Cell-Induced Conformational Change in Poliovirus: Externalization of the Amino Terminus of VP1 Is Responsible for Liposome Bindingt

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1 JOURNAL OF VIROLOGY, May 1990, p Vol. 64, No X/90/ $02.00/0 Copyright C) 1990, American Society for Microbiology Cell-Induced Conformational Change in Poliovirus: Externalization of the Amino Terminus of VP1 Is Responsible for Liposome Bindingt CARL E. FRICKS1 AND JAMES M. HOGLE2* Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, California 92037,2 and Los Angeles County USC Medical Center, Los Angeles, California 90033' Received 5 October 1989/Accepted 3 January 1990 Upon attachment to susceptible cells, poliovirus and a number of other picornaviruses undergo conformational transitions which result in changes in antigenicity, increased protease sensitivity, the loss of the internal capsid protein VP4, and a loss of the ability to attach to cells. These conformationally altered particles have been characterized by using a number of sequence-specific probes, including two proteases, a panel of antiviral monoclonal antibodies, and a panel of antisera against synthetic peptides which correspond to sequences from the capsid protein VP1. With these probes, cell-altered virus is clearly distinguishable from native and heat-inactivated virions. The probes also demonstrate that the cell-induced conformational change alters the accessibility of several regions of the virus. In particular, the amino terminus of VP1, which is entirely internal in the native virion, becomes externalized. Unlike native and heat-inactivated virus, cell-altered virions are able to attach to liposomes. The exposed amino terminus of VP1 is shown to be responsible for liposome attachment. We propose that during infection the amino terminus of VP1 inserts into endosomal membranes and thus plays a role in the mechanism of cell entry. To initiate a productive infection, an animal virus must first attach to a susceptible cell and then must transport all or a portion of its nucleocapsid across a lipid bilayer. The description of glycoprotein-mediated membrane fusion has provided a plausible mechanism for the entry of enveloped viruses into the cells (9, 20, 53). In contrast, the mechanism by which viruses which lack an external envelope cross the cell membrane and enter the cell is much less clear. The picornaviruses provide excellent models for the study of early events in infection. These viruses are relatively simple, being composed of a protein coat and a small (7,500- nucleotide) RNA genome. The three-dimensional structures of several members of the family, including poliovirus (13, 21), rhinovirus (45), mengo virus (34), and foot-and-mouth disease virus (1), are known at high resolution. The cellular receptors for several picornaviruses, including poliovirus (37) and the major group rhinoviruses (16, 49, 51), have been identified, and the early events in infection for several of these viruses have been relatively well characterized. Picornavirus infection is initiated by attachment to specific receptors (23, 24). Receptor attachment can readily be separated from subsequent stages of cell entry by performing the attachment at low temperature. A synchronous infection can then be initiated by warming the virus-cell complexes to physiological temperature (26). In a number of picornaviruses, including poliovirus (11, 26), coxsackieviruses (5, 6), and rhinoviruses (32, 40), a substantial fraction of the bound virus is released (eluted) from the cell in a significantly altered form. The altered particles sediment at 135S (versus 160S for native infectious particles), have lost the internal capsid protein VP4, have antigenic properties similar to those of heat-inactivated virus, are sensitive to proteases, and are no longer able to attach to susceptible cells (6, 8, 12, * Corresponding author. t Publication 6060-MB from the Research Institute of Scripps Clinic. 33). The conformational transition leading to the 135S particle also can be induced by extracts of membranes from susceptible cells (7, 8, 17). Although the significance of the eluted particles in the infection process has not been established, a particle with very similar properties is the dominant form of the virus found inside infected cells early after infection (10, 31). Like the eluted virus, this form of the particle sediments at 135S, has lost VP4, has altered antigenic properties, and is protease sensitive (10, 31). A number of antiviral drugs have been shown to interfere with the production of the intracellular 135S virus particles. These drugs include agents such as monensin and various weak bases which prevent acidification of intracellular vesicles (35) and agents such as arildone which bind the virus and apparently stabilize it against structural alterations (2, 14, 36). Compounds such as arildone also inhibit the formation of the eluted 135S particle and thermal inactivation of the virus (36). One of the major obstacles to characterizing cell entry in the picornavirus is the high particle-to-pfu ratio. Because physical and chemical characterizations deal with the bulk population, it can be difficult to assess whether observations are relevant to the productive infectious pathway. The correlation between antiviral activity and the inhibition of formation of the intracellular 135S particle, however, suggests that this conformational alteration is an essential step in the process of cell entry. In this article, we report the characterization of the eluted and intracellular 135S particles of the Sabin strain of type 1 poliovirus, using a series of sequence-specific probes. These probes include two specific proteases, a panel of antibodies to synthetic peptides, and a panel of antiviral monoclonal antibodies of known specificities. The reactivities of the eluted and the intracellular 135S particles have been compared with those of native and heat-inactivated virions. The results show that the eluted and intracellular 135S particles represent very similar, if not identical, conformational states 1934

2 VOL. 64, 1990 CELL-INDUCED CONFORMATIONAL CHANGE IN POLIOVIRUS 1935 p8. p 5 P 6 P P t 0 - " P 2. * P0P 2 - X~j1 P 1 FIG. 1. Residues corresponding to peptides P1 through P10 are shown in the context of the alpha carbon model of VP1 from poliovirus type 1 Mahoney (21). In the native virion, residues at the upper right of the figure are located on the outer surface of the protein shell and residues at the lower left are located on the inner surface. Residues shown in dark blue are not included in any of the peptides used in this study. Peptides in cyan (P3, P5, and P8) elicit antibodies which do not bind any of the forms of the virus described here. Peptides in red (P7 and P10) or orange (P9) are external in the native virus and elicit antibodies which recognize all of the conformationally altered particles. Peptides in white (P0, P2, and P6) or green (P1) are internal in the native virus but elicit antibodies that recognize one or more of the conformationally altered particles. Figure by David J. Filman, Research Institute of Scripps Clinic. of the virion which are clearly distinct from either native virus or heat-inactivated virus. The results further demonstrate that the release of VP4 which characterizes the native-to-135s transition is accompanied by the extrusion of the amino terminus of VP1 from the inside of the virion. In contrast, both VP4 and the amino terminus of VP1 are entirely internal in native virions and participate in forming the inner surface of the protein shell (21). Most significantly, the results demonstrate that the newly exposed amino terminus of VP1 specifically confers the ability to attach to liposomes. We propose that during infection the exposed lipophilic amino terminus of VP1 may insert into the endosomal membrane and either disrupt the membrane or form a pore to facilitate the transport of