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Studies on the in vitro uncoating of poliovirus II. Characteristics of the membrane-modified particle
VIROLOGY
78,
554-566
(1977)
Studies
on the in vitro
II. Characteristics JOHN Department
of Virology,
Uncoating
of Poliovirus
of the Membrane-Modified
DE SENA’ The Public Health New York, Accepted
BENJAMIN
AND
Research Institute New York 10016 February
Particle MANDEL
of The City
of New
York,
Inc.,
II,1977
Interaction of type I poliovirus with an extract of isolated HeLa cell membranes resulted in a modification of the viral capsid. As a consequence of the modification, the capsid became sensitive to proteases and detergents, and the sedimentation value of the virion was slightly diminished. Treatment of the modified particle with chymotrypsin reduced its sedimentation value and, although the RNA genome was still encapsidated, it was degradable by RNase. Treatment of the membrane-modified particle with detergent (sodium dodecyl sulfate, SDS) disengaged the viral RNA as an intact (i.e., 35 S) molecule. Each of the three serotypes of poliovirus was modified when treated with membrane extract. They differed, however, in the effect of secondary treatment with chymotrypsin, viz., type I was further modified, but types II and III underwent degradation. When secondary treatment was with SDS, all three released 35 S RNA. The above in vitro reactions of the membrane-modified particle are discussed as possible counterparts of the in viva uncoating phenomenon. INTRODUCTION
nothing is understood of the responsible cellular processes or events intervening between modification and the final stage of uncoating. It has, however, become increasingly evident that the initial early modification of the adsorbed vu-ion is attributable to a constituent of the plasma membrane, perhaps the viral receptor. This presumption is supported by the results of studies with isolated cellular membranes (Hoiland and Hoyer, 1962; and, more recently, Chan and Black, 1970; Roesing et al., 1975; De Sena and Mandel, 1976), and by the results of studies of the effect of metabolic inhibitors (LonbergHolm and Whiteley, 1976) and concanavalin A (Lonberg-Holm, 1975) on the early interactions of picornaviruses with host cells. In considering the events subsequent to early modification, Lonberg-Holm and Whiteley (1976) have proposed that early modification is the penultimate stage to final uncoating, and, intervening, is a period during which the modified virion intercalates with the plasma membrane of the cell. The latter suggestion is based on
Results of previous studies on the uncoating of picornaviruses (Holland and Hoyer, 1962; Mandel, 1962) suggested that uncoating is a multistage phenomenon. In support, recent observations (Crowell and Philipson, 1971; Lonberg-Holm and Kount-, 1972; Fenwick and Wall, 1973; Lonberg-Holm et al., 1975) have shown that after attaching to the surface of a susceptible cell, virus undergoes a preliminary modification characterized by (1) loss of the VP4 capsid polypeptide, (2) slight reduction in S value, (3) acquired sensitivity to proteases and detergents, and (4) loss of infectivity, although the particle still contains a functional genome which is inaccessible to nucleolytic enzymes. This early modification is considered to be a prerequisite stage to uncoating. Although some characteristics of the modified particle are known, virtually ’ Present address: Department and Biochemistry, Idaho State tello, Idaho 83209.
of Microbiology University, Poca554
Copyright All rights
8 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0042-6822
UNCOATING
OF
POLIOVIRUS
the observed lipophilic character of modified particles when exposed to artificially prepared liposomes (Lonberg-Holm et al., 1976). There is, however, no evidence to rule out the possibility of a process involving the progression of the early modified particles through subsequent stages of modification, thus constituting a series of transient unstable intermediates in the uncoating pathway. The induced sensitivity to proteolytic enzymes of the early modified particles suggests the possibility that proteolysis may play a role in uncoating. The same possibility can be considered for such a role for substances with detergent properties. These possibilities have been examined in the present study utilizing the previously described in vitro system (De Sena and Mandel, 1976) to generate modified particles for further characterization. MATERIALS
AND
METHODS
Virus and cells. HeLa cell monolayer cultures were used for the propagation of viral stocks and for plaque assay (Mandel, 19611, and 32P-labeled stocks were prepared and purified as previously described (Mandel, 1967). 13H] Amino acid labeled stocks were prepared by adding a mixture of 3H-labeled amino acids to the viral growth medium to a concentration of 0.035 mCilm1. Concentration and purification was as for 32P-labeled virus. Strains of poliovirus used were Brunhilde (type I), MEF, (type II), and Saukett (type III). Naturally occurring empty capsids were collected by centrifuging the 3H-labeled stock through a cesium chloride gradient. Two peaks of radioactivity were observed which were collected and assayed for infectivity. The peak of density 1.335 g/ml had more than loo-fold higher specific infectivity than the peak of density 1.285 g/ml. Each material, after dialysis, was centrifuged through a sucrose density gradient. Based on the relative sedimentation rates, the lower density material had an S value of 80 and this material was considered to be empty capsids. Reagents and solutions. The following reagents were obtained as indicated: al-
IN
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pha-chymotrypsin (EC 3.4.4.5) 3x crystallized, trypsin (EC 3.4.4.4) 2x crystallized, ribonuclease A (EC 2.7.7.16) from Worthington Biochemical Corp.; sodium dodecyl sulfate from Schwarz/Mann; dextran T-500 from Pharmacia Fine Chemicals, Inc.; polyethylene glycol (Carbowax 6000) from Union Carbide Corp.; carrier-free H,“2P0, and 3H-labeled amino acid mixture from New England Nuclear Corp. Preparation of HeLa cell membrane extract. The procedure for obtaining plasma membrane fragments was based on the method of Brunette and Till (1971) as modified by Weintraub and Dales (1974) and has been described in detail previously (De Sena and Mandel, 1976). Reaction of virus with membrane extract and biochemical analysis. An appropriate dilution of virus was added to undiluted membrane extract and held for 1 hr at 36”; variations of this procedure will be described in the text. Samples of the reaction mixture were taken for biochemical analysis which involved treatment with chymotrypsin and RNase, then determining the percentage of labeled RNA that was soluble in trichloroacetic acid (De Sena and Mandel, 1976). Treatment of membrane-modified virus. Following interaction of virus with membrane extract, 2.5 ml of the reaction mixture was added to 0.1 vol of reagent and held at 23” for 20 min in the case of enzymes, and 5 min in the case of detergent. Aliquots were taken immediately for centrifugation and in some instances for biochemical analysis. Density gradient centrifugation. Sucrose density gradients (10% to 25% in 0.58% NaCl, w/w) were prepared the day before use and held overnight at about 5”. Prior to preparing the gradients, about 15 drops of 1% agar were added to the tubes and, after solidification, 15 drops of glycerol were added, thus forming a double cushion. Linear gradients were formed over the cushion using 16.4 ml of the lo%, and 15.6 ml of the 25% sucrose solutions. Two milliliters of specimen was layered on the gradient solution and centrifuged in a Beckman SW27 rotor at 26,000 rpm for 2 hr at 5” in a Spinco model L2-65B centrifuge.
