Interactions of components of human rhinovirus type 2 with hela cells

61, 270-278 (1973)

VIROLOGY

interactions

OF Components with 5. NOBLE2

Central

Research

Department,

AND

of Human Hela

Rhinovirus

2

Cells’

K. LONBERG-HOLM

Experimental Station, E. I. du Pont WGmington, Delaware 19898 Accepted

Type

de Nemours

and Company,

October 16, 1972

Two particulate components were produced by acidification followed by neutralization of human rhinovirus type 2, an RNA-containing A component (135 S) and the B component (80 S), and these were found to lack the ability to attach to host cells. Components sedimenting at 135 S and/or at 80 S were also generated by acidification and neutralization of several other human rhinoviruses, suggesting that the ability to produce these may be of taxonomic significance. About u)$$ of the naturally occurring top component of human rhinovirus type 2 attached to the virus-specific receptors of host cells, and this population has the same polypeptide composition as natural top component particles incapable of interaction with host cells. It is postulated that the biologically active population differs only in its surface conformation from the nonat,taching fraction of natural top component. INTRODUCTION

There are at least 96 known serotypes of human rhinoviruses (Collaborative Report, 1971; J. M. Gwaltney, private communication). The physicochemical criteria for classifying the rhinoviruses are not entirely satisfactory. The capsid proteins of a few strains have been examined; like other picornaviruses, they consist of four polypeptides designated VPl, VP2, VP3, and VP4,3 and the relative sizes of these vary from strain to strain (Medappa et al., 1971; Korant et al., 1972). Although t’he rhinoviruses have a higher buoyant density in CsCl than does the enterovirus subgroup, this property is not unique among picornaviruses (Rowlands 1 Contribution No. 1951. 2 Present Address: Department of Biology, University of Delaware, Newark, Delaware 19711. 3 Abbreviations used: VP, virus polypeptide; HRV, human rhinovirus; ERV, equine rhinovirus; NTC, natural top component; Tris, tris (hydroxymethyl)aminomethane; EDTA, sodium ethylenediaminetetraacetic acid; HIFC, heat-inactivated fetal calf serum; PBS, phosphate-buffered saline without added magnesium or calcium.

et al., 1971). Another taxonomic characteristic of rhinoviruses is their loss of infectivity on exposure to pH 3-5 (Hamre, 1968; Tyrrell, 1968). However, other picornaviruses are also labile under certain conditions in weakly acidic solutions (see discussion in Rueckert, 1971). Previous studies have shown that the products of acidification and neutralization of human rhinovirus type 2 (HRV-2) are different than those of other acid-labile picornaviruses (Cowan and Graves, 1966; Dunker and Rueckert, 1971; Mak et al., 1971). Acidification and neutralization of HRV-2 in the presence of 0.2 M NaCl and 5 X 1O-4 M EDTA at pH 5.0 yields two particles: the A component (135 S) which contains RNA, and the B component (80 S) which is devoid of RNA (Korant et al., 1972). Both these particles contain the same relative amounts of VPl, VP2, and VP3 as infectious HRV-2, but are deficient in Dhe smallest virion polypeptide, VP4 (Korant et al., 1972). The purpose of this study is to extend these findings to several other rhinoviruses, and to investigate the biological basis for the 270

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@ 1973 by Academic Press. of reproduction in any form

Inc. reserved.

INTERACTIONS

OF RHINOVIRUS

loss of infectivity of HRV-2 at pH 5.0. We have also investigated the biological activity of’ the naturally occurring top component (NTC) of HRV-2, which resembles that of poliovirus in its relative sedimentat,ion rate (Schati et al., 1964; Hummeler et al., 1962; Korant et al., 1972), and in its polypeptide composition; i.e., it contains VPO, a larger polypeptide than those in infectious &ions, and lacks VP2 and VP4 (Maize1 et al., 1967; Korant et al., 1972). Poliovirus NTC does not attach to host cells (Katagiri et al., 1968), but it is shown in this report that a portion of HRV-2 NTC does attach and that this attachment is specific for the receptors (Lonberg-Holm and Korant, 1972) recognized by mature virions. The possible structural relationships between attaching and nonattaching virion-related particles arc discussed in the light of these studies. MATERIALS