viral components into the cytoplasm. MATERIALS AND METHODS Cells and viruses. Hi HeLa cells were propagated in suspension in Joklik minimal Eagle medium supplemented with 0.1 mm nonessential amino acids-50 U each of penicillin and streptomycin per ml-0.05% pluronic acid. Seed stock prepared from a plaque-purified isolate of the Sabin strain of type 1 poliovirus (P1/Sabin/Lsc2ab) (27) was obtained from Roland Rueckert, University of Wisconsin, Madison. The virus preparations used in this study were no more than three passages from these initial seed stocks. Antibodies. A panel of anti-poliovirus type 1 Sabin monoclonal antibodies was obtained from Anne Mosser, University of Wisconsin, Madison. The preparation of these antibodies and their mapping to specific neutralizing sites has been described elsewhere (42). The type 1-specific monoclonal antibody, C3 (52, 57), was obtained from Radu Crainic, Pasteur Institute. A panel of site-specific antibodies raised against peptides corresponding to residues 24 to 40 (P1), 61 to 80 (P2), 86 to 103 (P3), 161 to 181 (P5), 182 to 201 (P6), 202 to 221 (P7), 244 to 264 (P8), 270 to 287 (P9), and 286 to 302 (P10) of VP1 (4) was provided by Marie Chow, Massachusetts Institute of Technology. Mouse antiserum to a synthetic peptide corresponding to residues 7 to 24 of VP1 (P0) and a monoclonal antibody to P1 (residues 24 to 40 of VP1) p I were prepared by using the protocols described by Niman et al. (39). Figure 1 indicates the location of the residues corresponding to these peptides in the three-dimensional structure of VP1 in the native virion (21). Virus propagation. Virus was propagated in HeLa cells in Spinner flasks (Belco) in medium AL (Dulbecco modified Eagle medium containing calcium and magnesium, 10% calf serum [Irvine Scientific], 50 U each of penicillin and streptomycin per ml, 0.1 mm nonessential amino acids, 2.2 mm L-glutamine, and 25 mm HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid] buffer at ph 7.4). The cells were infected with 5 to 10 PFU/cell. Virus labeled with [35S]methionine or [3H]leucine was prepared by infecting cells in medium deficient in the amino acid carrying the label and adding the appropriate labeled amino acid [3H]leucine [specific activity, 60 Ci/mmol] to a final concentration of 0.02 mci/ml of infected cells or [35S]methionine [specific activity, 1,150 Ci/mmol] to a final concentration of 0.4 mci/ml of infected cells) at 3 h postinfection. Infected cells were harvested at 7.5 h postinfection and lysed by multiple cycles of freezing and thawing, and the virus was purified by differential centrifugation and CsCi density gradient centrifugation as described elsewhere (46). Purified virus was diluted in PBSA (Dulbecco phosphate-buffered saline containing 0.1% bovine serum albumin [BSA]), pelleted through a cushion of 1 M NaCl-30% sucrose in PBSA, and suspended in PBSA to yield a concentration of 107 cpm/ml for 3H-labeled virus (approximately 0.1 mg of virions per ml) or 6 x 107 cpm/ml for 35S-labeled virus (approximately 0.02 mg/ml). Purified virus was used in all experiments described below, including the preparation of conformationally altered virus particles. Preparation of heated virions. Radiolabeled purified virus was heated for 10 min at 56 C in the presence of 0.05% Nonidet P-40. The solution was cooled and layered onto a 15 to 30% sucrose gradient in 10 mm phosphate buffer (ph 7.4) containing 0.14 M NaCl and 0.1% BSA. The gradients were centrifuged for 2.5 to 3 h at 39,000 rpm in an SW40 rotor and fractionated. Heated virions (often referred to as H or C particles) were isolated as the 80S fraction of the gradient,

3 1936 FRICKS AND HOGLE diluted in PBSA-NP (PBSA containing 0.05% Nonidet P-40), and pelleted. The 80S particles were suspended in PBSA-NP in a volume appropriate to yield 106 cpm/ml. Preparation of extracellular 135S particles (eluted virus). Purified virions (approximately 1,000 virions per cell) were added to HeLa cells in medium AL at a density of 4 x 107 cells per ml and incubated for 30 min at 20 C. The cells were then washed to remove unattached virus, suspended at 2 x 106 cells per ml in fresh medium AL, and incubated at 37 C for 40 min. The cells were pelleted, and the supernatant was purified on a 15 to 30% sucrose gradient as described above. The 135S fraction was diluted in PBSA-NP, pelleted, and suspended in PBSA-NP to a final concentration of 106 cpm/ml. Preparation of intracellular 135S particles. Purified [35S] methionine-labeled virions were attached to cells and washed as described above. The cells were incubated for 5, 15, or 40 min at 37 C, pelleted, washed three times at 4 C to remove eluted virus, and dissolved in 0.5% Triton X-100 in PBSA. The cell lysate was clarified by centrifugation for 3 min in an Eppendorf centrifuge, and the supernatant was collected and adjusted to 0.2% sodium dodecyl sulfate (SDS). The particles were purified by sucrose gradient fractionation as described for the preparation of eluted virus. The 135S fraction was diluted in PBSA-NP, pelleted, and suspended in PBSA-NP at a concentration of 106 cpm/ml. Optimal yields of 135S particles were obtained when the infected cells were incubated for 40 min at 37 C before lysis. Proteolytic digestion of virus and related particles. The proteolytic sensitivities of virus and virus-derived particles were assessed by adding 5,ug of trypsin (Cooper Biomedical) or 10 Fg of Staphylococcus aureus V8 protease (Cooper Biomedical) to 50,000 cpm (approximately 25 [Lg) of the appropriate particle in 50 [L of PBSA. Native virus (which is quite resistant to proteolysis) was incubated at 25 C. Altered particles (heated virus and eluted virus) were digested at 4 C. The digests were stopped by dilution of the samples with an equal volume of 7 M urea-2% SDS-2% P-mercaptoethanol and then by boiling of the mixture for 2 min. The samples were subjected to SDS-polyacrylamide gel electrophoresis in the presence of 3.5 M urea (27), and the proteins were visualized by autoradiography or silver staining (56). Western immunoblots. Specific proteolytic fragments were identified in Western blots by using protein- or peptidespecific antibodies. Samples were loaded onto SDS-urea, electrophoresed as described above, and transferred to nitrocellulose, and the nitrocellulose was incubated for 1 h with 3% BSA-0.1% Triton X-100 (Sigma) in phosphatebuffered saline. Individual strips were incubated with 50 to 100 1LI of the appropriate antisera in 10 ml of the blocking buffer described above, washed three times with 0.5 M LiCl-0.025% Triton X-100 in 0.1 M Tris hydrochloride (ph 7.4), reblocked by incubation for 30 min in 3.0% BSA-0.1% Triton X-100 in phosphate-buffered saline, and developed by incubation with approximately 10,uCi of 125I-protein A for 1 h. The strips were washed three times with 1% BSA-0.1% Triton X-100, the nitrocellulose was dried, and the label was visualized by autoradiography. Immunoprecipitation assays. In 10 RI of PBSA, 10,000 cpm of [3H]leucine- or [35S]methionine-labeled virus or virusderived particle was mixed with equal volumes of known dilutions of antisera in PBSA and incubated for 2 h at room temperature. The complexes were adsorbed onto S. aureus cells by being mixed with 1 ml of a 10-fold dilution of Pansorbin (Calbiochem-Behring) in PBSA, and the adsorbed complexes were pelleted in an Eppendorf centrifuge. Bound counts were released by being boiled in 2% SDS-2% I- mercaptoethanol. The dilution of serum required to precipitate half the counts was determined by inspection of titration curves, and the titer was expressed as counts per minute of label precipitated per microliter of serum. Preparation of liposomes. Liposomes consisting of phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, [3H]cholesterol, and phosphatidic acid in molar ratios of 1:1:1:1.5:0.3 were prepared fresh for each experiment as described by White and Helenius (53). All lipids were