556
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Fractions were collected dropwise through a puncture in the bottom of the tube. Experience had shown that little or no radioactivity emerged in the first few fractions. Therefore, the first seven fractions were collected as 50-drop (l-ml) fractions, the next 38 fractions represented 25 drops, and the remainder were 50-drop fractions. The residue remaining in the tube was recovered by washing the tube with a small volume of 1 N NaOH and saving the wash for radioactivity count. RESULTS
Effect
of Modification by Membrane Extract, and Subsequent Modification by Chymotrypsin, on Sedimentation of Poliovirus in Sucrose Density Gradients
Previously (De Sena and Mandel, 1976), it was shown that treatment of type I poliovirus with an extract of isolated HeLa cell membranes induced a modification in the particles that rendered them sensitive to chymotrypsin. This conclusion was based on the observation that the viral RNA became accessible to RNase after sequential treatment with membrane extract (ME) and chymotrypsin. A study was undertaken to characterize further the TABLE EFFECT
Specimen
OF CHYMOTRYPSIN
ON POLIOVIRUS
Chymotrypsinb- (minii3”)
MANDEL
modification in the virion induced by ME. In view of the observation (Lonberg-Holm and Koran& 1972; Fenwick and Wall, 1973) that soon after penetration of rhino- and poliovirus into susceptible cells some of the virions show a slight reduction in S value, the effect of ME treatment on the sedimentation of poliovirus was examined. 32P-labeled type I poliovirus was treated with ME for 1 hr at 36”. Aliquots were then treated with chymotrypsin at room temperature for several time periods. Samples were then collected for biochemical assay and for centrifugation through sucrose density gradients. All dropwise-collected gradient fractions were assayed for radioactivity, and several fractions from several gradients were assayed for infectivity. As seen in Table 1, virus alone, or in the presence of ME for 1 hr at o”, was insensitive to the sequential treatments with chymotrypsin and RNase. Virus alone, held for 1 hr at 36, became moderately sensitive, but in the presence of ME sensitivity was considerably increased. It should be noted that ME-modified virus shows a moderate sensitivity to RNase directly. This sensitivity has varied considerably in these experiments and has been interpreted as a reflection of varying degrees of modification. 1
MODIFIED
Percentage Water
BY HeLa
acid-soluble Chymotrypsin
CELL
MEMBRANE
RNA
after RNase
EXTRACT
treatment? Chymotrypsin and RNase
Virus + ME 36”/1 hr
5 10 30 60
-e 11.4
14.2 14.8 15.2 16.3
47.2
65.3 65.3 62.3
Virus + H,O 36”/1 hr
30
16.7
18.9
22.3
25.1
Virus O”/l
+ ME hr
30
2.1
-
-
3.1
Virus + H,O On/1 hr
30
2.1
-
-
3.2
a See Materials and Methods for details b The final concentration was 0.5 mglml. c Not done.
of biochemical
analysis.
UNCOATING
OF
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The effects of ME and chymotrypsin on sedimentation rate are shown in Fig. 1. Treatment with ME produces a slower sedimenting particle (panel A) which, as will be shown below, is noninfectious in contrast to the faster sedimenting particle.
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These will be referred to hereafter as M (modified) and N (native) particles, respectively. Subsequent treatment with chymotrypsin results in additional modification (panels B and 0 to a particle of still lower S value, namely, C (chymotrypsin modi;I
;‘
EEL 8.5 IO-
5. > I-
60
70
FIG. 1. The reduction in sedimentation rate of poliovirus after modification by membrane extract and subsequent modification by chymotrypsin. Details of the centrifugation procedure are described under Materials and Methods. After centrifugation, each gradient was collected dropwise. Each symbol (0) represents 25 drops. Some fractions were collected as 50 drops but, to facilitate comparison, are represented as two equal 25-drop fractions (00). Results are shown as the percentage in each fraction of the total radioactivity in the gradient, including the residue remaining in the tube which is represented by the value of R shown in each panel. Since only trace amounts, at most, are present in the first 20 fractions, these are not shown. This scheme for presenting the results of centrifugation studies is used throughout this report. Standardization of this system has shown the peak positions of native (N) and empty (E) particles and 35 S viral RNA (R). The percentage distribution of sZP radioactivity is shown for poliovirus treated with membrane extract for 1 hr at 36” (A) and subsequently treated with chymotrypsin for 30 min (B) and for 60 min (C) at 23”. The peak positions of native particles are indicated by N, the modified particles resulting from exposure to membrane extract are indicated by M, and particles subsequently modified by chymotrypsin are designated C.
