Conformational Changes Required for Reovirus Cell Entry are Sensitive to pH

Abstract

During cell entry, reovirus particles disassemble to generate ISVPs. ISVPs undergo conformational changes to form ISVP* and this conversion is required for membrane penetration. In tissues where ISVP formation occurs within endosomes, ISVP-to-ISVP* conversion occurs at low pH. In contrast, in tissues where ISVP formation occurs extracellularly, ISVP-to-ISVP* transition occurs at neutral pH. Whether these two distinct pH environments influence the efficiency of cell entry is not known. In this study, we used Ouabain to lower the endosomal pH and determined its effect on reovirus infection. We found that Ouabain treatment blocks reovirus infection. In cells treated with Ouabain, virus attachment, internalization, and ISVP formation were unaffected but the efficiency of ISVP*s formation was diminished. Low pH also diminished the efficiency of ISVP-to-ISVP* conversion in vitro. Thus, the pH of the compartment where ISVP-to-ISVP* conversion takes place may dictate the efficiency of reovirus infection.

INTRODUCTION

Viruses can cross the host cell membrane barrier at the plasma membrane or from within intracellular compartments such as endosomes (Yamauchi and Helenius, 2013). The site of membrane bypass is largely governed by the presence of host factors needed for virus entry. For those viruses that enter cells via the endocytic compartment, virus entry is often dependent on the low pH environment of the endocytic pathway. For enveloped viruses such as influenza A virus (IAV) or vesicular stomatitis virus (VSV), low pH can trigger conformational changes in the viral glycoprotein and consequently permit fusion of viral and host membranes (White et al., 1981). For nonenveloped viruses such as adenovirus and rhinovirus, low pH can promote conformational changes in the viral capsid that allow exposure of the membrane-penetration machinery (Brabec et al., 2003; Greber et al., 1993). Some viruses such as Ebola virus, require the activity of low-pH dependent endosomal proteases for priming the envelope glycoproteins for membrane fusion (Chandran et al., 2005). Similarly, for nonenveloped viruses such as the mammalian reovirus, low pH-dependent proteases mediate disassembly of the viral capsid to expose membrane-active components (Ebert et al., 2002; Martinez et al., 1996; Sturzenbecker et al., 1987). The pH of the endocytic compartment progressively decreases from early endosomes (pH 6.8–6.0) to late endosomes (pH 6.0–5.0) to lysosomes (pH < 5.0) (Ohkuma and Poole, 1978; Tycko and Maxfield, 1982). Thus, the pH at which viral machinery for cell entry is optimally active determines when the virus exits the endocytic pathway (Lozach et al., 2011). Thus, viruses such as IAV, that require lower pH (~5.5–5.1) to complete events required for crossing the host membrane, exit the endocytic pathway from late endosomes whereas those, such as Semliki Forest virus (SFV), that require a less acidic pH (~6.0) exit the endocytic pathway from early endosomes (Lozach et al., 2010; White et al., 1981). In this study, we investigated how pH affects the reovirus cell penetration machinery and if it impacts the efficiency with which reovirus cores exit the endocytic pathway.

Particles of reovirus are comprised of two concentric protein shells, the outer-capsid and the inner core (Dryden et al., 1993). The reovirus core encapsidates 10 segments of dsRNA along with enzymes necessary for generating viral mRNA. The outer-capsid of reovirus is comprised of three major proteins (σ1, σ3 and μ1), which function at different stages of cell entry. Reovirus initiates infection by binding to carbohydrate and proteinaceous receptors on the host cell via the σ1 protein (Barton et al., 2001a; Barton et al., 2001b; Dermody et al., 1990; Reiss et al., 2012). Following attachment, the virus is internalized into endosomes via clathrin- or caveolin-mediated endocytosis (Boulant et al., 2013; Ehrlich et al., 2004; Maginnis et al., 2008; Schulz et al., 2012). Within cellular endosomes, the reovirus capsid is disassembled by the action of acid pH-dependent cathepsin B and L proteases (Ebert et al., 2002; Martinez et al., 1996; Sturzenbecker et al., 1987). The viral σ3 protein is completely digested by cathepsins resulting in exposure of the μ1 membrane penetration protein (Ebert et al., 2002). Cathepsins also cleave the μ1 protein into two particle-associated fragments, μ1δ and ϕ. This partially disassembled particle is referred to as the infectious subvirion particle (ISVP) (Baer et al., 1999; Borsa et al., 1973; Chang and Zweerink, 1971; Ebert et al., 2002; Silverstein et al., 1972; Sturzenbecker et al., 1987). ISVPs then undergo a conformational change, likely triggered by a host membrane component to form ISVP*s (Chandran et al., 2002). ISVP* formation is characterized by autocleavage of the μ1 protein near the N terminus to generate μ1N and by the release of μ1N and ϕ (Chandran et al., 2002; Ivanovic et al., 2008; Nibert et al., 2005). μ1N functions with ϕ to form pores in membranes, which are necessary for delivery of the viral core into the cytoplasm to continue the viral replication cycle (Agosto et al., 2006; Ivanovic et al., 2008; Odegard et al., 2004).

The exact compartment in which each of the reovirus cell entry events take place is not known. Reovirus disassembly is blocked by lysomotropic agents such as ammonium chloride and Bafilomycin A1 that prevent the lowering of endosomal and lysosomal pH (Martinez et al., 1996; Sturzenbecker et al., 1987). Cathepsin proteases are most active in late endosomes and lysosomes (Turk et al., 2012). Based on the requirement for transport of virus to the late endosomes and the requirement for low-pH dependent cathepsin activity for virus infection (Ebert et al., 2002; Mainou and Dermody, 2012; Martinez et al., 1996; Sturzenbecker et al., 1987), it is assumed that ISVP formation occurs in late endosomes. Under these conditions, ISVP* formation occurs in a low pH compartment. In some tissues, such as the intestinal tract or respiratory tract, reovirus disassembly can occur extracellularly (Bass et al., 1990; Bodkin et al., 1989; Nygaard et al., 2012). Serine proteases present in the lumen of these tissues can generate ISVPs by degradation of σ3 and cleavage of μ1 (Bass et al., 1990; Bodkin et al., 1989; Nygaard et al., 2012). ISVPs generated extracellularly can initiate infection even in the presence of lysomotropic agents (Martinez et al., 1996; Sturzenbecker et al., 1987). Moreover, they initiate infection at the plasma membrane or following internalization but prior to reaching the early endosomes (Borsa et al., 1979; Boulant et al., 2013; Lucia-Jandris et al., 1993; Schulz et al., 2012). Thus, ISVP* formation after infection is initiated by ISVPs occurs under neutral or near-neutral conditions. Though determinants that control ISVP-to-ISVP* formation have been extensively studied (Agosto et al., 2007; Agosto et al., 2008; Chandran et al., 2002; Chandran et al., 2003; Coffey et al., 2006; Madren et al., 2012; Middleton et al., 2007; Sarkar and Danthi, 2010, 2013), all of these studies have analyzed ISVP* formation in vitro, at neutral pH.