obtained from Avanti Polar Lipids (Birmingham, Ala.) except for cholesterol which was obtained from Sigma Chemical Co. Briefly, a lipid film was prepared by rotary evaporation of a solution containing the above molar ratio of lipids dissolved in chloroform-methanol (2:1 [vol/vol]) in an acidwashed tube. The film was washed twice with chloroformmethanol and once with anhydrous ether; then it was dried under reduced pressure for at least 90 min. A suspension of 1-mm-diameter glass beads in PBSA (0.05 to 0.5 ml per 1.25 plmol of phosphatidylethanolamine) was added to the dried film, and the mixture was vortexed for 1 min. The sample was then sonicated for 1 min, and the glass beads and large aggregates were removed by centrifugation for 15 min in an Eppendorf centrifuge. Liposome flotation. The 50,000 cpm of [35S]methioninelabeled virus or virus-derived particles was mixed with [3H]cholesterol-labeled liposomes in a total volume of 50 pli in the bottom of a centrifuge tube, and the sample was brought to 50% sucrose by the addition of 150,ul of 67% sucrose in PBSA. The sample was overlaid with a step gradient of 2.4 ml of 25% sucrose and 2.4 ml of 10% sucrose in PBSA. The gradients were centrifuged for 5 to 10 h at 45,000 rpm in an SW50 rotor, and fractions of 0.2 ml were taken from the top of the tube. All 35S-labeled material in the top half of the gradient was defined to be associated with liposomes. RESULTS J. VIROL. Purification of conformationally altered virus particles. Characterization of conformationally altered virus particles requires that the particles be obtained free of significant contamination with virus or other altered forms of the virus. In this study, all virus-related particles were prepared by modification of CsCI-purified native virions, and altered particles were purified on sucrose gradients. Representative sucrose gradients for native virions, heated virus, and eluted virus are shown in Fig. 2. Gradients for native virions and heated virions each typically contained a single peak. Eluted virus preparations typically had two well-resolved peaks, a larger peak at 135S and a smaller peak at 80S. Preparation of 135S particles from within cells early after infection was more problematic. A time course for the production of intracellular 135S particles is shown in Fig. 3. The yield of 135S particles was dependent on the duration of incubation at 37 C before cell lysis. The quantity of label sedimenting at 135S increased slowly with time, reaching a maximum at approximately 40 min. Longer incubations caused progressively more label to accumulate in the 80S fraction. At all time points, the gradients contained significant quantities of material sedimenting at 160S and 80S (Fig. 3). Consistent with a recent report by Everaert et al. (10), the intracellular 135S particles obtained at the later time points had undergone significant proteolytic degradation (data not shown). Proteolytic sensitivity of native and altered virions. In a previous report, we showed that many strains of poliovirus

4 VOL. 64, 1990 CELL-INDUCED CONFORMATIONAL CHANGE IN POLIOVIRUS n b FIG. 2. Sucrose density gradient purification of [35S]methioninelabeled poliovirus and virus-derived particles. The gradients were fractionated from the bottom. Representative gradients for native virus, heat-inactivated 80S particles, and eluted 135S particles are shown in panels A, B, and C, respectively. (including PI/Sabin and P3/Sabin) contain a unique trypsin cleavage site in the vicinity of amino acid 100 of VP1 (15). This tryptic site was subsequently shown to be in a highly exposed loop located near the particle fivefold axes on the outer surface of the virion (21). (The cleavage site is located at the carboxyl end of peptide P3 [Fig. 1]). We also showed that the patterns of tryptic digestion for native and heated virions were significantly different, thus demonstrating that proteolytic digests could serve as probes for conformational rearrangements in poliovirus (15). (i) Trypsin sensitivity. Limited tryptic digestion of native virus (Fig. 4, lane A) resulted in partial cleavage of the protein VP1 into two smaller fragments, VPlT1 and VP1T2, which we have previously shown to correspond to residues 100 to 302 and residues 1 to 99 of VP1, respectively (15). t Upon prolonged exposures (12 h), VP1 was completely cleaved to the two fragments and no further cleavages were seen. Under similar conditions (0.5 mg of particle and 0.01 mg of trypsin per ml at room temperature), VP1 and VP2 of both heated virus and eluted virus are completely degraded to unresolved fragments within 1 h (data not shown). If the digestion is performed at 4 C, however, in both heated virions (15) and eluted virus (Fig. 4, lanes C and D) VP1 and VP2 are cleaved to yield a pattern of fragments which differs significantly from the pattern of tryptic fragments characteristic of digests of native virus. In the trypsin digests of the eluted 135S particles, there are two prominent tryptic fragments: one which migrates similarly to VPlT1 in digests of native virus and a second which migrates to a position midway between that of VP1T2 (Mr, 10,000) and VP4 (Mr, 7,500) (Fig. 4, lane D). The relative positions of these two bands are similar to those of two prominent fragments seen previously in digests of heated virus (15). The comigration of the larger fragment with VPlT1 is probably fortuitous, since the fragment fails to react with a number of VP1-derived antipeptide antibodies which react strongly with VPlT1 in Western blots. The combined apparent molecular weights of the larger and smaller fragments are consistent with the possibility that the two are derived from cleavage of VP2. Trypsin digestion of eluted virus also generates two fragments which migrate between VP1 and VP2. Because the bands representing these fragments are too dense to be derived from cleavage of the low levels of VPO which are found in poliovirus (Fig. 4, lanes A to D), they must be derived from cleavage of VP1. This suggests that the conformational change associated with the production of the eluted virus has exposed novel tryptic sites very near one or both chain termini of VP1. (ii) Sensitivity to the V8 protease from S. aureus. Precise identification of the tryptic cleavage fragments is complicated by the low levels and transient appearance of many of the fragments in the time course of the digests. In contrast, digestion of eluted virions with the Staphylococcus V8 protease produces a much simpler pattern of proteolytic fragments. Indeed, even after prolonged incubations with the V8 protease, the only changes observed in the gels are the disappearance of the band corresponding to VP1 and a broadening of the bands corresponding to VP2 and perhaps VP3 (Fig. 4, lane E). The cleaved virions can easily be isolated as 135S sedimenting particles and used for further studies. The cleavage pattern for heated virions is indistinguishable from that shown for eluted virus. Native virus is not detectably cleaved under these conditions (data not shown). Western blots of the V8 digests with antisera to peptide P9 (residues 270 to 287 of VP1) highlight the band in the position corresponding to VP2 (Fig. 4, lane G). Because this antiserum does not cross-react with VP2, this result indicates that the broadening and darkening of the band at Mr 30,000 is due to the comigration of a proteolytic fragment of VP1 with VP2. The anti-p9 serum also reacts with a band migrating just below the position of VP2. This band does not appear in the autoradiogram of the [3H]leucine-labeled V8 digest (Fig. 4, lane E), from which we conclude that it represents a very minor proteolytic fragment with a very high affinity for anti-p9. The comigration of a proteolytic fragment of VP1 (Mr, 33,000) with VP2 (Mr, 30,000) indicates that the eluted virus has exposed a novel V8 protease site approximately 30 residues from either the carboxyl or amino terminus of VP1.