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fied) .particle. Based on standardization of this system with native virions, naturally occurring empties, and extracted viral 35 S RNA, the expected positions of these markers are indicated by N, E, and R, respectively. The M particles appear to be similar to the slower sedimenting particles described by Lonberg-Holm and Korant (1972) and Fenwick and Wall (1973). The nature of the C particles is puzzling since their sedimentation rate is similar to that of empties but they contain RNA. In this respect they resemble the 80 S ribonucleoprotein particles obtained by heating (Breindl, 1971). Both the 80 S particles of Breindl and the C particles described above are sensitive to RNase and also release 35 S RNA when treated with sodium dodecyl sulfate. The results of infectivity assays, Table 2, are expressed as specific infectivity (SD which is a measure of the relative proportion of infectious to total labeled particles, i.e., PFU/cpm. Virus in the N peak has the same SI as the control. In contrast, virus in the M and C peaks has SI’s of about 1% of the control. In order to determine if the reduction is the result of reversible binding to host receptor, the specimens were treated with sodium dodecyl sulfate. In
MANDEL
neither case did reactivation occur. Usually a variable amount of viral isotope is recoverable from the tube after the contents have been collected. Assay of this residual material shows a low SI which, however, approaches that of the control after treatment with SDS. It is likely that the residue consists of virus bound reversibly to high-molecular-weight receptor in contrast to N and M particles that may have eluted with or without modification.
Characteristics Particle
INFECTIVITY
2
OF POLIOVIRUS PARTICLES AFFER TREATMENT WITH MEMBRANE SEPARATION BY SUCROSE DENSITY GRADIENT CENTRIFUGATION
Specimen
Gradient fractions
Type of particle
PFU/ml
+ ME
36”/1
hr
Virus + ME chymotrypsin
36”/1
Virus
0”/3 hr
+ ME’
hr
+
EXTRACT Specific
cpm/ml
-
AND infectiv-
ity” No treatment
Virus
(M)
Treatment of poliovirus with ME results in chemical modification as shown by the induced sensitivity to chymotrypsin, and in physical modification as shown by the reduction in sedimentation rate. Further characterization of the M particle was undertaken. After treatment of 32P-labeled virus with ME, aliquots were subjected to secondary treatment by adding 0.1 vol of reagent for 20 min at room temperature. The following reagents were used: water, chymotrypsin, chymotrypsin followed by RNase, RNase, sodium dodecyl sulfate (SDS), and SDS followed by RNase. Each was then centrifuged through a sucrose gradient.
TABLE SPECIFIC
of the Modified
SDS treatmentb
No treatment
24 + 25 29 + 30 residue
N M N
3.0 x 106 I.3 x 105 1.3 x 105
1.6 x lo6 1.0 x 105 5.6 x lo5
4.2 x lo3 1.4 x 104 1.9 x 103
23 + 24 30 + 31 35 + 36 residue
N M C N
3.0 5.2 2.9 5.0
2.1 4.0 5.0 5.3
2.7 6.0 5.8 1.4
N
4.5 x 10’
x x x x
106 104 104 104
x x x x
106 104 103 105
2.5 x 10’
SDS treatment
714 9 68
381 7 294
lo3 lo3 103 103
1097 9 5 36
781 7 1 380
5.2 x lo4
861
486
x x x x
a Specific infectivity = PFU/cpm b Sodium dodecyl sulfate was added to a final concentration of 0.5%, the reaction was held temperature for 10 to 15 min and then diluted for assay. r An aliquot of the original virus-ME reaction mixture was held at 0” for 3 hr and then assayed having been subjected to centrifugation.
at room without
UNCOATING
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POLIOVIRUS
iY R&),H
IN
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559
II
r; ‘;
-4.2 0 1.5
6
8-
8-
FRACTION
NUMBER
FIG. 2. Characterization of type I poliovirus modified by membrane extract. 32P-labeled virus was treated with membrane extract for 1 hr at 36”. The reaction mixture was subdivided and treated with various reagents for 20 min at 23”. The percentage distribution in each gradient is shown in A after secondary treatment with water (0) or with 0.5 mglml chymotrypsin (0); in B after secondary treatment with chymotrypsin as above, followed by 10 @g/ml RNase (01, or with RNase alone (0); in C after secondary treatment with 1% sodium dodecyl sulfate (01, or with sodium dodecyl sulfate followed by RNase (01.
The results in Fig. 2 show that treatment with ME (panel A) produces a population of M particles which sediment slower than N particles. Secondary treatment with chymotrypsin (panel A) does not visibly affect N particles but converts M particles to the more slowly sedimenting C particle. Treatment with RNase after chymotrypsin (panel B) degrades the RNA of the C particles and also the RNA of some of the N particles. SDS causes the release of viral RNA from M particles, and the RNA is seen in its conserved (i.e., 35 S)
state and is degradable by RNase (panel C). A puzzling result is seen when RNase is used directly after ME treatment (panel B). A class of particles appears intermediate in S value between N and M, probably derived from the N population. It is possible that some particles considered to be N have been minimally modified without change in S value but with induced sensitivity to various reagents. The decrease in the proportion of particles in the N peak after chymotrypsin-RNase treatment and SDS treatment suggests this possibility.
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The peak appearing after RNase treatment may represent a population of pseudo-N particles. Such particles may therefore represent the earliest stage in modification which could be a simple rearrangement of the quaternary structure of the capsid. Effect
of Membrane Extract Serotypes II and III
on Poliovirus
In previous studies (De Sena and Mandel, 1976) it was reported that, unlike type I, poliovirus types II and III were insensi-
MANDEL
tive to the action of membrane extract. This conclusion was based on biochemical analysis which detects an induced state of sensitivity to chymotrypsin. Inasmuch as modification is also recognizable by a reduction in sedimentation rate, the effect of ME on serotypes II and III was reexamined for a possible change in this parameter. 32P-labeled virus of each type was treated either with water or ME for 1 hr at 36”. Aliquots were taken for biochemical analysis and gradient centrifugation analysis. Data depicted in Fig. 3 show that M
60
FRACTION
NUMBER
FIG. 3. Production of M particles by treatment of poliovirus serotypes extract. Each $*P-labeled virus was mixed either with water or membrane then centrifuged through sucrose density gradients. Percentage distribution (0) and virus-extract (0) mixtures is shown for types I, II, and III.
