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Poliovirus replication and spread in primary neuron cultures
Virology 340 (2005) 10 – 20 www.elsevier.com/locate/yviro
Poliovirus replication and spread in primary neuron cultures John K. Daley, Lisa A. Gechman, Jason Skipworth, Glenn F. Rall* Fox Chase Cancer Center, Institute for Cancer Research, 333 Cottman Avenue, Philadelphia, PA 19111, USA Received 24 November 2004; returned to author for revision 20 December 2004; accepted 23 May 2005 Available online 11 July 2005
Abstract While some neurotropic viruses cause rapid central nervous system (CNS) disease upon entry into the brain parenchyma, other viruses that are cytolytic in the periphery either result in little neuropathology or are associated with a protracted course of CNS disease consistent with persistent infection. One such virus, poliovirus (PV), is an extremely lytic RNA virus that requires the expression of CD155, the poliovirus receptor (PVR), for infection. To compare the kinetics of PV infection in neuronal and non-neuronal cell types, primary hippocampal neurons and fibroblasts were isolated from CD155+ transgenic embryos and infected with the Mahoney and Sabin strains of PV. Despite similar levels of infection in these ex vivo cultures, PV-infected neurons produced 100-fold fewer infectious particles as compared to fibroblasts throughout infection, and death of PV-infected neurons was delayed approximately 48 h. Spread in neurons occurred primarily by trans-synaptic transmission and was CD155-dependent. Together, these results demonstrate that the magnitude and speed with which PV replication, spread, and subsequent cell death occur in neurons is decreased as compared to non-neuronal cells, implicating cell-specific effects on replication that may then influence viral pathogenesis. D 2005 Elsevier Inc. All rights reserved. Keywords: Poliovirus; CD155; Primary neurons; Mouse embryonic fibroblasts; CNS; Viral spread; Trans-synaptic transmission
Introduction Poliovirus (PV) is an enterovirus belonging to the family Picornaviridae and is the etiological agent of poliomyelitis in humans. PV possesses a positive strand RNA genome of approximately 7.5 kb, packaged in a non-enveloped icosahedral protein capsid (Hogle et al., 1985). The capsid consists of 60 copies of each of four virally encoded structural proteins VP1, VP2, VP3, and VP4. The icosahedral structure formed by these capsid proteins produces deep surface depressions, or Fcanyons_, that have been identified as receptor binding sites (Colston and Racaniello, 1994, 1995; Hogle et al., 1985; Racaniello and Ren, 1996). PV infection initiates when the viral particle binds to its cellular receptor, CD155 (Belnap et al., 2000; He et al., 2000). Upon binding, the PV particle undergoes a conformational change in which the VP4 capsid protein is lost and the amino-
* Corresponding author. Fax: +1 215 728 2412. E-mail address: [email protected] (G.F. Rall). 0042-6822/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.virol.2005.05.032
terminus of the internal VP1 protein is extruded (Everaert et al., 1989; Fricks and Hogle, 1990; Gomez et al., 1993; Lonberg-Holm et al., 1975; Wien et al., 1996). It is believed that the exposure of the hydrophobic N-terminus of the VP1 protein forms a pore in the host cell membrane, allowing the viral particle to enter the cytoplasm. Additionally, these conformational changes in the virion result in a reduced sedimentation coefficient in the viral particle from the native 160S to an infectious intermediate or 135S sub-particle. It is thought that viral engagement with CD155 triggers this conformational change (Tsang et al., 2001); however, whether the loss of VP4 is the basis of the altered sedimentation coefficient remains debated (Basavappa et al., 1998). The normal route of PV infection in naturally permissive hosts begins with infection of the enteric system through oral ingestion of the virus. Viral particles initially replicate in the gastrointestinal system, but replication at this site does not result in any detectable pathology (Bodian, 1955; Sabin, 1956). In approximately 0.1 –1% of acute PV infections, the virus invades the CNS, producing the paralytic disease
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symptoms associated with poliomyelitis. This disease results from PV infection, translational inhibition, and destruction of motor neurons in the central nervous system (CNS) and is clinically manifested as limb paralysis. Interestingly, despite a rapid course of viremia that can be detected soon after viral exposure, PV appears to adopt a less productive course in the CNS (Girard et al., 2002; Minor, 1996). The ability of polio to spread in a somewhat restricted manner within the CNS may foster the establishment of a slow or persistent infection. While controversial, this observation has led to suggestions by others that chronic infection may be linked to diseases such as post-polio syndrome, in which patients develop signs of poliomyelitis months to years after primary exposure (Destombes et al., 1997; Pavio et al., 1996). The natural expression of CD155 in humans and some primates, but not in experimental animals such as mice, limited early studies of PV spread and pathogenesis. In 1990 –91, transgenic mice expressing CD155 were established; these mice were susceptible to PV infection by all three viral serotypes (Koike et al., 1991; Ren et al., 1990). The development of these valuable models was responsible for substantial advances in our understanding of PV replication and pathogenesis; however, important aspects of PV interaction with its natural host remain obscure. For example, while expression of CD155 is required for poliovirus entry (Koike et al., 1990; Mendelsohn et al., 1989), it is not sufficient to confer permissivity, as poliovirus cannot establish an infection in CD155-expressing mice by the intra-oral route, despite expression of CD155 within the gut (Ren et al., 1990). The pathway of PV spread from the periphery to the CNS in susceptible hosts is not fully understood. As viremia is a prerequisite for the development of poliomyelitis and dendritic cells and macrophages can be infected with poliovirus (Wahid et al., 2005; Buisman et al., 2003; Freistadt et al., 1993), polio may gain entry to the brain via transport of infected blood cells across the blood – brain barrier (BBB) (Yang et al., 1997). A second proposed mechanism of PV neurotropism is transport along neural pathways, perhaps provoked by muscle injury at the site of infection, as in the case of provocation poliomyelitis (Gromeier and Wimmer, 1998). In immunoelectron microscopy studies of spinal cord sections of mice infected with the type 2 Lansing strain, PV antigens were primarily detected in neuronal axons and synapses, suggesting that spread may be trans-synaptic (DalCanto et al., 1986). Subsequently, PV was shown to travel to the CNS from infected muscle tissue in transgenic mice via major nerves, such as the sciatic nerve (Ponnuraj et al., 1998; Ren and Racaniello, 1992), providing additional support for transsynaptic transmission. The speed of PV spread from the periphery to the CNS via neuron – neuron interactions was later shown to be consistent with fast retrograde axonal transport (Ohka et al., 1998). Interestingly, however, PV particles isolated from the sciatic nerve were of the intact
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160S form of the viral particle, not the 135S sub-particles thought to be required for infection (Arita et al., 1999). One possible interpretation of these results is that interaction with the viral receptor may not be required for PV transmission across the synapse, a theory for which evidence exists in a model of neuronal infection by another RNA virus, measles (Lawrence et al., 2000). Although PV has been studied extensively in vivo, its replication and spread in purified primary neuron cultures have not been extensively explored. Based on observations that PV infection of the CNS is distinct from infection of the periphery with respect to viral yield and cytopathicity, we undertook studies to address whether PV adopts an altered replication profile in purified primary neurons and to further explore the basis of PV spread in such cultures. Using highly enriched primary neuron cultures isolated from CD155+ transgenic embryos, as well as control mouse embryo fibroblasts (MEFs) obtained from the same animals, we compared the kinetics of infection of neurons with MEFs, established the mode of interneuronal viral spread, and clarified the role played by CD155 in neuronal PV transport.
Results Poliovirus growth kinetics in permissive neurons and mouse embryo fibroblasts To assess PV replication kinetics in different primary cell cultures, type 1 PV infection by the Sabin strain and the more virulent Mahoney strain of cultured CD155+ neurons was compared to infection of mouse embryo fibroblasts (MEFs) isolated from the same embryos. To confirm that CD155+ explant cultures indeed express this receptor, immunofluorescence using a monoclonal antibody to CD155 was performed on CD155+ and CD155 cells. As shown in Fig. 1, neurons (A – C) and MEFs (G –I) isolated from CD155+ transgenic mice were immunopositive, whereas neurons isolated from CD155 (c57Bl/6) embryos (D –F) were not. The slight background signal detected in nontransgenic neurons is likely due to nonspecific binding of the secondary antibody. PV replication and transmission in CD155+ primary neurons and MEFs were then examined. Cells were infected with PV-Sabin or PV-Mahoney (MOI = 0.1); at 8 h intervals, cells and supernatants were collected and analyzed for infection by flow cytometry and immunocytochemistry (Figs. 2A and B), extent of cell death by cellular morphology and counting of residual live cells (Fig. 2C), and virus production by plaque assay and infectious center assay (Fig. 3). As shown by flow cytometry (Fig. 2A) at 24 and 40 h post-infection (hpi), the extent of infection in MEFs and neurons was comparable. Although more MEFs were infected at each timepoint evaluated, both neurons and MEFs became heavily infected over time, and the rate of
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Fig. 1. CD155 expression in primary hippocampal neurons and mouse embryonic fibroblasts (MEFs). CD155+ primary hippocampal neurons (A – C) and mouse embryonic fibroblasts (G – I) were prepared from PVR transgenic mice. CD155 control neurons (D – F) were prepared from c57Bl/6 mice. All cells were immunostained for CD155 expression. Phase (A, D, G), fluorescence (B, E, H), and merged (C, F, I) are shown. Representative images were taken from one of three identical experiments. Magnification: 100.