5 1938 FRICKS AND HOGLE J. VIROL S l 20 A 0' at 370C of these antisera (aop7, up9, and otp10) were raised against amino acid sequences which are at least partly exposed on the surface of the native virus (22) (Fig. 1). The ability of these antisera to recognize the conformationally altered particles may be due to an increase in the exposure or mobility of these peptides upon conformational rearrange- E l 0 U) I (.0 160S _ C5 5' at 37 C n l35s Fraction Number Fraction Number FIG. 3. Sucrose density gradient fractionation of extracts of [35S]methionine-labeled infected cells. Purified native virions were attached to cells at 4 C and incubated at 37 C for 0 (A), 5 (B), 15 (C), or 40 (D) min before lysis. The lysates were overlaid on sucrose gradients, and the gradients were developed and fractionated from the bottom. The reactivity of this fragment with the anti-p9 serum and the failure of this (or any other band) to react with antisera to P1 (residues 24 to 40 of VP1), which have previously been shown to react with VP1 and VP1T2 in Western blots (15) (J. Icenogle and J. M. Hogle, unpublished data), suggests that the cleavage occurs near the amino terminus of VP1. Reactivity with monoclonal antibodies and antipeptide antisera. Panels of antiviral monoclonal antibodies and synthetic peptide antisera have been used to probe for conformational differences in the virus-derived particles. The panel of monoclonal antibodies includes antibodies specific for each of the major antigenic sites as defined by Page et al. (42). The antipeptide antisera were raised against a series of synthetic peptides which nearly span the entire sequence of VP1. The locations of these peptides are summarized in Table 2 and in Fig. 1. Because the conformational integrity of the virion is known to be disrupted by attachment to surfaces, we have used immunoprecipitation assays to determine relative titers for each of the antisera. Whenever possible, the titrations were carried out by using a range of dilutions sufficient to probe the entire immunoprecipitation profile. The immunization titers are summarized in Tables 1 and 2. Representative titrations for the monoclonal and antipeptide antibodies are shown in Fig. 5a and b, respectively, to demonstrate the significance of the differences in titers presented in Tables 1 and 2. (i) Immunoprecipitation with neutralizing monoclonal antibodies. The immunoprecipitation titers of the panel of neutralizing monoclonal antibodies versus native virus, eluted virus, V8-treated eluted virus, and heated virus are summarized in Table 1. Two of the monoclonal antibodies (McAb 10 and 13) are highly specific for native virus, showing no detectable reactivity with any of the conformationally altered forms of the virus. These monoclonal antibodies are specific for site 3B, which has been shown to span the boundary between pentamers in the virion structure and is therefore expected to be highly dependent on conformation (42). Two of the monoclonal antibodies show a broad and approximately equivalent specificity for native, eluted, and heated virions. These include monoclonal antibody C3 which is a site 1-specific neutralizing antibody originally raised by immunization with heated virions (52, 57), and McAb 6 which is specific for site 2b (VP2, 164 to 170) (42). The remaining three monoclonal antibodies have immunoprecipitation titers which differ significantly for eluted and heated virus (Fig. 5a, Table 1). These antibodies include McAb 4 which is specific for site 2ab (VP1, 221 to 226; VP2, 164 to 170; and VP2, 270), McAb 7 which is specific for site 2b (VP2, 164 to 170), and McAb 9 which is specific for site 3a (VP3, 58 to 60) (42). The ability of McAb 4 and 9 to distinguish between heated virus and eluted virus is particularly striking. The smaller difference in the titer of McAb 7 versus that of heated virus and eluted virus is reproducible, however, and is believed to be significant (Fig. Sa). The ability of these antibodies to distinguish heated and eluted forms of the virus provides a conclusive demonstration that these two forms represent different conformational states of the virus. (ii) Immunoprecipitation by antipeptide antibodies. The immunoprecipitation titers of the antipeptide antisera versus native virus, eluted virus, V8-treated eluted virus, and heated virus are summarized in Table 2. Under the conditions used in this study, none of the antipeptide antisera precipitated native virions. Three of the antipeptide antisera (xp3, (op5, and otp8) also failed to precipitate any of the conformationally altered forms of the virion (Table 2, Fig. 1). Several of the other antipeptide antisera (including apo, ap1i, McotPl, otp2, ap6, ap7, otp9, and ap10), however, showed significant immunoprecipitation titers with one or more conformationally altered forms of the virion (Table 2). Three

6 VOL. 64, 1990 CELL-INDUCED CONFORMATIONAL CHANGE IN POLIOVIRUS 1939 VPIT2 VP4 A B C D v,po * VP2 VP3 VPIT 1 E F G U NV EV EV+T EV+T EV+V8 EV+V8 EV+V8 + TV 1 min t hr api apq FIG. 4. SDS-polyacrylamide gel electrophoresis of trypsin and V8 protease digests of native virus and of eluted 135S particles. A [3H]leucine-labeled sample (50,000 cpm) was loaded in each lane and resolved by electrophoresis as described in the text. In lanes A through E, the gels were soaked in En3Hance (New England Nuclear Corp.) and the bands were visualized by autoradiography. Lanes: A, partial trypsin digest of native virions; B, eluted virus; C and D, eluted virus digested with trypsin for 1 min and 1 h, respectively; E to G, eluted virus digested with V8 protease for 30 min. In lanes F and G, material from lane E has been transferred to nitrocellulose and incubated with antisera to peptides P1 (lane F) and P9 (lane G); the complexes have been stained with 125I-labeled protein A; and the bands have been visualized by autoradiography. The positions of the viral capsid proteins and major tryptic fragments of native virus are indicated at the left. ment. The remaining four antisera (including apo, apl, ap2, and oap6) are specific for amino acid sequences which are located on the inner surface of the protein shell in the native virus (21) (Fig. 1). The ability of these antisera to recognize altered particles provides a strong indication that the conformational alterations result in the exposure or externalization of normally internal components in both the eluted and heat-inactivated virions. Note, however, that the titers of the apo and apl antisera, and especially the apl monoclonal antibody, are significantly higher for eluted virus (Fig. Sb, Table 2), once again demonstrating that the eluted and heat-inactivated particles represent two distinct conformational states of the virus. The observed binding to altered virions may provide an explanation for the previously published observation that the atp1, atp2, and ap6 antisera have significant, albeit weak, neutralizing titers (4). Neutralization of infectivity with these antisera may be related to their ability to induce conformational alterations, which results in loss of infectivity, or to trap the conformationally altered particles once they are formed. The substantial immunoprecipitation titers of the apo and apl antisera for eluted virus and heated virus confirm the indication from the protease sensitivity that at least a portion of the amino terminus of VP1 is externalized by the conformational alterations induced by heat and by attachment to cells. Moreover, the reactivity of the 135S (eluted) particle with ap0 and with the McaPl monoclonal antibody is completely abolished and the reactivity with the apl antisera is significantly reduced upon digestion of the 135S particles with V8 protease (Fig. Sb, Table 2). These results are consistent with a V8 protease-induced cleavage somewhere between residues 24 and 40 of VP1. Under the conditions used in these studies (digestion in the presence of 0.1 M ammonium bicarbonate [ph 8.0]), the V8 protease is specific for cleavage following glutamic acid. The sequence of VP1 for type 1 poliovirus has five glutamic acids within the first 50 residues of VP1, at amino acids 7, 16, 31, 40, and 48 (29, 41, 43). Of these possible cleavage sites, only cleavage following glutamic acid 31 would be consistent with the fragment sizes and with the lack of reactivity with ap0 (VP1, 7 to 24) and the partial reactivity with apl (VP1, 24 to 40) observed after V8 proteolysis of the 135S particles. Except for the altered reactivities with antibodies specific for the two peptides at or before the putative cleavage site (apo, apl and McaP1), the 135S (eluted) particle and the V8-treated 