I, II, and III with membrane extract, held for 1 hr at 36”, and of radioactivity of virus-water
UNCOATING
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POLIOVIRUS
particles are produced by ME treatment of all three types. Although type III is more resistant to modification, the M particles of all three appear to be alike in respect to S value. Of interest is the observation that in the absence of ME no M particles are produced indicating that the formation of M particles is not a spontaneous occurrence that is accelerated by ME. However, in view of the appearance of viral oligonucleotides in the absence of ME, there is the possibility that M particles are produced, but under these conditions they are unstable and are rapidly degraded. Biochemical data in Table 3 show that all three types, whether exposed to water or ME but held at O”, are insensitive to sequential treatment with chymotrypsin and RNase. At 36” spontaneous degradation of viral RNA to an acid-soluble state occurs but to a lesser extent in the presence than in the absence of ME. Treatment with RNase reveals in each case accessibility of viral RNA, which, only in the case of type I, is enhanced by prior treatment with chymotrypsin. The previous conclusion that types II and III viruses do not undergo modification (De Sena and Mandel, 1976) was TABLE BIOCHEMICAL
Specimen
ANALYSIS
FOR MODIFICATION
Incubated at
BASED
1 hr
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561
II
based on two observations: (1) that chymotrypsin did not enhance sensitivity to RNase, and (2) that there was no difference in degree of sensitivity to RNase between virus incubated in the presence of ME or water. Although this conclusion is supported by the data in Table 3, it is clear from the centrifugation results in Fig. 3 that all three types yield M particles when treated with ME. On the other hand, they differ in that the M particles of type I are sensitive to chymotrypsin unlike the M particles of types II and III (Table 3). The Effect of Chymotrypsin and Sodium Dodecyl Sulfate on Modified (M) Particles of Types II and III Viruses Treatment of type I M particles with chymotrypsin produces an additionally modified (C) particle, and treatment of M particles with SDS results in release of 35 S viral RNA. The effect of these reagents on types II and III M particles was examined. The data in Fig. 4 show that the M particles produced from types II and III viruses (panel A) are degraded by chymotrypsin with the release of viral oligonucleotides (panel B), whereas SDS promotes 3 ON INDUCED
Percentage Water
Type I virus + ME + H,O Type II virus + ME + H,O Type III virus i ME + H,O Type I virus + ME + H,O Type II virus + ME +H,O Type III virus + ME + H,O
IN
SENSITIVITY
acid-soluble
Chymotrypsin
TO CHYMOTRYPSIN
RNA RNase
after Chymotrypsin and RNase
36” 5.4 21.6
14.6 17.3
53.0 38.1
77.3 49.3
6.9
4.3 6.5
21.0 38.1
23.2 35.6
4.4 7.0
2.0 4.6
14.5 16.8
15.1 20.0
2.3 3.9
-
-
3.3 3.6
3.5 2.6
-
-
3.6 3.8
3.6 3.2
-
-
3.8 3.3
36 11.0
36”
0”
0”
0”
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DE
6
SENA
AND
MANDEL
I
R(%)
Q
030.0 6.0
t
8
0
40
FRACTION
50
60
70
NUMBER
FIG. 4. The effect of chymotrypsin and sodium dodecyl sulfate on M particles of poliovirus types II and III. After reacting with membrane extract for 1 hr at 36”, aliquots of each reaction were treated either with chymotrypsin (0.5 mg/ml) or with SDS (1%) for 20 min at room temperature. All specimens, including an untreated control, were centrifuged through density gradients. The results for types II (0) and III (0) viruses are shown without secondary treatment (A), after chymotrypsin (B), and after SDS treatment (0.
the release of 35 S viral RNA (panel C!). In respect to chymotrypsin, types II and III M particles differ from type I, while all three are affected similarly by SDS. Effect of Trypsin on M Particles of Types I, II, and III Poliovirus In view of the different effect of chymotrypsin on the M particles of the three serotypes, an examination of the effect of trypsin was undertaken. 32P-labeled virus of each type was held for 1 hr at 36” with ME. Each reaction was subdivided and
either held as is or treated with trypsin (0.5 mg/ml) for 20 min at room temperature, then centrifuged as usual. The results are depicted in Fig. 5. Unlike the effect of chymotrypsin, no particles corresponding to C particles are generated when type I is treated with trypsin. A portion of the M particles are digested with the formation of broadly heterogeneous material. As with type I, types II and III M particles are degraded by trypsin. Interpretation of the results of trypsin treatment of types II and III is made difficult by
UNCOATING
OF
IN VITRO.
POLIOVIRUS
77
7
II
563
‘;
L
8-
FRACTION FIG. held for trypsin sucrose (0) and
NUMBER
5. Effect of trypsin on M particles of types I, II, and III poliovirus. 32P-labeled virus of each type was 1 hr at 36” with membrane extract. Each reaction was subdivided and either held as is, or with (0.5 mg/ml) for 20 min at room temperature. Each reaction was subjected to centrifugation through a density gradient. The percentage distribution of radioactivity is shown for each virus type without with trypsin treatment (0).
the large reduction in the fraction of residual material. Possibly in the case of type II the increase in the proportion of N particles after trypsinization reflects the reduction in residual material. An alternative explanation is that some of the M particles are not in fact M particles but are N particles bound to host material and they are restored to their true sedimentation behavior after treatment. Such an effect has been described after detergent treatment of virus recovered from infected cells (Lonberg-Holm et al., 1975).
A Comparative Study of M Particle Formation, and the Effect of Chymotrypsin and Ribonuclease Treatment, Based on the Use of Viral RNA and Protein Tracers Since all the studies thus far were based on the use of a viral RNA label, the basic observations were repeated using a viral protein label. Singly-labeled 32P and 3H stocks were treated with ME and then with either chymotrypsin or RNase following the usual
564
DE
SENA
AND
MANDEL
-6
-6
FRACTION FIG. 6. Characterization of the products of the of RNA V*P) and protein c3H) labeled viruses. membrane extract, then subdivided and treated then centrifuged through sucrose density gradients. shown after reacting with membrane extract (A) with RNase (Cl.