spread within the cultures was similar. For the Mahoney strain, 72% and 84% of MEFs were immunopositive at 24 and 40 hpi respectively, compared to 36% and 58% infection of neurons (Fig. 2A; Table 1). Although the number of antigen-positive cells was lower following infection by the less virulent Sabin strain, a similar profile of infection was observed. Interestingly, two distinct peaks were observed in the FACScan histograms of Sabin-infected MEFs at 40 hpi (Fig. 2A); this was also observed as two immunopositive populations that differed in staining intensity (Fig. 2B, panels k and m). Despite efficient PV infection and spread in both cell types, virus-induced cytopathicity in neurons was reduced as compared with matched infected MEFs (Fig. 2C). For these studies, the number of live and dead cells was determined at each timepoint by morphology, TUNEL staining, or trypan blue exclusion. Mahoney infection of MEFs was highly lytic, killing virtually all infected cells by 24 hpi (Fig. 2B, panel e; Fig. 2C). In contrast, neuronal infection at this same timepoint was not associated with extensive cell loss (Fig.
2B, panel f; Fig. 2C), and even at 40 hpi, neuronal death was still less than 50% (Fig. 2B, panel j). Although MEF cell death was less pronounced with the Sabin strain, most infected MEFs showed signs of nuclear condensation and rounding (Fig. 2B, panels g and k), whereas neurons at the same timepoints appeared healthy, with intact processes (panels h and l). Thus, while neurons eventually do die as a result of PV infection with both strains, virus-induced cytopathicity was noted significantly earlier in MEFs than in neurons: dying fibroblasts were seen as early as 8– 16 hpi for both strains of PV; in contrast, neuronal cytopathicity was not observed until 36 – 48 hpi, and even at this late timepoint, viable neurons were abundant. The delay in PV-induced cytopathic effect (CPE) in neurons was consistent with a 100- to 500-fold decrease in extracellular infectious viral titers, relative to matched MEFs (Fig. 3A). For example, in the representative experiment shown (of five such experiments), at 24 hpi, MEF titers following PV-Mahoney challenge were 9 107 PFU/ml as compared to neuronal titers of 2 105 PFU/ml. Though
Fig. 2. PV spread and cytotoxicity in primary neurons and fibroblasts. (A) Flow cytometry was performed on single cell suspensions of PV-infected MEFs and neurons at 24 and 40 hpi. As described in Materials and methods, cells were labeled with anti-PV antibodies and visualized with an FITC-conjugated secondary antibody. PV-infected: blue lines; uninfected: red lines. (B) CD155+ MEFs (a, c, e, g, i, k, m) and neurons (b, d, f, h, j, l) were infected with either the Mahoney strain (a – b, e – f, i – j) or Sabin strain (c – d, g – h, k – m) of PV (MOI = 0.1) and collected at 16, 24, and 40 h post infection. PV antigen was detected by immunostaining using a monoclonal antibody to PV and visualized by DAB precipitation. For panel (m), arrows denote low antigen-expressing cells, whereas arrowheads denote high-expressing cells. Representative images from one of three identical experiments are shown. Magnification: (a – l): 100; (m): 400. (C) The total number of live and dead cells was ascertained, as determined by cellular morphology, TUNEL staining, and/or trypan blue exclusion. Viability was independently confirmed by trypan blue exclusion and absence of TUNEL staining (data not shown). Results are shown for both Mahoney (left) and Sabin (right) at 0, 16, 24, and 40 hpi. Black bars: MEFs; stippled bars: neurons.
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extent of infection and the magnitude of infectious particles produced were substantially reduced in neurons as compared to control fibroblasts. Interneuronal poliovirus spread is primarily trans-synaptic
Fig. 3. Poliovirus-infected primary neuron cultures produce less extracellular infectious virus than infected MEFs. (A) Equivalent numbers of MEFs and primary neurons were plated and infected with PV-Mahoney or Sabin, as described. At the indicated times post-infection, supernatants were collected, and plaque assays were performed; data are presented as log PFU/ml. A representative experiment of five is shown. Black circles: Mahoney titers in MEFs; white circles: Mahoney titers in neurons; black triangles: Sabin titers in MEFs; white triangles: Sabin titers in neurons. (B) The average number of viral particles produced per infected cell was calculated for each timepoint post-infection with PV-Mahoney. These values were obtained by dividing the amount of total extracellular virus by the total number of immunopositive cells in the cultures. Black bars: MEFs; stippled bars: neurons. ND = none detected.
infection with the Mahoney strain resulted in higher titers in both cell types, overall differences in titer between the two cell populations remained consistent. This difference was also apparent on a per-infected cell basis; as indicated in the representative Mahoney experiment shown in Fig. 3B (one of four such experiments), approximately 20-fold more infectious PV particles were produced in MEFs than in neurons. Similar results were obtained when identical comparisons were done with the Sabin strain of PV (data not shown). To confirm that antigen-positive cells were productively infected with PV, infectious center assays were performed. As shown in Table 1, at 32 h post-infection with PVMahoney, fibroblasts produced approximately 5 PFU/ infected cell, whereas neurons produced only 1 particle/ infected cell, mirroring the data obtained by histochemistry. Thus, by both immunochemical and functional assays, the
In a mouse model of neuronal infection by measles virus, reduced cytopathicity was associated with selective transmission at the synapse (Lawrence et al., 2000). To determine if a similar process was occurring for PV, we turned our attention to the basis of spread in these primary cultures. Immunocytochemical data indicated that clusters of infected neurons were connected by antigen-positive processes (e.g., Fig. 2B, panels b, f, j, and l), supporting studies that demonstrated trans-synaptic spread in vivo (Ohka et al., 1998; Ren and Racaniello, 1992). These immunopositive processes were not observed when poliovirus-challenged control PVR neurons were stained for PV antigen (data not shown). To ascertain the relative contributions of extracellular virus and trans-synaptic transfer to PV spread in infected neurons, neutralizing antibodies were employed. Serum from previously infected PVR+ transgenic mice (greater than 25 days post-infection) was used; similar experiments were repeated with a commercially available type 1 neutralizing antibody (BioSource) with identical results. The neutralizing antiserum was added to the cultures 1 h post-infection with PV-Sabin (MOI = 0.1); cells and supernatants from antibody-treated and non-treated cultures were collected at 24 and 32 h post-infection and were immunostained for PV antigens as described previously. As shown in Fig. 4A, addition of neutralizing antibodies reduced viral titers by greater than 2 orders of magnitude for both cell types. For neurons, this represented a decline in titers from 105 to 103, and for fibroblasts, a decline from 106 to 103.5. Different sources of antibodies, combinations of antibodies, or addition of higher doses did not appreciably decrease viral titers further (data not shown). As expected, the presence of PV neutralizing antibody in infected MEF cultures almost entirely inhibited viral spread, reducing the percent of immunopositive cells at 24 hpi from >50% to <10% (Figs. 4B and C, panels a and b). Note that, since the neutralizing antibody was added to the cultures after viral challenge, cells infected by the original inoculum Table 1 Infectious center analysis of PV-infected neurons and fibroblasts HPI
16 24 32
Neurons
Fibroblasts
% Infected
PFU/infected cell
% Infected
PFU/infected cell
22 32.6 46.3
0.13 0.33 1.1
64.9 77.6 85
1.13 0.92 4.95
Primary neurons and fibroblasts were cultured and infected with PVMahoney as described in Materials and methods. Infectious center assays were performed on the infected cells, and the percent of infected cells determined. The total PFU in the supernatants (determined by plaque assay) was then divided by this value to obtain PFU/infected cell.