135S particle are indistinguishable in their reactivities with the panels of antipeptide antisera and antiviral monoclonal antibodies (Tables 1 and 2). Therefore, it is unlikely that cleavage with the V8 protease produces gross structural changes in the 135S particle apart from the loss of the first 31 amino acids of VP1. Ability of virus and related particles to attach to liposomes. Several previous investigations have shown that the conformational rearrangements which produce the 135S particles result in an increase in the hydrophobicity of the particles. The increased hydrophobicity is evidenced by a tendency of the particles to aggregate in the absence of detergents, the ability of the altered particles to adsorb to liposomes (32), and partitioning of the particles in the detergent phase under conditions in which Triton X-114 separates from water (35). The hydrophobic properties of the 135S particles may be necessary for the passage of virus across membranes during cell entry (32, 35). The lipophilic potential of native virus, heated virus, eluted virus, and V8-treated eluted virus were compared by liposome flotation (Fig. 6). In these experiments, radiolabeled virions or conformationally altered particles were premixed with liposomes and overlaid with sucrose step gradients, and the extent of liposome association was evaluated as the fraction of label which floated with the liposomes to the top of the gradients during development in a centrifugal field. In the absence of liposomes, all forms of the virus remained at the bottom of the gradient. In the presence of liposomes, native virions and heat-treated virions remained at or near the bottom of the gradient (Fig. 6). In contrast, a substantial fraction (approximately 80%) of the radioactivity associated with the 135S particles floated to the top of the gradient along with the liposomes (Fig. 6). A similar percentage of 135S particles is associated with the

7 1940 FRICKS AND HOGLE J. VIROL. a MA C - c 0 m 0) 0. b ) a log [Ab] ap9 VP1: F. & & ) C 4,~,,==, o log [Ab] a _- pp1 Monoclonal A. McAb 13 * NV O HV * EV o V8/EV P1 VP1: log [Ab] -log [Ab] FIG. 5. Immunoprecipitation titrations of poliovirus and virus-derived particles with antiviral monoclonal antibodies (a) or with anti-peptide sera (b). In each panel, the percentage of label which is precipitated is plotted versus the negative log of the concentration of the indicated antibody. The titer of each antibody was determined versus that of native versus (0), heat-inactivated virus (0), eluted virus (-), and V8-treated eluted virus (0). Midpoint titers derived from inspection of the titration curves are summarized in Tables 1 and 2. \~& liposomes when the gradient is run at high ionic strength (1 M NaCI) (data not shown), indicating that the association is predominantly nonionic. The ability of 135S particles to associate with liposomes is abolished when the particles are treated with V8 protease prior to being mixed with the liposomes (Fig. 6). This indicates that the exposed amino terminus of VP1 (which is specifically removed by V8 digestion [see above]) is responsible for the liposome association. Further evidence for the role of the amino terminus of VP1 in liposome association is obtained when 135S particles are first mixed with liposomes and then digested with V8 protease (2 h at 4 C) prior to flotation. Under these conditions, far less label floats to the top of the gradient and SDS-polyacrylamide gel electrophoretic analysis of the top fractions indicates that the liposomes are highly enriched in a 3,000-molecular-weight peptide (Fig. 7). The relatively low levels of intact capsid proteins at the top of the gradient probably represent incompletely digested particles which have a sufficient content of intact VP1 to remain associated with the liposomes. The observation of an

8 VOL. 64, 1990 CELL-INDUCED CONFORMATIONAL CHANGE IN POLIOVIRUS 1941 TABLE 1. Immunoprecipitation titers of monoclonal antibodies to poliovirus and virus-derived particles Log titer against Monoclonal Sitea indicated particle' Specificity antibody NV EV V8-EV HV McAb 10 3b NV McAb 13 3b NV C General McAb General McAb NV>>HV>>EV McAb NV>HV>EV McAb 9 3a NV>>HV>EV a Site recognized by the monoclonal antibodies, using the nomenclature of Page et al. (42). b Titers against native virus (NV), eluted 135S particle (EV), eluted 135S particles after digestion with V8 protease for 30 min at 4 C (V8-EV), and heat-inactivated virus (HV). -, The antisera precipitated much less than 50% of the label at the lowest dilution tested. Titers are given as 3H-labeled counts per minute of label precipitated per microliter of serum. intact 3,000-molecular-weight peptide (not normally seen in V8 digests in the absence of liposomes) indicates that the putative association of the amino terminus of VP1 with the liposomes is sufficiently extensive to protect the aminoterminal peptide (which contains two additional glutamic acid residues) from further proteolytic processing. Curiously, despite the evidence that the amino terminus of VP1 is also exposed in heated virions, the 80S particles do not appear to associate with liposomes in these experiments. The failure of heated virions to attach to liposomes may reflect the decreased accessibility of the amino terminus of VP1 (as evidenced by the lower titers of the opo and aopl antisera for heated virions [Table 2]). Alternatively, the failure of heated virus to attach to liposomes may indicate that additional structural components which are unique to the eluted virus are required for binding to liposomes and that exposure of the amino terminus is a necessary but not sufficient condition for attachment. TABLE 2. Immunoprecipitation titers of antipeptide antibodies to poliovirus and virus-derived particles Log titer against Antipeptide Site' indicated particleb Exposure' antibody NV EV V8-EV HV apo ap McaPl cxp ND d - ap < ap ap aplo <2.6 < a Amino acid residues in VP1 that correspond to the sequence of peptide used b to raise the antibody. Titers against native virus (NV), eluted 135S particle (EV), eluted 135S particles after digestion with V8 protease for 30 min at 4 C (V8-EV), and heat-inactivated virus (HV). The symbol < preceding the titer indicates that the titration did not approach 1O0o precipitation at the lowest dilution, and the titer is estimated from the 50% precipitation point. -, The antisera precipitated much less than 50% of the label at the lowest dilution tested. Titers are given as 3H-labeled counts per minute of label precipitated per microliter of serum. Immunoprecipitation titers versus ap3 (VP1, 86 to 103), ap5 (VP1, 161 to 181), and otp8 (VP1, 244 to 264) were less than 2.0 for all forms of the particle. ' +, A significant portion of the sequence is exposed on the surface of the native virion; -, the corresponding sequence is totally buried in the structure of the native virion. d ND, Not determined. Characterization of intracellular 135S particles. Several previous investigations have demonstrated the presence of 135S particles inside cells early after infection with poliovirus. The intracellular 135S particle has been suggested to be an important intermediate in the cell entry pathway. The intracellular 135S particle and the eluted 135S particle widely have been assumed to be identical. In order to further characterize the relationship between the two forms of 135S particle, we compared the immunoprecipitation titers of five antibodies (McAb 7, tpo, oxpl, cp2, and McaPl) versus the intracellular 135S particle and the eluted 135S particle, with and without prior treatment with the V8 protease. In addition, we determined the ability of each type of particle to associate with liposomes in flotation experiments. The results are summarized in Table 3. For each of the five antibodies, the immunoprecipitation titers against the eluted and intracellular forms of 135S particle were indistinguishable. Similarly, the effect of V8 cleavage on the immunoprecipitation titers (namely, no effect on the titer of McAb 7, a 10-fold reduction in the titer of ap1, and a complete loss of affinity of apo and McaPl) was the same for the intracellular and eluted 135S particles. Finally, the intracellular 135S particle, like the eluted particle (but unlike native or heated virus), has significant affinity for liposomes which is abolished by pretreatment with V8 protease. Although the percentage of the label associated with liposomes (50%) was reproducibly lower than that observed for the eluted 135S particle (up to 80% binding), the difference may be due to residual detergent or cellular lipid in the intracellular 135S