AND

METHODS

T’irus and cells. The viruses used in this study were HRV-IA (strain 2060), HRV-2 (strain HGP), HRV-14 (strain 1059), HRV51 (strain FOl-4081), and ERV (strain Plummer). The source of each virus and the methods used in their propagation and radiolabeling have been reported, as have t’he methods of purifying virus and natural top component’ (Korant et al., 1972; Lonberg-Holm and Koran& 1972). CsCl-purified virus stabilized by addition of serum and by dialysis against spinner medium (Korant et al., 1972) is referred to here as dialyzed virus. CsCl-purified NTC particles were also dialyzed in the presence of serum, and it was found that they lost their biological act’ivity (see below) by either prolonged exposure to CsCl, or, more slowly, by aging at 4”; hence they were used within 24 hr after purification. Computation of the virus (or NTC) particles/ml has been described (Korant et al., 1972). In work with A and B components, which contain a fract’ion (ca. 93 %) of the protein mass of the virions, the reciprocal of this fraction was used in calculating particles/ml. Growth of HeLa cells (“rhino-HeLacells,” Flow Laboratories, Rockville, Maryland) used in at’tachment experiments was

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as described by Lonberg-Holm and Korant (1972). Solutions. Spinner medium is Eagle’s minimal essential medium, Joklik-modified for spinner culture (Grand Island Biological Co., Grand Island, New York). TNE is 0.02 M Tris-HCl, 0.2 M NaCl, and 5 X lOA M EDTA, pH 7.5, and ANE is 0.02 M sodium acetate, 0.2 A!?NaCl, and 5 X 10m4211 EDTA, pH 5.0. Production and isolation of acidification products of rhinovirus. Acidification of rhinovirus by dilution into ANE buffer and the isolation of the neutralized products of this reaction from sucrose-TNE gradients were previously described by Korant et al. (1972). Attachment of virus or viral components to cells. HeLa cells in suspension culture were washed twice with cold spinner medium containing 5 % HIFC and resuspended at 10’ cells/ml in spinner medium for attachment studies, which were carried out as previously described for virus (Lonberg-Holm and Korant, 1972). The reaction was stopped by addition of 2 ml of ice-cold PBS cont,aining 5 X lop4 d1 i\lgClz (PBS-Mg) to each sample. The cells were sedimented at low speed for 10 min at 4”C, resuspended in 2 ml of PBS-Mg, and resedimented. The radioactivity in the pelleted cells and in both supernatant solutions was determined, and the percentage of cell-associated radioactivity was calculated. Before use in at’tachment experiments, component’s or virus isolated from CsCl or sucrose gradients were made 5 % wit’h HIFC and were dialyzed for 16 hr at 4” against spinner medium. A and B components, after dialysis, maintained their original sedimentation characteristics. Isolation of attached and nonattaching NTC. After completion of the attachment reaction, the cells were sedimented from the medium by low speed cent’rifugation at 4”. NonaMached particles were pelleted from t’he supernatant medium by centrifugation in a Spinco type 50 t’itanium rotor at 50,000 rpm for 4 hr at 4”. The cells with attached NTC were washed once in four volumes of spinner medium containing 5 % HIFC, and the attached KTC was reisolated from them by the same method which has already been