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neurons (Figs. 4B and C, panels c and d). Similar observations were obtained following treatment of Mahoney virus-infected cultures (data not shown). Thus, the absence of an effect on viral spread in neurons, despite a significant impact on extracellular viral levels following neutralizing antibody treatment, supports the hypothesis that the predominant mode of spread in neurons is by trans-synaptic transmission. Because the neutralizing antibody did not completely eliminate extracellular virus, we cannot formally exclude the possibility that some spread in neuron cultures was due to residual infectious particles; however, this seems unlikely as spread in MEFs in the presence of the antibody was completely ablated and neuronal spread still occurred efficiently in antibody-treated neuron and MEF co-cultures (data not shown). Poliovirus spread between infected CNS neurons requires the expression of CD155
Fig. 4. Poliovirus spread in infected CD155+ neuron cultures occurs in the presence of type 1 PV neutralizing antibody. (A) Supernatants from PV Sabin-infected MEFs and neurons, in the presence or absence of type 1 PV neutralizing antibody, were collected and assayed for infectious particles by plaque assay through 32 hpi. Black bars: no neutralizing antibody; stippled bars: antibody added. The experiment shown is representative of three experiments. (B) CD155+ MEFs and neurons infected with PV-Sabin (MOI = 0.1) were incubated for 16 and 24 hpi in the presence or absence of type 1 PV neutralizing antibody, and the percent of infected cells was then determined by immunocytochemistry. (C) CD155+ MEFs (a, b) and neurons (c, d) were infected with PV-Sabin (MOI = 0.1) and were incubated for 24 h with (+) or without ( ) PV type 1 neutralizing antibody. PV antigen was visualized as described in Materials and methods. Magnification = 100.
would be expected. In contrast, the presence of PV neutralizing antibody in neuron cultures infected with PVSabin had virtually no impact on viral spread in infected
CD155 is required for initiation of PV infection in susceptible mice (Koike et al., 1991; Ren et al., 1990). However, it is not clear if subsequent PV spread between neurons requires CD155, as a recent study indicated that only the intact 160S, and not the ‘‘infectious’’ 135S subviral particle, could be found in the sciatic nerve of infected mice (Arita et al., 1999). To address this question, primary neurons from c57Bl/6 mice were loaded with an orange vital dye and were mixed in varying ratios with unlabeled CD155+ neurons. These ‘‘mixed’’ neuron cultures were then infected with either PV-Sabin or PV-Mahoney at an MOI = 0.1 and were collected and fixed 24 hpi. As shown in Figs. 5a, d, and g, neurons infected with either Sabin or Mahoney strains were connected by immunopositive processes. Nevertheless, no spread to nontransgenic neurons (Panels 5b and e) was observed when the images were merged (Figs. 5c and f). This was also true in cultures collected at 40 hpi, a relatively late timepoint post-infection (data not shown). As a control, CD155+ neurons were infected in suspension (Mahoney; MOI = 0.1) and plated with uninfected, vital-dye-labeled CD155+ neurons in the presence of neutralizing antibody. As shown in Figs. 5g– i, spread to vital-dye-labeled neurons occurred readily, as expected. Thus, we conclude from these experiments that expression of CD155 is required for both viral entry and subsequent trans-synaptic spread.
Discussion While many viruses can infect the human CNS, they do so rarely. For many of these viruses, once access to the CNS is achieved, the profile of replication is distinct from that in the periphery: often, a more chronic or persistent infection occurs, usually associated with a progressive course, as with measles and the rare but fatal disease, subacute sclerosing panencephalitis. Poliovirus is the causative agent of polio-
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Fig. 5. Interneuronal spread of PV requires the expression of the poliovirus receptor, CD155. c57Bl/6 neurons (b and e) or CD155+ neurons (h) were loaded with an orange vital dye and plated with unlabeled, PV-infected CD155+ neurons (Sabin: a; Mahoney: d, g). After 24 h, the neurons were fixed and immunostained for PV antigen using an FITC-conjugated secondary antibody. (a, d and g) PV-infected neurons; (b, e, and h) vital dye-labeled neurons; (c, f, and i) merge. Magnification = 100.
myelitis, a rare and progressive virus-induced degeneration of motor neurons that results in limb paralysis. In an effort to determine whether PV replication and spread were impacted upon neuronal infection, primary cultures from susceptible PV receptor-expressing transgenic mice were established, and the infection profiles compared. Two conclusions can be drawn from the experiments described in this work. First, the extent of viral cytopathicity and extracellular virus production is significantly reduced in neurons compared to MEFs, despite similar levels of infection. These data support previous observations in transgenic mice and in established neuroblastoma cells that PV replication is restricted upon neuronal infection (Couderc et al., 2002; Girard et al., 2002). Secondly, despite the production of some extracellular viral progeny, the predominant mechanism of PV spread is via trans-synaptic transfer, a process that is CD155-dependent. The ability of virus to spread via the synapse (rather than through cell lysis, as occurs in non-neuronal cells) may contribute to the reduced cytopathicity of PV within neurons. The reduced productivity and cytopathicity of both Sabin and Mahoney strains of PV in neurons are consistent with observations that many DNA and RNA viruses undergo a change in the viral replication cycle upon neuronal infection. For example, upon entry into spinal cord neurons, herpes simplex virus-1 undergoes a switch to a latent state in which viral gene expression is virtually ablated (reviewed in Johnson, 1998). Moreover, MV adopts a noncytolytic, nonfusogenic, trans-synaptic mode of spread in neurons that is not associated with the emergence of viral variants (Lawrence et al., 2000) (Gechman et al., in preparation). Thus, our observations using PV-infected neurons may reflect a common mechanism by which neurons restrict or
modulate viral replication. While the reduced productivity in neurons may be attributable to viral mutations, as previously shown for some strains of PV (LaMonica and Racaniello, 1989), other reasonable explanations exist. For example, it is possible that the cellular machinery available to viruses upon neuronal entry may be sufficiently unique to support a different mode of replication. Key differences in the availability of translation-associated proteins (Guest et al., 2004), the cellular proteins that are required for viral budding (Lawrence et al., 2000), the reduced mitotic capacity of parenchymal cells, and the level of cyclic nucleotides (Miller and Carrigan, 1982) may impact on viral replication and resultant pathogenesis. As these studies continue, it will be of interest to further identify the cellular factors that influence the poliovirus life cycle in neurons. Our studies support and extend those of other laboratories who have assessed the spread and pathogenesis of PV in neurons. While care must be taken not to infer too broadly from purified primary neurons to the more complex interaction of poliovirus with other cell types in vivo, studies from our laboratory and others indicate that such cultures can reproducibly parallel observations made in permissive mice. For example, work from Blondel and colleagues using permissive transgenic mice and mixed brain cell cultures showed that PV infection of neurons resulted in apoptosis, as caspase inhibitors prevented cell death in these cultures (Couderc et al., 2002; Girard et al., 1999). Consistent with our observations of delayed neuronal death, apoptosis in mixed cultures was apparent only after 28 hpi. We have since confirmed in our purified cultures that neuronal loss due to PV infection is attributable to apoptosis, whereas MEF death is more likely due to classic virus-induced CPE (data not shown).
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Work from other laboratories has begun to define the trafficking of PV within neurons. Wimmer and colleagues have reported that the cytoplasmic domain of the CD155 receptor binds to Tctex-1 (Mueller et al., 2002), a light chain of the dynein motor complex that governs retrograde transport in polarized cells. The apparent ‘‘hand-off’’ of the incoming viral particle to molecular motor proteins likely facilitates the fast retrograde axonal transport used by PV in vivo to spread from muscle to the CNS through the sciatic nerve (Ohka et al., 1998, 2004). Given that the mature 160S form of PV was isolated from infected nerves (Arita et al., 1999; Ohka and Nomoto, 2001; Ohka et al., 1998), it was possible that the interaction of PV with its receptor may be either different at the synapse than at the plasma membranes of non-neuronal cells or that CD155 may be dispensable for viral spread altogether. As support for the latter hypothesis, our laboratory previously demonstrated that measles virus, while requiring a receptor to initially infect neurons, could spread from infected neurons to uninfected neurons in a receptor-independent manner (Lawrence et al., 2000). Our studies here indicate that CD155 is required for neuron-to-neuron spread; therefore, if the interneuronal spread of poliovirus is distinct from infection by an extracellular particle, it is nevertheless CD155-dependent. The delayed course to PV-induced cytolysis and reduced viral productivity that we observed in cultured neurons may have direct bearing on the development of persistent infection within the CNS. Several studies have demonstrated that PV RNA can persist in susceptible mouse neurons, both in vivo (Destombes et al., 1997) and in vitro (Pavio et al., 1996). Moreover, PV RNA has been detected in human cerebrospinal fluid from individuals who previously had an acute case of poliomyelitis (Leparc-Goffart et al., 1996). PV remains high on the World Health Organization’s list of viruses to be eradicated in the near future, and some have argued that the risks of a sustained vaccination program are greater than the risks of viral recrudescence. Nevertheless, as recently seen with outbreaks of MV in England, viruses can make a rapid and serious recurrence in populations with declining vaccine coverage. The diseases associated with PV infection, including both the risk of poliomyelitis in the short term and post-polio syndrome in the long term, argue in favor of further studies to define the interaction of this virus with its cellular host.
