Transport to Late Endosomes Is Required for ... - Journal of Virology

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Jan 13, 2012 - Chen PI, Kong C, Su X, Stahl PD. 2009. Rab5 isoforms differentially regulate the trafficking and degradation of epidermal growth factor recep-.

Transport to Late Endosomes Is Required for Efficient Reovirus Infection Bernardo A. Mainoua,b and Terence S. Dermodya,b,c Departments of Pediatricsa and Pathology, Microbiology, and Immunologyc and Elizabeth B. Lamb Center for Pediatric Research,b Vanderbilt University School of Medicine, Nashville, Tennessee, USA

Rab GTPases play an essential role in vesicular transport by coordinating the movement of various types of cargo from one cellular compartment to another. Individual Rab GTPases are distributed to specific organelles and thus serve as markers for discrete types of endocytic vesicles. Mammalian reovirus binds to cell surface glycans and junctional adhesion molecule-A (JAM-A) and enters cells by receptor-mediated endocytosis in a process dependent on ␤1 integrin. Within organelles of the endocytic compartment, reovirus undergoes stepwise disassembly catalyzed by cathepsin proteases, which allows the disassembly intermediate to penetrate endosomal membranes and release the transcriptionally active viral core into the cytoplasm. The pathway used by reovirus to traverse the endocytic compartment is largely unknown. In this study, we found that reovirus particles traffic through early, late, and recycling endosomes during cell entry. After attachment to the cell surface, reovirus particles and JAM-A codistribute into each of these compartments. Transfection of cells with constitutively active and dominant-negative Rab GTPases that affect early and late endosome biogenesis and maturation influenced reovirus infectivity. In contrast, reovirus infectivity was not altered in cells expressing mutant Rab GTPases that affect recycling endosomes. Thus, reovirus virions localize to early, late, and recycling endosomes during entry into host cells, but only those that traverse early and late endosomes yield a productive infection.

A

s obligate intracellular parasites, viruses require host cell machinery to internalize, replicate, and disseminate. After attachment to target cells via the interactions of viral capsid components and cellular receptors, viruses must find a way to deliver their genetic payloads to intracellular sites suitable for the initiation of viral replication. Some viruses directly fuse their envelope coats with the cell membrane at the cell surface, while other viruses use the endocytic machinery to gain access to the cell interior. Viruses that utilize endocytic routes to enter cells must exit endocytic vesicles to begin viral replication and avoid transport to degradative lysosomes. Understanding how viruses are internalized and routed to appropriate organelles in the endocytic pathway will yield a more complete view of how cells import macromolecular cargoes and perhaps illuminate new targets for the development of antiviral therapeutics that impede viral uptake. Nonfusogenic mammalian orthoreoviruses (called reoviruses here) are nonenveloped, double-stranded RNA viruses belonging to the Reoviridae. Reoviruses have a broad host range and infect most mammalian species (50). Junctional adhesion molecule-A (JAM-A) serves as a receptor for each of the reovirus serotypes (5, 7, 20). After attachment to JAM-A, reovirus uses ␤1 integrin (33, 34) to enter cells via clathrin-dependent endocytosis (6, 18, 34, 48, 55). During internalization, reovirus activates Src family kinases (35) and undergoes acid-dependent, proteolytic disassembly catalyzed by cathepsin proteases (16, 36, 55). Disassembly results in the removal of outer-capsid protein ␴3 and conformational rearrangement of outer-capsid protein ␮1, which allows endosomal membrane penetration and release of transcriptionally active viral core particles into the cytoplasm (8, 9, 13, 40, 41). The cellular organelles that serve as sites for reovirus disassembly are largely unknown. Rab GTPases constitute the largest family of small GTPases in cells, with more than 60 members in humans (54). As coordinators of vesicular traffic, Rab GTPases regulate sorting of a variety

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of cellular cargoes through indirect interactions with vesicular coat components, intracellular motors, and soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) (54). The activation state of Rab GTPases is in part regulated by guanine nucleotide exchange factors, which catalyze the exchange of GDP for GTP to shift Rab GTPases into an active state (44, 54). GTPase-activating proteins, in conjunction with Rab GTPase activity, catalyze GTP hydrolysis and shift Rab GTPases into an inactive, GDP-bound state (26, 54). Rab GTPases localize to specific organelles (11) and distinct membrane microdomains (52). For example, early and recycling endosomes contain a mixture of Rab4, Rab5, and Rab11 microdomains (52, 54), whereas late endosomes have microdomains enriched in Rab7 and Rab9 (3, 54). As such, Rab GTPases serve as molecular markers for specific vesicular compartments and useful tools to investigate endocytic uptake and sorting mechanisms. In this study, we found that reovirus virions localize to early, late, and recycling endosomes during entry into host cells. Within these endosomal compartments, reovirus virions are associated with JAM-A. However, only virions that traffic through early and late endosomes are capable of establishing infection. These data indicate that reovirus virions traverse several endocytic compartments during cell entry, but only a fraction of these virions use a pathway that leads to productive infection.

Received 13 January 2012 Accepted 27 May 2012 Published ahead of print 6 June 2012 Address correspondence to Terence S. Dermody, [email protected] Supplemental material for this article may be found at http://jvi.asm.org/. Copyright © 2012, American Society for Microbiology. All Rights Reserved. doi:10.1128/JVI.00100-12

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Reovirus and the Endocytic Pathway