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described by Lonberg-Holm and Korant (1972) for the reisolation of “eclipsed” virus. This entailed sonic disruption of the cells followed by treatment with deoxyribonuclease, then detergent, followed by sucrose gradient centrifugation. The previously cellassociated NTC was pelleted from the appropriate fractions of high-salt sucrose gradi&s by centrifugation in a Spinco type 50 titanium rotor at 40,000 rpm for 18 hr at 4”. Polyacrylamide gel electrophoresis. Two methods of SDS-polyacrylamide gel electrophoresis were employed. Method A. Electrophoresis in 10% acrylamide-bisacrylamide gels as described by Korant et al. (1972). Method B. Electrophoresis in 15-c, gels containing 12 % acrylamide (Eastman Kodak Co., Rochester, New York), 0.5% ethylene diacrylate (Borden Chemicals Co., Philadelphia, Pennsylvania), 8 M urea, and 0.1 M phosphate buffer, pH 7.2. Gels were polymerized using 0.05 % N , N , N’ , N’-tetramethylethylenediamine (Eastman Kodak Co.) and 0.075 % ammonium persulfate, which were partially removed by electrophoresis at 5 mA/gel for 1 hr prior to sample application. The electrophoresis buffer contained 0.1% SDS and 0.1 M phosphate buffer. For analysis on 12 % gels, material was dispersed in 0.01 M phosphate buffer containing 8 M urea. Before electrophoresis, the samples were made 1% SDS and 5-20 % 2-mercaptoethanol (not in excess of 50 ~1 per sample), flushed with nitrogen, and boiled for 2 min. After electrophoresis of samples at 5 mA/gel for 36 hr, gels were frozen at -20°C and fractionated into 1.1 mm slices using an assembly of razor blades. Assay of radioactivity. The methods for measuring radioactivity in samples containing fragments of 10 % polyacrylamide gels and in other samples have been described previously (Korant et al., 1972; LonbergHolm and Korant, 1972). Slices of 12 % polyacrylamide gels were dissolved in 1.0 ml 0.5 M NH,OH at room temperature in 16 hr, and the radioactivity was counted aft,er addition of 10 ml of Bray’s solution (6 70 naphthalene, 0.4 70 2,5-diphenyloxa-

zole, 0.02 % p-bis[2(4-methyl-5-phenyloxazolyl)]benxene, 10% methanol, and 2 % ethylene glycol in dioxane) . Plaque assay of infectivity. The assay was performed as already described (Korant et al., 1972). Acidified virus was diluted lOO-fold into spinner medium containing 5 % HIFC prior to assay, and stored at. -70”. RESULTS

The Biological Basis for Acid-Inactivation HRV-2

of

Acidification of HRV-2 by dilution 20-fold into pH 5.0 buffer (ANE) at room temperature followed by neutralization causes at least a 99.9% loss of infectivity (Korant et al., 1972, and unpublished results). This reaction has previously been shown to produce a significant quantity (30-50%) of RNA-containing A component particles as well as non-RNA-containing B component. The basis for the extensive loss nf infectivity n-as studied further by testing the ability of A or B components to adsorb to host cells. The components, isolated from sucrose-TNE gradients, were dialyzed against medium prior to use as described in Materials and Methods. Figure 1 shows that 90% of untreated virus or virus reisolated from sucroseTNE attached to cells in 10 min. The A and B components, however, did not attach, and only 0.9Yo and 1.3% of the total acidprecipitable radioactivity in A and B component, respectively, were cell-associated after 20 minutes of incubation. Since A and B components are both ae ficient in VP4 and since VP4 from HRV-2 is readily obtainable from the top portion of sucrose-TNE gradients containing A and B components (Korant et al., 1972), attempts were made to study the interaction of VP4 with HeLa cells. It was, however, found that in addition to sticking to cells, VP4 readily sticks to the tubes employed in the attachment reaction. The attachment to cells (or tubes) is not affected by excess nonradioactive HRV 2 or by EDTA at a concentration which blocks the uptake of labeled native virions (data not shown, see Lonberg-Holm and Korant. 1972). A similar nonspecific “stickiness” has been observed for poliovirus VP4 by M. Breindl (private communica-

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PRODUCTS OF ACIDIFICATION AND NEUTRALIZBTION OF RHINOVIRTJSES~ Virions A 1.50 S Component 135 s HRV-2 HRV-14 HRV-51 HRV-1A ERV

0

20 TIM:Otmin)

FIG. 1. Interaction of A and B components with HeLa cells. Dialyzed %-amino acid-labeled virus was diluted 20-fold into either ANE or TNE, held 20 min at room temperature, and sedimented through sucrose-TNE gradients. The A and B components and virions isolated from gradients were pooled separately and dialyzed against medium as described in Materials and Met,hods. Attachment was carried out at lo4 particles/cell using untreated virus (n--n), virus reisolated from sucrose gradients (O-----O), A component (0.----•), and B component (X--X).

tion). The adhesive properties of VP4 make the interpretation of our data difficult, but at present there is no direct evidence that isolated VP4 can react specifically with the cellular receptors for intact virions, and indeed, we consider this to be unlikely. Acti Treatment of Other Rhinoviruses As indicated in Table 1, all four human rhinoviruses tested under uniform conditions yielded A and/or B components with only small amounts of amino acid-labeled material in the “soluble” (<40 S) area of the sucrose gradients. Equine rhinovirus (ERV), on the other hand, reacted differently to acidification and neutralization. Even after 30 min at room temperature in ANE, some virions remained. No A or B component was produced, but mat,erial accumulated which remained at the top of the sucrose gradient.

++

+

“Soluble” B Component material 80 s <40 s ++

++

+ -

++ ++ -

It f f f +

a Virions labeled with ‘%-amino acids were banded in CsCl gradients and diluted 1:25 into ANE buffer at 4”. The reaction mixtures were warmed to room temperature for 10-15 minutes and then neutralized, and the products were isolated on sucrose-TNE gradients. A score of ++ indicates that more than half of the products were found in the designated component. If less than half but. more than 10% of the label was found in a component it is scored as +, while less than lOye is indicated by f.

Attachment of Natural Top Component of HRV-2 to HeLa Cells Since acidification of virions causes concomit’ant loss of VP4 from A and B components and loss of t’heir ability to adsorb to cells, we investigated the interaction of cells with the natural top component (NTC) of HRV-2, w-hich lacks VP4 as a separable polypeptide (Korant et al., 1972). Figure 2 shows that purified NTC of HRV-2 adsorbs to cells under the same conditions used for virus attachment, and t’hat the kinetics of attachment of NTC were very similar to that of virus. However, the extent of adsorption was only 25 % that of virus. Figure 2 also shows that 5 mM EDTA inhibited NTC adsorption by 9“ 5%and t’hat the presence of excess homologous virions during attachment reduced NTC attachment by competing for the virus-specific receptor sites on the cells. The experiment shown in Fig. 3 tested whether the limited extent of adsorption of NTC seen in Fig. 2 was due to partial saturat.ion of receptors by NTC at lo4 particles/ cell. After 15 minutes of adsorption, when the reaction was essentially complete, approximately the same proportion of this

NOBLE 100

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LONBERG-HOLM

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1 5 IO 50 MULTIPLICITYOF NTC(PARTICLES/CELL x 1O-3)

0

10 20 TIME (min)

30

Attachment of NTC to HeLa cells. Suspensions of cells contained ‘%-labeled NTC of HRV-2 at lo4 particles/cell (@---a), Wlabeled virus at 104 particles/cell (A--A), NTC at lo4 particles/cell in the presence of 5 mM EDTA (O--O), and lo4 particles/cell 14C-labeled NTC plus 2 X 10” particles/cell 3H-labeled HRV-2 (X--X). Samples were incubated, washed, and analyzed as described in Materials and Methods.

preparation of NTC (17-23 %) became cellassociated after incubation at multiplicities of NTC ranging from 1 X lo3 to 5 X lo4 particles/cell. Presumably, the receptors for NTC particles can be saturated when more than 5 X lo4 particles, i.e., more than 1 X lo4 active particles, are added per cell, as is the case with virions (Lonberg-Holm and Korant, 1972), but we have not attempted to show this. Elution of Natural Top Component Figure 4 shows the results of a typical experiment measuring the extent. of spontaneous elution of NTC compared to that of virus. After 10 min of adsorption of HRV-2 virions or NTC, the cells were washed and reincubated in the original volume of medium. Samples were t,aken at various times t’o determine the proportion of acid-precipitable material which had dissociated. Although four times more radioactive virus than NTC became cell-associated in 10 min,

FIG. 3. Attachment of HRV-2 NTC at various multiplicities. Suspension of cells used for the attachment studies contained “C-labeled NTC at multiplicities of 1 X 103, 5 X 103, 1 X 104, and 5 X 104 particles/cell. Samples were incubated at 34.5” for 15 min and washed and analyzed as described in Materials and Methods.

the kinetics virions and proximately eluted within

and extent of elution of both NTC were very similar. Ap25 % of both types of particles 30 min.

The Polypeptides of NTC Attaching to HeLa Cells The polypeptide compositions of adsorbing and nonadsorbing NTC were studied after isolation as described in Materials and Methods. Cell-associated NTC, after isolation from disrupted cells, sedimented in 1 M NaCl sucrose gradients as 80 S particles similar to freshly purified NTC (not shown). Figure 5 shows the polypeptide profiles of adsorbing (Panel A) and nonadsorbing NTC (Panel B) on 10 % gels. Both types of particles contain large amounts of VP0 which is found in only small amounts in purified virions. Similar analysis in 12 % polyacrylamide gels, which do not resolve VP0 and VPl, but do resolve VP2 and VP3, is shown in Fig. 6. Electrophoresis of the polypeptides of adsorbing and nonadsorbing NTC was carried out for 36 hr, in which time VP4 moved completely through the gel. The results indicate that adsorbing NTC contains no significant amount of VP2.

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Fro. 4. Adsorption and elution of HRV-2 virions and NTC. ‘GLabeled HRV-2 virions (A--A) and NTC (O-----O) were adsorbed to HeLa cells at 34.5” for 10 min at multiplicities of 10” particles/cell. Cells with attached virus and NTC were washed twice with 4 volumes of spinner medium containing 5% HIFC at 4’ and resuspended in the original volume of medium. Samples were reincubated at 34.5” for 0, 10, 20, and 30 min, after which they were analyzed for residual cellassociated radioactivity as described in Materials and Methods. DISCUSSION

The acidification-neutralization reaction of HRV-2 is presented schematically in Fig. 7. The primary product of acidification is an aggregate which was isolated by centrifugation through sucrose-ANE (pH 5.0) gradients (unpublished data). Upon aggregation, about 50% of the RNA and up to 90 5; of the VP4 of the virions are solubilized. Independently, it has been found that acidification produces aggregation of HRV-2 (RI. Fiala, personal communication) and of HRV-1A (Medappa et al., 1971). Figure 7 depicts the aggregate as composed of relatively intact subviral particles. However, it is possible that under more stringent conditions of acidification, fibrous structures are formed such as reported from electron microscopic studies (Reeves and Mayor, 1969). Neutralization of acid-aggregated HRV-2 produces subviral component A (135 S) which contains RNA, and B (80 S) which does not. As shown in Table 1, this is not

J FIG. 5. Polypeptide analysis of adsorbed (A) and nonadsorbed (B) populations of NTC. The purified NTC of HRV-2 was attached to cells at lo4 particles/cell for 10 min at 34.5”, and the cellassociated and supernatant particles were isolated and prepared for polypeptide analysis as described in Materials and Methods. Electrophoresis was carried out for 18 hr in 15-cm 10% polyacrylamide gels.

unique to HRV-2 and all the human rhinoviruses examined produce A and/or B components under the conditions outlined. This may be of taxonomic significance and may separate the human rhinoviruses from other acid-labile picornaviruses (see below). HRV-IA and HRV-51 did not produce significant amounts of A component under these conditions; but it is probable that they would do so under other conditions, since it has been found in other unpublished experiments that the presence of l-2 Ail NaCl or high concentrations of sucrose during the acidificat,ion and neutralization of HRV-2 greatly increases the ratio of A: B. ERV, on the other hand, behaves entirely differently from all the human rhinoviruses studied, and its acid degradat’ion ma) resemble that’ of ot’her acid-labile picornadisease viruses, such as foot-and-mouth virus (Cowan and Graves, 1966), mengovirus (Mak et al., 1971), and ME-virus (Dunker and Rueckert, 1971), which, upon acidification, dissociate entirely to protein components which sediment at less than 15 S.

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In our experiments, acidification and neutralization of HRV-2 usually caused a loss of more than 99.9 % of the original viral infectivity and thus both A and B components must be essentially noninfectious. The I

I

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“PI

FRACTION

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VP2 VP3

NUMBER

FIG. 6. Polypeptide analysis of adsorbed and nonadsorbed populations of NTC. The NTC of HRV-2 was attached to cells at lo4 particles/cell for 15 min at 34.5’, and the particles were isolated and prepared for electrophoresis as described in Materials and Methods. Electrophoresis was carried out in separate 12% polyacrylamide gels for 36 hr at 5 mA/gel, and the gels were fractionated and analyzed as described in Materials and Methods. The positions of VPl, VP2, and VP3 in such a gel, determined by electrophoresis of the polypeptides of purified HRV-2 in a parallel gel, are indicated by arrows. Solid lines, adsorbed NTC; dashed line, nonadsorbed NTC.

VIRIONS 150s RNA.VPI,VPZ,VPJ,VW

FIG. 7. Schematic

representation

B component, lacking RNA, is a priori noninfective. A sufficient explanation for the lack of infectivity of the RNA-containing A component is that these particles, like the B particles, are unable to attach to host cells (Fig. 1). The loss of ability of A or B components to attach to cells is concomitant with the loss of most of their VP4 (Korant et al., 1972). Similarly, treatment of poliovirus type 1 with heat (Breindl, 1971a) or with alkali (Maize1 et al., 1967; Katagiri et al., 1971) produces subviral particles lacking VP4 and lacking the ability to attach to host cells (Katagiri et al., 1968; Breindl, 1971a). It has been suggested that the native antigenicity of poliovirus and its ability to interact with host cells are both primary attributes of VP4 (Breindl, 1971a, b). The establishment of a relationship between VP4 content and the ability of picornaviruses to attach to host cells would be strengthened further if it were possible to demonstrate that VP4 itself attaches specifically to cellular virus receptors. Our attempts to demonstrate this were unsuccessful (Results). An alternative hypothesis is that a native conformation of the entire surface of the picornavirus virion is required for biological activity. The antigenicity of VP4-deficient poliovirus particles (Roizman et al., 19.59; ScharfI et al., 1964; Katagiri et aZ., 1971) and of VP4-deficient rhinovirus particles (F. H. Yin, K. Lonberg-Helm, and J. Noble, in preparation) is greatly altered, suggesting that the entire surface of the particle has been changed. Also, it has recently been

ACID AGGREGATE > 150s RNA,VPl,VPL,VP3 *RNA + VP4

of the acidification

135s “A” COMPONENT:RNA,VPI,VPL,VP3 80s “B” COMPONENT.VPI. VP2.VP3 + RNA + VP4

and neutralization

reaction

of HRV-2.

INTERACTIONS

OF RHINOVIRUS

reported by Breindl and Koch (1972) that it is possible to destroy both t’he native antigenicity of infectious poliovirus and its ability to at’tach to cells, wit,hout causing loss of VP4. It is of interest, that approximately 20% of the freshly purified NTC of HRV-2 at,taches to host cells (Fig. 2). This limited attachment appears to be due to a subpopulation of active particles. The limitation is not due to saturation of viral receptors by KTC particles (Fig. 3) nor does it result from a dynamic equilibrium between attachment and elution of NTC which favors the non-cell-associated state. The latter is unlikely because elution of cell-associated NTC proceeds to the same extent (20-25s) as that of virus (Fig. 4). Furthermore, the XTC particles which fail to attach (approximately 80 %) are incapable of attaching to fresh cells (unpublished data). The finding that some r\‘TC particles of HRV-2 can interact with virus specific recrptors (Fig. 2) is in contrast to the published findings that purified poliovirus NTC particles do not attach t#ohost, cells (Iiatagiri et al., 1968). It has brhrn shown that t’hc largest polypeptide, VPO, of poliovirus NTC contains the polypeptidc sequences present in VP2 and 4 (Jacobson et al., 1970), and it has been postulat’ed that during virion morphogenesis VP0 is cleaved to form VP2 and VP4 of the virion (Jacobson and Baltimore, 1968). The possibility existed that attachment of the XTC of HRV-2 was due t,o a population of particles with viral polypeptide composition, but lacking RNA, generated either in viuo by abortive morphogenesis (Jacobson and Baltimore, 1968) or by degradation during purification. However, t#hishypothesis is untenable because the polypeptide compositions of attaching and nonattaching NTC show qualitative identity, i.e., the attaching particles contain large amounts of VP0 (Fig. 5) and no significant amount of VP2 (Fig. 6). These results taken together favor a hypothesis that there are two conformational populations of NTC particles; about 20 % of the particles have a surface structure resembling that of intact virions and hence are able to attach to host cells, although they lack cleaved VPO.