NUMBER
reaction of virus with membrane extract based on the use Singly-labeled viruses were held for 1 hr at 36” with with either chymotrypsin or RNase. The specimens were The percentage distributions of 32P (@) and 3H (0) are and after treatment with chymotrypsin (B) or treatment
protocol, then analyzed by the usual centrifugation procedure. As seen in Fig. 6, the azPtracer showed the expected results, namely, the formation of M particles which were converted to C particles by chymotrypsin. RNase had no effect, unlike the results shown in Fig. 2. As mentioned previously, after treatment of virus with ME, the resultant particles showed a variable sensitivity to RNase, probably reflecting different degrees of modification. Basically, the results seen via the protein tracer show that the sedimentation
rate of native virions is reduced by ME and that the resulting M particle is sensitive to chymotrypsin. The ratio of protein to RNA decreases when M particles are converted to C particles, indicating the possibility of some loss of capsid material which is consistent with the lowered sedimentation rate. DISCUSSION
Recent studies have implicated a modification in the viral capsid as the forerunner stage in the uncoating of picornaviruses
UNCOATING
OF
POLIOVIRUS
(Crowell and Philipson, 1971; LonbergHolm and Korant, 1972; Fenwick and Wall, 1973; Lonberg-Holm et al., 1975). The modification very likely is the result of virus interacting with the plasma membrane of the intact cell (Lonberg-Holm, 1975; Lonberg-Holm and Whiteley, 1976), or with isolated membranous material derived from susceptible cells (Holland and Hoyer, 1962; Chan and Black, 1970; Roesing et al., 1975; De Sena and Mandel, 1976). The modification in the capsid seems to be the same whether the particle (1) is found intracellularly, (2) has eluted from the surface of cells, (3) is produced in vitro by extracted membranes, or (4) is produced in vitro by physicochemical stress such as heat or acid. The principal characteristics of the modified particle are loss of VP, polypeptide, lo-20% loss in S value, loss of infectivity, retention of functional genome in a RNase-resistant state, and sensitivity to proteolytic enzymes and detergents. Little is known of the events that lead from modification to uncoating. Uncoating, for the purpose of this report, is taken to mean release of the viral genome from the enveloping capsid. In a study of the early stages of infection of cells with poliovirus, Fenwick and Wall (1973) showed that three forms of the parental virus can be recovered from disrupted cells: native and modified particles, and viral RNA associated with endoplasmic reticulum. Similarly, in studies of polio- and rhinoviruses (Lonberg-Holm and Korant, 1972; Lonberg-Holm and Noble-Harvey, 1973; Lonberg-Holm et al., 1975) only one type of modified particle was found intracellularly early in infection. Lonberg-Holm and Whiteley (1976) have proposed that the uncoating process involves early modification, followed by intercalation in the plasma membrane, followed by final uncoating. However, failure to find particles modified beyond the stage of early modification, but not yet uncoated, does not rule out the occurrence of transitory unstable intermediates between early modification and final uncoating. The inaccessibility of viral RNA to RNase, and the sensitivity to pro-
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tease of the early modified particle indicate that although the capsid has been modified it is still fairly intact, but now is vulnerable to attack by intracellular proteases. The possibility that proteolytic attack represents the event following early modification has been investigated in the present study utilizing the in vitro system previously described (De Sena and Mandel, 1976) to produce modified particles. The results have shown that chymotrypsin treatment of early modified (i.e., M) particles produces a further modified (i.e., C) particle characterized by a decreased S value and RNase sensitivity. One puzzling aspect of the C particle is that its S value is about the same as that of empties. It has, however, been shown (Breindl, 1971) that it is possible for ribonucleoprotein particles of poliovirus to occur with an S value of about 80. The low S value of the C particle may be a composite result of loss of capsid material as well as rearrangement of the residual structure. The present studies have also shown that treatment of M particles with a detergent (sodium dodecyl sulfate) resulted in the release of 35 S viral RNA. It is therefore possible to envision two alternate pathways for uncoating: (1) M particles are uncoated directly by a detergent-like cellular constituent, or (2) M particles are further modified by an intracellular protease, prior to uncoating. Roesing et al. (1975) have reported that a distinction can be made between plasma membrane receptors that induce modification, and receptors that induce uncoating of coxsackievirus B3. The criterion for uncoating in these studies was sensitivity to RNase. These results raise the possibility that the “uncoated” virus corresponds to the C particles described in the present report. There is the further intriguing possibility that the two receptors described by Roesing et al. (1975) are responsible for the successive production of M and C particles. In a previous report (De Sena and Mandel, 1976) it was stated that types II and III poliovirus were unlike type I since they resisted in vitro modification. The present study shows that all three types are in fact modified, if the reaction is analyzed by
566
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AND
gradient centrifugation. However, types II and III M particles are sensitive to RNase which obviates the potentiating effect of chymotrypsin on RNA sensitivity to RNase. These types also differ in that chymotrypsin does not produce C particles but rather causes extensive degradation. A constant source of uncertainty in the interpretation of results of studies on picornaviruses is the low efficiency of infection and the occurrence of extensive degradation of intracellular uncoated viral RNA (Joklik and Darnell, 1961). Against this background, the present study may indicate two distinct pathways open to incoming parental genomes, one leading to productive, the other to abortive infection. If this consideration is valid, it is not possible at present to relate which of the two proposed possible pathways terminates in productive or abortive infection. ACKNOWLEDGMENTS These studies were supported in part by Grant No. AI-10429 from the National Institute of Allergy and Infectious Diseases, U. S. Public Health Service. The authors wish to acknowledge the excellent technical assistance of Regina Krivickas. REFERENCES BREINDL, M. (1971). The structure of heated poliovirus particles. J. Gen. Virol. 11, 147-156. BRUNETTE, D. M., and TILL, J. E. (1971). A rapid method for the isolation of L-cell surface membranes using an aqueous two-phase polymer system. J. Membrane Biol. 5, 215-224. CHAN, V. F., and BLACK, F. L. (1970). Uncoating of poliovirus by isolated membranes. J. Virol. 5, 309-312. CROWELL, R. L., and PHILIPSON, L. (1971). Specific alterations of coxsackievirus B3 eluted from HeLa cells. J. Virol. 8, 509-515. DE SENA, J., and MANDEL, B. (1976). Studies on the in vitro uncoating of poliovirus. I. Characterization of the modifying factor and the modifying reaction. Virology 70, 470-483.
MANDEL FENWICK, M. L., and WALL, M. J. (19731. Factors determining the site of synthesis of poliovirus proteins: The early attachment of virus particles to endoplasmic membranes. J. Cell Sci. 13, 403-413. HOLLAND, J. J., and HOYER, B. H. (1962). Early stages of enterovirus infection. Cold Spring Harbor Symp. Quant. Biol. 27, 101-111. JOKLIK, W. K., and DARNELL, J. E., JR. (1961). The adsorption and early fate of purified poliovirus in HeLa cells. Virology 13, 439-447. LONBERG-HOLM, K. (1975). The effects of concanavalin A on the early events of infection by rhinovirus type 2 and poliovirus type 2. J. Gen. Viral. 28, 313-327. LONBERG-HOLM, K., and KORANT, B. D. (1972). Early interaction of rhinovirus with host cells. J. Virol. 9, 29-40. LONBERG-HOLM, K., and NOBLE-HARVEY, J. (1973). Comparison of in vitro and cell-mediated alteration of a human rhinovirus and its inhibition by sodium dodecyl sulfate. J. Virol. 12, 819-826. LONBERG-HOLM, K., and WHITELEY, N. M. (1976). Physical and metabolic requirements for early interaction of poliovirus and human rhinovirus with HeLa cells. J. Virol. 19, 857-870. LONBERG-HOLM, K., GOSSER, L. B., and KAUER, J. C. (1975). Early alteration of poliovirus in infected cells and its specific inhibition. J. Gen. Virol. 27, 329-342. LoNaxaG-Holm, K., GOSSER, L. B., and SHIMSHICK, E. J. (1976). Interaction of liposomes with subviral particles of poliovirus type 2 and rhinovirus type 2. J. Virol. 19, 746-749. MANDEL, B. (1961). Reversibility of the reaction between poliovirus and neutralizing antibody. Virology 14, 316-328. MANDEL, B. (1962). Early stages of virus-cell interaction as studied by using antibody. Cold Spring Harbor Symp. Quant. Biol. 27, 123-131. MANDEL, B. (1967). The interaction of neutralized poliovirus with HeLa cells. I. Adsorption. Virology 31, 238-247. ROESING, T. G., TOSELLI, P. A., and CROWELL, R. L. (1975). Elution and uncoating of coxsackievirus B3 by isolated HeLa cell plasma membranes. J. Virol. 15, 654-667. WEINTRAUB, S., and DALES, S. (1974). Biogenesis of poxviruses: Genetically controlled modifications of structural and functional components of the plasma membrane. Virology 60, 96-127.
78,
554-566
(1977)
Studies
on the in vitro
II. Characteristics JOHN Department
of Virology,
Uncoating
of Poliovirus
of the Membrane-Modified
DE SENA’ The Public Health New York, Accepted
BENJAMIN
AND
Research Institute New York 10016 February
Particle MANDEL
of The City
of New
York,
Inc.,
II,1977
Interaction of type I poliovirus with an extract of isolated HeLa cell membranes resulted in a modification of the viral capsid. As a consequence of the modification, the capsid became sensitive to proteases and detergents, and the sedimentation value of the virion was slightly diminished. Treatment of the modified particle with chymotrypsin reduced its sedimentation value and, although the RNA genome was still encapsidated, it was degradable by RNase. Treatment of the membrane-modified particle with detergent (sodium dodecyl sulfate, SDS) disengaged the viral RNA as an intact (i.e., 35 S) molecule. Each of the three serotypes of poliovirus was modified when treated with membrane extract. They differed, however, in the effect of secondary treatment with chymotrypsin, viz., type I was further modified, but types II and III underwent degradation. When secondary treatment was with SDS, all three released 35 S RNA. The above in vitro reactions of the membrane-modified particle are discussed as possible counterparts of the in viva uncoating phenomenon. INTRODUCTION
nothing is understood of the responsible cellular processes or events intervening between modification and the final stage of uncoating. It has, however, become increasingly evident that the initial early modification of the adsorbed vu-ion is attributable to a constituent of the plasma membrane, perhaps the viral receptor. This presumption is supported by the results of studies with isolated cellular membranes (Hoiland and Hoyer, 1962; and, more recently, Chan and Black, 1970; Roesing et al., 1975; De Sena and Mandel, 1976), and by the results of studies of the effect of metabolic inhibitors (LonbergHolm and Whiteley, 1976) and concanavalin A (Lonberg-Holm, 1975) on the early interactions of picornaviruses with host cells. In considering the events subsequent to early modification, Lonberg-Holm and Whiteley (1976) have proposed that early modification is the penultimate stage to final uncoating, and, intervening, is a period during which the modified virion intercalates with the plasma membrane of the cell. The latter suggestion is based on
Results of previous studies on the uncoating of picornaviruses (Holland and Hoyer, 1962; Mandel, 1962) suggested that uncoating is a multistage phenomenon. In support, recent observations (Crowell and Philipson, 1971; Lonberg-Holm and Kount-, 1972; Fenwick and Wall, 1973; Lonberg-Holm et al., 1975) have shown that after attaching to the surface of a susceptible cell, virus undergoes a preliminary modification characterized by (1) loss of the VP4 capsid polypeptide, (2) slight reduction in S value, (3) acquired sensitivity to proteases and detergents, and (4) loss of infectivity, although the particle still contains a functional genome which is inaccessible to nucleolytic enzymes. This early modification is considered to be a prerequisite stage to uncoating. Although some characteristics of the modified particle are known, virtually ’ Present address: Department and Biochemistry, Idaho State tello, Idaho 83209.
of Microbiology University, Poca554
Copyright All rights
8 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0042-6822
UNCOATING
OF
POLIOVIRUS
the observed lipophilic character of modified particles when exposed to artificially prepared liposomes (Lonberg-Holm et al., 1976). There is, however, no evidence to rule out the possibility of a process involving the progression of the early modified particles through subsequent stages of modification, thus constituting a series of transient unstable intermediates in the uncoating pathway. The induced sensitivity to proteolytic enzymes of the early modified particles suggests the possibility that proteolysis may play a role in uncoating. The same possibility can be considered for such a role for substances with detergent properties. These possibilities have been examined in the present study utilizing the previously described in vitro system (De Sena and Mandel, 1976) to generate modified particles for further characterization. MATERIALS
AND
METHODS
Virus and cells. HeLa cell monolayer cultures were used for the propagation of viral stocks and for plaque assay (Mandel, 19611, and 32P-labeled stocks were prepared and purified as previously described (Mandel, 1967). 13H] Amino acid labeled stocks were prepared by adding a mixture of 3H-labeled amino acids to the viral growth medium to a concentration of 0.035 mCilm1. Concentration and purification was as for 32P-labeled virus. Strains of poliovirus used were Brunhilde (type I), MEF, (type II), and Saukett (type III). Naturally occurring empty capsids were collected by centrifuging the 3H-labeled stock through a cesium chloride gradient. Two peaks of radioactivity were observed which were collected and assayed for infectivity. The peak of density 1.335 g/ml had more than loo-fold higher specific infectivity than the peak of density 1.285 g/ml. Each material, after dialysis, was centrifuged through a sucrose density gradient. Based on the relative sedimentation rates, the lower density material had an S value of 80 and this material was considered to be empty capsids. Reagents and solutions. The following reagents were obtained as indicated: al-
IN
VITRO.
II
555
pha-chymotrypsin (EC 3.4.4.5) 3x crystallized, trypsin (EC 3.4.4.4) 2x crystallized, ribonuclease A (EC 2.7.7.16) from Worthington Biochemical Corp.; sodium dodecyl sulfate from Schwarz/Mann; dextran T-500 from Pharmacia Fine Chemicals, Inc.; polyethylene glycol (Carbowax 6000) from Union Carbide Corp.; carrier-free H,“2P0, and 3H-labeled amino acid mixture from New England Nuclear Corp. Preparation of HeLa cell membrane extract. The procedure for obtaining plasma membrane fragments was based on the method of Brunette and Till (1971) as modified by Weintraub and Dales (1974) and has been described in detail previously (De Sena and Mandel, 1976). Reaction of virus with membrane extract and biochemical analysis. An appropriate dilution of virus was added to undiluted membrane extract and held for 1 hr at 36”; variations of this procedure will be described in the text. Samples of the reaction mixture were taken for biochemical analysis which involved treatment with chymotrypsin and RNase, then determining the percentage of labeled RNA that was soluble in trichloroacetic acid (De Sena and Mandel, 1976). Treatment of membrane-modified virus. Following interaction of virus with membrane extract, 2.5 ml of the reaction mixture was added to 0.1 vol of reagent and held at 23” for 20 min in the case of enzymes, and 5 min in the case of detergent. Aliquots were taken immediately for centrifugation and in some instances for biochemical analysis. Density gradient centrifugation. Sucrose density gradients (10% to 25% in 0.58% NaCl, w/w) were prepared the day before use and held overnight at about 5”. Prior to preparing the gradients, about 15 drops of 1% agar were added to the tubes and, after solidification, 15 drops of glycerol were added, thus forming a double cushion. Linear gradients were formed over the cushion using 16.4 ml of the lo%, and 15.6 ml of the 25% sucrose solutions. Two milliliters of specimen was layered on the gradient solution and centrifuged in a Beckman SW27 rotor at 26,000 rpm for 2 hr at 5” in a Spinco model L2-65B centrifuge.
556
DE
SENA
AND
Fractions were collected dropwise through a puncture in the bottom of the tube. Experience had shown that little or no radioactivity emerged in the first few fractions. Therefore, the first seven fractions were collected as 50-drop (l-ml) fractions, the next 38 fractions represented 25 drops, and the remainder were 50-drop fractions. The residue remaining in the tube was recovered by washing the tube with a small volume of 1 N NaOH and saving the wash for radioactivity count. RESULTS
Effect
of Modification by Membrane Extract, and Subsequent Modification by Chymotrypsin, on Sedimentation of Poliovirus in Sucrose Density Gradients
Previously (De Sena and Mandel, 1976), it was shown that treatment of type I poliovirus with an extract of isolated HeLa cell membranes induced a modification in the particles that rendered them sensitive to chymotrypsin. This conclusion was based on the observation that the viral RNA became accessible to RNase after sequential treatment with membrane extract (ME) and chymotrypsin. A study was undertaken to characterize further the TABLE EFFECT
Specimen
OF CHYMOTRYPSIN
ON POLIOVIRUS
Chymotrypsinb- (minii3”)
MANDEL
modification in the virion induced by ME. In view of the observation (Lonberg-Holm and Koran& 1972; Fenwick and Wall, 1973) that soon after penetration of rhino- and poliovirus into susceptible cells some of the virions show a slight reduction in S value, the effect of ME treatment on the sedimentation of poliovirus was examined. 32P-labeled type I poliovirus was treated with ME for 1 hr at 36”. Aliquots were then treated with chymotrypsin at room temperature for several time periods. Samples were then collected for biochemical assay and for centrifugation through sucrose density gradients. All dropwise-collected gradient fractions were assayed for radioactivity, and several fractions from several gradients were assayed for infectivity. As seen in Table 1, virus alone, or in the presence of ME for 1 hr at o”, was insensitive to the sequential treatments with chymotrypsin and RNase. Virus alone, held for 1 hr at 36, became moderately sensitive, but in the presence of ME sensitivity was considerably increased. It should be noted that ME-modified virus shows a moderate sensitivity to RNase directly. This sensitivity has varied considerably in these experiments and has been interpreted as a reflection of varying degrees of modification. 1
MODIFIED
Percentage Water
BY HeLa
acid-soluble Chymotrypsin
CELL
MEMBRANE
RNA
after RNase
EXTRACT
treatment? Chymotrypsin and RNase
Virus + ME 36”/1 hr
5 10 30 60
-e 11.4
14.2 14.8 15.2 16.3
47.2
65.3 65.3 62.3
Virus + H,O 36”/1 hr
30
16.7
18.9
22.3
25.1
Virus O”/l
+ ME hr
30
2.1
-
-
3.1
Virus + H,O On/1 hr
30
2.1
-
-
3.2
a See Materials and Methods for details b The final concentration was 0.5 mglml. c Not done.