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(Banker and Goslin, 1991; Pasick et al., 1994; Rall et al., 1995), with the exception that cells were maintained in neurobasal medium (Life Technologies, Grand Island, NY) containing 4 Ag/ml glutamate, in the absence of an astrocyte feeder layer. These mixed sensory and motor neuron cultures are routinely >95% pure, as determined by staining with microtubule-associated protein-2 (MAP-2) (data not shown). For all studies in this report, plated neurons were allowed to differentiate for 72 – 96 h before infection, coinciding with the formation of functional synapses and expression of neuron-specific markers (Rall et al., 1995). Mouse embryonic fibroblasts (MEFs) were prepared from the same day 15.5 CD155+ transgenic embryos. Briefly, livers were removed, and the remaining tissues were then finely minced in 10 ml of 0.4% trypsin followed by trituration with a 10 ml pipette. The single cell suspension was incubated at 37 -C for 10 min, washed and re-trypsinized. After large tissues pieces were allowed to settle and were discarded, the resulting cell suspension was pelleted by centrifugation at 1100 rpm for 5 min. The resulting pellet was resuspended in complete DMEM (Life Technologies, supplemented with 5% FCS, 2 mM lglutamine, 100 U/ml penicillin, and 100 ng/ml streptomycin) and plated into culture flasks. Vero fibroblasts were also cultured in complete DMEM. All cells were maintained at 37 -C in a humidified incubator with 5% CO2 and were routinely tested for mycoplasma contamination. The Sabin and Mahoney strains of type 1 poliovirus (PV) were a gift from Luis Sigal and were passaged and titered in Vero fibroblasts. Infections
Materials and methods
Primary neurons and MEFs were plated onto poly-llysine-treated 15-mm-diameter glass coverslips at a density of 500 cells/mm2. Neurons were differentiated for 72 –96 h prior to infection, whereas MEFs were plated 24 h before infection. Cells were infected with type 1 PV, Sabin or Mahoney strain, at a multiplicity of infection (MOI) = 0.1. The coverslips were covered with the viral inoculum and incubated for 1 h at 37 -C, after which the inoculum was replaced with either conditioned neurobasal medium (primary neurons) or fresh complete DMEM (MEFs). Slips and supernatants were collected every 8 h after infection. Slips were washed briefly in fresh PBS, and the cells were fixed for 5 min in a 1:1 solution of ice-cold methanol: acetone. After fixation and drying, the slips were stored at 4 -C in PBS.
Cells and viruses
Immunofluorescence
Poliovirus receptor (PVR; CD155+) transgenic mice, originally developed by Racaniello and colleagues (Ren et al., 1990), were obtained from Luis Sigal at Fox Chase. Primary hippocampal neurons were prepared from day 15.5 CD155+ transgenic embryos, as previously described
To detect CD155 expression, a mouse antibody to CD155 (BioSource International, Camarillo, CA) was used at a 1:100 dilution followed by an Alexa-Fluor 488 conjugated anti-mouse IgG (Molecular Probes, Eugene, OR; 1:500).
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For viral spread studies, CD155-negative neurons were isolated from c57Bl/6 day 15.5 embryos as described above, counted, and loaded with Vybrant CMDil cell labeling solution (Molecular Probes). The vital dye was added to neurons in suspension at a concentration of 5 Al/2.0 106 cells/ml in serum-free neurobasal media and incubated at 37 -C for 30 min. Labeled neurons were then washed twice in fresh serum-free neurobasal medium and centrifuged through a 2 ml FCS cushion. Immunofluorescence detection of PV antigen utilized a type 1 PV-specific mouse monoclonal (Accurate, Westbury NY; 1:200 dilution in PBS) followed by incubation with a biotinylated anti-mouse IgG2a (Serotec, Raleigh NC; 1:300 dilution in PBS). Finally, an FITC-conjugated avidin (1:500 dilution; Vector, Burlingame, CA) was incubated with the slips for 30 min followed by three 5 min washes. All slides were visualized using a Nikon fluorescence microscope model TE300, equipped with appropriate filters for the fluorescence studies. For the viral trafficking studies, fluorescent orange and green images from identical fields were captured and merged using Adobe Photoshop. Immunohistochemistry Fixed cells were blocked with 1% (vol/vol) goat serum in PBS, further blocked with avidin and biotin (Vector), and stained for PV antigens as described above. However, after incubation with the secondary antibody, a streptavidin – peroxidase conjugate (Vector) was added, and PV was visualized by diaminobenzidine precipitation (0.7 mg/ml DAB with 1.6 mg/ml H2O2 in 60 mM Tris; Sigma Chemical). In some cases, cells were counterstained with hematoxylin to visualize nuclei. To quantify cell death in infected cultures, live and dead cells (as determined by morphology) were counted from 5 representative fields at 400 for each timepoint post-infection. Samples were coded until after counting.
at 4 -C. Cells were centrifuged and resuspended in 400 Al of 0.5% formaldehyde in PBS and analyzed on a BD FACScan fluorescence-activated cell scanner. Plaque assay Supernatants from infected cells were serially diluted and added to Vero fibroblasts plated in 6-well cell culture dishes in complete DMEM at a cell density of 106 cells/well. After incubation at 37 -C for 1 h, the viral inoculum was removed, and the cells were covered with a 1:1 mixture of 2 Medium 199 (Life Technologies) and 1% molten agarose in water. The agarose was allowed to solidify, and the plates were then returned to the 37 -C incubator. Two days later, the cells were fixed with 10% formaldehyde in PBS. Agarose overlays were removed, and plaques were visualized by staining the remaining fibroblasts with 0.1% crystal violet. Infectious center assay Infectious center assays (ICA) were performed using PVR+ neurons and MEFs that were infected with the Mahoney strain of poliovirus at an MOI = 0.1. At 16, 24, and 32 hpi, supernatants were removed and assayed for infectious PV particles by plaque assay (see above). The attached cells from each timepoint were subsequently washed with PBS and removed with 0.4% trypsin (2 ml final volume). The cells were counted (to obtain the total number of cells), serially diluted, and added to Vero fibroblasts plated on 6-well cell culture dishes. Two days post-plating, the cells were fixed and subsequently visualized with crystal violet. The percentage of infected cells was calculated using the following equation: # of plaques (from ICA) dilution factor / total number of cells. Infectious virus per-infected cell was calculated using the following equation: total amount of infectious virus released (determined by plaque assay) / # of infected cells (determined by ICA).
Flow cytometry Neutralizing antibody assay Primary PVR+ hippocampal neurons and MEFs were plated on poly-l-lysine-treated 6-well culture plates at a cell density of 500 cells/mm2. Cells were infected with the Mahoney or Sabin strain of PV at an MOI = 0.1 or were mock-infected. Cells were removed from the plates at 24 and 40 hpi using Cell Stripper (Cellgro Mediatech, Inc., Herndon, VA). Cells were pelleted at 1200 rpm for 5 min and resuspended in 2 ml cytofix/cytoperm buffer (BD Biosciences/Pharmingen, San Diego, CA) and incubated for 20 min at 4 -C. Cells were then washed twice with perm/ wash buffer and transferred to wells of a 96-well microtiter plate. Poliovirus type 1 antibody at a 1:100 dilution or PBS (for no primary control) was added to each well for 30 min at 4 -C. An anti-mouse IgG FITC-conjugated secondary antibody (Vector; 1:500) was added to all wells for 30 min
CD155+ MEFs and neurons, plated on coverslips, were infected with the Sabin strain of PV at an MOI = 0.1 as described above. For some slips, 30 Al of type 1 PV neutralizing antibody (1:320; BioSource) was added 1 h post-infection. Infected cells and corresponding supernatants were collected at various times post-infection. Coverslips were fixed and immunostained for PV antigen as described above; supernatants were subjected to plaque assay. The percent of infected cells at each timepoint was calculated by counting 5 representative fields/slip at 400 magnification; identification of the slips was coded until after counting. Viral titers from each timepoint were determined by plaque assay of supernatants from infected cells as described above.