particle, to partial proteolytic degradation of the intracellular particle, or to low levels of contamination with 160S (native) or 80S (heat-inactivated) particles. Thus, with the probes used in these experiments, the intracellular and eluted forms of the 135S particle appear to be very similar. In particular, in both forms of the 135S particle, the amino terminus of VP1 is externalized and serves as a signal for association with liposomes. DISCUSSION A receptor-mediated conformational alteration is thought to be an essential step in the process by which poliovirus and related viruses attach to and enter cells. The conformational change is known to result in the loss of the normally internal capsid protein VP4 and in changes in the sedimentation behavior, antigenicity, and protease sensitivity of the particle (6, 8, 12, 33). A substantial fraction of the conformationally altered virions elutes away from the cell upon incubation at 37 C and can readily be obtained in relatively pure form (11, 26). A similar if not identical particle is the major form of the virus within infected cells early after infection (10, 31). We have used a number of sequence-specific probes, including proteases, synthetic peptide antibodies, and monoclonal antibodies with known specificities, to further characterize both the intracellular and extracellular forms of the altered virions. With respect to these probes, the intracellular and extracellular forms of the particle are indistinguishable from one another, but both can readily be distinguished from either native infectious or heat-inactivated virions. The probes also demonstrate that the conformational alteration results in the increased accessibility of several portions of the capsid proteins. In some cases (for example residues 202 to 221, 268 to 287, and 288 to 302 of the capsid protein VP1), the increased accessibility may simply correspond to an increase in the exposure or mobility of peptide segments which are normally exposed on the exterior surface of the

9 1942 FRICKS AND HOGLE J. VIROL. I EV-L Vs I E 40 a. 1 S U I o- - 20~~~~~~~~ 10..* 0~~ ~ ~ ~ ~. b Fraction Number FIG. 6. Association of poliovirus and virus-derived particles with liposomes as measured by liposome flotation. For native virus (U), heat-inactivated virus (0), eluted virus (A), V8-treated eluted virus (*), and liposomes (0), the percentage of the total label present in each fraction is plotted versus fraction present in the gradient. Label which is located in the top half of the gradient is considered to be associated with liposomes. virion. In other cases, for example residues 7 to 24, 24 to 40, 61 to 80, and 182 to 201 of VP1, the increased accessibility is more significant because it requires the externalization of polypeptide segments which are buried on the inside of native virions. The externalization of the amino terminus of VP1 may be particularly significant. The results presented in this report indicate that the exposure of the first 31 amino acids of VP1 is responsible for the ability of the 135S particles to attach to liposomes. Liposome attachment occurs at both physiological and high ionic strength and is abolished by the removal of approximately 30 residues from the amino terminus of VP1 by the Staphylococcus V8 protease. Moreover, if V8 protease digestion is performed subsequent to the formation of the virus-liposome complex, the majority of the virus is released and the liposome is enriched in a 3-kilodalton peptide which presumably corresponds to the detached amino terminus of VP1. Although the exposure of the amino terminus of VP1 is required for liposome attachment, other features of the conformationally altered particles also appear to be important since heat-inactivated virions, in which the amino terminus of VP1 is at least partially exposed, are incapable of attaching to liposomes. A recent study by Kirkegaard, which shows that two poliovirus variants which have deletions in the amino terminus of VP1 are defective in the late stages of cell entry, indicates that exposure of the amino terminus of VP1 may also play a role in membrane penetration during normal infection (28). The conformational alteration that produces the 135S particle appears to have striking structural and functional homology to receptor- or ph-induced conformational rearrangements which have been implicated in the exposure of fusion peptides for a number of enveloped viruses. These peptides, which are generally highly hydrophobic, are thought to form helices that insert into the host cell membranes and induce the viral envelope to fuse with the host cell membrane. The conformational rearrangement which exposes the fusion peptide of the influenza virus hemagglutinin has been particularly well characterized. The hemagglutinin is a trimer, and in the native structure (55) the fusion peptide is buried in the trimer interface. Characterization t VPl l VP2-- VP3 3KD I FIG. 7. SDS-polyacrylamide gel showing enrichment of liposomes with a 3,000-molecular-weight peptide. [35S]methionine-labeled eluted virus particles were mixed with liposomes and subsequently digested with V8 protease. The mixture was brought to 50% sucrose and overlaid with a sucrose step gradient, and the gradient was developed and fractionated. The top fraction of the gradient was loaded on an SDS-polyacrylamide gel, electrophoresed as described in the text, and the bands were visualized by autoradiography. The low levels of VP1, VP2, and VP3 (which presumably result from incomplete digestion) serve as internal markers. KD, Kilodaltons; EV-L, eluted virus-liposome complex. using proteolytic (48) and immunological probes (54) shows that upon acidification the hemagglutinin undergoes a stepwise conformational alteration resulting in partial disruption of the trimer interface and exposure of the fusion peptide. The fusion peptides of a number of enveloped viruses share considerable sequence homology. In contrast, the amino-terminal sequence of VP1 is very poorly conserved among the picornaviruses. However, the sequences of a number of members of the enterovirus and related rhinovirus genera of the picornavirus family exhibit a periodicity of hydrophobic amino acids which is characteristic of sequences which are capable of forming an amphipathic helix. The sequences of several representative virus strains are plotted schematically on a helical wheel (47), demonstrating that in each case the distribution of amino acids would result in a helix with one distinctly hydrophobic side (Fig. 8). The existence of an amphipathic helix would be consistent with a mechanism in which the exposed amino termini of several subunits of VP1 (either from a single particle or from more than one particle) formed a pore. Such a pore might also include the capsid protein VP4, which is also externalized during the conformational rearrangement and is expected to partition into membranes because of the presence of myristate at its amino terminus (3). Alternatively, the amphipathic helices (perhaps in concert with VP4) could destabilize the endosomal membrane, resulting in its disruption and the release of its contents directly into the cytoplasm. Note, however, that a mechanism of this type might not be shared by all members of the picornavirus family. In particular, in the aphthoviruses (foot-and-mouth disease virus) and the cardioviruses, the amino terminus of VP1 is significantly shorter and lacks residues which are structurally homologous to the first 30 residues in poliovirus and rhinovirus (1, 34). It may, therefore, be significant that there

