surface staining and no VP6 intracellular staining (data not shown). These data indicate that the MDCK-associated VP6 immunostaining observed was dependent on an infectious virus entry pathway requiring both the presence of the surface proteins VP4 and VP7 and trypsin activation. These observations support the notion that a polarized MDCK cell epithelium offers a tractable model system for visualizing rotavirus entry, as well as the roles of VP4 and VP7, during the earliest period of viral infection. Distinct VP4 and VP7 conformations are visualized during RRV entry. We next examined changes in VP4 and VP7 occurring during the early steps of RRV infection. To monitor the fate of VP4, we used MAbs that specifically recognize regional or conformation-dependent epitopes on VP8* (the amino-terminal cleavage segment of VP4), conformational epitopes on VP5* (the carboxy-terminal cleavage segment of VP4), or soluble monomeric VP5* (Fig. 1E to G) (46, 52). Interestingly, immunostaining for VP8* increased from 10 min (not shown) to 1 h but remained limited exclusively to the actin network at 1 h p.i. and was never observed in the cytoplasm as seen with early VP6 staining (Fig. 1E, white arrows). An actin network staining pattern was also observed for VP5* at both 10 min (not shown) and 1 h postinfection. However, discrete VP5* cytoplasmic staining inside cells was also seen at 1 h p.i. (Fig. 1F, black arrows). Finally, soluble monomeric VP4 staining was detected only in the cytoplasm at 1 h (Fig. 1G, black arrows). Similar experiments were next performed for VP7 by using either a MAb that recognizes a conformational trimer-dependent epitope of VP7 uniquely associated with intact TLPs (18) or a MAb specific for unassembled VP7 (58), not readily detectable on the virion. Substantial staining, limited to the actin network, was observed with the TLP-specific VP7 MAb, increasing from 10 min to 1 h after infection (Fig. 1H and data not shown), while unassembled VP7 staining was seen only in the cell cytoplasm as early as 10 min p.i. (Fig. 1I).

2496 WOLF ET AL. J. VIROL. FIG. 2. Quantification of RV antigen staining during infection. MDCK cell monolayers were infected with RRV at an MOI of 500 for 30 min, washed 3 times, and incubated at 37 C for another 30 min (total infection time, 1 h). After staining for the indicated RV antigens and confocal acquisition, signals corresponding to VP6, VP4, or VP7 were quantified by using Volocity software. Quantification was based on fluorescence intensity and the size of every dot under each condition. Total staining was compared to cell surface staining (A) or to actin network-associated staining (B) under each condition. Membrane, total, and actin network-associated stainings were defined as described in Materials and Methods. Results are mean numbers of indicated immunostained dots ( standard errors of the means [SEM]) quantified from four separate experiments., P 0.05;, P 0.01;, P 0.001. In order to more accurately assess whether these qualitative analyses of the micrographs were accurate, data from the confocal images were examined by using an automated quantification software program (Volocity; Improvision). This allowed direct comparisons of both the degrees and localizations of the different RV antigens during the entry process (Fig. 2). We used two different but complementary approaches to quantify the staining for each viral protein. First, we used nonpermeabilizing conditions to quantify viral antigens bound at the cell surface. Permeabilizing conditions were then used with different fluorochromes (see Materials and Methods) on the same sample to quantify total cell-associated antigen staining (which included cell surface-bound as well as internalized antigens) (Fig. 2A). Cell surface-specific staining of trimeric VP7 and VP8* accounted for about half of the total cell-associated staining, whereas these two antigens were never detected deep in the cytoplasm. We thus used a second quantification method to distinguish bound and entering viral particles from uncoated virions present deep in the cytoplasm. To do so, we cropped actin-positive sections at the top of the cells and quantified the amount of specific viral antigens present in these selected sections in close proximity to the actin network (Fig. 2B). For each antigen, the actin-positive fraction was composed of both cell surface-bound and entering particles but not antigens deep within the cytoplasm. Taken together, these data give a more complete picture of both the location and quantity of each viral antigen at a specific time postinfection. The quantitative analysis confirmed that intracellular VP8* and trimer-specific VP7 signals were present exclusively in close proximity to the actin network and were not found in the cell cytoplasm. Indeed, there was no significant difference between the actin network and the total cell-bound staining for these two antigens (Fig. 2B). On the other hand, the amount of VP5* actin-associated staining was significantly different from that of total cell-bound VP5* staining (P 0.01), indicating that VP5* was also found in substantial amounts in the cell cytoplasm (Fig. 2B). VP4 fold-back occurs during RRV entry. The RRV entry process is likely to involve several VP4 and VP7 conformational changes that take place in both parallel and sequential manners (20). In order to detect VP4 conformational changes, and in particular folded-back VP5, we used, in addition to the VP4-specific MAbs described above, a MAb generated against folded-back VP5, designated 1E2. 