MATERIALS AND METHODS Cells, viruses, chemical inhibitors, and antibodies. Spinner-adapted murine L929 cells were grown in either suspension or monolayer cultures in Joklik modified Eagle minimal essential medium (Lonza) supplemented to contain 5% fetal bovine serum (FBS; Invitrogen), 2 mM L-glutamine (Invitrogen), 100 U of penicillin per ml, 100 ␮g of streptomycin (Invitrogen) per ml, and 0.25 mg of amphotericin B (Sigma) per ml. HeLa CCL2 cells (HeLa, obtained from Carolyn Coyne, University of Pittsburgh) were grown in Dulbecco modified Eagle medium (Invitrogen) supplemented to contain 10% FBS, minimal essential medium nonessential amino acid solution (Sigma), 0.11 mg of sodium pyruvate (Sigma) per ml, and penicillin, streptomycin, and amphotericin B. Reovirus strain type 1 Lang (T1L) is a laboratory stock. Working stocks of virus were prepared by plaque purification and passage using L929 cells (57). Purified virions were generated from second-passage L929 cell-lysate stocks. Virus was purified from infected cell lysates by Freon extraction and CsCl gradient centrifugation as described previously (21). The band corresponding to the density of reovirus particles (1.36 g/cm3) was collected and dialyzed exhaustively against virion-storage buffer (150 mM NaCl, 15 mM MgCl2, 10 mM Tris-HCl [pH 7.4]). Reovirus particle concentration was determined from the equivalence of 1 U of optical density at 260 nm to 2.1 ⫻ 1012 particles (51). Virus titers were determined by plaque assay using L929 cells (57). Ammonium chloride (NH4Cl; Gibco) was resuspended in water according to the manufacturer’s instructions. The immunoglobulin G (IgG) fraction of a rabbit antiserum raised against T1L (59) was purified by protein A-Sepharose as described previously (4). JAM-A-specific monoclonal antibody (J10.4; provided by Charles Parkos, Emory University) and human ␤1 integrin-specific monoclonal antibody MAB2253Z (Millipore) were used in indirect immunofluorescence and flow cytometry analyses. Alexa Fluor-conjugated antibodies (Invitrogen) were used as secondary antibodies. Plasmids. Rab GTPase plasmids encoding wild-type, dominant-negative, or constitutively active Rab4A and Rab4B (Marci Scidmore, Cornell University), Rab5A and Rab7 (Walter Atwood, Brown University), Rab5C (Philip Stahl, Washington University), Rab9 (Suzanne Pfeffer, Stanford University), Rab11 (James Goldenring, Vanderbilt University), and Rab34 (Carolyn Coyne, University of Pittsburgh) were appended at the amino terminus with enhanced green fluorescent protein (EGFP). Rab13 and human RILP cDNAs were obtained from Open Biosystems. EGFP was fused to the amino terminus of Rab13 by subcloning Rab13 cDNA into pEGFP-C1 (Clontech) by PCR amplification using Platinum Pfx (Invitrogen) and Rab13-FWD and Rab13-REV oligonucleotide primers. PCR amplification was followed by restriction enzyme digestion and insertion of the Rab13 fragment into the EcoRI and BamHI sites of pEGFP-C1. A linker was inserted between the amino terminus of Rab13 and carboxy terminus of EGFP. The RILP cDNA was amplified by PCR using RILP-FWD and RILP-REV oligonucleotide primers, followed by restriction enzyme digestion and insertion into the EcoRI site in EGFPC1. RILP lacking the amino-terminal 212 amino acids (RILP⌬212N) was amplified by PCR using RILP-C33 FWD and RILP-C33 REV oligonucleotide primers, followed by restriction enzyme digestion and insertion into the EcoRI and NotI sites in EGFP-C1. Plasmid sequences were confirmed to ensure the fidelity of cloning. Point mutations were inserted into EGFP fused wild-type Rab GTPases using Pfu Ultra (Agilent Technologies) and construct-specific degenerate primers to engineer constitutively active forms of Rab5A (Q79L), Rab5C (Q80L), Rab7 (Q67L), and Rab11 (Q70L) and dominantnegative versions of Rab5C (S35N) and Rab13 (T22N), in each case fused to EGFP. Plasmid sequences were confirmed to ensure the fidelity of cloning and mutagenesis. Primer sequences are available from the corresponding author upon request. Succinimidyl ester labeling. Reovirus virions were labeled with succinimidyl ester Alexa Fluor 546 (A546) or pHrodo SE (pHrodo; Invitrogen) as described previously (19, 27). Succinimidyl esters preferentially

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label reovirus proteins ␭2, ␮1, ␴2, and ␴3 (19). Reovirus particles (3 ⫻ 1012) were diluted into fresh 0.05 M sodium bicarbonate (pH 8.5) and incubated with 10 ␮M succinimidyl ester A546 or pHrodo at room temperature for 90 min in the dark. Virus particles were dialyzed against phosphate-buffered saline (PBS) at 4°C overnight and stored at 4°C. Virus titers following labeling were determined by plaque assay using L929 cells. Confocal microscopy of reovirus internalization. HeLa cells were plated on glass coverslips (#1.5; Thermo Scientific) in 24-well plates at 37°C overnight. Cells were transfected with plasmids using Fugene 6 (Roche) according to the manufacturer’s instructions. After incubation at 37°C for 24 h, the cells were chilled at 4°C for 1 h. Virus was adsorbed at 4°C for 1 h, the inoculum was removed, and the cells were washed three times with PBS and either fixed with 5 to 10% formalin or supplemented with complete medium and incubated at 37°C for various intervals. Cells were washed once with PBS and fixed for 20 min with 5 to 10% formalin, quenched with 0.1 M glycine, and washed three times with PBS. Cells were incubated with 1% Triton X-100 for 5 min and PBS-BGT (PBS, 0.5% bovine serum albumin [BSA], 0.1% glycine, and 0.05% Tween 20) for 10 min. The cells were incubated with reovirus-specific polyclonal antiserum (1:1,000) in PBS-BGT for 1 h, washed with PBS-BGT, and incubated with Alexa Fluor 488 (A488), A546, or Alexa Fluor 633 (A633) IgG (1:1,000) in PBS-BGT for 1 h. Nuclei were visualized by incubating cells with TOPRO-3 (Invitrogen) conjugated to Alexa Fluor 642 (A642; 1:1,000) in PBS-BGT for 20 min. The cells were washed with PBS-BGT, and coverslips were removed from wells and placed on slides using Aqua-Poly/ Mount mounting medium (Polysciences, Inc.). Images were captured using a Zeiss LSM 510 Meta laser-scanning confocal microscope using a ⫻63 Plan-Apochromat objective lens. Images were thresholded for pixel intensity, and the pinhole size used was identical for all fluors. All images represent single sections and were adjusted for brightness and contrast to the same extent. Colocalization analysis was performed using the profile function of Zeiss LSM image software (Zeiss) taking into consideration endosomal vesicle size to try to isolate individual endosomes. Virions within the boundary of single cells were quantified. The percentage of internalized viral particles was determined using HeLa cells plated on #1.5 glass coverslips adsorbed with A546-labeled reovirus. Cells were fixed with 5% formalin and stained as described except that PBS-BG (PBS, 0.5% BSA, and 0.1% glycine) was used instead of PBS-BGT. Internalized virions were quantified by enumerating viral particles that were labeled with A546 but not stained by reovirusspecific antiserum. Extracellular virions were quantified by enumerating viral particles that were labeled with A546 and stained by reovirusspecific antiserum. Live microscopy experiments. Cells were plated on 35-mm glass-bottom microwell dishes (MatTek Co.) and adsorbed with A546-labeled reovirus at 4°C for 1 h. Cells were transferred to a Zeiss LSM 510 Meta laser-scanning confocal microscope, washed three times with cold PBS, and supplemented with warm complete medium. Images were captured every 30.5 s (10.5-s scan time and 20-s intervals between scans) for 30 min with a ⫻63 Plan-Apochromat objective lens. Dwell time was calculated by analyzing time lapses frame by frame and assessing colocalization of reovirus particles with individual Rab GTPases. Assessment of receptor expression and reovirus binding by flow cytometry. HeLa cells were washed once with PBS and detached with Cellstripper (Cellgro) at 37°C, quenched with fluorescence-activated cell sorting (FACS) buffer (PBS with 2% FBS), pelleted at 1,000 ⫻ g, washed once with PBS, and pelleted a second time at 1,000 ⫻ g. The cells were adsorbed with reovirus (105 particles/cell) at 4°C for 1 h, washed once in FACS buffer, pelleted, and stained in FACS buffer with JAM-A-specific antibody (J10.4), ␤1 integrin-specific antibody (MAB2253Z), or reovirusspecific polyclonal antiserum at 4°C for 30 min. The cells were washed twice in FACS buffer, pelleted, and stained in FACS buffer containing Alexa Fluor-conjugated antisera or antibodies at 4°C for 30 min, pelleted, washed twice in FACS buffer, and fixed in FACS fix (PBS with 1% electron microscopy-grade paraformaldehyde [Electron Microscopy Sciences]).