10 VOL. 64, 1990 CELL-INDUCED CONFORMATIONAL CHANGE IN POLIOVIRUS 1943 TABLE 3. Antibody Immunoprecipitation titers and flotation of eluted and intracellular 135S particles Log titer against indicated particlea EV IV V8-EV V8-IV McAb apo <2.0 <2.0 alpi McaPl <2.0 <2.0 ap2 NDb ND Coxsackie 83 Residues 1-20 of VP1 Sabin 1; Mahoney 1 (N-terminus) Residues 1-23 of VP1 % Flotation' <2 <2 a Eluted 135S particle (EV), intracellular 135S particle (IV), eluted 135S particle after V8 protease digestion for 30 min at 4'C (V8-EV), intracellular 135S particle after V8 protease digestion for 30 min at 4'C (V8-IV). Titers are given as 35S-labeled counts per minute of label precipitated per microliter of serum. b ND, Not determined. C Percentage of total label which floats to the top half of the gradient in liposome flotation experiment. is no evidence for the presence of 135S intermediates in the cell entry of these viruses. Instead, the conformational change associated with cell entry appears to result in pentameric subunits (18, 52). The conformational alteration leading to formation of the 135S particle is widely believed to occur at the cell surface as a prelude to cell entry. This view is consistent with observations that the structural transition can be induced by extracts of (presumably cytoplasmic) membranes from susceptible cells (7, 8, 17). We have attempted to utilize synthetic peptide antibodies which are specific for the altered particles to detect conformationally rearranged particles on the cell surface. Although similar experiments with native-specific probes have readily detected the small number of native virions which remain at the cell surfaces after attachment and incubation at 37 C, we have never detected altered virus at the cell surface (C. E. Fricks, Ph.D. thesis, University of California, San Diego, 1989). This finding may simply reflect a lack of sensitivity in the assay, or it may suggest that the altered particle once formed is rapidly internalized or released from the cell. Alternatively, the failure to detect altered virus attached to the cell surface may indicate that the conformational alteration occurs inside the cell at some point in the endosomal pathway. The altered virus which is eluted from the -cewould then represent the fraction of virions which are in endosomes that are recycled to the cell surface. This interpretation would explain the parallel time course for the appearance of eluted and intracellular 135S particles and is consistent with previously published observations that induction of the conformational rearrangement in paraformaldehyde-fixed cells requires acidification (35). The exposure of the amino terminus of VP1 and the loss of VP4 raise the question of how internal components are externalized during the conformational transition. An analogy with a well-characterized conformational transition in plant viruses may indicate how such externalization occurs. A number of icosahedral plant viruses have been shown to undergo an expansion of approximately 10% in radius upon exposure to chelators of divalent cations at slightly alkaline ph in vitro (25, 30). These conditions (low calcium and magnesium, alkaline ph) are characteristic of the conditions in the cytoplasm of plant cells, and the expanded forms of the plant viruses have been proposed as intermediates in assembly and uncoating (19). The expansion has been particularly well characterized in Sabin 3, Leon 3 Residues 1-18 of VP1 Rhino 14 FIG. 8. Helical wheels (47) indicate that the amino-terminal sequence of VP1 in several enteroviruses is consistent with the possible formation of an amphipathic helix. Each panel represents an alpha helix viewed along the helix axis, with side chains corresponding to the amino-terminal sequences of VP1 in coxsackievirus B3, poliovirus type 1 Mahoney and Sabin, poliovirus type 3 Leon and Sabin, and human rhinovirus 14. Residue numbers and the identities of side chains are indicated. Hydrophobic side chains are stippled. Charged or highly polar side chains are white. Amino acids which could be accommodated either on the polar or on the nonpolar side of an amphipathic helix (Gly, Ala, Ser, and Thr [47]) are hatched if they are located on the nonpolar side and white if they are located on the polar side of the helix. a group of related plant viruses (for example tomato bushy stunt virus and turnip crinkle virus) which bear striking structural similarities to the picornaviruses. In these viruses, expansion is controlled by an interface between capsid proteins which contains a pair of divalent cation-binding sites (22). Upon removal of the divalent cations at basic ph, the interface is destabilized by local concentration of negative charges from the acidic residues which bind the cations (22). Biochemical characterization of tomato bushy stunt virus and turnip crinkle virus has shown that the expansion of the plant viruses also results in the externalization of residues near the amino termini of the capsid proteins (19). In the structure of the expanded form of tomato bushy stunt virus, which has been solved at low resolution (44), the newly exposed portions of the amino termini of the capsid proteins are extruded through fenestrations caused by the disruption of this interface. An analysis of the location of residues which contribute to the thermostability of variants of the Sabin strain of type 3 poliovirus (38) suggests that the analogous interface is also important in controlling stability and conformational rearrangements in poliovirus (13). Furthermore, the point at which the amino-terminal extension of VP1 joins the beta

11 1944 FRICKS AND HOGLE barrel is located immediately below this interface in the native virus particle. The location of these residues and the analogy with plant virus expansion suggest that the cellmediated conformational change in poliovirus might involve disruption of this interface and that the amino terminus of VP1 might be externalized through the disrupted interface. The same interface might also be the portal for the externalization of VP4. Indeed, the native virus structure provides evidence that the externalization of VP4 and of the amino terminus of VP1 may be coordinated. Most of the amino terminus of VP1 up to residue 20 is disordered. During refinement of the poliovirus structure, however, we identified an electron density feature corresponding to five residues from the extreme amino terminus of VP1. These residues form a short segment of extended chain which clearly adds a third beta strand to the two-stranded sheet formed by the amino terminus of VP4 (13). The interaction between the amino termini of VP1 and VP4 could provide a mechanism for coordinating their externalization during the formation of the 135S particle. Finally, there is growing evidence that the conformationally altered virus may play an important role in the immune recognition of poliovirus. In a previous study, we have shown that synthetic peptides corresponding to two regions of the amino terminus of VP1 (residues 24 to 40 and 61 to 80) were capable of eliciting neutralizing antibodies in mice and in rats (4). The observation that these sequences are accessible to antibody binding in a form of the virus associated with cell entry suggests several plausible mechanisms for how these antibodies might neutralize the virus. We have obtained preliminary evidence that these normally internal sequences are recognized in the human response to poliovirus immunization. Comparisons of the immunoprecipitation titers against native virus, 135S eluted virus, and V8-treated 135S eluted virus, indicate that sera from two human vaccinees contain high levels of antibodies which are specific for the 135S particles. In one of the two vaccinees, the overwhelming majority of the 135S-specific response was directed against the first 30 residues of VP1 (Fricks, Ph.d. thesis). A more extensive study by Roivainen et al., using the peptide-scanning approach, has confirmed the presence of high titers to residues 1 to 30 of VP1 in some human vaccinees and has revealed a second epitope, comprising residues 40 to 53 of VP1, which is present in the sera of more than 90% of human vaccinees (M. Roivainen, A. Narvanen, M. Korkolainen, M.-L. Huhtala, and T. Hovi, Virology, in press). These results demonstrate that receptor-mediated conformational changes may expose novel neutralizing epitopes in susceptible hosts. ACKNOWLEDGMENTS This work was supported by Public Health Service grant A (to J.M.H.) from the National Institutes of Health. The authors thank Margie Kubitz for technical assistance; Joseph Icenogle, Ian Wilson, Judy White, and Marie Chow for helpful discussions; Margaret Graber for assistance in preparing the manuscript; and David Filman for helpful discussions and critical reading of the manuscript. LITERATURE CITED 1. Acharya, R., E. Fry, D. Stuart, G. Fox, D. Rowlands, and F. Brown The three-dimensional structure of foot-andmouth disease virus at 2.9 A resolution. Nature (London) 337: Caliguiri, L. A., J. J. McSharry, and G. W. Lawrence Effect of aridone on modification of poliovirus in vitro. Virology J. VIROL. 105: Chow, M., J. F. E. Newman, D. Filman, J. M. Hogle, D. J. Rowlands, and F. Brown Myristylation of picornavirus capsid protein VP4 and its structural significance. Nature (London) 327: Chow, M., R. Yabrov, J. Bittle, J. M. Hogle, and D. Baltimore Synthetic peptides from four separate regions of the poliovirus capsid protein VP1 induce neutralizing antibodies. Proc. Natl. Acad. Sci. USA 82: Crowell, R. L., B. J. Landau, and L. Philipson The early interaction of coxsakie Be with HeLa cells. Proc. Soc. Exp. Biol. Med. 137: Crowell, R. L., and L. Philipson Specific alteration of coxsackievirus B3 eluted from HeLa cells. J. Virol. 