Toward the goal of assessing the effect of pH on the function of the reovirus membrane penetration apparatus, we sought to alter the pH of the endosomal compartment. Blocking endosomal acidification prevents ISVP formation (Martinez et al., 1996; Sturzenbecker et al., 1987), and therefore would not allow us to determine the effect of pH on membrane penetration steps that occur subsequent to ISVP formation. We therefore elected to increase the acidity of the endosomal compartment. Endosomal pH is controlled by the vacuolar ATPase (Forgac, 2007; Van Dyke, 1996). The function of the vacuolar ATPase is dependent on the positive charges present in the lumen of the endosome (Rudnick, 1986). Positive charge within the endosome is controlled by the electrogenic Na+/K+ pump (Cain et al., 1989; Fuchs et al., 1989). Disruption of the function of Na+/K+ pump by agents such as Ouabain octahydrate (referred to as Ouabain) further lowers the pH of the endosomes (Cain et al., 1989; Feldmann et al., 2007). We therefore tested the effect of Ouabain on the capacity of reovirus to initiate infection of host cells. We observed that further acidification of cellular endosomes using Ouabain diminishes the efficiency of virus infection. A decrease in pH does not affect virus attachment, uptake, or ISVP formation but blocks conversion of ISVPs to ISVP*s. These data indicate that the reovirus cell penetration apparatus is sensitive to pH. Thus, the pH of the compartment from which reovirus initiates infection can influence the efficiency of infection.

MATERIALS AND METHODS

Cells

ATCC L929 cells were maintained in Eagle’s MEM (EMEM)(Lonza) supplemented to contain 5% fetal bovine serum (FBS) (Invitrogen) and 2 mM L-glutamine (Invitrogen). Spinner-adapted murine L929 cells were maintained in Joklik’s MEM (Lonza) supplemented to contain 5% FBS, 2 mM L-glutamine, 100 U/ml penicillin (Invitrogen), 100 μg/ml streptomycin (Invitrogen), and 25 ng/ml amphotericin B (Sigma-Aldrich). CHO cells were maintained in HAM’s F12 medium (Lonza) supplemented to contain 10% FBS and 2 mM L-glutamine. All experiments were performed in ATCC L929 cells or CHO cells. Spinner-adapted L929 cells were used for cultivating and purifying viruses.

Reagents

Ouabain octahydrate, Bafilomycin A1 and proteinase K were purchased from Sigma-Aldrich. TLCK-treated Chymotrypsin (CHT) and proteinase K was purchased from Worthington. Anti-reovirus polyclonal antibodies, anti-μ1 4A3 mAb, and anti-σNS antibodies were a gift from Dr. T. Dermody (Vanderbilt University).

Viruses

Purified reovirus virions were prepared using second- or third-passage lysates stocks of recombinantly generated reovirus T1L or T1L/T3DM2 (Kobayashi et al., 2010; Sarkar and Danthi, 2010). Viral particles were Vertrel-XF (Dupont) extracted from infected cell lysates, layered onto 1.2 to 1.4 g/cm³ CsCl gradients, and centrifuged at 187,183 × g for 4 h. Bands corresponding to virions (1.36 g/cm³) were collected and dialyzed in virion-storage buffer (150 mM NaCl, 15 mM MgCl₂, 10 mM Tris-HCl [pH 7.4]) (Berard and Coombs, 2009). The concentration of reovirus particles in purified preparations was determined from an equivalence of one OD unit at 260 nm equals 2.1 × 10¹² virions per ml (Smith et al., 1969). The particle to PFU of ratios of each virus preparation used for infections was between 200–400.

For pHrodo labeling, reovirus particles were diluted 10-fold into fresh 0.05 M sodium bicarbonate buffer (pH 8.5) to attain a final concentration of 3 × 10¹² particles/ml and incubated with 10μM pHrodo Green STP (Invitrogen) at room temperature for 90 min in the dark. Virus particles were dialyzed against PBS and stored at 4°C.

Infections

Monolayers of ATCC L929 or CHO cells were pretreated with media containing different concentrations of Ouabain at 37°C for 1 h. Cells were washed with PBS, and adsorbed with virions or ISVPs at 4°C (for attachment and in cell ISVP formation) or room temperature (for infectivity measurement, internalization, σNS expression, and RNA synthesis) for 1h. Cells were washed with PBS and incubated in media containing different concentrations of Ouabain at 37°C for the indicated time intervals. For experiments using Bafilomycin, the inhibitor was included in the pretreatment media 1 h prior to Ouabain treatment and replaced in the media following virus adsorption. For analyzing ISVP formation, media added post adsorption also included 10 μg/ml cycloheximide. Cycloheximide was used to ensure that signal from only incoming viral proteins is detected. For attachment and internalization assays, cells were detached using PBS supplemented with 20 mM EDTA and virus adsorption was performed by continuous rotation. MOIs for indirect immunofluorescence were selected to obtain 200–1000 foci per field of view. 1000 foci per field represent ~ 25% of infected cells. Fewer particles were used for T1L/T3DM2 than T1L because T1L/T3DM2 produces ~ 10 times as many infected cells as T1L. A higher dose of virus was used to infect CHO cells because CHO cells are poorly permissive to reovirus due to the absence of function JAM-A (Danthi et al., 2006). MOIs for other experiments were selected based on the sensitivity of the assay used.

Assessment of infectivity by indirect immunofluorescence

Monolayers were fixed with chilled methanol at −20°C for a minimum of 30 min, washed twice with PBS, blocked with 2.5% Ig-free bovine serum albumin (BSA)(Invitrogen), and incubated with polyclonal rabbit anti-reovirus serum at a 1:5000 dilution in PBS containing 0.25% Triton X-100 (TX-100) at room temperature for 30 min. Monolayers were washed twice with PBS and incubated with a 1:5000 dilution of Alexa Fluor 488-labeled anti-rabbit IgG (Invitrogen) in PBS containing 0.25% TX-100. The infected cells were visualized by indirect immunofluorescence using an Olympus IX71 microscope. Infected cells were identified by the presence of intense cytoplasmic fluorescence that was excluded from the nuclei. No background staining of uninfected control monolayers was observed. Reovirus antigen-positive cells were quantified by counting fluorescent cells in random fields in three independent wells at a magnification of 20×.

To assess infectivity of viral cores, cells in each well of a 96 well plate were transfected with 3.3 × 10⁸ cores using 0.05 μl of Lipofectamine 2000 (Invitrogen). The transfection was incubated at 37°C for 12 h. Infectivity was assessed using indirect immunofluorescence as described above. Incubation of cells with an equivalent number of cores without addition of lipofectamine was used as a control and produced no reovirus-positive cells.

Growth assay

After removal of the inoculum, the cells were washed with PBS and incubated at 37°C for 0 h or 24 h. Cells were frozen and thawed twice prior to determination of viral titer by plaque assay using L929 cells. Viral yields were calculated according to the following formula: log₁₀ = log₁₀(PFU/ml)_{24 h} − log₁₀(PFU/ml)_{0 h}.

Cell viability assay

Uninfected cells grown in black walled 96-well plates were incubated with the indicated concentration of Ouabain for 18 h. Cell lysates were prepared using Cell-titer Glo reagents (Promega) according to manufacturer’s instructions and luminescence was measured using a Biotek Synergy H1 plate reader. Uninfected cells grown in 96-well plates were incubated with the indicated concentration of Ouabain for 18 h and imaged by light microscopy using an Olympus IX71 microscope at 20× magnification.

Assessment of viral attachment by flow cytometry

After removal of the inoculum, the cells were harvested and resuspended in PBS supplemented with 5% BSA (PBS-BSA). Reovirus specific rabbit polyclonal antiserum at 1:2500 was added and incubated at 4°C for 30 min with continuous rotation. The cells were washed twice with PBS-BSA followed by incubation at 4°C for 30 min with the Alexa fluor 488-labeled goat anti rabbit antibody at 1:1000 in PBS-BSA with continuous rotation. The cells were washed twice with PBS-BSA prior to being fixed with cold 1% paraformaldehyde in PBS. Viral attachment to cells was quantified using BD FACSCalibur Cell Analyzer and the CellQuestPro software.

Measurement of reovirus internalization

ATCC L929 cells plated on coverslips were washed with chilled PBS and then pretreated with the indicated concentration of Ouabain and 10 μg/ml cycloheximide at 37°C for 1h. Cells were adsorbed at 4°C for 1 h with T1L/T3DM2 virus (8 × 10⁵ particles/cell). Media containing the indicated concentration of Ouabain and 10 μg/ml cycloheximide was added and incubated for 3 h. Cells were washed with PBS and fixed with 4% formaldehyde for 20 min at room temperature. Excess formaldehyde was quenched with an equal amount of 0.1 M glycine, followed by washing with PBS. Cells were treated with 1% Triton X-100 for 5 min and incubated with PBS-BGT (PBS, 0.5% BSA, 0.1% glycine, and 0.05% Tween 20) for 10 min. Cells were incubated with reovirus-specific polyclonal antiserum (1:2000) in PBS-BGT for 1 h and washed with PBS-BGT. Cells were stained for 1 h with donkey anti-rabbit immunoglobulin conjugated to Alexa594 (Molecular probes) and Acti-stain (Cytoskeleton, Inc) to visualize actin. Coverslips were removed and placed on slides with Aqua Polymount (Polysciences, Inc.). Images were captured on a Leica laser-scanning confocal microscope using LAS-AF software. The intensity of reovirus stain in an area of the cells internal to the boundary marked by Acti-stain was quantified using the LAS-AF software.

Assessment of pHrodo fluorescence intensity by flow cytometry

After removal of the inoculum, cells were washed with PBS, and resuspended in pre-warmed EMEM containing 0 or 500 μM Ouabain. The cells were placed back in plates and incubated at 37°C for 3 h to allow for internalization. The attached and unattached cells were collected with PBS-EDTA and resuspended in cold 1% paraformaldehyde in PBS. Fluorescence emitted by the pHrodo-labeled virus was quantified using BD FACSCalibur Cell Analyzer and the CellQuestPro software.

Evaluating ISVP-to-ISVP* formation in cells

ATCC L929 cells plated in 96 well plates were washed with chilled PBS and then pretreated at 37°C with indicated concentration of Ouabain and 10 μg/ml cycloheximide for 1 h. Cells were adsorbed at 4°C for 1 h with T1L ISVP with 10⁴ particles per cell. Media containing indicated concentration of Ouabain and 10 μg/ml cycloheximide was added and incubated for 0 or 2 h. Cells were washed with PBS and fixed with 4% formaldehyde at room temperature for 20 min. Excess formaldehyde was quenched with an equal amount of 0.1 M glycine, followed by washing with PBS. Cells were treated with 1% Triton X-100 for 5 min and incubated with PBS-BGT (PBS, 0.5% BSA, 0.1% glycine, and 0.05% Tween 20) for 10 min. Cells were incubated with reovirus-specific 4A3 antiserum (1:500) in PBS-BGT for 1 h and washed with PBS-BGT three times. Cells were stained with anti-mouse IgG conjugated to Alexa 594 (Molecular probes) and DAPI for 1 h and washed with PBS-BGT three times. The infected cells were visualized by indirect immunofluorescence using an Olympus IX71 microscope. Reovirus antigen-positive cells were quantified by counting fluorescent cells in random fields in three independent wells at a magnification of 20×

Generation of ISVPs and cores in vitro

ISVPs of T1L or T1L/T3DM2 were generated by incubation of 2 × 10¹² virions or 2 × 10¹³ virions with 200 μg/ml of CHT in a total volume of 0.1 ml at 32°C in virion storage buffer or Sodium Citrate buffer (150 mM NaCl, 15 mM sodium citrate [pH 7.5]) for 60 min (Nibert et al., 1995). Proteolysis was terminated by addition of 2 mM phenylmethylsulphonyl fluoride (PMSF) and incubation of reactions on ice. Viral cores were generated by incubating 2 × 10¹³ particles with 200 μg/ml TLCK-treated CHT and 400 mM CsCl in a total volume of 0.05 ml at 37°C for 2 h. Proteolysis was terminated by addition of 2 mM PMSF. Cores were dialyzed against PBS prior to use. Generation of ISVPs or cores was confirmed by SDS-polyacrylamide gel electrophoresis (PAGE) and Coomassie Brilliant Blue staining.

Preparation of infected cell lysates and immunoblotting

Cells were harvested, washed with PBS, and lysed using RIPA lysis buffer (50 mM Tris [pH 7.5], 50 mM NaCl, 1% TX-100, 1% DOC, 0.1% SDS, and 1 mM EDTA) containing a protease inhibitor cocktail (Roche), 500 μM DTT and 500 μM PMSF followed by centrifugation at 13,000 rpm at 4°C for 5 min to remove cell debris. The clarified lysates were subjected to immunoblot analysis.

The cell lysates were resolved by electrophoresis in SDS-PAGE gels and transferred to nitrocellulose membranes. Membranes were blocked for at least 1 h in blocking buffer (TBS containing 5% milk or 2.5% BSA) and incubated with anti-reovirus polyclonal antibody (1:1000) or σNS (1:2000 each) and PSTAIR (1:10000) at 4°C overnight. Membranes were washed three times for 5 min each with washing buffer (TBS containing 0.1% Tween-20) and incubated with 1:20000 dilution of Alexa Fluor conjugated goat anti-rabbit IgG (for reovirus polyclonal), anti-mouse IgG (for PSTAIR), or IRDye-conjugated anti-guinea pig IgG (for σNS) in blocking buffer. Following three washes, membranes were scanned using an Odyssey Infrared Imager (LI-COR).

RT-qPCR

RNA was extracted from infected cells using Tri-reagent (Molecular Research Center). 0.5 to 2 μg of RNA was reverse transcribed using gene-specific primers complementary to T1L S1 and GAPDH mRNA using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). A 1:10 dilution of the cDNA was subjected to PCR using SYBR Select Master Mix (Applied Biosystems). Ct values for each cDNA sample were calculated by subtracting Ct values for GAPDH from Ct values of T1L S1. Fold change in gene expression with respect to samples harvested at 0 h following infection was measured using the Ct method (Schmittgen and Livak, 2008).

In vitro ISVP-to-ISVP* conversion

To assess the effect on Ouabain on in vitro ISVP* formation, ISVPs of T1L/T3DM2 at a concentration of 2 × 10¹² per ml reaction were diluted 1:10 into sodium citrate buffer (150 mM NaCl, 15 mM sodium citrate [pH 7.5]) with 2mM PMSF and with 0 or 500 μM Ouabain. To assess the effect of pH on ISVP* formation, ISVPs of T1L/T3DM2 at a concentration of 2 × 10¹² per ml reaction were diluted 1:10 into Sodium Citrate buffers (150 mM NaCl, 15 mM sodium citrate) of pH 5.0, 6.0, or 7.5 containing 2 mM PMSF. The reaction was divided into aliquots and incubated at temperatures ranging from 33°C to 50°C in a gradient thermal cycler for 20 min, transferred to ice for 20 min, and incubated with 30 μg/ml proteinase K at 4°C for 30 min. Proteinase K digestion was terminated by addition of SDS-PAGE loading buffer and boiling the samples at 95°C for 5 min. Generation of ISVP*s was confirmed by SDS-PAGE and Coomassie Brilliant Blue staining.

Hemolysis

Citrated calf red blood cells (RBCs) (Colorado Serum Company) were washed extensively with chilled PBS supplemented to contain 2 mM MgCl₂ (PBS-Mg) and resuspended at a concentration of 30% v/v in PBS-Mg. Hemolysis efficiency was analyzed by mixing a 2.22 μl aliquot of resuspended RBCs with T1L/T3DM2 ISVPs in a total volume of 22.22 μl sodium citrate buffer at the desired pH in the presence of 0 or 300 mM CsCl, followed by incubation at 37°C for 60 min. RBCs mixed with sodium citrate buffers without ISVPs or with 1% Triton X-100 (TX-100) were used as control for 0 and 100% lysis, respectively. Samples were placed on ice for 30 min to prevent further hemolysis and centrifuged at 500 × g at 4°C for 3 min. The extent of hemoglobin release was quantified by measuring the A₄₀₅ of a 1:5 dilution of the supernatant in a microplate reader (Molecular Devices). Percentage hemolysis was calculated using the following formula: % hemolysis = 100 × (A_sample−A_buffer)/(A_TX-100 − A_buffer).

Statistical analysis

Statistical significance between experimental groups was determined using the unpaired t test function of the Graphpad Prism software. P values less than 0.05 were considered to be significant and are indicated by asterix (*).

RESULTS

Ouabain blocks reovirus infection

To test the effect of Ouabain on reovirus infection, cells were infected with two virus strains T1L and T1L/T3DM2 in the presence of increasing concentrations of Ouabain for 18 h. T1L and T1L/T3DM2 contain an identical σ1 attachment protein but distinct μ1 proteins (Sarkar and Danthi, 2010). Thus, these two viruses should be expected to traffic via the same route but it is anticipated that T1L/T3DM2 will exit the endosomal compartment with greater efficiency than T1L (Chandran et al., 2002; Lucia-Jandris et al., 1993; Sarkar and Danthi, 2010). Using indirect immunofluorescence to detect reovirus antigen, we observed a dose-dependent decrease in cells positive for reovirus proteins following infection with each reovirus strain (Figure 1A). These data suggest that Ouabain treatment affects reovirus infectivity. To rule out the possibility that the inhibitory effect of Ouabain on reovirus infection is restricted to L929 cells, we assessed the effect of Ouabain on reovirus infection of CHO cells (Figure 1B). We observed that Ouabain was also inhibitory to infection by both viruses in CHO cells. Because Ouabain blocked infection by both viruses in two different cell lines with equivalent efficacy, we performed the remainder of our experiments with T1L/T3DM2 in L929 cells.

FIG. 1. Ouabain inhibits reovirus infection.

ATCC L929 cells (A) or CHO cells (B) were pretreated with the indicated concentration of Ouabain. The cells were adsorbed with 10³ particles per cell of T1L or 10² particles per cell of T1L/T3DM2. (B) CHO cells were adsorbed with 10⁵ particles per cell of T1L or 10⁴ particles per cell of T1L/T3DM2. After incubation at 37°C for 18 h in the presence of the indicated concentration of Ouabain, reovirus positive cells were identified by indirect immunofluorescence. Infectivity equivalent to the number of fluorescent foci obtained in cells treated with 0 μM Ouabain was considered 100%. Results are expressed as mean infectivity relative to that obtained in the presence of 0 μM Ouabain for three independent samples. Error bars indicate SD. *, P < 0.05 as determined by Student’s t-test in comparison to cells treated with 0 μM Ouabain. (C) ATCC L929 cells pretreated with Ouabain were adsorbed with 10² particles per cell of T1L/T3DM2. Titers of virus in cell lysates at 0 and 24 h in the presence of the indicated concentration of Ouabain were determined by plaque assay. Results are expressed as yield of virus for three independent samples. Error bars indicate SD. *, P < 0.05 as determined by Student’s t-test in comparison to cells treated with 0 μM Ouabain. (D) ATCC L929 cells were treated with the indicated concentration of Ouabain for 18 h. Cell viability was assessed by CellTiter-Glo chemiluminiscent assay. Results are expressed as mean relative light units for three independent samples. Error bars indicate SD. (E) Bright field images of cells treated with 0 or 500 μM Ouabain for 18 h are shown. Scale bar indicates 100 μm.

We next measured the capacity of Ouabain to affect the production of infectious reovirus following a single round of replication. We observed a 2.2 log₁₀ increase in reovirus titer over 24 h following infection in control cells (Figure 1C). In contrast, only a 1.3 log₁₀ increase in viral titer was observed in the presence of Ouabain (Figure 1C). To ensure that the inhibitory effect of Ouabain on reovirus infection was not due to deleterious effect on cell division or due to toxicity to cells, we determined if Ouabain altered intracellular ATP levels in uninfected cells (Crouch et al., 1993; Saunders et al., 2014). We observed no difference in the intracellular ATP levels in Ouabain-treated and control cells (Figure 1D) indicating that Ouabain treatment did not alter metabolic activity in the treated cells. Uninfected cells treated with 0 or 500 μM Ouabain also did not demonstrate any obvious difference in morphology (Figure 1E). These data indicate that Ouabain does not compromise cell health and suggest that Ouabain treatment has an inhibitory effect on reovirus replication.

Ouabain does not affect virus attachment or uptake into a low pH compartment

To identify how Ouabain affects reovirus replication, we assessed the effect of Ouabain on each stage of virus replication. Using flow cytometry, we compared both the number of cells staining positive for cell-associated reovirus (% reovirus-positive cells) and the amount of virus bound to each cell (Mean fluorescence intensity, MFI) in the presence or absence of Ouabain. Under the conditions used, we found that ~ 70–80 % of control and Ouabain-treated cells were reovirus positive and each displayed a MFI of ~ 500–600 (Figure 2A). These data indicate that Ouabain does not influence reovirus attachment.

FIG. 2. Ouabain allows efficient viral attachment, internalization and delivery into a low pH compartment.

(A) ATCC L929 cells pretreated with 0 or 500 μM Ouabain were adsorbed with 5 × 10⁴ particles per cell of T1L/T3DM2. Attached virions were detected by staining with anti-reovirus sera using a flow cytometer. The results are expressed as the mean percentage of reovirus-positive cells and the mean fluorescence intensity for three independent samples. Error bars indicate SD. (B) ATCC L929 cells pretreated with 0 or 500 μM Ouabain were adsorbed with 8 × 10⁵ particles per cell of T1L/T3DM2. Following infection for 3 h in the presence of CHX and 0 or 500 μM Ouabain, the cells were fixed and stained with anti-reovirus sera and Acti-stain. Mean intensity of reovirus antigen normalized to the area from 10 randomly selected cells is shown. Error bars indicate SD. (C) ATCC L929 cells pretreated with 0 or 500 μM Ouabain were adsorbed with 5 × 10⁴ virions per cell of pHrodo-labeled T1L/T3DM2. Following infection at 37°C for 3 h, the mean fluorescence intensity of reovirus-positive cells was determined using a flow cytometer. Mean fluorescence intensity of infected cells in the presence of 0 μM Ouabain was considered to be 100%. The results are expressed as mean fluorescence intensity for three independent samples relative to cells treated with 0 μM Ouabain. Error bars indicate SD. *, P < 0.05 as determined by Student’s t-test in comparison to cells treated with 0 μM Ouabain.

Following attachment to host cells, reovirus particles are internalized by clathrin- or caveolin-mediated endocytosis into early endosomes (Boulant et al., 2013; Ehrlich et al., 2004; Maginnis et al., 2008; Schulz et al., 2012). To determine whether Ouabain treatment affects endocytosis of reovirus, we allowed internalization of attached reovirus particles for 3 h and imaged infected cells using confocal microscopy. Reovirus particles were found internalized in both untreated and Ouabain treated cells. Quantification of internalization indicated that the extent of internalization was not affected by the presence of Ouabain (Figure 2B). Following internalization, particles traverse through early and late endosomal compartments prior to delivery of viral cores into the cytoplasm (Mainou and Dermody, 2012). Thus, the virus is delivered into a low pH compartment. To determine if Ouabain treatment influences uptake and delivery of reovirus particles into a low pH compartment, we used reovirus virions chemically coupled to pHrodo, a pH sensitive dye. An increase in fluorescence of pHrodo-labeled reovirus during early stages of infection is indicative of delivery of virus particles into a low pH environment (Anafu et al., 2013; Mainou and Dermody, 2012). Consistent with efficient internalization of the particles, pHrodo fluorescence increased following infection in control or Ouabain-treated cells (data not shown). Moreover, we observed a modest (~ 15 %), yet significant increase in the mean fluorescence intensity of the internalized virus 3 h following infection of Ouabain-treated cells in comparison to untreated cells (Figure 2C). Thus, in agreement with previous observations, Ouabain treatment leads to a lowering of the endosomal pH. Though pHrodo and other available pH indicators display large differences in fluorescence between neutral and acidic pH, they exhibit only a small change in fluorescence at acidic pHs below pH 6. Thus, the modest increase in fluorescence could be due to a small change in pH, low sensitivity of pHrodo in that pH range, or both. Regardless, our results indicate that in Ouabain-treated cells, reovirus particles traffic through a compartment with below-normal pH.

Ouabain influences reovirus infection at a step after ISVP formation

In the endosomal compartment, reovirus virions are disassembled by the action of low pH-dependent cathepsin proteases to form ISVPs (Ebert et al., 2002). To determine if Ouabain treatment influences ISVP formation, we monitored cleavage of the reovirus μ1 protein into the δ fragment over the first few hours after infection. We found that μ1 cleavage occurred with similar kinetics both in control and Ouabain-treated cells (Figure 3A). These data indicate that Ouabain treatment does not affect ISVP formation. Together, the results presented thus far suggest that Ouabain lowers the pH of the compartments accessed by reovirus during cell entry and affects a step in reovirus infection subsequent to ISVP formation.

FIG. 3. Ouabain influences infection subsequent to ISVP formation.

(A,B) ATCC L929 cells pretreated with 0 or 500 μM Ouabain were adsorbed with 10⁴ particles per cell of T1L/T3DM2. Cell lysates prepared following infection in the presence of CHX and the indicated concentration of Ouabain for various time intervals were immunoblotted for μ1 and PSTAIR loading control. μ1 resolves as μ1C on SDS-PAGE gels (Nibert et al., 2005). (B) ATCC L929 cells pretreated with the indicated concentration of Ouabain were adsorbed with 10³ ISVPs per cell of T1L/T3DM2. After incubation at 37°C for 8,12, or 18 h in the presence of the indicated concentration of Ouabain, reovirus positive cells were identified by indirect immunofluorescence. Infectivity equivalent to the number of fluorescent foci obtained in cells treated with 0 μM Ouabain was considered 100%. Results are expressed mean infectivity relative to that obtained in the presence of 0 μM Ouabain for three independent samples. Error bars indicate SD. *, P < 0.05 as determined by Student’s t-test in comparison to cells treated with 0 μM Ouabain. (C) ATCC L929 cells pretreated with 0 or 500 μM Ouabain were adsorbed with 10³ ISVPs per cell of T1L/T3DM2. Cell lysates prepared following infection in the presence of the indicated concentration of Ouabain for various time intervals were immunoblotted for σNS and PSTAIR loading control. (D) ATCC L929 cells pretreated with 0 or 200 nM Bafilomycin were treated with 0 or 500 μM Ouabain. Cells were adsorbed with 10³ ISVPs per cell of T1L/T3DM2. After incubation at 37°C for 8 h in the presence (+) or absence (−) of Bafilomycin, Ouabain or both, reovirus positive cells were identified by indirect immunofluorescence. Infectivity equivalent to the number of fluorescent foci obtained in cells treated with 0 μM Ouabain was considered 100%. Results are expressed as mean infectivity relative to that obtained in the presence of 0 μM Ouabain for three independent samples. Error bars indicate SD. *, P < 0.05 as determined by Student’s t-test in comparison to cells treated with 0 μM Ouabain. **, P < 0.05 as determined by Student’s t-test in comparison to cells treated with 500 μM Ouabain.

In vitro treatment of reovirus virions with proteases results in formation of ISVPs that are identical in composition to ISVPs generated within infected cells (Baer et al., 1999; Borsa et al., 1973; Chang and Zweerink, 1971; Ebert et al., 2002; Silverstein et al., 1972; Sturzenbecker et al., 1987). The capacity of in vitro generated ISVPs to efficiently attach to host cells and initiate infection allowed us to directly test the impact of Ouabain on infection by ISVPs using indirect immunofluorescence. Since ISVPs initiate infection with kinetics faster than those observed during virus infection, these experiments were terminated at 8 h following infection. We observed that infection initiated using ISVPs was insensitive to Ouabain at concentrations up to 200 μM. A reduction in infectivity of ISVPs was observed in the presence of 500 μM Ouabain (Figure 3B). Comparison of our results here and those presented in Figure 1 indicate that ISVPs are sensitive to Ouabain but to a lower extent than virions. Upon incubation of infected cells for an extended time, we observed that the inhibitory effect of Ouabain on infection by ISVPs was less marked at 12 h and Ouabain did not influence infectivity of ISVPs by 18 h following infection (Figure 3B). Consistent with this timing, we observed that though expression of the reovirus nonstructural protein σNS was lower in Ouabain-treated cells at 6 h following infection with ISVPs, the levels of σNS in cells were equivalent in control and Ouabain-treated cells by 9–12 h following infection (Figure 3C). These data suggest that Ouabain treatment delays but does not prevent infection by ISVPs.

The lowering of endosomal pH by Ouabain requires the function of the vacuolar ATPase (Cain et al., 1989; Feldmann et al., 2007). Thus, blockade of activity of the vacuolar ATPase should prevent Ouabain-mediated decrease in the pH of cellular endosomes and therefore diminish the effect of Ouabain treatment on infection with ISVPs. To test this possibility, we determined whether Ouabain diminished the infectivity of ISVPs in cells pretreated with vacuolar ATPase inhibitor, Bafilomycin A1 (Yoshimori et al., 1991). Consistent with previous studies and our results above, infection by ISVPs was not affected by Bafilomycin A1 but diminished by Ouabain (Martinez et al., 1996). In cells treated with Bafilomycin A1 prior to addition of Ouabain, the infectivity of ISVPs was unaffected (Figure 3D). These data further suggest that inhibitory effect of Ouabain on infection is due to lowering of the pH.

Ouabain affects ISVP-to-ISVP* conversion

The μ1 protein exposed on the surface of the ISVPs undergoes a conformational change to form ISVP* and release pore forming μ1 fragments that facilitate the delivery of viral cores into the cytoplasm (Chandran et al., 2003). As a consequence of these events, viral cores begin RNA synthesis. To determine the effect of Ouabain on viral RNA synthesis, we measured viral RNA synthesis in ISVP infected cells using reverse transcription quantitative PCR (RT-qPCR). In untreated cells, we detected an ~ 1800-fold increase in + strand specific for the viral S1 gene segment. In contrast, Ouabain treatment resulted in only an ~ 60-fold increase (Figure 4A). These data suggest that Ouabain treatment affects viral RNA synthesis, indicating that either ISVP-to-ISVP* conversion, core delivery across membranes, RNA synthesis from cores, or each of these steps are sensitive to Ouabain.

FIG. 4. Ouabain treatment diminishes efficiency of ISVP* formation.

(A) ATCC L929 cells pretreated with 0 or 500 μM Ouabain were adsorbed with 10⁴ ISVPs per cell of T1L/T3DM2. Following incubation for 6 h in the presence of the indicated concentration of Ouabain, RNA extracted from infected cells was subjected to RT-qPCR for reovirus S1 + strand RNA and GAPDH mRNA at 0 and 6 h post infection. The results are presented as the mean fold increase in viral S1 + strand RNA over 6 h for three independent samples. Error bars indicate SD. *, P < 0.05 as determined by Student’s t-test in comparison to cells treated with 0 μM Ouabain. (B) ATCC L929 cells pretreated with 0 or 500 μM Ouabain were transfected with reovirus cores. Infectivity 12 h following transfection was measured using indirect immunofluorescence. Infectivity equivalent to the number of fluorescent foci obtained in cells treated with 0 μM Ouabain was considered 100%. Results are expressed as mean infectivity relative to that obtained in the presence of 0 μM Ouabain for three independent samples. Error bars indicate SD. (C) ATCC L929 cells pretreated with 0 or 500 μM Ouabain were adsorbed with 2.5 × 10⁴ ISVPs per cell of T1L. Following incubation for 0 or 2 h in the presence of CHX and the indicated concentration of Ouabain, the cells were fixed, permeabilized, and immunostained with 4A3 and DAPI. Scale bar indicates 20 μm (D) Percentage of total cells (DAPI positive) that also stain with 4A3 at 2 h following infection were quantified by counting ~ 150 cells from randomly selected fields in three independent wells. No 4A3 positive cells were detected at 0 h. *, P < 0.05 as determined by Student’s t-test in comparison to cells treated with 0 μM Ouabain.

Due to the absence of outer-capsid proteins required for cell entry, reovirus cores are noninfectious. However, cores transfected into cells can bypass the usual cell entry events and complete the remaining stages of virus infection (Jiang and Coombs, 2005). To identify if Ouabain affects virus infection after the delivery of viral cores into the cytoplasm, we measured infectivity of transfected reovirus cores in the presence and absence of Ouabain. At 12 h, the first time point at which a significant fraction of reovirus antigen-positive cells were detected following transfection, the infectivity of cores was equivalent in the presence and absence of Ouabain (Figure 4B). In addition to the results presented in Figures 1D and 1E, the absence of an inhibitory effect of Ouabain on establishment of infection by transfected cores rules out the possibility that Ouabain treatment adversely affects the viability of host cells (Figure 1D). Moreover, the data demonstrating that infection initiated by transfected cores is unaffected by Ouabain suggest that a step in reovirus infection subsequent to ISVP formation but prior to core transcription is affected by Ouabain treatment. These results point to ISVP-to-ISVP* conversion or cytoplasmic delivery of cores as Ouabain-sensitive events.

To directly test the effect of Ouabain on ISVP-to-ISVP* conversion in infected cells, we stained infected cells with a monoclonal antibody, 4A3, that detects μ1 in ISVP* but not in an ISVP conformation (Chandran et al., 2003). Consistent with previous studies, 4A3 did not label cells immediately following attachment (0 h) (Figure 4C) (Chandran et al., 2003). Upon infection of cells in the absence of Ouabain for 2 h, we observed diffuse cytoplasmic staining by 4A3 in ~ 15% of cells (Figure 4C, 4D). Because the altered conformer of δ is released from core particles following ISVP-to-ISVP* conversion and distributes into the cytoplasm of infected cells, 4A3 positivity is indicative of ISVP* conversion (Chandran et al., 2003). Following incubation of cells in the presence of Ouabain, we found significantly fewer 4A3-positive cells. Thus, our studies demonstrate that Ouabain treatment inhibits ISVP-to-ISVP* conversion.

Efficiency of ISVP-to-ISVP* conversion is diminished at low pH

Conversion of reovirus virions-to-ISVP and ISVP-to-ISVP* can also be recapitulated in vitro. We therefore decided to determine if in vitro conversion of ISVPs-to-ISVP*s is affected directly by Ouabain or indirectly by the effect of Ouabain on the pH of the endosomal compartment. Incubation of ISVPs at elevated temperatures in vitro triggers their conversion to ISVP*s (Agosto et al., 2007; Middleton et al., 2007; Sarkar and Danthi, 2013). ISVP-to-ISVP* conversion is characterized by conformational changes to the particle-associated δ fragment that render the δ fragment sensitive to proteases. We found that inclusion of Ouabain during in vitro experiments did not affect the temperature at which δ became protease sensitive (data not shown). These data suggest that the inhibitory effect of Ouabain on ISVP* formation in cells is not due to the direct effect on Ouabain on the virus capsid but likely a consequence of Ouabain-mediated decrease in endosomal pH. To directly test whether ISVP-to-ISVP* conversion is affected by pH, ISVPs were placed in buffers of decreasing pHs and triggered to form ISVP* by incubation at elevated temperatures. CsCl-generated ISVP*s were used in parallel to control for protease activity. We found that heat-triggered ISVP* formation occurs most efficiently at pH 7.5. Further acidification to a pH of 6.0 or 5.0, reminiscent of the pH encountered in the early or late endosomes (Ohkuma and Poole, 1978; Tycko and Maxfield, 1982), required increasingly higher temperatures to complete ISVP* formation (Figure 5A). Because the δ fragment of ISVP* generated using CsCl as a trigger was protease-sensitive in buffers of each pH, these data indicate that the effect of pH on the digestion susceptibility of δ was a function of its conformation.

FIG. 5. Low pH affects ISVP-to-ISVP* conversion.

(A) ISVPs in different buffers of the indicated pH were heated at a range of temperatures between 33°C and 50°C for 20 min and treated with proteinase K at 4°C for 30 min. Samples were resolved by SDS-PAGE and stained using Coomassie Brilliant Blue. Virus (V), ISVP (I) and ISVP* (I*) were resolved to indicate the protein composition of each type of particle. I* samples were generated by heating ISVPs to 39.7°C in the presence of 300 mM CsCl (Chandran et al., 2002). This sample also serves as a control for efficient proteolysis under each buffer condition. The positions of reovirus capsid proteins are shown. ϕ is too small to resolve on the gel. (B) ISVPs were incubated with erythrocytes in buffers of the indicated pH with 0 mM or 300 mM CsCl. Hemolysis was quantified by determining absorbance of the supernatant at 405 nm. Results are expressed as mean percent hemolysis for three independent samples. Error bars indicate SD. *, P < 0.05 as determined by Student’s t-test in comparison to hemolysis at pH 7.5.

ISVP-to-ISVP* conversion in cells is likely triggered by interaction of the exposed μ1 protein with a membrane component (Chandran et al., 2002; Liemann et al., 2002). Therefore, incubation of ISVPs with model membranes such as those of bovine erythrocytes results in ISVP-to-ISVP* conversion and the concomitant release of μ1-derived pore-forming peptides under physiological conditions (Agosto et al., 2006; Chandran et al., 2002; Sarkar and Danthi, 2010, 2013). Because the released peptides perforate erythrocyte membranes and cause hemolysis, hemolysis can be used as a readout for the generation of ISVP*s (Agosto et al., 2006; Chandran et al., 2002; Ivanovic et al., 2008). Though hemolysis was significantly less efficient in citrate buffer than the Tris-based buffer typically used for hemolysis (Sarkar and Danthi, 2010, 2013), a significant amount (~ 25%) of hemolysis was observed at a pH of 7.5 (Figure 5B). In contrast, minimal hemolysis was observed at a pH of 5.0. Thus, hemolysis efficiency is diminished at acidic pH. To rule out the possibility that the pore-forming activity of μ1 peptides is sensitive to pH, we assessed the capacity of ISVPs to hemolyze erythrocytes under conditions where ISVP* formation is efficient. For these experiments, CsCl, was included in the hemolysis reaction (Chandran et al., 2002). We observed that in the presence of CsCl, hemolysis was equivalently efficient at pH 7.5 and 5.0. These data indicate that pore formation by μ1-derived peptides is not affected by acidic pH. Therefore, our studies demonstrate that ISVP-to-ISVP* conversion is sensitive to acidic pH.

DISCUSSION

During entry of reovirus into host cells, ISVPs can be formed intracellularly at low pH or extracellularly at neutral pH. Thus, subsequent steps in reovirus replication including ISVP-to-ISVP* conversion and pore formation to deliver viral cores can occur in distinct environments. In this study, we investigated the effect of pH on cell entry events that occur subsequent to ISVP formation. We found that enhanced acidification of cellular endosomes using Ouabain diminishes the capacity of reovirus to initiate infection by blocking ISVP-to-ISVP* conversion. In vitro conversion of ISVP-to-ISVP* was also affected by low pH. Thus, our data indicate that the efficiency with which reovirus initiates infection may depend on the pH of the compartment where ISVP-to-ISVP* conversion occurs.

How pH influences ISVP-to-ISVP* conversion is unknown. During ISVP-to-ISVP* conversion, the μ1 protein undergoes structural changes that result in exposure of buried μ1 hydrophobic regions and antibody epitopes (Chandran et al., 2002). Concomitantly, the viral μ1N and ϕ fragments are released (Agosto et al., 2006; Ivanovic et al., 2008). Trimers of μ1 form a stabilizing lattice on the surface of ISVPs (Dryden et al., 1993; Hooper and Fields, 1996; Middleton et al., 2002; Wessner and Fields, 1993; Zhang et al., 2005). The structural changes needed to complete ISVP-to-ISVP* transition require disruption of interactions of μ1 molecules within and between μ1 trimers, and between μ1 and the core proteins (Agosto et al., 2007; Middleton et al., 2007; Sarkar and Danthi, 2010, 2013; Zhang et al., 2006). It would therefore be anticipated that pH affects ISVP* conversion by changing the strength of one or more these interactions between viral capsid proteins.

Previously, it has been demonstrated that a reovirus mutant that is inefficient in the formation of ISVP* is less infectious and ISVPs of this virus accumulate in a compartment positive for lysosomal markers (Chandran et al., 2003). Localization of virions or an entry intermediate to the lysosomes has also been reported when viral uptake mechanisms or viral trafficking routes are perturbed in cells lacking the cytoplasmic tail of β1 integrins or in cells treated with Src kinase inhibitors (Maginnis et al., 2008; Mainou and Dermody, 2011). Because cathepsins should be active in this compartment (Turk et al., 2012), we think it likely that the particle trapped in the lysosomes is an ISVP. Why ISVPs fail to escape lysosomes is not understood. Our results presented here indicating that ISVPs cannot complete ISVP* formation efficiently at low pH offer a possible explanation for these observations.

In many cell types, including the L929 cells used in our studies here, ISVPs initiate infection much more readily than virions (Golden et al., 2002). While in most cases, the poorer infectivity of virions can be explained by the absence of sufficient amounts of mature cathepsins, a subset of these cell types are unable to support virion infection despite the presence of mature cathepsins (Golden et al., 2002). Thus, it is proposed that the nonpermissivity of these cell lines is related to the absence of other host factors needed for infection by virions. However, an alternate explanation for this observation could be that these cell lines present an environment that is restrictive to infection by viruses but not ISVPs. L929 cells are among the most permissive to reovirus infection and our data indicating that reovirus titers increase by two orders of magnitude in 24 h support this idea (Figure 1C). Thus, it is unlikely that the deficiency in cathepsin activity contributes to the lower infectivity of virions in comparison to ISVPs. Following infection by virions, particles traffic via early endosomes to late endosomes (Boulant et al., 2013; Mainou and Dermody, 2012). In contrast, depending on the cell type, following infection by ISVPs, the particles either penetrate at the plasma membrane or are internalized but exit the endocytic pathway prior to reaching early endosomes (Borsa et al., 1979; Boulant et al., 2013; Lucia-Jandris et al., 1993; Schulz et al., 2012). Thus, virions and ISVPs exit the endosomal system from distinct pH environments. We therefore propose that infection by virions is less efficient because virions convert to ISVPs in a low pH compartment where the next step in the infection process – ISVP-to-ISVP* conversion is less efficient.

While we did not attempt to identify which cellular membrane is bypassed by ISVPs, the sensitivity of ISVPs to the pH lowering effect of Ouabain is supportive of the idea that infection initiated by ISVPs requires their internalization through a compartment that contains both the Na+/K+ pump and the vacuolar ATPase. Virions may be more sensitive to Ouabain than ISVPs because they transit through a more distal compartment within the endosomal uptake pathway that contains either more of the Na+/K+ pump, more vacuolar ATPase, or both. In some cell types, ISVPs penetrate the host membrane directly at the plasma membrane (Lucia-Jandris et al., 1993). The lower sensitivity of ISVPs can therefore also be explained by the possibility that a fraction of the ISVPs enter directly at the plasma membrane and do not traverse a compartment that has any Na+/K+ pumps or vacuolar ATPase and consequently are not exposed to a low pH environment. Given our in vitro data, we reasoned that ISVPs entering at the plasma membrane could be blocked for ISVP* formation by initiating infection in buffers of lower pHs. Though we attempted to initiate infection of ISVPs in such conditions, the effect of low pH treatment on cells for the amount of time needed to allow initiation of infection by ISVPs severely compromised the health of the cells and did not allow us to address this possibility.

Ouabain has been previously demonstrated to block infection by a variety of enveloped viruses including Ebola virus, Herpes Simplex virus, Sendai virus, Sindbis virus and Porcine Reproductive and Respiratory Syndrome virus (PRRSV) (Dodson et al., 2007; Garcia-Dorival et al., 2014; Karuppannan et al., 2012; Mento and Stollar, 1978; Nagai et al., 1972). In a majority of these studies, it has been demonstrated that Ouabain treatment inhibits viral gene expression. However, the stage at which Ouabain blocks viral infection is not known. The inhibitory effect of Ouabain on disparate viruses argues that Ouabain targets a cellular component. In the current study we focused on the pH lowering effect of Ouabain (Cain et al., 1989; Feldmann et al., 2007). Our analyses of each stage of virus infection, the neutralization of the effect of Ouabain by blocking the vacuolar ATPase, and in vitro analysis of stages of viral entry indicate that enhanced acidification of the endosomes blocks reovirus infection. However, at least some of the viruses (Herpesvirus and Sendai virus) that are sensitive to Ouabain do not require endocytosis for cell entry (Fuller and Spear, 1987; White et al., 1980). Thus, it seems unlikely that the pH lowering effect of Ouabain blocks infection by these viruses. In addition to the endsomal pH, Ouabain can also affect endocytosis and cellular signaling due to the inhibition of the Na+/K+ pump (Feldmann et al., 2007; Haas et al., 2002; Liu et al., 2000; Wang et al., 2004). Detailed analyses of how blockade of the Na+/K+ pump influences infection by these viruses could aid in the development of antiviral agents against these pathogenic viruses.

Highlights.

• Early events in reovirus entry are inefficient in presence of Ouabain

• Ouabain affects the capacity for ISVP-to-ISVP* conversion

• Effect of Ouabain on ISVP-to-ISVP* conversion is due to lowering of endosomal pH