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The cells were analyzed with a BD LSRII flow cytometer, and cell staining was quantified using FlowJo software (FlowJo). Assessment of pHrodo fluorescence intensity by flow cytometry. HeLa cells were chilled at 4°C for 1 h and adsorbed with A546- or pHrodolabeled reovirus (5 ⫻ 103 particles/cell) at 4°C for 1 h. The inoculum was removed, and the cells were washed three times with cold PBS to remove unbound virus and supplemented with warm Opti-MEM-I (Gibco) for various intervals. The cells were washed once with PBS, detached with Cellstripper, quenched with FACS buffer, washed once with FACS buffer, and fixed in FACS fix. The cells were analyzed with a BD LSRII flow cytometer, and cell staining was quantified using FlowJo software. Quantification of reovirus infectivity. HeLa cells were transfected with various Rab constructs using Fugene 6, incubated at 37°C for 24 to 48 h, and adsorbed with reovirus at a multiplicity of infection (MOI) of 0.5 PFU per cell at room temperature for 1 h. The inoculum was removed, and the cells were washed once with PBS, supplemented with complete medium, and incubated at 37°C for 20 h. The cells were then washed once with PBS, detached with Cellstripper, and quenched with FACS buffer. Next, the cells were washed once with PBS and stained with Live/Dead fixable violet dead cell stain (Invitrogen) at room temperature for 30 min. The cells were washed once with PBS, fixed and permeabilized with Cytofix/Cytoperm (BD Biosciences) at 4°C for 20 min, washed twice with Perm/Wash (BD Biosciences), and incubated with reovirus-specific polyclonal antiserum (1:20,000) at 4°C for 30 min. Then, the cells were washed twice with Perm/Wash and incubated with A546 or Alexa Fluor 647 (A647) IgG (1:1,000) at 4°C for 30 min. Finally, the cells were analyzed with a BD LSRII flow cytometer, and cell staining was quantified using FlowJo software. Statistical analysis. Mean values for at least triplicate samples were compared using unpaired Student’s t test (GraphPad Prism). P values of ⬍0.05 were considered to be statistically significant.

RESULTS

Reovirus traffics through the endocytic compartment during cell entry. To determine whether reovirus accesses the endocytic compartment during cell entry, HeLa cells were transfected with EGFP or EGFP-tagged versions of Rab4, Rab5A, Rab5C, Rab7, Rab9, Rab11, or Rab-interacting lysosomal protein (RILP) 24 h prior to adsorption. The cells were chilled at 4°C for 1 h, adsorbed at 4°C with A546-labeled reovirus particles, incubated at 37°C over a time course of reovirus entry, and imaged by confocal microscopy (Fig. 1). Quantification of spectral overlap between reovirus and Rab4 (Fig. 1C), which labels early and fast recycling compartments, showed statistically significant colocalization at 20, 60, and 120 min. Colocalization of reovirus and the two Rab5 isoforms tested, Rab5A (Fig. 1D) and Rab5C (Fig. 1E), peaked at 20 min after infection, with statistically significant colocalization also observed at 60 min with Rab5C but not Rab5A. These findings indicate that reovirus particles traffic through the early and recycling compartments during entry and that reovirus particles in Rab5A- and Rab5C-marked endosomes traffic with different kinetics. We also observed modest, but significant, colocalization of reovirus with Rab11 at 20, 60, and 120 min (Fig. 1H), suggesting that reovirus accesses the slow recycling endocytic compartment during entry. Some colocalization between reovirus and Rab GTPases, as high as 17% with Rab4, was observed at 0 min. The colocalization observed at 0 min may represent viral particles that have internalized despite adsorption at 4°C or spectral overlap between reovirus virions and Rab GTPases adjacent to the cell membrane. Of note, electron micrographs of cells infected with reovirus also show internalized particles as early as 0 min (34). As a control, the bulk of internalized transferrin colocalized with EGFP-Rab5A, and very little transferrin was detected in Rab7-

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marked compartments (Fig. 1B), findings consistent with the internalization of transferrin into early endosomes (32). The acidic environment of late endosomes or endolysosomes, where pH ranges from 4.5 to 6 (28, 38, 42) and active cathepsin proteases reside (15, 22, 45), provides a likely intracellular compartment for the reovirus disassembly events. To determine whether reovirus accesses late endosomes during cell entry, we assayed the colocalization of reovirus particles with Rab7 (Fig. 1F), Rab9 (Fig. 1G), and RILP (Fig. 1I). Quantification of confocal micrographs showed statistically significant colocalization of reovirus with each marker at 60 and 120 min. Statistically significant colocalization with Rab9 also was observed at 20 min, suggesting that reovirus particles access Rab9-marked endosomes with faster kinetics than endosomes marked by Rab7 and RILP. Although Rab7 preferentially labels the late endosomal compartment, some Rab7-marked organelles are also labeled by early endosomal makers (28, 46). Reovirus-infected cells transfected with Rab7 and stained for early endosomal antigen 1 (EEA1) revealed that 60 min after infection, 19% of reovirus particles that colocalize with Rab7 also distribute with EEA1 (data not shown). These particles likely delineate a population of virions that are transitioning from early to late endosomes. Thus, reovirus accesses the early, recycling, and late endosomal compartments during cell entry, with viral particles requiring between 20 and 60 min to reach late endosomes. Reovirus particles distribute with JAM-A during cell entry. To determine whether reovirus particles and JAM-A colocalize during cell entry, HeLa cells were transfected with JAM-A and EGFP-tagged versions of Rab4, Rab5A, Rab5C, Rab7, Rab11, or Rab13 for 24 h. Cells were chilled at 4°C for 1 h, adsorbed, incubated at 37°C for 0, 20, or 60 min, stained for reovirus and JAM-A, and imaged by confocal microscopy. Quantification of the spectral overlap of reovirus particles and JAM-A in cells exogenously expressing JAM-A and EGFP-tagged versions of various Rab GTPases showed consistent colocalization of reovirus particles with JAM-A over time (Fig. 2A). Overexpression of Rab4, Rab5A, Rab5C, and Rab7 did not affect colocalization of reovirus and JAM-A. Interestingly, overexpression of Rab13, which regulates tight-junction formation (37) and colocalizes with JAM-A (61), diminished the colocalization of reovirus and JAM-A at 60 min, suggesting that Rab13 affects reovirus trafficking with JAM-A following internalization. These data suggest that reovirus particles bind to JAM-A at the cell surface and remain associated with JAM-A during endocytic transport. To determine whether JAM-A-associated reovirus particles localize to specific endosomes during uptake into cells, we assessed the distribution of reovirus particles and JAM-A within distinct Rab-marked vesicles by confocal microscopy (Fig. 2B). Similar to the overall colocalization with endosomes marked with Rab4 and Rab11 (Fig. 1), JAM-A-associated reovirus particles distributed into Rab4- and Rab11-marked endosomes by 20 min, and the levels remained relatively unchanged at 60 min. Also similar to the distribution of reovirus particles into Rab7-marked endosomes, reovirus and JAM-A localization with Rab7 peaked at 60 min of infection (Fig. 2C). Interestingly, JAM-A-associated viral particles distributed to Rab5A-marked vesicles to a greater extent than to those marked by Rab5C, suggesting that viral particles found within Rab5A-marked endosomes are associated with JAM-A, while viral particles found in Rab5C-marked endosomes are not. In addition, JAM-A and reovirus particles were observed in Rab5A-marked endosomes at 0 min, suggesting that soon after

Journal of Virology

Reovirus and the Endocytic Pathway

FIG 1 Reovirus particles colocalize with markers of endocytic compartments during cell entry. (A) HeLa cells were transfected with EGFP-Rab7 (green) 24 h prior to infection. Cells were chilled and adsorbed with 5,000 particles/cell of reovirus-A546 (red) at 4°C for 1 h. The inoculum was removed, unbound virus was washed away, and cells were either fixed with 10% formalin or supplemented with complete medium and incubated at 37°C for the times shown. Cells were imaged by confocal microscopy. Insets depict enlarged areas of boxed regions. Scale bars, 10 ␮M. (B) HeLa cells were transfected with EGFP-Rab5A or EGFP-Rab7 for 24 h and incubated with 10 ␮g of transferrin-A546/ml at 37°C for 20 min. The cells were acid washed to remove cell surface transferrin, fixed with 10% formalin, and imaged by confocal microscopy. The quantification of spectral overlap of transferrin-A546 with RabGTPases is shown as the percent colocalization. (C to I) Quantification of spectral overlap of reovirus-A546 particles with EGFP-tagged versions of Rab4 (C), Rab5A (D), Rab5C (E), Rab7 (F), Rab9 (G), Rab11 (H), and RILP (I) after adsorption of reovirus for the times shown. Spectral overlap is shown as percent colocalization (n ⫽ 12 cells per time point, average of 136 particles per time point). Error bars indicate standard deviations. *, P ⬍ 0.05 in comparison to 0 min.

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FIG 2 JAM-A and reovirus particles traffic to similar endocytic compartments during cell entry. HeLa cells were transfected with JAM-A and EGFP-tagged versions of Rab4, Rab5A, Rab5C, Rab7, Rab11, or Rab13 24 h prior to infection. Cells were chilled and adsorbed with 20,000 particles/cell of reovirus at 4°C for 1 h. The inoculum was removed, the unbound virus was washed away, and the cells were either fixed with 10% formalin or supplemented with complete medium and incubated at 37°C for the times shown. The cells were stained for JAM-A and reovirus, imaged by confocal microscopy, and analyzed for spectral overlap between JAM-A and reovirus particles (A) or JAM-A, reovirus particles, and Rab-marked organelles (B). The spectral overlap is shown as the percent colocalization (n ⫽ 11 cells per time point, average of 468 particles per time point). Error bars indicate standard deviations. (C) Representative images of HeLa cells transfected with EGFP-Rab7 (green), adsorbed with reovirus-A546 (red), and stained for JAM-A (blue) at the times shown. Cells were imaged by confocal microscopy. Insets depict enlarged areas of boxed regions. Arrows indicate areas of JAM-A, EGFP-Rab7, and reovirus colocalization. Scale bars, 10 ␮m.

binding to cell surface JAM-A, reovirus is targeted to Rab5Amarked endosomes. Furthermore, reovirus and JAM-A colocalized with endosomes containing Rab 13, with little change over time, despite the fact that Rab13 overexpression decreases the percentage of viral particles associated with JAM-A at 60 min (Fig. 1). These results indicate that following attachment to JAM-A on the cell surface, reovirus particles are internalized and traffic with

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JAM-A through early, late, and recycling endocytic compartments. Reovirus particles transiently traverse through endosomes marked by Rab5A, Rab5C, or Rab11. Endocytic transport is a dynamic process in which molecules move quickly through intracellular compartments to reach a specific destination. To determine the residence time of reovirus particles in early and recycling

Journal of Virology

Reovirus and the Endocytic Pathway

FIG 3 Reovirus particles transiently distribute to endosomes marked by Rab5A, Rab5C, or Rab11. (A) HeLa cells were transfected with EGFP-Rab5A, EGFP-Rab5C, or EGFP-Rab11 (green) 24 h prior to infection. Cells were chilled and adsorbed with 10,000 particles/cell of reovirus-A546 (red) at 4°C for 1 h. The inoculum was removed, the unbound virus was washed away, and the cells were supplemented with complete medium and imaged by confocal microscopy every 30.5 s for 30 min. Shown is a single cell with boxed regions highlighting areas enlarged in panel B. Scale bar, 10 ␮m. (B) Successive 30.5-s time frames of boxed regions indicated in panel A. Arrows label reovirus particles distributing to endosomes marked with EGFP-Rab5A or EGFP-Rab11. (C) Dwell time of reovirus particles in endosomes marked by Rab5A and Rab11 from time lapse (n ⫽ 3 cells, 20 particles per cell). The mean dwell time is shown for each Rab GTPase.

endosomes, HeLa cells were transfected with EGFP-tagged (green) Rab5A (see Movie S1 in the supplemental material), Rab5C (see Movie S2 in the supplemental material), or Rab11 (see Movie S3 in the supplemental material) for 24 h, chilled at 4°C for 1 h, adsorbed with A546-labeled reovirus particles (red), supplemented with warm complete medium, and imaged by confocal

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microscopy every 30.5 s for 30 min (Fig. 3A). Reovirus particles transiently colocalized with Rab5A, Rab5C, and Rab11 for the first 30 min of infection (Fig. 3B), providing further evidence that reovirus traverses early and recycling compartments during cell entry. To determine the interval in which reovirus particles reside in endosomes marked by Rab5A, Rab5C, or Rab11, time lapses were

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FIG 5 Expression of dominant-negative Rab5A and Rab7 impairs reovirus virions from accessing an acidified compartment. HeLa cells were transfected with EGFP, EGFP-tagged versions of dominant-negative Rab4, Rab5A, Rab7, Rab11, Rab13, or Rab34 or left untransfected 24 h prior to infection. Untransfected cells were treated with 20 mM NH4Cl at 37°C for 1 h. Cells were chilled and adsorbed with 5,000 particles of reovirus-pHrodo/cell at 4°C for 1 h. The inoculum was removed, the unbound virus was washed away, and the cells were incubated in complete medium at 37°C for 90 min. Cells were fixed with 1% PFA and analyzed by flow cytometry. The data are presented as mean fluorescence intensity (MFI) relative to EGFP from duplicate experiments. Error bars indicate standard errors of the mean. *, P ⬍ 0.05 in comparison to EGFP.

FIG 4 Internalized reovirus particles access an acidified cellular compartment. (A) HeLa cells were chilled at 4°C for 1 h and adsorbed with pHrodo dye (Mock), 5,000 particles/cell of reovirus-pHrodo, or 5,000 particles/cell reovirus-A546 at 4°C for 1 h. The inoculum was removed, the unbound virus was washed away, and the cells were either fixed with 1% paraformaldehyde (PFA) or supplemented with complete medium and incubated at 37°C for the times shown. The cells were analyzed by flow cytometry. The data are shown as the mean fluorescence intensity (MFI) from triplicate samples. Error bars indicate standard deviations. (B) HeLa cells were incubated in complete medium (Untreated) or medium supplemented with 20 mM NH4Cl at 37°C for 1 h, chilled at 4°C for 1 h, and adsorbed with either complete medium (Mock) or 5,000 particles of reovirus-pHrodo/cell at 4°C for 1 h. The inoculum was removed, the unbound virus was washed away, and the cells were either fixed with 1% PFA or supplemented with complete medium and incubated at 37°C for the times shown. The cells were analyzed by flow cytometry. The data are presented as the MFI over mock from triplicate samples. Error bars indicate standard deviations. *, P ⬍ 0.05 in comparison to Mock.

analyzed to track individual viral particles during residence in specific endosomes (Fig. 3C). Viral particles had an average dwell time of 102.7 s in Rab5A-marked endosomes, 88.5 s in Rab5Cmarked endosomes, and 96.1 s in Rab11-marked endosomes. These data suggest that reovirus particles distribute into endosomal compartments marked by Rab5A, Rab5C, and Rab11 in a temporal manner, with particles residing within these compartments for less than 2 min. Reovirus particles access an acidified intracellular compartment during cell entry. To determine the kinetics with which reovirus particles reach endocytic compartments with a pH of 5 or less, HeLa cells were chilled at 4°C and adsorbed with reovirus particles labeled with either A546 or pHrodo at 4°C. Cells were supplemented with warm medium, incubated at 37°C for various intervals, and assayed for fluorescence intensity by flow cytometry (Fig. 4A). A546-labeled reovirus particles maintained signal intensity throughout the time course, since A546 fluorescence emission is not pH dependent. In contrast, pHrodo-labeled reovirus particles displayed increased fluorescence over time, with maximum fluorescence observed at 90 min. Labeling of reovirus particles with either A546 or pHrodo did not significantly alter the viral particle/PFU ratio (data not shown), indicating that the fluorescent tags do not adversely affect viral infectivity.

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To ensure that the fluorescence of pHrodo-labeled reovirus virions was attributable to distribution within an intracellular compartment with acidic pH, HeLa cells were incubated with complete medium in the presence or absence of 20 mM NH4Cl at 37°C for 1 h, chilled at 4°C, and adsorbed with pHrodo-labeled reovirus particles. Cells were supplemented with warm medium with or without 20 mM NH4Cl, incubated at 37°C for 90 min, and assayed for fluorescence intensity by flow cytometry (Fig. 4B). In untreated cells, fluorescence intensity increased over time, reaching a maximum at 90 min after adsorption. In cells treated with 20 mM NH4Cl, the fluorescence intensity was decreased in comparison to untreated control cells at all time points tested. These data indicate that NH4Cl quenches the acidification of intracellular compartments reached by reovirus particles during cell entry. Furthermore, reovirus particles distribute to acidified compartments between 20 and 60 min after adsorption and remain in an acidified environment for at least 90 min. Rab7 regulates reovirus access to acidic intracellular organelles during cell entry. To identify the endocytic pathway required for reovirus particles to reach an acidic compartment during cell entry, HeLa cells were transfected with EGFP or EGFPtagged versions of dominant-negative Rab4, Rab5A, Rab7, Rab11, Rab13, and Rab34 for 24 h prior to infection. As a control, nontransfected cells were incubated in the presence of 20 mM NH4Cl at 37°C for 1 h. The cells were at chilled at 4°C for 1 h, adsorbed with pHrodo-labeled reovirus at 4°C for 1 h, supplemented with warm medium in the presence or absence of 20 mM NH4Cl at 37°C for 90 min, and assayed for fluorescence intensity by flow cytometry (Fig. 5). The expression of dominant-negative Rab4, Rab 11, Rab13, and Rab34 had minimal effect on reoviruspHrodo fluorescence. Expression of dominant-negative Rab5A yielded a modest but statistically significant decrease in reoviruspHrodo fluorescence, whereas the expression of dominant-negative Rab7 had the largest inhibitory effect on fluorescence of reovirus-pHrodo. Treatment with NH4Cl impaired reovirus pHrodo fluorescence, again indicating that the labeled virions require access to an acidified compartment to emit fluorescence. Taken to-

Journal of Virology

Reovirus and the Endocytic Pathway

FIG 6 Cell surface expression of JAM-A and ␤1 integrin and binding of reovirus virions are not affected by expression of dominant-negative Rab-GTPases. HeLa cells were transfected with EGFP (white peaks in all plots) or EGFP-tagged versions of dominant-negative Rab4, Rab5A, Rab7, or Rab11 24 h prior to infection. Cells were adsorbed with 10,000 particles/cell of reovirus at 4°C for 1 h, stained with JAM-A-specific antibody (A), ␤1 integrin-specific antibody (B), or reovirus-specific antiserum (C), and analyzed by flow cytometry. The data are presented as the fluorescence intensity from a representative experiment of three performed. Gray peaks represent JAM-A, ␤1 integrin, or reovirus fluorescence intensity in EGFP-positive cells transfected with the respective dominant-negative Rab GTPase.

gether, these data suggest that Rab5A and Rab7 regulate reovirus particle transport to acidified organelles. Dominant-negative Rab GTPases do not affect the cell surface expression of JAM-A or ␤1 integrin. After attachment to JAM-A (5), reovirus is internalized and sorted in the endocytic pathway using a process dependent on ␤1 integrin (33, 34). To determine whether expression of dominant-negative Rab GTPases affects the cell surface expression of JAM-A or ␤1 integrin or alters attachment of reovirus to cells, HeLa cells were transfected with EGFP or EGFP-tagged versions of dominant-negative Rab4, Rab5A, Rab7, or Rab11 for 24 h prior to infection. Cells were adsorbed with reovirus at 4°C for 1 h, stained with antibodies specific for JAM-A, ␤1 integrin, or reovirus, and analyzed by flow cytometry (Fig. 6). Expression of dominant-negative Rab4, Rab5A, Rab7, or Rab11 did not alter cell surface expression of JAM-A or ␤1 integrin, nor did expression of these constructs hinder reovirus attachment to cells. These data indicate that dominant-negative Rab GTPases do not regulate the steady-state cell surface expression of either JAM-A or ␤1 integrin or inhibit reovirus attachment to the cell surface. Effect of dominant-negative Rab GTPases on internalization of reovirus particles. To determine whether Rab GTPases govern the internalization of reovirus particles from the cell surface, HeLa cells were transfected with EGFP or EGFP-tagged versions of

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dominant-negative Rab4, Rab5A, Rab7, Rab9, Rab11, or ECFPtagged dominant-negative Rab5C for 24 h prior to infection. Cells were chilled at 4°C for 1 h, adsorbed with A546-labeled reovirus particles at 4°C for 1 h, supplemented with warm medium for 0, 20, 60, or 120 min, stained with reovirus-specific antiserum under nonpermeabilizing conditions, and imaged by confocal microscopy (Fig. 7A). Internalized fluorescently labeled viral particles (red, arrows) were distinguished from extracellular viral particles that were stained with reovirus antiserum (blue) (Fig. 7B). We observed an increased percentage of internalized viral particles over time, from 4% at 0 min to greater than 53% by 120 min. Cells expressing dominant-negative Rab5A and Rab5C displayed modest reductions in the percentage of internalized particles, although the observed decreases were statistically significant only at 120 min after adsorption in cells expressing dominant-negative Rab5A. In contrast, expression of dominant-negative Rab7 resulted in a slightly higher percentage of internalized particles at 60 min but did not affect the percentage of internalized particles at 120 min, suggesting a temporal disruption of internalization kinetics by expression of this mutant Rab construct. Dominantnegative Rab4, Rab9, and Rab11 did not affect internalization of reovirus particles at any time point tested. Collectively, these data suggest that dominant-negative Rab GTPases do not alter internalization of reovirus particles. Subtle effects of Rab GTPase over-

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FIG 7 Dominant-negative Rab GTPases do not significantly alter internalization of reovirus particles. (A) HeLa cells were transfected with EGFP, EGFP-tagged versions of dominant-negative Rab4, Rab5A, Rab7, Rab9, or Rab11, or ECFP-tagged dominant-negative Rab5C 24 h prior to infection. The cells were chilled at 4°C for 1 h and adsorbed with 1,000 particles of reovirus-A546/cell at 4°C for 1 h. The inoculum was removed, the unbound virus was washed away, and the cells were either fixed with 5% formalin or supplemented with complete medium and incubated at 37°C for the times shown. The cells were stained with reovirusspecific antiserum without permeabilization, imaged by confocal microscopy, and quantified for spectral overlap between internalized viral particles (red, white arrows, panel B) and extracellular viral particles (blue, panel B). The data are expressed as the percentage of internalized particles (n ⫽ 12 cells per time point, average of 391 particles per time point). Error bars indicate standard deviations. *, P ⬍ 0.05 in comparison to 0 min. (B) Representative images of cells at 0, 20, 60, or 120 min after adsorption. Insets depict enlarged areas of boxed regions. Reovirus-A546 (red), viral particles stained with reovirus-specific antiserum (blue), EGFP (green), and merged images are shown. Scale bars, 10 ␮m.

expression on reovirus internalization might be due to dysregulation of the endocytic compartment or altered intracellular transport of host molecules required for reovirus uptake. Early and late endosomes, but not recycling endosomes, are required for efficient reovirus infection. To test whether Rab GTPases are required by reovirus to establish productive infection, HeLa cells were transfected with EGFP-tagged versions of

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wild-type, constitutively active, or dominant-negative versions of Rab5A and Rab5C (Fig. 8A), Rab7, Rab9, and RILP (Fig. 8B), or Rab4 and Rab11 (Fig. 8C) for 24 h prior to infection. Cells were adsorbed with reovirus at an MOI of 0.5 PFU per cell, incubated at 37°C for 20 h, stained with reovirus-specific antiserum, and scored for new viral protein synthesis by flow cytometry. Expression of wild-type Rab5A or Rab5C did not affect reovirus infec-

Journal of Virology

Reovirus and the Endocytic Pathway

(Q67L) significantly increased reovirus infectivity, whereas infectivity was significantly decreased in cells expressing dominantnegative Rab7 (N125I), Rab9 (S21N), or RILP (⌬212N). Reovirus infectivity was minimally affected by expression of wild-type, dominant negative, or constitutively active forms of Rab4 or Rab11. These data suggest that reovirus requires early endosomes marked by Rab5A and Rab5C and late endosomes marked by Rab7 and Rab9 to efficiently infect host cells. Moreover, the increased infectivity observed with overexpression of wild-type Rab7 and Rab9 suggests that late endosomes regulate the rate of reovirus infection and that these organelles play a pivotal role in reovirus cell entry. DISCUSSION

FIG 8 Reovirus requires early and late endosomal compartments for efficient infection. HeLa cells were transfected with EGFP-tagged versions of Rab5A, Rab5A-Q79L, Rab5A-S34N, Rab5C, Rab5C-Q80L, or Rab5C-S35N (A), Rab7, Rab7-Q67L, Rab7-N125I, Rab9, Rab9-S21N, or RILP⌬212N (B), or Rab4, Rab4-Q61L, Rab4-S22N, Rab11, Rab11-Q79L, or Rab11-S25N (C) at 24 h prior to infection. The cells were adsorbed with reovirus at an MOI of 0.5 PFU/cell. The inoculum was removed, the unbound virus was washed away, and the cells were supplemented with complete medium and incubated for 20 h. Cells were permeabilized, stained with reovirus-specific antiserum, and assayed for infection and EGFP expression by flow cytometry. The data are expressed as the percentage of infected cells in EGFP-positive populations normalized to the percentage of infected cells in the nontransfected, EGFPnegative population from triplicate samples. Error bars indicate standard deviations. *, P ⬍ 0.05 in comparison to the nontransfected population.

tion, while expression of dominant-negative Rab5A (S34N) or Rab5C (S35N) diminished reovirus infectivity minimally. In contrast, reovirus infectivity was significantly decreased in cells expressing constitutively active Rab5A (Q79L) or Rab5C (Q80L), which each impair early endosome maturation (28, 46). Cells expressing wild-type Rab7 or Rab9 or constitutively active Rab7

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In this study, we identified endosomal compartments traversed by reovirus to establish productive infection. After adsorption to the cell surface, reovirus particles are detected in early, late, and recycling endosomes. However, access to early and late endosomes, but not recycling endosomes, is required by reovirus to productively infect host cells. Reovirus and JAM-A codistribute to various endocytic organelles, including those marked by Rab7, which are the likely site for viral disassembly. Since reovirus requires ␤1 integrin to sort to organelles that provide an environment suitable for disassembly (33, 34), we hypothesize that reovirus, JAM-A, and ␤1 integrin cotraffic to the endocytic compartments traversed by reovirus during cell entry (Fig. 9). The distribution of reovirus particles into endocytic compartments that segregate productive (early and late endosomes) versus nonproductive (recycling endosomes) routes of infection suggests that not all internalized particles are targeted to the same endocytic compartment. The presence of viral particles in recycling endosomes during cell entry also has been observed for other viruses, including foot-and-mouth disease virus (FMDV) (29), Kaposi’s sarcoma-associated herpesvirus (KSHV) (23), and vesicular stomatitis virus (VSV) (39). In contrast to FMDV, KSHV, and VSV, which exit the endocytic compartment before reaching late endosomes, reovirus must access the acidic environment of late endosomes to infect cells. Interestingly, FMDV (29), KSHV (23), VSV (39), and reovirus (34) can enter cells via receptor-mediated endocytosis using clathrin-dependent uptake mechanisms. As such, it is possible that the presence of viral particles in the recycling compartment is a by-product of this process. The fate of viral particles observed in the recycling compartment is not known. It is possible that virions in recycling endosomes fail to induce an appropriate signal during receptor binding or endocytic uptake to route the virus to organelles in the endocytic compartment that serve as sites of viral disassembly. Instead, such particles would be recycled along with JAM-A to the cell surface. Whether these virions are then released from the cell or reinternalized after reaching the cell surface, or whether these virions remain infectious, is unclear. However, expression of dominant-negative Rab4 and Rab11 did not affect the percentage of extracellular virions, suggesting that particles present in the recycling compartment do not remain attached to cells. Moreover, the distribution of internalized virions to a noninfectious pathway may explain why some reovirus particles are not infectious. Particle/PFU ratios for reovirus are frequently in excess of 100:1 (14, 47), indicating that the majority of viral particles do not give rise to productive infection. Although particles that traverse early and late endosomes are found in an infectious route, it remains un-

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JAM-A

Rab7

Integrins

?

?

Rab9

Rab4

Rab11

Endocytic vesicle Late endosome

Fast recycling

Slow recycling

Rab4

Rab5A Rab5C

Rab11

Early endosome

Virion

ISVP

Core

FIG 9 Model of reovirus internalization. After attachment to cell surface glycans and JAM-A, reovirus is internalized via receptor-mediated endocytosis using

a mechanism dependent on ␤1 integrin. Reovirus traffics to early endosomes marked by Rab5A or Rab5C, where viral particles are sorted into productive or nonproductive entry pathways. Virions in the nonproductive pathway enter recycling endosomes marked by Rab4 or Rab11 and may return to the cell surface. Virions in the productive pathway enter endosomes marked by Rab7 or Rab9, where viral disassembly takes place. The disassembly intermediate penetrates endosomal membranes, releasing the transcriptionally active viral core into the cytoplasm.

clear what percentage of these particles will productively infect cells. It is likely that the success of a viral entry event is determined by viral and cellular factors in addition to distribution to an appropriate endocytic organelle. The requirement for reovirus to traverse early endosomes for efficient infection indicates that the virus is targeted to endocytic vesicles that follow a maturation pathway from early to late endosomes. Reovirus particles localize to organelles marked by Rab5A and Rab5C, although the means by which virions access these compartments appears to differ. Reovirus and JAM-A were detected in Rab5A-marked endosomes, whereas the bulk of reovirus particles in Rab5C-marked endosomes were not associated with JAM-A. Distribution of reovirus to endosomes marked by Rab5A or Rab5C may depend on the cell surface receptor engaged during attachment. JAM-A is the only proteinaceous receptor known for reovirus. However, reovirus infection of some tissues does not require JAM-A (1), raising the possibility that engagement of yet unidentified reovirus receptors preferentially targets viral particles to Rab5C-marked endosomes. Despite the observed differences in the association of viral particles with JAM-A, expression of constitutively active forms of Rab5A or Rab5C, which each impair early endosome maturation (28, 46), diminished reovirus

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infectivity to similar levels. The modest effect on reovirus infectivity observed following expression of dominant-negative Rab5A or Rab5C might be attributable to redundancy in early endosome biogenesis. If so, blocking endosomal trafficking with one dominant-negative Rab5 isoform would not be expected to impede the capacity of the virus to access early endosomes. Distinct tissues express a subset of Rab5 isoforms (25). Although functional redundancy exists between these isoforms, in some contexts, Rab5 isoforms differentially regulate endosomal trafficking of specific receptors (12). Thus, the capacity to internalize into early endosomes marked by different Rab5 isoforms might serve as a fitness advantage for reovirus, allowing it to infect a wider range of host cells and tissues. In contrast to the limited colocalization of reovirus and JAM-A in Rab5C-marked endosomes, we observed substantial colocalization of reovirus and JAM-A in Rab7-marked late endosomes. These observations are consistent with transient transport of reovirus through early endosomes and accumulation over time in late endosomes, where colocalization of reovirus and JAM-A is more apparent. Although it is possible that the relatively more abundant colocalization of reovirus and JAM-A is due to JAM-A overexpression, it is likely that following reovirus attachment to the cell

Journal of Virology

Reovirus and the Endocytic Pathway

surface, both virus and receptor are targeted to a specific intracellular site where viral disassembly takes place. The affinity of reovirus attachment protein ␴1 for JAM-A (24) may be sufficient to allow the virus-receptor complex to traffic to a site of viral disassembly. Reovirus requires an acidic environment for cathepsin-mediated proteolytic disassembly (2, 16), which is required for release of the transcriptionally active viral core into the cytoplasm (Fig. 9). Cathepsin proteases are synthesized as zymogens and require two autocatalytic cleavage events and an acidic pH for optimum activity (14, 15, 22, 56). Interestingly, Rab7 is required to deliver cathepsin D to late endosomes (22). Although cathepsin D is dispensable for reovirus disassembly (17, 31), it is possible that dominant-negative Rab7 impairs reovirus infectivity by blocking transport to late endosomes of both reovirus virions and other cathepsin proteases. The enhanced infectivity observed in cells expressing wild-type versions of Rab7 or Rab9 or constitutively active Rab7 also is consistent with a model in which late endosomes are required for reovirus cell entry. Whether this effect is due to Rab GTPases regulating cathepsin loading into endosomes or another step that enhances viral disassembly remains to be defined. Our findings that reovirus accesses early, late, and recycling endosomal compartments were based on colocalization data gathered via confocal microscopy. A potential limitation of this technique is the requirement for high MOIs (ranging from 1,000 to 20,000 particles per cell) to visualize viral particles after adsorption, sample processing, and staining. A high MOI also is required to overcome the fact that only a fraction of the adsorbed virus actually attaches to cells and is internalized (B. A. Mainou and T. S. Dermody, unpublished observations). Although direct labeling of particles with succinimidyl ester dyes yields a greater number of detectable particles compared to indirect labeling techniques, relatively high MOIs are still required for microscopy-based experiments. An important aspect of reovirus entry that remains unresolved by our study is whether reovirus distributes with JAM-A, and possibly ␤1 integrin, in the endocytic compartment by following physiologic uptake mechanisms used by these molecules. JAM-A (60) and ␤1 integrin (10) are internalized via clathrin-dependent endocytosis, the preferred method of entry for reovirus (18, 34). Interestingly, the cytoplasmic tail of JAM-A is not required for productive reovirus infection in some types of cells (33), suggesting that JAM-A is unlikely to mediate signaling events that target the virus to a productive entry route. However, NPXY motifs in the ␤1 integrin cytoplasmic tail are required for efficient reovirus entry into cells. In fact, mutation of the ␤1 integrin NPXY tyrosine residues to phenylalanine direct reovirus to lysosomes where virions are degraded (34). Reovirus also requires activation of Src kinase to efficiently enter cells. Interestingly, like ␤1 integrin NPXY mutants, inhibition of Src kinase activity directs reovirus to lysosomes (35). Although reovirus induces signaling events during entry (34, 35), it is unclear whether these events are similar to those induced by native ligand engagement of JAM-A or ␤1 integrin, or whether reovirus induces unique signals that target viral particles to specific endosomal compartments. Intracellular pathogens must specifically engage host cells, traverse cell membranes, and deliver their infectious payloads to specific intracellular sites to establish infection. In the present study, we found that reovirus utilizes the cellular endocytic sorting ma-

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chinery to gain access to an acidic endocytic compartment marked by Rab7 or Rab9 to productively enter cells. Our findings also illustrate that viral cell entry is not strictly a linear pathway but instead involves both productive and nonproductive routes. Several virulent human pathogens, including Ebola virus (49), Old World arenaviruses (43), and SARS coronavirus (30, 53, 58), require access to late endosomes to productively infect cells. Therefore, it is possible that transient inactivation of endosomal maturation may have broad-spectrum antiviral efficacy. ACKNOWLEDGMENTS We thank Josh Doyle, Jennifer Konopka, Caroline Lai, and Danica Sutherland for critical review of the manuscript. We are grateful to members of the Dermody lab for useful suggestions during the course of this study. The flow cytometry experiments were performed in the Vanderbilt Cytometry Shared Resource. The confocal microscopy experiments were conducted in the Vanderbilt Cell Imaging Shared Resource. This study was supported by Public Health Service awards T32 HL07751 and F32 A1801082 (B.A.M.) and R01 AI32539 (T.S.D.) and the Elizabeth B. Lamb Center for Pediatric Research. Additional support was provided by Public Health Service awards P30 CA68485 for the Vanderbilt-Ingram Cancer Center and P60 DK20593 for the Vanderbilt Diabetes Research and Training Center.

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