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Acknowledgments The authors acknowledge support from the following sources: NIH grants MH 56951 (G.R.) and T32-AI07492 (J.D.) and the F.M. Kirby Foundation. We would also like to thank Isabelle Cruz for secretarial support and Drs. Kerry Campbell, Wesley Rose, Christine Matullo, and Luis Sigal for helpful suggestions. References Arita, M., Ohka, S., Sasaki, Y., Nomoto, A., 1999. Multiple pathways for establishment of poliovirus infection. Virus Res. 62, 97 – 105. Banker, G., Goslin, K., 1991. Culturing Nerve Cells. MIT Press, Cambridge. Basavappa, R., Gomez-Yafal, A., Hogle, J.M., 1998. The poliovirus empty capsid specifically recognizes the poliovirus receptor and undergoes some, but not all, of the transitions associated with cell entry. J. Virol. 72, 7551 – 7556. Belnap, D.M., McDermott, B.M., Filman, D.J., Cheng, N., Trus, B.L., Zuccola, H.J., Racaniello, V.R., Hogle, J.M., Steven, A.C., 2000. Three dimensional structure of poliovirus receptor bound to poliovirus. Proc. Natl. Acad. Sci. 97, 73 – 78. Bodian, D., 1955. Emerging concept of poliomyelitis infection. Science 122, 105 – 108. Buisman, A.M., Sonsma, J.A., Wijk, M.G., Vermeulen, J.P., Roholl, P.J., Kimman, T.G., 2003. Pathogenesis of poliovirus infection in PVRTg mice: poliovirus replicates in peritoneal macrophages. J. Gen. Virol. 84, 2819 – 2828. Colston, E.M., Racaniello, V.R., 1994. Soluble receptor-resistant poliovirus mutants identify surface and internal capsid residues that control interaction with the cell receptor. EMBO J. 13, 5855 – 5862. Colston, E.M., Racaniello, V.R., 1995. Poliovirus variants selected on mutant receptor-expressing cells identify capsid residues that expand receptor recognition. J. Virol. 69, 4823 – 4829. Couderc, T., Guivel-Benhassine, F., Calaora, V., Gosselin, A., Blondel, B., 2002. An ex vivo murine model to study poliovirus-induced apoptosis in nerve cells. J. Gen. Virol. 83, 1925 – 1930. DalCanto, M.C., Barbano, R.L., Jubelt, B., 1986. Ultrastructural immunohistochemical localization of poliovirus during virulent infection of mice. J. Neuropathol. Exp. Neurol. 45, 613 – 618. Destombes, J., Couderc, T., Thiesson, D., Girard, S., Wilt, S.G., Blondel, B., 1997. Persistent poliovirus infection in mouse motoneurons. J. Virol. 71, 1621 – 1628. Everaert, L., Vrijsen, R., Boeye, A., 1989. Eclipse products of poliovirus after cold-synchronized infection of HeLa cells. Virology 171, 76 – 82. Freistadt, M.S., Fleit, H.B., Wimmer, E., 1993. Poliovirus receptor on human blood cells: a possible extraneural site of poliovirus replication. Virology 195, 798 – 803. Fricks, C.E., Hogle, J.M., 1990. Cell-induced conformational change in poliovirus: externalization of the amino terminus of VP1 responsible for liposome binding. J. Virol. 64, 1934 – 1945. Girard, S., Couderc, T., Destombes, J., Thiesson, D., Delpeyroux, F., Blondel, B., 1999. Poliovirus induces apoptosis in the mouse central nervous system. J. Virol. 73, 6066 – 6072. Girard, S., Gosselin, A.S., Pelletier, I., Colbere-Garapin, F., Couderc, T., Blondel, B., 2002. Restriction of poliovirus RNA replication in persistently infected nerve cells. J. Gen. Virol. 83, 1087 – 1093. Gomez, Y.A., Kaplan, G., Racaniello, V.R., Hogle, J.M., 1993. Characterization of poliovirus conformational alteration mediated by soluble cell receptors. Virology 197, 501 – 505. Gromeier, M., Wimmer, E., 1998. Mechanism of injury-provoked poliomyelitis. J. Virol. 72, 5056 – 5060.
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Poliovirus replication and spread in primary neuron cultures John K. Daley, Lisa A. Gechman, Jason Skipworth, Glenn F. Rall* Fox Chase Cancer Center, Institute for Cancer Research, 333 Cottman Avenue, Philadelphia, PA 19111, USA Received 24 November 2004; returned to author for revision 20 December 2004; accepted 23 May 2005 Available online 11 July 2005
Abstract While some neurotropic viruses cause rapid central nervous system (CNS) disease upon entry into the brain parenchyma, other viruses that are cytolytic in the periphery either result in little neuropathology or are associated with a protracted course of CNS disease consistent with persistent infection. One such virus, poliovirus (PV), is an extremely lytic RNA virus that requires the expression of CD155, the poliovirus receptor (PVR), for infection. To compare the kinetics of PV infection in neuronal and non-neuronal cell types, primary hippocampal neurons and fibroblasts were isolated from CD155+ transgenic embryos and infected with the Mahoney and Sabin strains of PV. Despite similar levels of infection in these ex vivo cultures, PV-infected neurons produced 100-fold fewer infectious particles as compared to fibroblasts throughout infection, and death of PV-infected neurons was delayed approximately 48 h. Spread in neurons occurred primarily by trans-synaptic transmission and was CD155-dependent. Together, these results demonstrate that the magnitude and speed with which PV replication, spread, and subsequent cell death occur in neurons is decreased as compared to non-neuronal cells, implicating cell-specific effects on replication that may then influence viral pathogenesis. D 2005 Elsevier Inc. All rights reserved. Keywords: Poliovirus; CD155; Primary neurons; Mouse embryonic fibroblasts; CNS; Viral spread; Trans-synaptic transmission
Introduction Poliovirus (PV) is an enterovirus belonging to the family Picornaviridae and is the etiological agent of poliomyelitis in humans. PV possesses a positive strand RNA genome of approximately 7.5 kb, packaged in a non-enveloped icosahedral protein capsid (Hogle et al., 1985). The capsid consists of 60 copies of each of four virally encoded structural proteins VP1, VP2, VP3, and VP4. The icosahedral structure formed by these capsid proteins produces deep surface depressions, or Fcanyons_, that have been identified as receptor binding sites (Colston and Racaniello, 1994, 1995; Hogle et al., 1985; Racaniello and Ren, 1996). PV infection initiates when the viral particle binds to its cellular receptor, CD155 (Belnap et al., 2000; He et al., 2000). Upon binding, the PV particle undergoes a conformational change in which the VP4 capsid protein is lost and the amino-
* Corresponding author. Fax: +1 215 728 2412. E-mail address: [email protected] (G.F. Rall). 0042-6822/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.virol.2005.05.032
terminus of the internal VP1 protein is extruded (Everaert et al., 1989; Fricks and Hogle, 1990; Gomez et al., 1993; Lonberg-Holm et al., 1975; Wien et al., 1996). It is believed that the exposure of the hydrophobic N-terminus of the VP1 protein forms a pore in the host cell membrane, allowing the viral particle to enter the cytoplasm. Additionally, these conformational changes in the virion result in a reduced sedimentation coefficient in the viral particle from the native 160S to an infectious intermediate or 135S sub-particle. It is thought that viral engagement with CD155 triggers this conformational change (Tsang et al., 2001); however, whether the loss of VP4 is the basis of the altered sedimentation coefficient remains debated (Basavappa et al., 1998). The normal route of PV infection in naturally permissive hosts begins with infection of the enteric system through oral ingestion of the virus. Viral particles initially replicate in the gastrointestinal system, but replication at this site does not result in any detectable pathology (Bodian, 1955; Sabin, 1956). In approximately 0.1 –1% of acute PV infections, the virus invades the CNS, producing the paralytic disease
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symptoms associated with poliomyelitis. This disease results from PV infection, translational inhibition, and destruction of motor neurons in the central nervous system (CNS) and is clinically manifested as limb paralysis. Interestingly, despite a rapid course of viremia that can be detected soon after viral exposure, PV appears to adopt a less productive course in the CNS (Girard et al., 2002; Minor, 1996). The ability of polio to spread in a somewhat restricted manner within the CNS may foster the establishment of a slow or persistent infection. While controversial, this observation has led to suggestions by others that chronic infection may be linked to diseases such as post-polio syndrome, in which patients develop signs of poliomyelitis months to years after primary exposure (Destombes et al., 1997; Pavio et al., 1996). The natural expression of CD155 in humans and some primates, but not in experimental animals such as mice, limited early studies of PV spread and pathogenesis. In 1990 –91, transgenic mice expressing CD155 were established; these mice were susceptible to PV infection by all three viral serotypes (Koike et al., 1991; Ren et al., 1990). The development of these valuable models was responsible for substantial advances in our understanding of PV replication and pathogenesis; however, important aspects of PV interaction with its natural host remain obscure. For example, while expression of CD155 is required for poliovirus entry (Koike et al., 1990; Mendelsohn et al., 1989), it is not sufficient to confer permissivity, as poliovirus cannot establish an infection in CD155-expressing mice by the intra-oral route, despite expression of CD155 within the gut (Ren et al., 1990). The pathway of PV spread from the periphery to the CNS in susceptible hosts is not fully understood. As viremia is a prerequisite for the development of poliomyelitis and dendritic cells and macrophages can be infected with poliovirus (Wahid et al., 2005; Buisman et al., 2003; Freistadt et al., 1993), polio may gain entry to the brain via transport of infected blood cells across the blood – brain barrier (BBB) (Yang et al., 1997). A second proposed mechanism of PV neurotropism is transport along neural pathways, perhaps provoked by muscle injury at the site of infection, as in the case of provocation poliomyelitis (Gromeier and Wimmer, 1998). In immunoelectron microscopy studies of spinal cord sections of mice infected with the type 2 Lansing strain, PV antigens were primarily detected in neuronal axons and synapses, suggesting that spread may be trans-synaptic (DalCanto et al., 1986). Subsequently, PV was shown to travel to the CNS from infected muscle tissue in transgenic mice via major nerves, such as the sciatic nerve (Ponnuraj et al., 1998; Ren and Racaniello, 1992), providing additional support for transsynaptic transmission. The speed of PV spread from the periphery to the CNS via neuron – neuron interactions was later shown to be consistent with fast retrograde axonal transport (Ohka et al., 1998). Interestingly, however, PV particles isolated from the sciatic nerve were of the intact
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160S form of the viral particle, not the 135S sub-particles thought to be required for infection (Arita et al., 1999). One possible interpretation of these results is that interaction with the viral receptor may not be required for PV transmission across the synapse, a theory for which evidence exists in a model of neuronal infection by another RNA virus, measles (Lawrence et al., 2000). Although PV has been studied extensively in vivo, its replication and spread in purified primary neuron cultures have not been extensively explored. Based on observations that PV infection of the CNS is distinct from infection of the periphery with respect to viral yield and cytopathicity, we undertook studies to address whether PV adopts an altered replication profile in purified primary neurons and to further explore the basis of PV spread in such cultures. Using highly enriched primary neuron cultures isolated from CD155+ transgenic embryos, as well as control mouse embryo fibroblasts (MEFs) obtained from the same animals, we compared the kinetics of infection of neurons with MEFs, established the mode of interneuronal viral spread, and clarified the role played by CD155 in neuronal PV transport.
Results Poliovirus growth kinetics in permissive neurons and mouse embryo fibroblasts To assess PV replication kinetics in different primary cell cultures, type 1 PV infection by the Sabin strain and the more virulent Mahoney strain of cultured CD155+ neurons was compared to infection of mouse embryo fibroblasts (MEFs) isolated from the same embryos. To confirm that CD155+ explant cultures indeed express this receptor, immunofluorescence using a monoclonal antibody to CD155 was performed on CD155+ and CD155 cells. As shown in Fig. 1, neurons (A – C) and MEFs (G –I) isolated from CD155+ transgenic mice were immunopositive, whereas neurons isolated from CD155 (c57Bl/6) embryos (D –F) were not. The slight background signal detected in nontransgenic neurons is likely due to nonspecific binding of the secondary antibody. PV replication and transmission in CD155+ primary neurons and MEFs were then examined. Cells were infected with PV-Sabin or PV-Mahoney (MOI = 0.1); at 8 h intervals, cells and supernatants were collected and analyzed for infection by flow cytometry and immunocytochemistry (Figs. 2A and B), extent of cell death by cellular morphology and counting of residual live cells (Fig. 2C), and virus production by plaque assay and infectious center assay (Fig. 3). As shown by flow cytometry (Fig. 2A) at 24 and 40 h post-infection (hpi), the extent of infection in MEFs and neurons was comparable. Although more MEFs were infected at each timepoint evaluated, both neurons and MEFs became heavily infected over time, and the rate of
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Fig. 1. CD155 expression in primary hippocampal neurons and mouse embryonic fibroblasts (MEFs). CD155+ primary hippocampal neurons (A – C) and mouse embryonic fibroblasts (G – I) were prepared from PVR transgenic mice. CD155 control neurons (D – F) were prepared from c57Bl/6 mice. All cells were immunostained for CD155 expression. Phase (A, D, G), fluorescence (B, E, H), and merged (C, F, I) are shown. Representative images were taken from one of three identical experiments. Magnification: 100.
spread within the cultures was similar. For the Mahoney strain, 72% and 84% of MEFs were immunopositive at 24 and 40 hpi respectively, compared to 36% and 58% infection of neurons (Fig. 2A; Table 1). Although the number of antigen-positive cells was lower following infection by the less virulent Sabin strain, a similar profile of infection was observed. Interestingly, two distinct peaks were observed in the FACScan histograms of Sabin-infected MEFs at 40 hpi (Fig. 2A); this was also observed as two immunopositive populations that differed in staining intensity (Fig. 2B, panels k and m). Despite efficient PV infection and spread in both cell types, virus-induced cytopathicity in neurons was reduced as compared with matched infected MEFs (Fig. 2C). For these studies, the number of live and dead cells was determined at each timepoint by morphology, TUNEL staining, or trypan blue exclusion. Mahoney infection of MEFs was highly lytic, killing virtually all infected cells by 24 hpi (Fig. 2B, panel e; Fig. 2C). In contrast, neuronal infection at this same timepoint was not associated with extensive cell loss (Fig.
2B, panel f; Fig. 2C), and even at 40 hpi, neuronal death was still less than 50% (Fig. 2B, panel j). Although MEF cell death was less pronounced with the Sabin strain, most infected MEFs showed signs of nuclear condensation and rounding (Fig. 2B, panels g and k), whereas neurons at the same timepoints appeared healthy, with intact processes (panels h and l). Thus, while neurons eventually do die as a result of PV infection with both strains, virus-induced cytopathicity was noted significantly earlier in MEFs than in neurons: dying fibroblasts were seen as early as 8– 16 hpi for both strains of PV; in contrast, neuronal cytopathicity was not observed until 36 – 48 hpi, and even at this late timepoint, viable neurons were abundant. The delay in PV-induced cytopathic effect (CPE) in neurons was consistent with a 100- to 500-fold decrease in extracellular infectious viral titers, relative to matched MEFs (Fig. 3A). For example, in the representative experiment shown (of five such experiments), at 24 hpi, MEF titers following PV-Mahoney challenge were 9 107 PFU/ml as compared to neuronal titers of 2 105 PFU/ml. Though
Fig. 2. PV spread and cytotoxicity in primary neurons and fibroblasts. (A) Flow cytometry was performed on single cell suspensions of PV-infected MEFs and neurons at 24 and 40 hpi. As described in Materials and methods, cells were labeled with anti-PV antibodies and visualized with an FITC-conjugated secondary antibody. PV-infected: blue lines; uninfected: red lines. (B) CD155+ MEFs (a, c, e, g, i, k, m) and neurons (b, d, f, h, j, l) were infected with either the Mahoney strain (a – b, e – f, i – j) or Sabin strain (c – d, g – h, k – m) of PV (MOI = 0.1) and collected at 16, 24, and 40 h post infection. PV antigen was detected by immunostaining using a monoclonal antibody to PV and visualized by DAB precipitation. For panel (m), arrows denote low antigen-expressing cells, whereas arrowheads denote high-expressing cells. Representative images from one of three identical experiments are shown. Magnification: (a – l): 100; (m): 400. (C) The total number of live and dead cells was ascertained, as determined by cellular morphology, TUNEL staining, and/or trypan blue exclusion. Viability was independently confirmed by trypan blue exclusion and absence of TUNEL staining (data not shown). Results are shown for both Mahoney (left) and Sabin (right) at 0, 16, 24, and 40 hpi. Black bars: MEFs; stippled bars: neurons.
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extent of infection and the magnitude of infectious particles produced were substantially reduced in neurons as compared to control fibroblasts. Interneuronal poliovirus spread is primarily trans-synaptic
Fig. 3. Poliovirus-infected primary neuron cultures produce less extracellular infectious virus than infected MEFs. (A) Equivalent numbers of MEFs and primary neurons were plated and infected with PV-Mahoney or Sabin, as described. At the indicated times post-infection, supernatants were collected, and plaque assays were performed; data are presented as log PFU/ml. A representative experiment of five is shown. Black circles: Mahoney titers in MEFs; white circles: Mahoney titers in neurons; black triangles: Sabin titers in MEFs; white triangles: Sabin titers in neurons. (B) The average number of viral particles produced per infected cell was calculated for each timepoint post-infection with PV-Mahoney. These values were obtained by dividing the amount of total extracellular virus by the total number of immunopositive cells in the cultures. Black bars: MEFs; stippled bars: neurons. ND = none detected.
infection with the Mahoney strain resulted in higher titers in both cell types, overall differences in titer between the two cell populations remained consistent. This difference was also apparent on a per-infected cell basis; as indicated in the representative Mahoney experiment shown in Fig. 3B (one of four such experiments), approximately 20-fold more infectious PV particles were produced in MEFs than in neurons. Similar results were obtained when identical comparisons were done with the Sabin strain of PV (data not shown). To confirm that antigen-positive cells were productively infected with PV, infectious center assays were performed. As shown in Table 1, at 32 h post-infection with PVMahoney, fibroblasts produced approximately 5 PFU/ infected cell, whereas neurons produced only 1 particle/ infected cell, mirroring the data obtained by histochemistry. Thus, by both immunochemical and functional assays, the
In a mouse model of neuronal infection by measles virus, reduced cytopathicity was associated with selective transmission at the synapse (Lawrence et al., 2000). To determine if a similar process was occurring for PV, we turned our attention to the basis of spread in these primary cultures. Immunocytochemical data indicated that clusters of infected neurons were connected by antigen-positive processes (e.g., Fig. 2B, panels b, f, j, and l), supporting studies that demonstrated trans-synaptic spread in vivo (Ohka et al., 1998; Ren and Racaniello, 1992). These immunopositive processes were not observed when poliovirus-challenged control PVR neurons were stained for PV antigen (data not shown). To ascertain the relative contributions of extracellular virus and trans-synaptic transfer to PV spread in infected neurons, neutralizing antibodies were employed. Serum from previously infected PVR+ transgenic mice (greater than 25 days post-infection) was used; similar experiments were repeated with a commercially available type 1 neutralizing antibody (BioSource) with identical results. The neutralizing antiserum was added to the cultures 1 h post-infection with PV-Sabin (MOI = 0.1); cells and supernatants from antibody-treated and non-treated cultures were collected at 24 and 32 h post-infection and were immunostained for PV antigens as described previously. As shown in Fig. 4A, addition of neutralizing antibodies reduced viral titers by greater than 2 orders of magnitude for both cell types. For neurons, this represented a decline in titers from 105 to 103, and for fibroblasts, a decline from 106 to 103.5. Different sources of antibodies, combinations of antibodies, or addition of higher doses did not appreciably decrease viral titers further (data not shown). As expected, the presence of PV neutralizing antibody in infected MEF cultures almost entirely inhibited viral spread, reducing the percent of immunopositive cells at 24 hpi from >50% to <10% (Figs. 4B and C, panels a and b). Note that, since the neutralizing antibody was added to the cultures after viral challenge, cells infected by the original inoculum Table 1 Infectious center analysis of PV-infected neurons and fibroblasts HPI
16 24 32
Neurons
Fibroblasts
% Infected
PFU/infected cell
% Infected
PFU/infected cell
22 32.6 46.3
0.13 0.33 1.1
64.9 77.6 85
1.13 0.92 4.95
Primary neurons and fibroblasts were cultured and infected with PVMahoney as described in Materials and methods. Infectious center assays were performed on the infected cells, and the percent of infected cells determined. The total PFU in the supernatants (determined by plaque assay) was then divided by this value to obtain PFU/infected cell.
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neurons (Figs. 4B and C, panels c and d). Similar observations were obtained following treatment of Mahoney virus-infected cultures (data not shown). Thus, the absence of an effect on viral spread in neurons, despite a significant impact on extracellular viral levels following neutralizing antibody treatment, supports the hypothesis that the predominant mode of spread in neurons is by trans-synaptic transmission. Because the neutralizing antibody did not completely eliminate extracellular virus, we cannot formally exclude the possibility that some spread in neuron cultures was due to residual infectious particles; however, this seems unlikely as spread in MEFs in the presence of the antibody was completely ablated and neuronal spread still occurred efficiently in antibody-treated neuron and MEF co-cultures (data not shown). Poliovirus spread between infected CNS neurons requires the expression of CD155
Fig. 4. Poliovirus spread in infected CD155+ neuron cultures occurs in the presence of type 1 PV neutralizing antibody. (A) Supernatants from PV Sabin-infected MEFs and neurons, in the presence or absence of type 1 PV neutralizing antibody, were collected and assayed for infectious particles by plaque assay through 32 hpi. Black bars: no neutralizing antibody; stippled bars: antibody added. The experiment shown is representative of three experiments. (B) CD155+ MEFs and neurons infected with PV-Sabin (MOI = 0.1) were incubated for 16 and 24 hpi in the presence or absence of type 1 PV neutralizing antibody, and the percent of infected cells was then determined by immunocytochemistry. (C) CD155+ MEFs (a, b) and neurons (c, d) were infected with PV-Sabin (MOI = 0.1) and were incubated for 24 h with (+) or without ( ) PV type 1 neutralizing antibody. PV antigen was visualized as described in Materials and methods. Magnification = 100.
would be expected. In contrast, the presence of PV neutralizing antibody in neuron cultures infected with PVSabin had virtually no impact on viral spread in infected
CD155 is required for initiation of PV infection in susceptible mice (Koike et al., 1991; Ren et al., 1990). However, it is not clear if subsequent PV spread between neurons requires CD155, as a recent study indicated that only the intact 160S, and not the ‘‘infectious’’ 135S subviral particle, could be found in the sciatic nerve of infected mice (Arita et al., 1999). To address this question, primary neurons from c57Bl/6 mice were loaded with an orange vital dye and were mixed in varying ratios with unlabeled CD155+ neurons. These ‘‘mixed’’ neuron cultures were then infected with either PV-Sabin or PV-Mahoney at an MOI = 0.1 and were collected and fixed 24 hpi. As shown in Figs. 5a, d, and g, neurons infected with either Sabin or Mahoney strains were connected by immunopositive processes. Nevertheless, no spread to nontransgenic neurons (Panels 5b and e) was observed when the images were merged (Figs. 5c and f). This was also true in cultures collected at 40 hpi, a relatively late timepoint post-infection (data not shown). As a control, CD155+ neurons were infected in suspension (Mahoney; MOI = 0.1) and plated with uninfected, vital-dye-labeled CD155+ neurons in the presence of neutralizing antibody. As shown in Figs. 5g– i, spread to vital-dye-labeled neurons occurred readily, as expected. Thus, we conclude from these experiments that expression of CD155 is required for both viral entry and subsequent trans-synaptic spread.
Discussion While many viruses can infect the human CNS, they do so rarely. For many of these viruses, once access to the CNS is achieved, the profile of replication is distinct from that in the periphery: often, a more chronic or persistent infection occurs, usually associated with a progressive course, as with measles and the rare but fatal disease, subacute sclerosing panencephalitis. Poliovirus is the causative agent of polio-
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Fig. 5. Interneuronal spread of PV requires the expression of the poliovirus receptor, CD155. c57Bl/6 neurons (b and e) or CD155+ neurons (h) were loaded with an orange vital dye and plated with unlabeled, PV-infected CD155+ neurons (Sabin: a; Mahoney: d, g). After 24 h, the neurons were fixed and immunostained for PV antigen using an FITC-conjugated secondary antibody. (a, d and g) PV-infected neurons; (b, e, and h) vital dye-labeled neurons; (c, f, and i) merge. Magnification = 100.
myelitis, a rare and progressive virus-induced degeneration of motor neurons that results in limb paralysis. In an effort to determine whether PV replication and spread were impacted upon neuronal infection, primary cultures from susceptible PV receptor-expressing transgenic mice were established, and the infection profiles compared. Two conclusions can be drawn from the experiments described in this work. First, the extent of viral cytopathicity and extracellular virus production is significantly reduced in neurons compared to MEFs, despite similar levels of infection. These data support previous observations in transgenic mice and in established neuroblastoma cells that PV replication is restricted upon neuronal infection (Couderc et al., 2002; Girard et al., 2002). Secondly, despite the production of some extracellular viral progeny, the predominant mechanism of PV spread is via trans-synaptic transfer, a process that is CD155-dependent. The ability of virus to spread via the synapse (rather than through cell lysis, as occurs in non-neuronal cells) may contribute to the reduced cytopathicity of PV within neurons. The reduced productivity and cytopathicity of both Sabin and Mahoney strains of PV in neurons are consistent with observations that many DNA and RNA viruses undergo a change in the viral replication cycle upon neuronal infection. For example, upon entry into spinal cord neurons, herpes simplex virus-1 undergoes a switch to a latent state in which viral gene expression is virtually ablated (reviewed in Johnson, 1998). Moreover, MV adopts a noncytolytic, nonfusogenic, trans-synaptic mode of spread in neurons that is not associated with the emergence of viral variants (Lawrence et al., 2000) (Gechman et al., in preparation). Thus, our observations using PV-infected neurons may reflect a common mechanism by which neurons restrict or
modulate viral replication. While the reduced productivity in neurons may be attributable to viral mutations, as previously shown for some strains of PV (LaMonica and Racaniello, 1989), other reasonable explanations exist. For example, it is possible that the cellular machinery available to viruses upon neuronal entry may be sufficiently unique to support a different mode of replication. Key differences in the availability of translation-associated proteins (Guest et al., 2004), the cellular proteins that are required for viral budding (Lawrence et al., 2000), the reduced mitotic capacity of parenchymal cells, and the level of cyclic nucleotides (Miller and Carrigan, 1982) may impact on viral replication and resultant pathogenesis. As these studies continue, it will be of interest to further identify the cellular factors that influence the poliovirus life cycle in neurons. Our studies support and extend those of other laboratories who have assessed the spread and pathogenesis of PV in neurons. While care must be taken not to infer too broadly from purified primary neurons to the more complex interaction of poliovirus with other cell types in vivo, studies from our laboratory and others indicate that such cultures can reproducibly parallel observations made in permissive mice. For example, work from Blondel and colleagues using permissive transgenic mice and mixed brain cell cultures showed that PV infection of neurons resulted in apoptosis, as caspase inhibitors prevented cell death in these cultures (Couderc et al., 2002; Girard et al., 1999). Consistent with our observations of delayed neuronal death, apoptosis in mixed cultures was apparent only after 28 hpi. We have since confirmed in our purified cultures that neuronal loss due to PV infection is attributable to apoptosis, whereas MEF death is more likely due to classic virus-induced CPE (data not shown).
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Work from other laboratories has begun to define the trafficking of PV within neurons. Wimmer and colleagues have reported that the cytoplasmic domain of the CD155 receptor binds to Tctex-1 (Mueller et al., 2002), a light chain of the dynein motor complex that governs retrograde transport in polarized cells. The apparent ‘‘hand-off’’ of the incoming viral particle to molecular motor proteins likely facilitates the fast retrograde axonal transport used by PV in vivo to spread from muscle to the CNS through the sciatic nerve (Ohka et al., 1998, 2004). Given that the mature 160S form of PV was isolated from infected nerves (Arita et al., 1999; Ohka and Nomoto, 2001; Ohka et al., 1998), it was possible that the interaction of PV with its receptor may be either different at the synapse than at the plasma membranes of non-neuronal cells or that CD155 may be dispensable for viral spread altogether. As support for the latter hypothesis, our laboratory previously demonstrated that measles virus, while requiring a receptor to initially infect neurons, could spread from infected neurons to uninfected neurons in a receptor-independent manner (Lawrence et al., 2000). Our studies here indicate that CD155 is required for neuron-to-neuron spread; therefore, if the interneuronal spread of poliovirus is distinct from infection by an extracellular particle, it is nevertheless CD155-dependent. The delayed course to PV-induced cytolysis and reduced viral productivity that we observed in cultured neurons may have direct bearing on the development of persistent infection within the CNS. Several studies have demonstrated that PV RNA can persist in susceptible mouse neurons, both in vivo (Destombes et al., 1997) and in vitro (Pavio et al., 1996). Moreover, PV RNA has been detected in human cerebrospinal fluid from individuals who previously had an acute case of poliomyelitis (Leparc-Goffart et al., 1996). PV remains high on the World Health Organization’s list of viruses to be eradicated in the near future, and some have argued that the risks of a sustained vaccination program are greater than the risks of viral recrudescence. Nevertheless, as recently seen with outbreaks of MV in England, viruses can make a rapid and serious recurrence in populations with declining vaccine coverage. The diseases associated with PV infection, including both the risk of poliomyelitis in the short term and post-polio syndrome in the long term, argue in favor of further studies to define the interaction of this virus with its cellular host.
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(Banker and Goslin, 1991; Pasick et al., 1994; Rall et al., 1995), with the exception that cells were maintained in neurobasal medium (Life Technologies, Grand Island, NY) containing 4 Ag/ml glutamate, in the absence of an astrocyte feeder layer. These mixed sensory and motor neuron cultures are routinely >95% pure, as determined by staining with microtubule-associated protein-2 (MAP-2) (data not shown). For all studies in this report, plated neurons were allowed to differentiate for 72 – 96 h before infection, coinciding with the formation of functional synapses and expression of neuron-specific markers (Rall et al., 1995). Mouse embryonic fibroblasts (MEFs) were prepared from the same day 15.5 CD155+ transgenic embryos. Briefly, livers were removed, and the remaining tissues were then finely minced in 10 ml of 0.4% trypsin followed by trituration with a 10 ml pipette. The single cell suspension was incubated at 37 -C for 10 min, washed and re-trypsinized. After large tissues pieces were allowed to settle and were discarded, the resulting cell suspension was pelleted by centrifugation at 1100 rpm for 5 min. The resulting pellet was resuspended in complete DMEM (Life Technologies, supplemented with 5% FCS, 2 mM lglutamine, 100 U/ml penicillin, and 100 ng/ml streptomycin) and plated into culture flasks. Vero fibroblasts were also cultured in complete DMEM. All cells were maintained at 37 -C in a humidified incubator with 5% CO2 and were routinely tested for mycoplasma contamination. The Sabin and Mahoney strains of type 1 poliovirus (PV) were a gift from Luis Sigal and were passaged and titered in Vero fibroblasts. Infections
Materials and methods
Primary neurons and MEFs were plated onto poly-llysine-treated 15-mm-diameter glass coverslips at a density of 500 cells/mm2. Neurons were differentiated for 72 –96 h prior to infection, whereas MEFs were plated 24 h before infection. Cells were infected with type 1 PV, Sabin or Mahoney strain, at a multiplicity of infection (MOI) = 0.1. The coverslips were covered with the viral inoculum and incubated for 1 h at 37 -C, after which the inoculum was replaced with either conditioned neurobasal medium (primary neurons) or fresh complete DMEM (MEFs). Slips and supernatants were collected every 8 h after infection. Slips were washed briefly in fresh PBS, and the cells were fixed for 5 min in a 1:1 solution of ice-cold methanol: acetone. After fixation and drying, the slips were stored at 4 -C in PBS.
Cells and viruses
Immunofluorescence
Poliovirus receptor (PVR; CD155+) transgenic mice, originally developed by Racaniello and colleagues (Ren et al., 1990), were obtained from Luis Sigal at Fox Chase. Primary hippocampal neurons were prepared from day 15.5 CD155+ transgenic embryos, as previously described
To detect CD155 expression, a mouse antibody to CD155 (BioSource International, Camarillo, CA) was used at a 1:100 dilution followed by an Alexa-Fluor 488 conjugated anti-mouse IgG (Molecular Probes, Eugene, OR; 1:500).
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For viral spread studies, CD155-negative neurons were isolated from c57Bl/6 day 15.5 embryos as described above, counted, and loaded with Vybrant CMDil cell labeling solution (Molecular Probes). The vital dye was added to neurons in suspension at a concentration of 5 Al/2.0 106 cells/ml in serum-free neurobasal media and incubated at 37 -C for 30 min. Labeled neurons were then washed twice in fresh serum-free neurobasal medium and centrifuged through a 2 ml FCS cushion. Immunofluorescence detection of PV antigen utilized a type 1 PV-specific mouse monoclonal (Accurate, Westbury NY; 1:200 dilution in PBS) followed by incubation with a biotinylated anti-mouse IgG2a (Serotec, Raleigh NC; 1:300 dilution in PBS). Finally, an FITC-conjugated avidin (1:500 dilution; Vector, Burlingame, CA) was incubated with the slips for 30 min followed by three 5 min washes. All slides were visualized using a Nikon fluorescence microscope model TE300, equipped with appropriate filters for the fluorescence studies. For the viral trafficking studies, fluorescent orange and green images from identical fields were captured and merged using Adobe Photoshop. Immunohistochemistry Fixed cells were blocked with 1% (vol/vol) goat serum in PBS, further blocked with avidin and biotin (Vector), and stained for PV antigens as described above. However, after incubation with the secondary antibody, a streptavidin – peroxidase conjugate (Vector) was added, and PV was visualized by diaminobenzidine precipitation (0.7 mg/ml DAB with 1.6 mg/ml H2O2 in 60 mM Tris; Sigma Chemical). In some cases, cells were counterstained with hematoxylin to visualize nuclei. To quantify cell death in infected cultures, live and dead cells (as determined by morphology) were counted from 5 representative fields at 400 for each timepoint post-infection. Samples were coded until after counting.
at 4 -C. Cells were centrifuged and resuspended in 400 Al of 0.5% formaldehyde in PBS and analyzed on a BD FACScan fluorescence-activated cell scanner. Plaque assay Supernatants from infected cells were serially diluted and added to Vero fibroblasts plated in 6-well cell culture dishes in complete DMEM at a cell density of 106 cells/well. After incubation at 37 -C for 1 h, the viral inoculum was removed, and the cells were covered with a 1:1 mixture of 2 Medium 199 (Life Technologies) and 1% molten agarose in water. The agarose was allowed to solidify, and the plates were then returned to the 37 -C incubator. Two days later, the cells were fixed with 10% formaldehyde in PBS. Agarose overlays were removed, and plaques were visualized by staining the remaining fibroblasts with 0.1% crystal violet. Infectious center assay Infectious center assays (ICA) were performed using PVR+ neurons and MEFs that were infected with the Mahoney strain of poliovirus at an MOI = 0.1. At 16, 24, and 32 hpi, supernatants were removed and assayed for infectious PV particles by plaque assay (see above). The attached cells from each timepoint were subsequently washed with PBS and removed with 0.4% trypsin (2 ml final volume). The cells were counted (to obtain the total number of cells), serially diluted, and added to Vero fibroblasts plated on 6-well cell culture dishes. Two days post-plating, the cells were fixed and subsequently visualized with crystal violet. The percentage of infected cells was calculated using the following equation: # of plaques (from ICA) dilution factor / total number of cells. Infectious virus per-infected cell was calculated using the following equation: total amount of infectious virus released (determined by plaque assay) / # of infected cells (determined by ICA).
Flow cytometry Neutralizing antibody assay Primary PVR+ hippocampal neurons and MEFs were plated on poly-l-lysine-treated 6-well culture plates at a cell density of 500 cells/mm2. Cells were infected with the Mahoney or Sabin strain of PV at an MOI = 0.1 or were mock-infected. Cells were removed from the plates at 24 and 40 hpi using Cell Stripper (Cellgro Mediatech, Inc., Herndon, VA). Cells were pelleted at 1200 rpm for 5 min and resuspended in 2 ml cytofix/cytoperm buffer (BD Biosciences/Pharmingen, San Diego, CA) and incubated for 20 min at 4 -C. Cells were then washed twice with perm/ wash buffer and transferred to wells of a 96-well microtiter plate. Poliovirus type 1 antibody at a 1:100 dilution or PBS (for no primary control) was added to each well for 30 min at 4 -C. An anti-mouse IgG FITC-conjugated secondary antibody (Vector; 1:500) was added to all wells for 30 min
CD155+ MEFs and neurons, plated on coverslips, were infected with the Sabin strain of PV at an MOI = 0.1 as described above. For some slips, 30 Al of type 1 PV neutralizing antibody (1:320; BioSource) was added 1 h post-infection. Infected cells and corresponding supernatants were collected at various times post-infection. Coverslips were fixed and immunostained for PV antigen as described above; supernatants were subjected to plaque assay. The percent of infected cells at each timepoint was calculated by counting 5 representative fields/slip at 400 magnification; identification of the slips was coded until after counting. Viral titers from each timepoint were determined by plaque assay of supernatants from infected cells as described above.
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