8: De Sena, J., and B. Mandel Studies on the in vitro uncoating of poliovirus. I. Characterization of the modifying factor and the modifying reaction. Virology 70: De Sena, J., and B. Mandel Studies on the in vitro uncoating of poliovirus. II. Characteristics of the membranemodified particle. Virology 78: Doms, R. W., A. Helenius, and J. White Membrane fusion activity of the influenza virus hemagglutinin. J. Biol. Chem. 260: Everaert, L., R. VriJsen, and A. Boeye Eclipse products of poliovirus after cold-synchronized infection of HeLa cells. Virology 171: Fenwick, M. L., and P. D. Cooper Early interactions between poliovirus and ERK cells. Some observations on the nature and significance of the rejected particles. Virology 18: Fenwick, M. L., and M. J. Wall Factors determining the site of synthesis of poliovirus proteins: the early attachment of virus particles to endoplasmic membranes. J. Cell Sci. 13: Filman, D. J., R. Syed, M. Chow, A. J. Macadam, P. D. Minor, and J. M. Hogle Structural factors that control conformational transitions and serotype specificity in type 3 poliovirus. EMBO J. 8: Fox, M. P., J. J. Otto, and M. A. McKinlay Prevention of rhinovirus uncoating by WIN 51711, a new antiviral drug. Antimicrob. Agents Chemother. 30: Fricks, C., J. Icenogle, and J. M. Hogle Trypsin sensitivity of the Sabin strain of type 1 poliovirus: trypsin cleavage sites in virus and related antigens. J. Virol. 54: Greve, J. M., G. Davis, A. M. Meyer, C. P. Forte, S. C. Yost, C. W. Marlor, M. E. Kamarck, and A. McClelland The major human rhinovirus receptor is ICAM-1. Cell 56: Guttman, N., and D. Baltimore A plasma membrane component able to bind and alter virions of poliovirus type 1: studies on cell-free alteration using a simplified assay. Virology 82: Hall, L., and R. Rueckert Infection of mouse fibroblasts by cardioviruses. Premature uncoating and its prevention by elevated ph and magnesium chloride. Virology 43: Harrison, S. C., P. K. Sorger, P. G. Stockley, J. M. Hogle, R. Altman, and R. K. Strong Mechanism of RNA virus assembly and disassembly. UCLA Symp. Mol. Cell. Biol. 54: Helenius, A., K. Kartenbeck, K. Simons, and E. Fries On the entry of Semliki Forest virus into BHK-21 cells. J. Cell Biol. 84: Hogle, J. M., M. Chow, and D. J. Filman Three-dimensional structure of poliovirus at 2.9 A resolution. Science 229: Hogle, J. M., T. Kirchhausen, and S. C. Harrison The divalent cation binding sites of tomato bushy stunt virus: differ ence maps at 2.9 A resolution. J. Mol. Biol. 171: Holland, J. J Receptor affinities as major determinants of enterovirus tissue tropisms in humans. Virology 15: Holland, J. J., and B. H. Hoyer Early stages of enterovirus infection. Cold Spring Harbor Symp. Quant. Biol. 27: Incardona, N. L., and P. Kaesberg A ph-induced struc-

12 VOL. 64, 1990 CELL-INDUCED CONFORMATIONAL CHANGE IN POLIOVIRUS 1945 tural change in bromegrass mosaic virus. Biophys. J. 4: Joklik, W. K., and J. E. Darnell The adsorption and early fate of purified poliovirus in HeLA cells. Virology 13: Kew, 0. M., M. A. Pallansch, D. R. Omilianowski, and R. R. Rueckert Changes in three of four coat proteins of oral polio vaccine strain derived from type 1 poliovirus. J. Virol. 33: Kirkegaard, K Mutations in VP1 of poliovirus specifically affect both encapsidation and release of viral RNA. J. Virol. 64: Kitamura, N., B. L. Semler, P. G. Rothberg, G. R. Larsen, C. J. Adler, A. J. Dorner, E. A. Emini, R. Hanecak, J. J. Lee, S. van der Werf, C. W. Anderson, and E. Wimmer Primary structure, gene organization and polypeptide expression of poliovirus RNA. Nature (London) 291: Kruse, J., K. M. Kruse, J. Witz, C. Chauvin, B. Jacrot, and A. Tardieu Divalent ion-dependent reversible swelling of tomato bushy stunt virus and organization of the expanded version. J. Mol. Biol. 162: Lonberg-Holm, K., L. B. Gosser, and J. C. Kauer Early alteration of poliovirus in infected cells and its specific inhibition. J. Gen. Virol. 27: Lonberg-Holm, K., L. B. Gosser, and E. J. Shimshick Interaction of liposomes with subviral particles of poliovirus type 2 and rhinovirus type 2. J. Virol. 19: Lonberg-Holm, K., and B. D. Korant Early interaction of rhinovirus with host cells. J. Virol. 9: Luo, M., G. Vriend, G. Kamer, I. Minor, E. Arnold, M. G. Rossmann, U. Boege, D. G. Scraba, G. M. Duke, and A. C. Palmenberg The atomic structure of Mengo virus at 3.0 A solution. Science 235: Madshus, I. H., S. Olsnes, and K. Sandvig Requirements for entry of poliovirus into cells at low ph. EMBO J. 3: McSharry, J. J., L. A. Caliguiri, and H. J. Eggers Inhibition of uncoating of poliovirus by arildone, a new antiviral drug. Virology 97: Mendelsohn, C. L., E. Wimmer, and V. R. Racaniello Cellular receptor for poliovirus: molecular cloning, nucleotide sequence, and expression of a new member of the immunoglobulin superfamily. Cell 56: Minor, P. D., G. Dunn, D. M. A. Evans, D. I. Magrath, A. John, J. Howlett, A. Phillips, G. Westrop, K. Wareham, J. W. Almond, and J. M. Hogle The temperature sensitivity of the Sabin type 3 vaccine strain of poliovirus: molecular and structural effects of a mutation in the capsid protein VP3. J. Gen. Virol. 70: Niman, H., R. Houghten, L. Walker, R. Reisfeld, I. Wilson, J. M. Hogle, and R. Lerner Generation of protein reactive antibodies by short peptides is an event of high frequency: implication for the structural basis of immune recognition. Proc. Natl. Acad. Sci. USA 80: Noble, J., and K. Lonberg-Holm Interactions of components of human rhinovirus type 2 with HeLa cells. Virology 51: Nomoto, A., T. Omata, H. Toyoda, S. Kuge, H. Horie, Y. Kataoka, Y. Genba, Y. Nakano, and N. Imuara Complete nucleotide sequence of the attenuated poliovirus Sabin 1 strain genome. Proc. Natl. Acad. Sci. USA 79: Page, G. S., A. G. Mosser, J. M. Hogle, D. J. Filman, R. R. Rueckert, and M. Chow Three-dimensional structure of the poliovirus serotype 1 neutralizing determinants. J. Virol. 62: Racaniello, V. R., and D. Baltimore Molecular cloning of poliovirus cdna and determination of the complete nucleotide sequence of the viral genome. Proc. Natl. Acad. Sci. USA 78: Robinson, I. K., and S. C. Harrison Structure of the expanded state of tomato bushy stunt virus. Nature (London) 297: Rossmann, M. G., E. Arnold, J. W. Erickson, E. A. Frankenberger, J. P. Griffith, H.-J. Hecht, J. E. Johnson, G. Kamer, M. Luo, A. G. Mosser, R. R. Rueckert, B. Sherry, and G. Vriend Structure of a human common cold virus and functional relationship to other picornaviruses. Nature (London) 317: Rueckert, R. R., and M. A. PaHlansch Preparation and characterization of encephalomyocarditis (EMC) virus. Methods Enzymol. 78: Schiffer, M., and A. B. Edmunson. 1%7. Use of helical wheels to represent the structures of proteins and to identify segments with helical potential. Biophys. J. 7: Skehel, J. J., P. M. Bayley, E. B. Brown, S. R. Martin, M. D. Waterfield, J. M. White, I. A. Wilson, and D. C. Wiley Changes in the conformation of influenza virus hemagglutinin at the ph optimum of virus-mediated membrane fusion. Proc. Natl. Acad. Sci. USA 79: Staunton, D. E., V. J. Merluzzi, R. Rothlein, R. Barton, S. D. Marlin, and T. A. Springer A cell adhesion molecule, ICAM-1, is the major surface receptor for rhinoviruses. Cell 56: Talbot, P., and F. Brown A model for foot-and-mouth disease virus particles. J. Gen. Virol. 15: Tomassini, J. E., D. Graham, C. M. DeWitt, D. W. Lineberger, J. A. Rodkey, and R. J. Colonno cdna cloning reveals that the major group rhinovirus receptor on HeLa cells is intercellular adhesion molecule 1. Proc. Natl. Acad. Sci. USA 86: van der Werf, S., C. Wychowski, P. Bruneau, B. Blondle, R. Crainic, R. Horodniceanu, and M. Girard Localization of a poliovirus type 1 neutralization epitope in viral capsid polypeptide VP1. Proc. Natl. Acad. Sci. USA 80: White, J., and A. Helenius ph-dependent fusion between the Semliki Forest virus membrane and liposomes. Proc. Natl. Acad. Sci. USA 77: White, J. M., and I. A. Wilson Anti-peptide antibodies detect steps in a protein conformational change: low-ph activation of the influenza hemagglutinin. J. Cell Biol. 105: Wilson, I. A., J. J. Skehel, and D. C. Wiley Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 A resolution. Nature (London) 289: Wray, W., T. Boulikas, V. P. Wray, and R. Hancock Silver staining of proteins polyacylamide gels. Analyt. Biochem. 118: Wychowski, C., S. van der Werf, 0. Siffert, R. Crainic, P. Bruneau, and M. Girard A poliovirus type 1 neutralization epitope is located within amino acid residues 93 to 104 of viral capsid polypeptide VP1. EMBO J. 11: