Vaccinia virus exhibits cell-type-dependent entry characteristics

Abstract

Differing and sometimes conflicting data have been reported regarding several aspects of vaccinia virus (VV) entry. To address this, we used a β-galactosidase reporter virus to monitor virus entry into multiple cell types under varying conditions. Entry into HeLa, B78H1 and L cells was strongly inhibited by heparin whereas entry into Vero and BSC-1 cells was unaffected. Bafilomycin also exhibited variable and cell-type-specific effects on VV entry. Entry into B78H1 and BSC-1 cells was strongly inhibited by bafilomycin whereas entry into Vero and HeLa cells was only partially inhibited suggesting the co-existence of both pH-dependent and pH-independent VV entry pathways in these cell types. Finally, entry into HeLa, B78H1, L and BSC-1 cells exhibited a lag of 6–9 min whereas this delay was undetectable in Vero cells. Our results suggest that VV exploits multiple cell attachment and entry pathways allowing it to infect a broad range of cells.

Introduction

Vaccinia virus (VV) is the prototypical member of the orthopox-virus genus (Moss, 2001) and shares a high degree of DNA sequence similarity and gene conservation with the human pathogen, variola virus (Gubser et al., 2004). Because of its extensive antigenic similarity to variola and its relatively low pathogenic potential, VV was used with great success as a vaccine against variola. Although variola was eradicated from the global human population some 30 years ago, there remains considerable concern regarding the possible re-introduction of variola virus as an act of bioterrorism (Mayr, 2003).

Two forms of enveloped infectious virus particles are produced in cells infected with VV (Appleyard et al., 1971), the mature virus (MV) and the enveloped virus (EV). MV forms within the infected cell and are released upon cell lysis. EV begins as MV particles, but go on to acquire an additional, trans-Golgi-derived envelope prior to being delivered to the host cell surface (Moss, 2001). Importantly, EV and MV contain distinct subsets of viral proteins in their outer envelopes (Appleyard et al., 1971; Moss, 2006) and thus present different targets to the host cell as well as to the host immune system. Several hypotheses have been proposed regarding the entry pathways of these two infectious forms. Because significantly less EV is produced in infected cells and its outer membrane is relatively fragile (Ichihashi, 1996), this form of the virus has proven more difficult to purify. For this reason, most studies of vaccinia virus entry have focused on MV.

Studies on the role of cell-surface glycosaminoglycans (GAGs) in MV attachment and entry have been reported by a number of laboratories. Chung et al. (1998) first reported the ability of purified VV virions to bind heparin suggesting a role for heparan sulfate binding in virus infection. To date, two MV proteins, A27 and H3, have been reported to bind cell-surface heparan sulfate (Chung et al., 1998; Lin et al., 2000). A third MV protein, D8, has been reported to bind another abundant cell-surface GAG, chondroitin sulfate (Hsiao et al., 1999). The importance of heparin sulfate for virus infection is highlighted by the observation that soluble heparin reduces virus attachment and entry (Carter et al., 2005; Chung et al.,1998). Second, soluble forms of A27, D8 and H3 bind to cells and block virus attachment and infection (Chung et al.,1998; Ho et al., 2005; Hsiao et al.,1998,1999; Lai et al.,1990; Lin et al., 2000; Maa et al., 1990). Third, cells lacking sulfated GAGs are more resistant to VV infection (Chung et al.,1998; Hsiao et al.,1999; Lin et al., 2000). Additionally, antibodies raised against each of these proteins blocks VV infection (Hsiao et al., 1999; Lin et al., 2000), although this effect was not shown to be entirely attributable to interference with cell-surface GAG interactions.

Numerous studies have also addressed the role(s) of endocytosis and low pH in VV entry. The earliest electron microscopic (EM) studies revealed the appearance of enveloped MV particles within vesicles inside L cells suggesting that VV entered cells via an endocytic route (Dales and Siminovitch, 1961; Dales and Kajioka, 1964). Subsequent EM studies of MV entry into HeLa and L cells revealed direct fusion of the virion envelope with the host cell plasma membrane (Armstrong et al., 1973; Chang and Metz, 1976). The authors suggested that if endocytic entry does occur, it is not the major route of virus entry. More recently, biochemical data have provided evidence for entry by either pH-dependent or pH-independent routes. Janeczko et al. (1987) showed that the lysosomotropic agents, methylamine and chloroquine had no effect on VV entry into BSC-40 cells suggesting a pH-independent entry mechanism. Consistent with this, reports by Doms et al. (1990), Ichihashi (1996) and Vanderplasschen et al. (1998) showed that lysosomotropic agents also failed to diminish MV entry into BSC-1, Vero and RK13 cells, respectively. Furthermore, both Doms et al. (1990) and Ichihashi (1996) reported that low pH treatment of MV did not enhance membrane fusion or entry activity providing further support for a pH-independent entry mechanism. In contrast to the largely consonant earlier biochemical data, it was reported by Townsley et al. (2006) that inhibitors of endosomal acidification, such as bafilomycin A1 and concanamycin A, significantly diminished MV entry into BSC-1, RK13 and HuTK-cells. These authors concluded that MV entry is, in fact dependent upon low pH. Even more recently, it was reported that in at least one cell type (BSC40) vaccinia virus appears to enter via macropinocytosis (Mercer and Helenius, 2008).

Although the origin and natural host species of VV are unknown (Moss, 2001), it infects a variety of cell types from a wide range of host species. The apparently conflicting results obtained in previous studies of virus entry prompted us to consider the possibility that VV entry is cell-type-dependent. To test this, we examined several aspects of virus entry using a variety of host cells. For the purposes of this study, we define entry as the early events in virus infection that culminate in the introduction of a virion core into the cytoplasm of a host cell. Thus, we consider attachment, receptor binding, endocytosis, membrane fusion and core uncoating to be steps in the entry process. To facilitate these studies, we developed an entry assay based on β-galactosidase expression from a reporter virus. We validated this entry assay by showing that similar results were obtained using plaque-reduction and core-uncoating assays. Our results show the importance of cell surface HSPG and the need for low endosomal pH, vary considerably depending upon the cell type used by the virus.

Results

Synthesis of β-galactosidase in VV reporter-virus-infected cells

Various methods of measuring VV entry have been reported. Although the classical method of plaque formation has been widely used, it may not always be the best way of measuring virus entry per se as a number of downstream events in virus replication must take place in order for plaque formation to occur. Perhaps the most sensitive method described to date for studying strictly entry is to follow virus uncoating. In this assay, the viral core is detected within the cytoplasm of a host cell using core-specific antibodies (Locker et al., 2000; Vanderplasschen et al., 1998). However, because this assay involves fluorescence microscopy, data acquisition is tedious and is not amenable to processing large sample numbers. Additionally, this method does not readily lend itself to quantitative measurements of entry. Increasingly, reporter viruses that express β-galactosidase, GFP or firefly luciferase under the control of an early viral promoter have been employed as rapid, sensitive and quantitative means of measuring VV entry (Ho et al., 2005; Hsiao et al., 1998; Townsley et al., 2006). To develop such an entry assay, we tested a recombinant VV carrying β-galactosidase under the p7.5 early/late viral promoter. Virus entry was indicated by the induction of β-galactosidase activity following inoculation of various cell types in a 96-well plate. Fig. 1A shows a typical time course of β-galactosidase expression in BSC-1, B78H1, HeLa and Vero cells infected with the reporter virus using a multiplicity of infection (MOI) of 1 pfu/cell. Although our virus stocks are routinely titered on BSC-1 cells, we have observed that stock titers vary by no more than 2-fold on B78H1, Vero and HeLa cells. A low level of β-galactosidase activity was detectable as early as 2 h post-infection (inset) and enzyme activity increased thereafter in a time-dependent manner in each cell type. To assess the amount of virus required to induce readily detectable β-galactosidase activity, we infected cells for 6 h using various MOI. Here, β-galactosidase activity was detectable following infection with as little as 0.1 pfu/cell. However, β-galactosidase activities induced by 1 pfu/cell were somewhat more reproducible. Therefore, with the goal of achieving a reasonable level of sensitivity to inhibitors of virus entry while allowing reliable detection in a relatively short period of time, we chose to carry out our entry studies using a multiplicity of 1 pfu/cell and 6 h of infection.

Fig. 1.

β-galactosidase expression following infection by the vaccinia reporter virus, vSIJC-20. Cells were inoculated at 37 °C and maintained at this temperature for the duration of the infection. (A) B78H1, BSC-1, HeLa and Vero cells were infected with vSIJC-20 at a multiplicity of 1 pfu/cell for various times prior to assaying β-galactosidase activity. Enzyme activity present in cells infected for 0 h was subtracted from the activity measured in cells infected for 2, 4 or 6 h. Inset: Expanded scale showing β-galactosidase activity at 2 h post-infection. (B) β-galactosidase activity induced in B78-H1, BSC-1, HeLa and Vero cells infected with vSIJC-20 for 6 h at the indicated multiplicities of infection (pfu/cell).

Virus neutralization

As one way to establish the validity of the β-galactosidase reporter virus assay for studies of VV entry, we performed a virus neutralization experiment and compared the results with those of a standard plaque reduction neutralization test (PRNT). We previously reported the preparation of monoclonal and polyclonal antibodies to a truncated form of the VV L1 protein (Aldaz-Carroll et al., 2005b). Many of these anti-L1 antibodies exhibited strong MV-neutralizing activity in a PRNT. We tested several anti-L1 IgG’s in parallel using a PRNT as well as the β-galactosidase entry assay. A representative neutralization assay using one of these antibodies (VMC-2) is shown in Fig. 2. Both the PRNT (Fig. 2A) and the reporter virus entry assay (Fig. 2B) resulted in nearly the same neutralization titers defined as the IgG concentration required to achieve a 50% reduction in virus entry (plaque number or β-galactosidase activity). Although data from experiments using HeLa cells are shown, similar results were also observed using BSC-1 cells (data not shown). As an additional validation of the β-galactosidase reporter virus entry assay, we examined the effect of VMC-2 on the appearance of uncoated VV cores via immunofluorescence microscopy (Fig. 3) similar to that first described by Vanderplasschen et al. (1998). In this experiment, purified vaccinia virus was mock-treated or pre-incubated with the virus-neutralizing monoclonal antibody VMC-2 (Aldaz-Carroll et al., 2005b) prior to infection of HeLa cells. After 90 min at 37 °C, uncoated intracellular vaccinia virus cores were detected using a rabbit polyclonal antiserum (R236) raised against 2 synthetic peptides mimicking portions of the vaccinia virus A4 (p39) polypeptide. No vaccinia virus cores were detected in infected, but non-permeabilized cells (Fig. 3A). However, following permeabilization, numerous cores were clearly visible in the cytoplasm of infected cells (Fig. 3B). When virus was pre-incubated with a neutralizing monoclonal antibody (Panel C), the appearance of intracellular cores was greatly diminished indicating that VMC-2 had blocked infection at a step prior to virus uncoating. Based on these results, we used the reporter virus assay to examine other aspects of MV entry as well.

Fig. 2.

Effect of a virus-neutralizing antibody on entry of vSIJC-20 as measured by plaque reduction (Panel A) and β-galactosidase expression (Panel B). vSIJC-20 was incubated with various concentrations of an anti-L1 monoclonal antibody (VMC-2) for 1 h at 37 °C. Virus was then either plated on HeLa cells for plaque assay or in a 96-well plate for β-galactosidase expression. An anti-myc monoclonal antibody was used as a control.

Fig. 3.

Effects of an MV-neutralizing MAb and heparin on vaccinia virus core uncoating. HeLa cells on glass coverslips were infected with MV and non-permeabilized (Panel A), infected and permeabilized (Panel B) or infected with MV that had been preincubated for 1 h. at 37 °C with VMC-2 MAb IgG (Panel C) or with heparin (Panel D). After 90 min infection at 37 °C, cells were fixed, permeabilized with 0.1% saponin (Panels B – D only) and incubated with R236 anti-A4-peptide IgG. R236 staining was revealed using goat anti-rabbit (green). To reveal cell contours, cell surface glycoproteins were stained with wheat germ agglutinin coupled to a red fluorescent dye.

Inhibition of VV entry by heparin is cell-type dependent

VV carries at least two proteins capable of binding cell-surface heparan sulfate (A27 and H3) and one protein that binds chondroitin sulfate (D8). Many viruses utilize cell-surface GAGs as a means of attachment to host cells (Marsh and Helenius, 2006; Olofsson and Bergstrom, 2005). In such instances, virus attachment and, as a result, entry can be inhibited to some extent by pre-incubation of virus with heparin, heparan sulfate or chondroitin sulfate. Under these conditions, heparin or soluble GAGs presumably bind to virions and interfere with the attachment to GAGs on the host cell surface. VV entry exhibits variable sensitivity to inhibition by soluble heparin (Carter et al., 2005; Chung et al., 1998). The greatest reported effect of soluble heparin occurred using BSC-40 cells wherein VV infection was reduced by up to 60%. In contrast, heparin reduced virus infection of HeLa cells by only 27% and had little or no effect on infection of BSC-1, RK-13 and PtK-2 cells. Since these earlier data were obtained using a plaque reduction assay, we questioned whether soluble heparin might exhibit a different pattern of inhibition using the β-galactosidase reporter virus entry assay. Similar to previously published results, we found that VV entry into BSC-1 and Vero cells was only weakly inhibited by the highest concentrations of heparin (Fig. 4A). In contrast, virus entry into B78H1 and HeLa cells was reduced to a much greater extent by heparin (40% and 70% inhibition, respectively). To test the possibility that heparin might have affected post-entry events (ie. early viral gene expression) we performed 2 additional experiments. First, we examined the effect of adding heparin at 1 h post-infection (Fig. 4B). If heparin had altered the ability of infected cells to express β-galactosidase, we would expect to see a decrease in its expression under these conditions. Instead, we found that addition of heparin 1 h after infection had no effect on β-galactosidase activity, indicating that the heparin-sensitive step had already occurred. As a second means of verifying the point at which heparin inhibition of VV infection occurs, we examined the effect of heparin on VV core uncoating in HeLa cells by immunofluorescence microscopy (Fig. 3D). Here, the appearance of uncoated VV cores was markedly reduced following pre-incubation of virus with heparin (100 μg/mL) indicating that infection had been blocked at a step prior to virus penetration. In a separate experiment, heparin also inhibited virus entry into L cells (Fig. 4C) suggesting that HSPG play a role in VV attachment to these cells as well. Gro2C and Sog9 are L cell derivatives deficient in heparan sulfate or heparan sulfate and chondroitin sulfate synthesis, respectively (Banfield et al., 1995; Gruenheid et al., 1993). Soluble heparin showed a diminished ability to block VV infection of Gro2C cells and had little effect on infection of Sog9 cells. These data suggest that in L cells, both heparan sulfate as well as chondroitin sulfate play roles in VV attachment as previously reported. Based on the studies shown here, we conclude that HSPG are important for VV entry into some cells (ie. HeLa, B78H1 and L), but are of lesser importance on others (ie. BSC-1 and Vero). Although the GAG-binding proteins of MV (ie. A27, H3 and D8) are commonly referred to as attachment factors, the importance of GAGs for virion attachment to cells has never been directly assessed. To examine the effect of GAGs on the binding of MV’s to host cells, we used a cell-based ELISA (Fig. 4D) to detect virions bound to cells. In this experiment, L, gro2c and sog9 cells were grown in wells of a 96-well plate for 24 h. Cells were chilled (4 °C) to prevent virus entry and various amounts of gradient-purified MV were added. Unbound virions were then washed away and the remaining (cell-bound) MV were fixed to the cell surface and detected using an anti-L1 monoclonal antibody. Interestingly, MV bound to gro2c and L cells with similar efficiency, but bound much more weakly to sog9 cells. Since gro2c cells lack HSPG, but sog9 cells lack both HSPG and chondroitin sulfate proteoglycans (CSPG), these results support the notion that CSPG can compensate for the absence of HSPG and allow MV attachment.

Fig. 4.

Effect of heparin on vaccinia entry. vSIJC-20 was either mock-treated (Panel B) or incubated with various concentrations of soluble heparin for 1 h at 37 °C (Panels A and C). Virus was then plated on BSC-1, Vero, B78H1 and HeLa cells (Panel A) or on sog9, BSC-1, gro2c and L cells (Panel C). After 1 h of infection, the inoculum was removed and replaced with medium containing heparin. After 6 h of infection, cells were lysed and assayed for β-galactosidase expression. Results are plotted as a percentage of β-gal activity induced in the absence of heparin. Panel D: L, gro2c and sog9 cells were incubated (at 4 °C) with various concentrations of purified MV for 1 h. Cells were washed and fixed with 3% paraformaldehyde. Bound virions were detected via indirect immunoperoxidase colorimetric assay.

VV entry into murine B78H1 cells is pH-dependent while entry into Vero cells is pH independent

EM studies of MV entry have shown virus entering cells either by direct fusion with the plasma membrane or by fusion with endocytic vesicles. Studies using lysosomotropic agents have also led to seemingly contradictory results. In light of recent studies showing that herpes simplex virus (HSV) can utilize different entry pathways in a cell-type-dependent manner, we asked whether there might be cell-type differences in the pH-dependency of VV entry as well. To test for pH-dependent entry of MV, we used baflomycin, a drug that inhibits the endosomal Na/H+ pump and thereby prevents acidification of the endosomal compartment. The effects of bafilomycin on VV entry were examined in several different cell lines. The data in Fig. 5A show that bafilomycin inhibited virus entry to some extent in each of the cell types tested. Entry into Vero cells was least sensitive to bafilomycin. Interestingly, in these cells entry was reduced by about 35% at 25 nM, but higher bafilomycin concentrations did not further reduce virus entry suggesting that some portion of virus entry occurred via a pH-independent mechanism. Similarly, bafilomycin only partially inhibited VV entry into BSC-1 cells, although the effect was greater than in Vero cells. Virus entry into the mouse melanoma cell line, B78H1 was very sensitive to bafilomycin inhibition. In this cell type, entry was reduced by about 90% at 12.5 nM. Bafilomycin exhibited a somewhat different pattern of entry inhibition in HeLa cells. Here, vaccinia virus entry was drastically reduced following bafilomycin treatment, but required much higher concentrations of the drug. This result may reflect different sensitivities of these two cell types to the effects of bafilomycin. Consistent with the notion that bafilomycin inhibits virus entry into HeLa cells, a dramatic reduction in VV core uncoating was observed in cells treated with bafilomycin (Huang et al., 2008). As in the previous set of experiments using soluble heparin, we wanted to ensure that bafilomycin did not interfere with reporter gene expression following virus entry. To address this, we infected cells with vSIJC-20 for 1 h prior to adding bafilomycin (Fig. 5B). Under these conditions, the drug had a modest effect on virus entry into B78H1 cells and very little effect on entry into the other cell types examined indicating that the bafilomycin-sensitive step of virus infection had largely occurred during the first hour of infection. Although we attempted to confirm the effect of bafilomycin on virus entry into B78H1 cells using the immunofluorescent core uncoating assay, we were unable to obtain definitive results due to a high level of background staining in these cells.

Fig. 5.

Effect of bafilomycin on vaccinia virus entry. BSC-1, Vero, B78H1 and HeLa cells were pre-incubated with the indicated concentrations of bafilomycin for 90 min (Panel A) or mock-treated (Panel B). Cells were then infected with vSIJC-20 in the presence (A) or absence (B) of bafilomycin. At 1 h post infection, the virus inoculum was removed and fresh medium containing bafilomycin was added. Six hours post-infection, the cells were lysed and assayed for β-galactosidase expression. Results are plotted as a percentage of β-gal activity induced in the absence of bafilomycin.

Inactivation of VV by low pH treatment

A number of enveloped viruses can be rapidly inactivated by incubation at low pH. In many cases (such as with influenza virus), such inactivation is due to irreversible triggering (conformational change) of the viral fusion protein. Although the basis for low pH inactivation of some viruses (ie. HSV) is unknown, this property nevertheless provides a simple means by which to inactivate extracellular virions to augment studies of virus entry (Highlander et al., 1987). Because of the potential utility of such a technique, we tested VV for low pH inactivation (Fig. 6). Virions were first incubated for different lengths of time at various pH levels. Treated virus samples were then neutralized and tested for viability using the β-galactosidase reporter virus entry assay. Virus was relatively insensitive to pH levels of 5.0 and above although there was a slight loss of infectivity compared with untreated virions (pH 7.3) perhaps due to the change in buffer composition. At pH 4.0 and 4.5, there was an apparent enhancement of virus infectivity similar to that originally reported by Ichihashi (1996) and more recently by Townsley et al. (2006) and Townsley and Moss (2007). In contrast, incubation at pH 3.5 and 3.0 resulted in a dramatic loss of infectivity. Interestingly, this is very similar to the profile of pH inactivation of HSV. Perhaps inactivation of these two viruses at low pH occurs by a similar mechanism. By employing this low pH inactivation step, we were then able to examine the rate of virus entry into Vero, BSC-1, HeLa, B78H1 and L cells.

Fig. 6.

Inactivation of vaccinia virus following brief low-pH treatment. vSIJC-20 was incubated for various times (from 1 to 8 min) in citrate-saline buffer at 37 °C. The pH of the virus-containing samples was then rapidly neutralized by addition of an excess of DMEM–HEPES (pH7). Virus was then plated on BSC-1 cells and incubated for 6 h. Cells were then lysed and assayed for β-galactosidase expression.

The rate of VV entry is also cell-type dependent

Because VV entry exhibited cell-type-dependent sensitivity to bafilomycin (suggesting different entry pathways), we wondered whether the kinetics of virus entry also varied with cell type. To assess the rate of entry, virus was added to Vero, BSC-1, B78H1, HeLa and L cells and allowed to attach for 1 h at 4 °C. In contrast to previous reporter virus experiments, these studies were done using a multiplicity of infection of 0.1 pfu/cell. This was done to minimize the possibility of saturating the cellular machinery required for virus entry. This may be particularly important in light of the high particle to pfu ratios typical of MV preparations (Moss, 2006). Synchronous entry was initiated by rapidly shifting the temperature of the plates to 37 °C. Entry was allowed to proceed at 37 °C for varying lengths of time prior to low-pH-inactivation of any remaining extracellular virions. Following the inactivation step, infected cells were re-fed with fresh growth medium and incubated for a total of 6 h at 37 °C. As with other aspects of virus infection, the kinetics of VV entry were also cell-type dependent (Fig. 7). Entry into Vero cells occurred most rapidly and was detected within 3 min. In contrast, entry into BSC-1, B78H1, HeLa and L cells exhibited similar kinetics and occurred more slowly than entry into Vero cells during the initial 6–9 min of infection. Thus, the slowest entry kinetics were observed in cells wherein virus entry was sensitive to bafilomycin whereas the fastest entry correlated with relative insensitivity to bafilomycin (Vero cells). Perhaps the rate of entry into BSC-1, B78H1, HeLa and L cells reflects the rate of endocytosis (leading to subsequent pH-dependent infection) while the faster rate of entry into Vero cells reflects the rate of virus entry via a different pathway/mechanism.

Fig. 7.

Measurement of the rate of vaccinia virus entry into different cell types. vSIJC-20 was added to the indicated cells (grown in 96-well plates) and incubated for 1 h at 4 °C to allow virus attachment, but not entry. The temperature of the culture plates was rapidly shifted to 37 °C to initiate entry. At the indicated times following temperature shift, the virus inoculum was removed and any remaining extracellular virions were inactivated by addition of citrate-saline buffer (pH 3.0) for 2 min. Following inactivation, the cells were re-fed with pre-warmed DMEM–HEPES (pH 7.0). All plates were incubated for a total of 6 h following temperature shift to 37 °C. Cells were then lysed and assayed for β-galactosidase expression.

Discussion

As obligate intracellular parasites, viruses must first gain access to the interior of the host cell in order to execute their replicative cycle. The earliest steps of virus infection are host cell attachment and entry. We are currently studying a number of VV proteins that are present in the virion envelope and are targets of the host protective immune response to the virus (Aldaz-Carroll, et al., 2005a, 2005b). Antibodies raised against some of these VV envelope proteins block virus infection suggesting that the proteins to which they bind play roles in attachment and/or entry.

To develop a simple, rapid and sensitive method for examining VV entry in our laboratory, we tested the applicability of an assay based on β-galactosidase expression in cells infected for several hours with a reporter virus carrying the lacZ gene under the control of the p7.5 early/late VV promoter. We found that appreciable levels of β-galactosidase expression could be detected within a few hours following infection with the reporter virus. We further established the sensitivity and validity of this assay by comparing the results of a virus neutralization experiment done using the reporter virus entry assay with a similar experiment done in parallel via the standard plaque reduction neutralization test (PRNT). Virus neutralization was carried out using a MAb raised against the VV L1 protein (Aldaz-Carroll et al., 2005b). L1 has recently been shown to be required for virion penetration of the host cell and to at least transiently associate with members of the entry-fusion complex of MV envelope proteins (Bisht et al., 2008; Chang and Metz, 1976; Moss, 2006). We were encouraged by the observation that the reporter virus entry assay gave nearly the same endpoint as the PRNT for virus neutralization by a previously characterized monoclonal antibody.

A recently introduced assay, the detection of uncoated, cytoplasmic VV cores via immunofluorescence microscopy, has become a useful qualitative tool for analyzing virus entry (Locker et al., 2000; Vanderplasschen et al., 1998). The advantage of this assay is that it directly measures one of the earliest events in virus infection. To further validate the results obtained using our β-galactosidase reporter virus, we also analyzed the effect of an MV-neutralizing antibody on the appearance of virus cores within infected cells. As anticipated, mock-treated MV readily entered cells giving rise to numerous intracellular cores whereas cores were only rarely detected inside cells infected with MV that had been pre-incubated with a neutralizing anti-L1 MAb. Despite its advantages, the core uncoating assay is not ideal for quantitative analyses of virus entry as individual cores must be identified within multiple microscopic fields and then individually counted. Because of this, the core-uncoating assay is not nearly as convenient for quantitative or high-throughput applications as the reporter virus assay.

Many viruses are able to bind to one or more types of sulfated, cell-surface proteoglycans (Marsh and Helenius, 2006; Olofsson and Bergstrom, 2005). Although many studies have addressed the roles of HSPG, CSPG and other polyanionic cell surface molecules in VV attachment and entry (Carter et al., 2005; Chung et al., 1998; Ho et al., 2005; Hsiao et al., 1998, 1999; Lai et al., 1990; Lin et al., 2000; Maa et al., 1990; Moss, 2006), we re-examined the effects of soluble heparin on virus entry into several different cell types. Unlike previously published data based on plaque reduction, the β-galactosidase reporter virus entry assay allowed us to monitor the effects of heparin during the very early stages of virus infection. Interestingly, we observed a marked inhibition of entry (70%) into HeLa cells by soluble heparin whereas Carter et al. (2005) reported only a modest (27%) inhibition. Since the previous result was obtained using a plaque assay, we suspect that the difference reflects the increased sensitivity of the β-galactosidase reporter virus assay for studies of virus entry. The conclusion that this effect resulted from a reduction in virion entry was confirmed using the immunofluorescent core uncoating assay. In contrast to HeLa cells and in agreement with previous reports (Carter et al., 2005; Chung et al., 1998), infection of BSC-1 and Vero cells was relatively insensitive to heparin. These large differences in the effect of soluble heparin on VV infection suggest that HSPG play a variable and cell-type-dependent role as virion attachment factors. Since the efficiency of VV plaque formation (plating efficiency) is similar on HeLa, B78H1, Vero and BSC-1 cells (our unpublished observations), it seems likely that other molecules present on the surface of these cell types promote virus attachment. Consistent with this interpretation are previously published data showing that the MV envelope proteins D8 and A26 mediate attachment to cell surface chondriotin sulfate and laminin, respectively (Chiu et al., 2007; Hsiao et al., 1999). We conclude from our data that in certain cell types, virus attachment mediated by HSPG is very important for efficient infection.

With respect to pH, there are two broad pathways by which enveloped viruses enter cells (Marsh and Helenius, 2006). One subset of enveloped viruses enters cells in a pH-independent manner. In this pathway, viruses may either fuse directly with the plasma membrane or with an endosomal membrane following internalization. Entry of these viruses is insensitive to drugs that perturb endosomal pH. Another subset of enveloped viruses enters by fusion with an endosomal membrane in a pH-dependent manner. Depending upon the virus, low pH alone may or may not be sufficient to trigger membrane fusion. In some cases, engagement of a specific receptor and/or proteolytic activation of a virion component may be required within the low pH compartment in order for fusion to occur. In any case, entry of viruses requiring a low pH step is markedly reduced by compounds that prevent endosomal acidification.

Seemingly contradictory reports have been published on the entry pathway used by the MV form of VV (the most abundant form produced during its replicative cycle). Some have provided evidence of entry via direct fusion with the plasma membrane of the host cell (Armstrong et al., 1973; Chang and Metz, 1976; Doms et al., 1990; Ichihashi 1996; Janeczko et al., 1987; Vanderplasschen et al., 1998) while more recent reports have provided evidence that MV uses a pH-dependent endosomal entry pathway in HeLa, RK-13, BSC-1 and HuTK cells (Huang et al., 2008; Townsley et al., 2006). In light of emerging evidence of multiple, cell-type-dependent entry pathways for herpes simplex virus, we hypothesized that the pH-dependence of VV entry may also exhibit cell-type-dependent differences. Using the reporter virus entry assay, we examined the effects of bafilomycin A1 on VV entry into several different cell types. Remarkably, our results varied widely from one cell type to another. Entry into B78H1 and HeLa cells was greatly reduced in the presence of bafilomycin (albeit at very different drug concentrations) suggesting that MV entry occurs via a pH-dependent mechanism in these cells. In contrast, MV entry into both African green monkey kidney cell lines (Vero and BSC-1) exhibited only partial sensitivity to bafilomycin. This could be interpreted as evidence for the co-existence of both pH-dependent as well as pH-independent VV entry pathways in these cells. If, indeed MV can enter some cells via multiple pathways, it would be of particular interest to determine whether the same or different virion proteins are required for entry via each pathway. Similarly, it is conceivable that distinct cellular receptors are involved in entry via pH-dependent and pH-independent pathways. Chung et al. (2005) showed that VV entry into BSC-40 and L cells can be blocked by depletion of cell membrane cholesterol suggesting a role for lipid rafts in vaccinia virus entry. It will be of interest in future experiments to determine whether this aspect of virus entry is also cell-type-dependent.

Another aspect of virus entry that can be experimentally measured is the rate of entry. In order to stop virus entry at defined intervals, a method of rapidly inactivating extracellular virions was needed. Previous studies of VV entry relied on inactivation by neutralizing antibodies (Payne and Norrby, 1978). However, virus neutralization with antibodies may not occur quickly enough to allow sensitive measurements of entry kinetics. Since herpes simplex virus has been shown to be rapidly inactivated by treatment at pH 3.0 (Highlander et al., 1987), we tested the effect of low pH treatment on MV infectivity. Like HSV, VV was inactivated following a brief incubation at pH 3.0. Using the β-galactosidase reporter virus entry assay coupled with a low pH inactivation step, we examined the kinetics of MV entry into several cell types. Although only modest differences were observed in the rates of virus entry into different cell types, we did note a short, but reproducible lag in entry into BSC-1, B78H1, HeLa and L cells. This entry lag was diminished or absent in Vero cells. The correlation of delayed entry with increased bafilomycin sensitivity (in BSC-1, B78H1, HeLa and L cells) and faster entry with reduced bafilomycin sensitivity (Vero cells) suggested to us that perhaps slower entry reflects the rate at which virions are endocytosed (leading to subsequent pH-dependent infection) while faster entry reflects the rate of entry via a different, pH-independent pathway.

We recently reported the results of a series of experiments evaluating the efficacy of a subunit vaccine against infection with VV (Fogg et al., 2004, 2007; Xiao et al., 2007). These studies showed that immunization of mice with various combinations of purified, recombinant A33, B5, L1 and A27 afforded protection from a lethal dose of VV comparable to the protection provided by immunization with live VV (Dryvax). Interestingly, a subunit vaccine consisting of all or a subset of these VV proteins also protected animals against heterologous orthopoxvirus challenges. To wit, vaccinated mice were completely protected against a lethal ectromelia virus challenge (Xiao et al., 2007) and vaccinated monkeys were protected against a lethal monkeypox challenge (Fogg et al., 2007). Notwithstanding the effectiveness of this subunit vaccine in monkeys, use of the replication-deficient VV strain MVA, afforded more complete protection against the appearance of lesions associated with monkeypox challenge (Earl et al., 2004). One explanation for this difference is that there may be additional, potentially important viral targets of the host protective immune response to VV and, by extension, monkeypox virus and variola virus. Based on the studies reported here showing that some important characteristics of VV entry differ depending upon the host cell, we suspect that there may be differences in the importance of certain viral proteins for entry into cells of different tissues or host species. If this is true, the targets of virus-neutralizing antibodies may depend, at least to some extent, upon the cell type or species being infected. Future subunit vaccine protein formulations may be improved based upon studies of VV entry into relevant human tissues.

Materials and methods

Cells and virus

BSC-1, B78H1, Vero, HeLa, L, Sog9 and gro2c cells were grown in DMEM supplemented with 10% fetal bovine serum and penicillin– streptomycin (Invitrogen). The vaccinia virus used throughout these studies was vSIJC-20, a β-galactosidase reporter virus constructed in the background of strain WR (a gift of Stuart N. Isaacs, University of Pennsylvania). This virus was constructed, as described elsewhere (Girgis et al., 2008), by targeted recombination of the lacZ ORF under transcriptional control of the vaccinia early/late p7.5 promoter into the thymidine kinase locus of the vaccinia genome. A His-tagged form of the vaccinia VCP protein (Girgis et al., 2008) was also recombined into the tk locus of vSIJC-20. Reporter virus stocks were propagated in BSC-1 cells. For MV stock preparation, cells were infected at a multiplicity of 0.05 pfu/cell and incubated at 37 °C until cytopathic effect was complete (3–4 days). Infected cell cultures were frozen at −80 °C and then thawed completely. This freeze–thaw cycle was repeated 2 times to effect release of MV particles. The culture supernatant was cleared of debris by centrifugation at 2000 ×g for 30 min at 4 °C. MV stocks prepared in this way were stored at −80 °C and typically had titers around 10⁷ pfu/mL. Gradient-purified virus was obtained by pelleting virus particles from clarified tissue culture supernatant through a 36% sucrose (w/v) cushion (in 10 mM Tris (pH 9.0)) for 2 h at 25,000 ×g. Pelleted virus was then resuspended in 10 mM Tris (pH 9.0), sonicated and loaded atop a stepwise gradient of 60 %, 36 % and 15% sucrose (prepared in 10 mM Tris (pH 9.0)). Gradients were centrifuged at 55,000 ×g for 2 h and banded virus was collected via side puncture of the tube. Recovered virus was then further concentrated by pelleting through a cushion of 36% sucrose for 2 h at 5000 ×g. The resulting pellet was resuspended in 1 mL of PBS, sonicated and stored at −80 °C. Virus titers were routinely determined by plaque assay on BSC-1 cells. However, we have noted in separate experiments that similar titers (less than 2-fold differences) are obtained upon plaque assay using B78H1, Vero and HeLa cells. Plaques were visualized using immunoperoxidase staining of infected cell monolayers. Infected cell mono-layers (24–48 h post-infection) were fixed for 30 min in methanol: acetone (2:1, vol/vol) and allowed to dry completely. Fixed monolayers were then re-hydrated in PBS for 5 min and blocked in PBS containing 2% BSA (PBS-BSA). Monolayers were then incubated for 1 h with rabbit polyclonal antisera raised against B5 and L1 (Aldaz-Carroll, et al., 2005a, 2005b) diluted 1:1000 in PBS-BSA. Monolayers were then washed 3 times with PBS and incubated for 1 h. in Protein A-HRP (Amersham) conjugate diluted 1:1000 in PBS-BSA. Monolayers were then washed 3 times with PBS and incubated in PBS containing 100 mg/mL 4-chloro-1-naphthol and 0.1% (vol/vol) hydrogen peroxide until the development of color (typically 10–30 min).

Antibodies

Rabbit polyclonal antiserum R236 was generated by immunizing a rabbit with 2 synthetic peptides mimicking portions of the vaccinia A4 core polypeptide (strain WR, GenBank accession number: NC_006998). The A4 sequences withinin the peptides were: YYSEEKDPDTKKDEAI and SKFNKDQKTTTPPSTQP. Synthetic peptides (Biosynthesis, Lewisville, TX) were coupled to keyhole limpet hemocyanin (KLH) for immunization of rabbits. Monoclonal antibody VMC-2 was obtained following immunization of a mouse with a recombinant form of vaccinia L1 (Aldaz-Carroll et al., 2005b).

Immunofluorescence microscopy of intracellular VV cores

HeLa cells were seeded onto glass coverslips and incubated for 20 h at 37 °C in a humidified CO₂ incubator. Prior to the addition of virus, cells were chilled on ice for 30 min. For studies on the effects of a neutralizing MAb, virus was pre-incubated for 1 h at 37 °C in the presence of various concentrations of MAb VMC-2 (Aldaz-Carroll et al., 2005b), chilled on ice and added to cells. For studies on the effects of soluble heparin, virus was pre-incubated for 1 h at 37 °C in the presence of various concentrations of heparin (Sigma), chilled on ice and added to cells. After 1 h on ice, the plates were warmed by floatation in a 37 °C water bath for 10 min. The plates were then transferred to a 37 °C, humidified CO₂ incubator for 90 min. The plates were then chilled on ice, washed 3 times with ice-cold PBS and fixed in ice-cold 3% paraformaldehyde. All subsequent steps were performed at room temperature. Cells were washed 3 times in PBS, quenched 10 min in 50 mM NH4Cl, permeabilized using 0.1% saponin (in PBS) and blocked for a minimum of 1 h in blocking solution (PBS containing 10% goat serum). Cells were then incubated 45 min in blocking solution containing 0.25 μg/mL R236 IgG, washed with PBS and further incubated for 30 min in blocking solution containing fluorescent-tagged antibody (Alexa-488, Molecular Probes) and wheat-germ agglutinin (Alexa 594, Molecular Probes) each diluted 1:1000. Coverslips were then washed 3 times in PBS, once in distilled water and mounted on microscope slides using ProLong Antifade mounting solution (Molecular Probes).

Virus neutralization assay

IgG was prepared from ascitic fluid containing MAb VMC-2 via protein G sepharose chromatography as peviously described (Aldaz-Carroll et al., 2005b). IgG was diluted in DMEM containing 10% heat-inactivated FBS and then mixed with an equal volume of vaccinia virus (vSIJC-20). The virus-IgG mixture was incubated for 1 h at 37 °C, added to HeLa cells and incubated at 37 °C for 72 h (plaque assay) or for 6 h (for reporter gene expression). Plaques were visualized by staining infected monolayers with 0.1% crystal violet. β-galactosidase expression following reporter virus infection was determined as previously described (Whitbeck et al., 1997). Briefly, virus-infected and control (uninfected) cells were lysed by addition of an equal volume of PBS containing 1% NP-40 (final concentration of NP-40 in each well is 0.5%). Cell lysates were then mixed in a separate 96-well ELISA plate with the β-galactosidase substrate, ONPG. Plates were read in a BioTek Synergy 2 plate reader at 570 nm. Data were collected at 5-minute intervals over 50 min. The data shown reflect the rate of substrate conversion over time.

Heparin inhibition of vaccinia virus entry

vSIJC-20 was incubated in the presence of soluble heparin (various concentrations) for 1 h at 37 °C. Virus and heparin were then added to cells grown in 96-well plates and incubated for 6 h at 37 °C in a humidified CO₂ incubator. Cells were then lysed by addition of an equal volume of PBS containing 1% NP-40. β-galactosidase activity was determined as described above.

Bafilomycin inhibition of vaccinia virus entry

Cells grown in 96-well plates were incubated for 1 h at 37 °C with various concentrations of bafilomycin A1 (Sigma) diluted in growth medium containing 0.1% DMSO. Medium containing bafilomycin A1 was removed and cells were then infected with vaccinia virus at 1 pfu/ cell and incubated at 37 °C for 6 h. Cells were then lysed by addition of an equal volume of PBS containing 1% NP-40. β-galactosidase activity was determined as described above.

Low pH inactivation of vaccinia virus

Gradient-purified virus was diluted and divided into multiple aliquots of 10 μL (each aliquot contained 1×10⁶ pfu). Aliquots were then shifted to different pH levels by adding 90 μL of either PBS (pH 7.3) or citrate-saline buffers (pH 6.0, 5.0, 4.5, 4.0, 3.5 and 3.0) and incubated at 37 °C for various times (Highlander et al., 1987). Following low pH treatment, samples were neutralized by adding 0.9 mL cell growth medium containing 40 mM HEPES (pH 7) and used to inoculate BSC-1 cells in a 96-well plate. Plates were incubated for 6 h at 37 °C in a humidified CO₂ incubator. Cells were then lysed by addition of an equal volume of PBS containing 1% NP-40. β-galactosidase activity was determined as described above.

Vaccinia virus penetration assay

Cells grown in 96-well plates were incubated for 1 h at 4 °C with vaccinia virus (0.1 pfu/cell) to allow virus attachment, but not entry. Plates were then rapidly shifted to 37 °C by floating atop a 37 °C water bath for 3 min. Virus-containing medium was then removed and cells were incubated in pre-warmed (37 °C) citrate-saline buffer (pH 3) for 2 min at 37 °C. Citrate-saline buffer was then removed and cells were re-fed with pre-warmed growth medium supplemented with 40 mM HEPES (pH 7). Plates were incubated for a total of 6 h at 37 °C in a humidified CO₂ incubator. Cells were then lysed by addition of an equal volume of PBS containing 1% NP-40. β-galactosidase activity was determined as described above.