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Similarly, the lack of ability of the A and B components to attach to host cells might simply be the result of a basic alteration of the particle surface which also allows for the loss of VP4. An immunochemical investigation of subviral particles related to HRV-2 will be reported elsewhere (F. H. Yin, I<. Lonberg-Holm, and J. Noble, in preparation). ACKNOWLEDGMENT We thank Dr. R. Z. Lockart for his generous support and valued criticism and Dr. B. D. Korant for advice and stimulating discussions. We also tha,nk V. Kiloren for valuable technical assistance. REFERENCES BREINDL, M. (1971a). The structure of heated poliovirus particles. J. Gcn. Viral. 11, 147-156. BREINDL, M. (1971b). VP4, the D-reactive part of poliovirus. Virology 46, 962-964. BREINDL, M., and KOCH, G. (1972). Competence of suspended HeLa cells for infection by inactivated poliovirus particles and by isolated viral RNA. Virology 48, 13&144. COLLABORATIVE REPORT (1971). A collaborative report: Rhinoviruses--extension of the numbering system. vi’iroloqu 43, 524-526. Cowah-, K. M., and GR.\VES, J. H. (1966). A third antigenic component associated with foot and mouth disease infection. Virology 30, 528-540. DUNKER, A. K., and RUECKERT, R. R. (1971). Fragments generated by pH dissociation of ME-virus and their relat,ion to the structure of the virion. J. Mol. Biol. 58, 217-235. HAMRE, D. (1968). In “Monographs in Virology” (J. L. Melnick, ed.), Vol. 1. Karger, New York, HUMMELER, K., ANDERSON, T. F., and BROWN. R. A. (1962). Identification of poliovirus particles of different antigenicity by specific agglutination as seen in the electron microscope. Virology 16, 84-90. J.~COBSON, M. F., and BALTIMORE, D. (1968). Morphogenesis of poliovirus I. Association of the viral RNA with coat protein. J. Mol. Biol. 33, 369378. JACOBSON, M. F., Asso, J., and BaLTrMORE, 1). (1970). Further evidence on the formation of poliovirus proteins. J. Mol. BioZ. 49,657-669. JOKLIK, W. K., and DBRNELL, J. E. (1961). The adsorption and early fate of purified poliovirus in HeLa cells. Virology 13, 439447. KAT~GIRI, S., HINUM.~, Y.,and ISHIDA,N. (1968). Relation between the adsorption to cells and antigenic properties in poliovirus particles. Virology 34, 797-799.

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KAT~GIRI, S., AIKAWA, S., and HINUM.~, Y. (1971). Stepwise degradation of poliovirus capsid by alkaline treatment. J. Gcn. Vi&. 13, 101-109. KOR.
degradation of the rhinovirion as studied by high resolution stereo electron microscopy. Proc. Electron Microsc. Sot. Amer. 27th Ann. Meet. (C. Arceneaux, ed.), pp. 404305. ROIZM.\N, B., MOYER, M. M., and ROANE, P. R., JR. (1959). Immunochemical studies of poliovirus. IV. Alteration of the immunologic specificity of purified poliomyelitis virus by heat and ultraviolet light. J. Immunol. 82, 19-25. ROWLANDS, D.J., SANGAR, D. V., and BROWN, F. (1971). Buoyant density of picornaviruses in cesium salts. J. Gen. Viral. 13, 141-152. RUECKERT, R. R. (1971). In “Comparative Virology” (K. Maramorosch and E. Kurstak, eds.), pp. 255-306. Academic Press, New York. SCHARFF,M. D., MAIZEL, J. V., and LEVINTO~, L. (1964). Physical and immunological properties of a soluble precursor of the poliovirus cs.psid. Proc. Nat. Acad. Sci. U.S. 51, 329-337. TYRRELL, D. A. J. (1968). In “Virology Monographs” (S. Gard, C. Hallauer, and K. F. Meyer, eds.), Vol. 2, pp. 67-124. Springer-Verlag, Berlin and New York.