1E2 was previously demonstrated to react with recombinant VP5CT (containing only the N-terminal half of VP5* in a folded-back state) in an enzymelinked immunosorbent assay (ELISA) but not with native VP4 present on trypsin-activated TLPs (65). Since the fold-back of VP5 is proposed to take place at some point during cell entry, we stained cells at very early times postinfection (1 h) and, under these conditions, were able to detect the generation of folded-back VP5 staining in and very close to the actin network (Fig. 3). In contrast to VP6 staining, which appeared very quickly in the cytoplasm (Fig. 1C and D), folded-back VP5 was visualized only in close proximity to the actin network but never deep in the cytoplasm (Fig. 3). Quantitative analysis showed that little folded-back VP5 was present at the cell surface (Fig. 2A). The folded-back VP5 signal was associated mainly with the actin network at 1 h postinfection, but a significant amount was also found in the cell cytoplasm (P 0.05) (Fig. 2B). Unlike the other RV MAbs that stained newly synthesized RV antigens in RV-infected cells at 16 h p.i. (Fig. 4A to D and F) as well as viral antigens associated with trypsinactivated input virus (Fig. 1B to F and H), the 1E2 epitope appears to be specific for the folded-back form of VP5 associated with input virus, as indicated by the absence of cell immunostaining at 16 h postinfection (Fig. 4E and F). This suggests that the folded-back form of VP5 is indeed associated with input virus, specific for very early steps of RRV entry and not present on newly synthesized intracellular VP4. VP5* and VP7 colocalize with early endosome markers during entry. To better visualize the RRV entry pathway, we next performed colocalization experiments with VP5* and VP7 and specific markers of various endocytic compartments and trafficking routes. We examined primary antibodies directed against (i) Rab5, a specific early endosomal marker present during endocytic vesicle formation at the cell membrane and

VOL. 85, 2011 VISUALIZATION OF ROTAVIRUS ENTRY 2497 FIG. 3. Detection of folded-back VP5 in a polarized epithelium. Polarized MDCK cells were infected with tissue culture-derived RRV at a high MOI (MOI of 500) for 1 h and stained with monoclonal antibodies (MAbs) specific to folded-back VP5 (MAb 1E2). Optical sections were taken at a 0.2- m resolution through the cell monolayers, and z stacks were reconstructed into three dimensions. RV antigens are visualized in green, while red staining corresponds to the actin network visualized using labeled phalloidin. Black arrows indicate intracellular staining. This micrograph is representative of at least 3 separate experiments, and in each experiment, at least 3 fields were quantified. Scale bar, 10 m. present on early endosome vesicles; (ii) Rab4, a GTPase also associated with the early endosome; and (iii) Rab7, a marker present specifically on late endosomes. These cellular markers were visualized at various times postinfection simultaneously with VP5* and VP7. VP5* and VP7 clearly colocalized with Rab5 (Fig. 5A and D) and Rab4 (Fig. 5B and E), both early endosome markers, in close proximity to the cell membrane. There was no colocalization of VP5* or VP7 with Rab7, a late endosome marker, indicating that RRV is likely to enter the cell using an endocytic route limited to the early endosomes. We did not observe a colocalization of VP6 with any early endosomal marker (not shown). RRV entry is impaired by endosomal acidification inhibitors. A major technical issue for all studies that attempt to visualize the viral entry process is the theoretical concern that the particles being visualized are not actually those that initiate a productive infection. To begin to address this issue and to confirm the involvement of an endocytic process, as indicated by the Rab costaining data, we performed a series of experiments to correlate our micrographic observations with a variety of pharmacological interventions that have been associated with the blocking of entry processes. We tested several pharmacological interventions known to block major viral entry pathways: (i) bafilomycin A1 (BafA1) and concanamycin A, two strong inhibitors of the vacuolar-type H -ATPase in vitro which have been used to block infection resulting from endocytosis and related acidification in other studies of viruses (8); (ii) chloroquine and ammonium chloride, which have been shown to inhibit the infection of several viruses that require a ph-dependent entry step but which did not appear to inhibit RV infection in previously reported studies (26, 39); (iii) methyl- -cyclodextrin, which blocks raft-mediated virus entry by sequestering cholesterol and which has been shown to reduce RV infection (55); (iv) dynasore, an inhibitor of dynamindependent entry; (v) leupeptin, a broad-spectrum inhibitor of lysosomal proteases; and (vi) cathepsin inhibitor III, which blocks cathepsin, a major lysosomal protease. Each of these pharmacological approaches was used to interrogate various possible steps in the early phase of RV infection. The results are summarized in Fig. 6A. Polarized MDCK monolayers were treated with various concentrations of these drugs 30 min to 1 h prior to, as well as during, infection. Observations of the infected cells by confocal microscopy showed no significant difference between untreated cells and those treated with most of the drugs used, i.e., chloroquine (Fig. 6A and 7A). Surprisingly, dynasore and methyl- -cyclodextrin, which have both been described to inhibit RV entry (33, 55), did not reduce RRV entry at 1hor16hpostinfection (Fig. 6A). As a positive control for these negative findings, we examined the entry of Listeria monocytogenes into MDCK cells under identical conditions (47, 57). As opposed to RRV and consistent with data in the literature, the internalization of L. monocytogenes was reduced over 70% by both drugs (Fig. 6B). Interestingly, 10 nm BafA1 substantially reduced cytoplasmic VP6 staining at 1 h postinfection (not shown), whereas a 100 nm concentration abolished over 95% of VP6 staining (Fig. 6A and 7B and E). Moreover, a similar reduction was observed for the folded-back VP5 signal, further suggesting that the creation of this epitope is specific to entry (Fig. 7E). On the other hand, BafA1 treatment induced a 4-fold increase in the actin network staining of VP7 (Fig. 7C and E) and VP8* (Fig. 7D and E). Comparable immunostaining effects were seen when concanamycin A was used (data not shown). A traditional 16-h replication assay showed that both BafA1 and concanamycin A treatments led to a 90% infectivity reduction, arguing that these drugs are indeed blocking productive infection as well as the trafficking of VP6 into the cytoplasm (Fig. 6A). BafA1 and concanamycin A are more specific and efficient than chloroquine or ammonium chloride in blocking endocytic acidification, and a cell type-specific action of these drugs was suggested previously (38). The fact that ammonium chloride and chloroquine have no effect on RRV infectivity combined with the substantial effects of BafA1 and concanamycin A suggested that although RRV entry is likely to involve endocytosis, the direct role of endosomal acidification was less clear. In order to further investigate the role of ph on RRV entry, we took advantage of the nonreversible nature of BafA1 and attempted to rescue the inhibition of VP6 staining by a low-ph pulse treatment (14). The low-ph rescue experiment was challenging because the binding of RRV to cells was relatively inefficient at 4 C in our hands. Similar limitations were described previously for other viruses (41) as well as for RV: after 1 h at 4 C, only 10% of the input virus was observed to bind to MA104s cells, a cell line highly permissive for RRV (40). We thus chose to take advantage of the relatively large amount of triple-layered RRV particles still attached at the cell surface of BafA1-treated cells at 37 C at 1 h p.i. (Fig. 7C and E). Our hypothesis was that if RRV requires a low-ph-induced conformational change for entry, the cell surface-bound particles in BafA1-treated cells would undergo enhanced entry fol-

2498 WOLF ET AL. J. VIROL. Downloaded from http://jvi.asm.org/ FIG. 4. Detection of RV antigens synthesized at 16 h postinfection. (A to E) Polarized MDCK monolayers were infected with RRV at an MOI of 1 for 16 h and stained for VP6 (MAb 1E11) (A); trimeric VP7 (MAb 159) (B); VP8* (MAb 7A12) (C); VP5* (MAb 2G4) (D); and folded-back VP5 (MAb 1E2) (E), shown in green. The actin network is stained in red. Scale bar, 10 m. (F) Total fluorescence was quantified under each condition as described in Materials and Methods. on July 7, 2018 by guest lowing a pulsed low-ph treatment. Interestingly, the application of pulsed exogenous ph acidification exposure was not able to rescue membranous or cytoplasmic VP6 staining in BafA1-treated cells (Table 1), suggesting that the BafA1 action on RRV entry potentially involves a secondary consequence of the ph changes inside the endosomal vesicle rather than the ph change itself. RRV entry is impaired by an elevated endosomal Ca 2 concentration. Vacuolar-type H -ATPase inhibitors such as BafA1 are often used to demonstrate a low-ph requirement for infection, but they can also cause defective receptor recycling, endosomal maturation inhibition, or the inhibition of Ca 2 efflux from the endosome (45). In particular, it was demonstrated that BafA1, while inhibiting ph acidification, prevents Ca 2 diffusion from the endosome. This results in an increased Ca 2 concentration in the endosome (27). To more directly investigate the importance of the Ca 2 concentration on RRV entry, we artificially raised the endosomal calcium concentration by treating the cells with 10 mm CaEGTA at the time of infection and for 15 min following infection. These conditions lead to an augmentation of the intraendosomal calcium concentration and to a delay in the dissipation of the Ca 2 gradient (8). The cells were then stained for VP6 and trimeric VP7. Under control conditions, at 15 min p.i., both antigens were restricted to the actin network (not shown) and were detected only in limited amounts (Fig. 8). Very little VP6 staining was detected at 15 min p.i. either with or without CaEGTA treatment, consistent with the above-described observations (Fig. 1B). On the other hand, CaEGTA treatment clearly led to a significant accumulation of total TLP-associ-

VOL. 85, 2011 VISUALIZATION OF ROTAVIRUS ENTRY 2499 FIG. 5. VP5 and trimeric VP7 colocalize with early endosome markers but not with late endosome markers. MDCK cells were infected at an MOI of 500, and 1 h later the epithelium was stained for VP7 (MAb 159) (red) (A to C) or VP5* (MAb 2G4) (red) (D to F) and the early endosome markers Rab5 (rabbit anti-rab5) (A and D) and Rab4 (rabbit anti-rab4) (B and E) or the late endosome marker Rab7 (rabbit anti-rab7) (green) (C and F). Monoclonal antibodies and Rab proteins were visualized by using donkey anti-mouse Alexa 647 antibodies and goat anti-rabbit Alexa 488 antibodies, respectively. The actin staining was removed for a better visualization of colocalization events (yellow), indicated by the open arrows. Scale bar, 10 m. ated VP7 immunostaining, suggesting a delay in RV decapsidation in the presence of an elevated endosomal Ca 2 concentration. DISCUSSION In this study, we performed an imaging analysis of RRV entry into a polarized epithelium. We were able to detect input virus by using a high MOI and monitoring the location and quantity of the major RRV structural antigens VP4, VP6, and VP7 at early times postinfection prior to the de novo synthesis of detectable rotaviral proteins. A similar strategy was previously used to examine the early events associated with bacterial interactions with target monolayers (48). Each antigen was quantified, and its specific cellular localization was determined at selected time points during the entry process. Based on this analysis, we provide evidence that RRV entry is a multistep program that can be monitored by observing the differing fates of its major structural proteins. Contrary to previously reported data (7), we did not detect any surface or cytoplasmic staining when DLP preparations were applied to the cells. Differences between that previous study and ours include the use of infectious TLP-derived DLPs instead of recombinant VLP DLPs and MDCK cells instead of MA104 cells. Based on EM observations confirming that no TLPs were present in our purified DLP preparations (not shown) and given the well-documented facts that DLPs are not infectious unless lipofected into permissive cells (3, 25) and that there is currently no DLP-specific receptor identified, the FIG. 6. Effects of pharmacological interventions on RV entry and infectivity. (A) Polarized MDCK cells were pretreated for 1 h with the indicated concentrations of each drug and infected with RV for 1 h at an MOI of 500. The total amount of cell-associated VP6 was quantified by using Volocity software and compared to that of nontreated cells. Infectivity was assessed by pretreating MDCK cell monolayers with each drug for 1 h, infecting them for 1 h, and counting the number of RV-positive foci at 16 h p.i. Nontreated cells were used as a reference and assigned a relative value of 100%. (B) Polarized MDCK cells were pretreated for 1 h with the indicated drug and infected with RRV or GFP-Listeria monocytogenes (Lm) for 1 h. Intracellular staining was quantified by using Volocity software. lack of DLP staining that we observed seems biologically plausible and likely to accurately reflect what takes place during the early stages of infection. We demonstrated that decapsidation occurs directly after cell membrane penetration, as assessed by the rapid loss of trimeric VP7 staining from TLPs and the gain of a VP6 signal very close to the actin network (Fig. 1). By monitoring various components and conformations of VP4 using epitope-specific MAbs, we established the sequence of structural changes occurring during entry: VP8*, VP5*, and folded-back VP5 can be detected on the cell surface as entry initiates, but only the VP8* signal is lost following cell membrane penetration. This suggests that the VP8 fragment of VP4 likely dissociates from the viral particle at this time, as was hypothesized previously (64). We found that the cell staining patterns of trimeric VP7 and VP8* are quite similar: they both increase over time (i.e., from 10 min [not shown] to 1 h postinfection) and are both restricted to the actin network. However, VP8* was never visualized in the cell cytoplasm, while soluble VP7 (as well as VP5*) was (Fig. 1I). Hence, the fates of the two surface proteins during entry are not identical. These findings suggest that decapsidation with the inherent elimination of trimeric VP7 and the loss of VP8* from the virion are linked events necessary for DLP trafficking into the cytoplasm. The proposed in vitro model of VP4 conformational changes (64) suggests that VP8* is present on soluble monomeric and cleaved spike (following trypsin activation) VP4 but that the VP5 fold-back requires a major rearrangement, potentially leading to a loss of VP8*. Our findings are consistent with this model. The VP5 fold-back, in vitro, can partially be promoted by VP4-mem-

2500 WOLF ET AL. J. VIROL. Downloaded from http://jvi.asm.org/ FIG. 7. Effects of pharmacological treatments on early events of RV infection. (A to E) Polarized MDCK cells were either pretreated or not with 50 mm chloroquine (A) or 100 nm bafilomycin A1 (B to D) for 1 h, infected by RRV (MOI of 500) for 1 h, and imaged to detect VP6 (MAb 1E11) (A and B), trimeric VP7 (MAb 159) (C), or VP8* (MAb 7A12) (green) (D). Red staining corresponds to the actin network. Scale bar, 10 m. (E) The effect of each treatment on RV input antigens was quantified by using Volocity software. Results are mean numbers of indicated immunostained dots ( SEM) quantified from four separate experiments., P 0.001. brane interactions and/or calcium chelation leading to VP7 decapsidation (63, 65). The biological relevance of these in vitro findings is supported by our data showing that VP5* localized first at the cell surface and then in the cell cytoplasm as well as the observation that folded-back VP5 is found both TABLE 1. The effect of BafA1 on input RV antigens is not rescued by low-ph pulses a Treatment Mean no. of immunostained dots SEM at ph: 7 6 5 4 3 VP6 100 108 3 102 4 99 5 95 5 VP6 BafA1 5 3 9 4 12 2 10 2 8 2 a MDCK cells were pretreated for 1 h with BafA1 (100 nm), infected for 1 h with RRV, exposed to the indicated low-ph buffers for 3 min, and then incubated for 2 additional hours. The effect of low-ph pulses on total VP6 immunostaining was quantified by using Volocity software and compared to that of nontreated cells used as a reference. at the cell membrane and in the cytoplasm at 1 h postinfection (Fig. 3). The folded-back VP5 conformation was the only RRV conformation detectable on input virus but not present on de novo-synthesized viral proteins and particles visualized at 16 h postinfection (Fig. 4). This finding further suggests that the folded-back VP5 conformation is limited exclusively to the entry phase of replication. This is the first study to demonstrate the generation of the folded-back form of VP5 during the actual infectious process. Since the VP4 conformational changes appear to resemble the steps used by enveloped viruses during entry and membrane fusion, one hypothesis is that the folded-back VP5 forms the virion structures responsible for membrane penetration and the release of DLPs in the cytoplasm. Based on data from colocalization studies and the use of pharmacological interventions, we provide additional evidence that RRV enters the cell and undertakes these membrane- on July 7, 2018 by guest

VOL. 85, 2011 VISUALIZATION OF ROTAVIRUS ENTRY 2501 FIG. 8. Effect of the endosomal Ca 2 gradient on RV entry. Polarized MDCK cells were treated with 10 mm CaEGTA at the time of infection and infected with RRV (MOI of 500) for 15 min. The effect of the treatment on total VP6 (MAb 1E11) and trimeric VP7 (MAb 159) staining was quantified by using Volocity software. Results are the mean numbers of indicated immunostained dots ( SEM) quantified from four separate experiments., P 0.001. interactive events through an endocytosis pathway. We first demonstrate that input RRV proteins colocalize with the early endosome markers Rab4 and Rab5 but not with the late endosome marker Rab7. Interestingly, an interaction between VP4 and Rab5 was previously demonstrated in vitro, suggesting a specific role of the early endosome in RV trafficking (23). We have now extended those previous studies to document the interaction during an actual infection. Our study is the first to measure the effect of pharmacological treatments specifically on RRV entry by measuring the impact of these treatments on input virus trafficking at 1 h postinfection. In most previously reported studies, indirect or direct measurements were performed at 6 to 16 h p.i. (33, 55), when the readout was indirect and relied on measurements of viral replication following entry. We did not measure any significant effect on RRV entry or infectivity following the treatment of MDCK cells with dynasore, an inhibitor of the dynamin GTPase domain. Dynamin was previously implicated in RV entry using a dominant negative mutant of the dynamin GTPase domain in an MA104 cell model system (55) or using dynasore (33). Of note, in our hands, dynasore treatment did not lead to a reduction in the infectivity to MA104 cells (data not shown), suggesting that the absence of an effect on the MDCK cells was not a cell typespecific phenomenon. We also did not observe a significant effect of methyl- -cyclodextrin, a drug that sequesters cholesterol and was also shown previously to reduce RV infectivity (33, 55), suggesting that cholesterol may not be critical for RRV entry in the polarized epithelium model that we used. At this point, we are not able to explain these differences. However, the data that we present seem to support the notion that RRV is likely to enter MDCK cells through a dynamin-independent endocytosis mechanism. Among the panel of drugs known to inhibit endocytic pathways, only BafA1 and concanamycin were shown to strongly inhibit RRV entry. This was documented both by the absence of intracellular VP6 staining at 1 h p.i. and by a 90% reduction in infectivity at 16 h p.i. Of interest, RRV decapsidation was completely abolished by BafA1 treatment, as assessed by the absence of VP6 immunostaining both at the cell surface and in the cytoplasm. This finding correlated with the absence of folded-back VP5 staining under the same conditions, supporting the conclusion that the VP5 fold-back requires RV decapsidation. Additionally, this observation is consistent with the hypothesis that trimeric VP7 capsid integrity prevents VP4 conformational changes. The fold-back of VP4 would thus be among the latter events of entry, following binding, cell penetration, and decapsidation. BafA1 and concanamycin are inhibitors of endosomal acidification and do not block virus internalization (50). The colocalization of VP7 with Rab5 in the actin network compartment increased in BafA1-treated cells, suggesting that, under these conditions, the RRV entry pathway is blocked at the level of Rab5-containing endocytic structures closely linked to the actin network. Since chloroquine and ammonium chloride did not have an effect on RRV entry or infectivity, a strictly phdependent effect on viral entry seemed questionable. We further investigated the possible role of ph on RRV entry directly by exposing cell surface-bound RRV to a brief low-ph pulse. This treatment failed to reverse the effect of BafA1 pretreatment. BafA1 and concanamycin have been described to be more specific and more potent inhibitors of endosomal ph than chloroquine and ammonium chloride, and a cell typespecific action was previously suggested (38). Of note, the BafA1 concentration that we used to block 90% of RRV entry (100 nm) is somewhat higher than the concentrations used previously to inhibit well-characterized ph-dependent virus entry pathways (5 to 100 nm) (4, 62). We thus hypothesized that BafA1, by very efficiently raising the ph, was also modifying the balance of other molecules present in the endosome. Interestingly, BafA1 treatment was shown to raise both the ph and the calcium concentration in the endosome (27). Hence, the effect of BafA1 on RRV entry could be explained by an elevated endosomal calcium concentration preventing viral uncoating and the conformational changes in VP7 and VP4 leading to VP4 trimerization. The importance of the calcium concentration has been demonstrated at many steps in the RV replication cycle (53), and in particular, previous studies suggested the importance of a calcium gradient in the endosome for RV entry (8). We tested this hypothesis by increasing the extracellular calcium reservoir by the addition of 10 mm CaEGTA to the cell culture medium. These conditions promote an increase in the endosomal Ca 2 concentration and a delay in the dissipation of the Ca 2 gradient (8). Of note, this treatment also led to the accumulation of TLP-associated VP7 in the actin network, suggesting a delay in RV decapsidation following entry. This effect was similar to the effect of BafA1 treatment, supporting the hypothesis that the primary effect of BafA1 on RRV infection is mediated indirectly through changes in the endosomal Ca 2 gradient. Taken together, these experimental observations support a model of RRV entry in which, after membrane binding and internalization, the low Ca 2 concentration in the endosome triggers VP7 decapsidation and a VP5 fold-back, ultimately leading to the release of DLPs in the cell cytoplasm. Importantly, these studies provide the first demonstration of the formation of folded-back VP5 during natural infection. Studies