of biochemical
analysis.
UNCOATING
OF
POLIOVIRUS
The effects of ME and chymotrypsin on sedimentation rate are shown in Fig. 1. Treatment with ME produces a slower sedimenting particle (panel A) which, as will be shown below, is noninfectious in contrast to the faster sedimenting particle.
IN VITRO.
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557
These will be referred to hereafter as M (modified) and N (native) particles, respectively. Subsequent treatment with chymotrypsin results in additional modification (panels B and 0 to a particle of still lower S value, namely, C (chymotrypsin modi;I
;‘
EEL 8.5 IO-
5. > I-
60
70
FIG. 1. The reduction in sedimentation rate of poliovirus after modification by membrane extract and subsequent modification by chymotrypsin. Details of the centrifugation procedure are described under Materials and Methods. After centrifugation, each gradient was collected dropwise. Each symbol (0) represents 25 drops. Some fractions were collected as 50 drops but, to facilitate comparison, are represented as two equal 25-drop fractions (00). Results are shown as the percentage in each fraction of the total radioactivity in the gradient, including the residue remaining in the tube which is represented by the value of R shown in each panel. Since only trace amounts, at most, are present in the first 20 fractions, these are not shown. This scheme for presenting the results of centrifugation studies is used throughout this report. Standardization of this system has shown the peak positions of native (N) and empty (E) particles and 35 S viral RNA (R). The percentage distribution of sZP radioactivity is shown for poliovirus treated with membrane extract for 1 hr at 36” (A) and subsequently treated with chymotrypsin for 30 min (B) and for 60 min (C) at 23”. The peak positions of native particles are indicated by N, the modified particles resulting from exposure to membrane extract are indicated by M, and particles subsequently modified by chymotrypsin are designated C.
558
DE
SENA
AND
fied) .particle. Based on standardization of this system with native virions, naturally occurring empties, and extracted viral 35 S RNA, the expected positions of these markers are indicated by N, E, and R, respectively. The M particles appear to be similar to the slower sedimenting particles described by Lonberg-Holm and Korant (1972) and Fenwick and Wall (1973). The nature of the C particles is puzzling since their sedimentation rate is similar to that of empties but they contain RNA. In this respect they resemble the 80 S ribonucleoprotein particles obtained by heating (Breindl, 1971). Both the 80 S particles of Breindl and the C particles described above are sensitive to RNase and also release 35 S RNA when treated with sodium dodecyl sulfate. The results of infectivity assays, Table 2, are expressed as specific infectivity (SD which is a measure of the relative proportion of infectious to total labeled particles, i.e., PFU/cpm. Virus in the N peak has the same SI as the control. In contrast, virus in the M and C peaks has SI’s of about 1% of the control. In order to determine if the reduction is the result of reversible binding to host receptor, the specimens were treated with sodium dodecyl sulfate. In
MANDEL
neither case did reactivation occur. Usually a variable amount of viral isotope is recoverable from the tube after the contents have been collected. Assay of this residual material shows a low SI which, however, approaches that of the control after treatment with SDS. It is likely that the residue consists of virus bound reversibly to high-molecular-weight receptor in contrast to N and M particles that may have eluted with or without modification.
Characteristics Particle
INFECTIVITY
2
OF POLIOVIRUS PARTICLES AFFER TREATMENT WITH MEMBRANE SEPARATION BY SUCROSE DENSITY GRADIENT CENTRIFUGATION
Specimen
Gradient fractions
Type of particle
PFU/ml
+ ME
36”/1
hr
Virus + ME chymotrypsin
36”/1
Virus
0”/3 hr
+ ME’
hr
+
EXTRACT Specific
cpm/ml
-
AND infectiv-
ity” No treatment
Virus
(M)
Treatment of poliovirus with ME results in chemical modification as shown by the induced sensitivity to chymotrypsin, and in physical modification as shown by the reduction in sedimentation rate. Further characterization of the M particle was undertaken. After treatment of 32P-labeled virus with ME, aliquots were subjected to secondary treatment by adding 0.1 vol of reagent for 20 min at room temperature. The following reagents were used: water, chymotrypsin, chymotrypsin followed by RNase, RNase, sodium dodecyl sulfate (SDS), and SDS followed by RNase. Each was then centrifuged through a sucrose gradient.
TABLE SPECIFIC
of the Modified
SDS treatmentb
No treatment
24 + 25 29 + 30 residue
N M N
3.0 x 106 I.3 x 105 1.3 x 105
1.6 x lo6 1.0 x 105 5.6 x lo5
4.2 x lo3 1.4 x 104 1.9 x 103
23 + 24 30 + 31 35 + 36 residue
N M C N
3.0 5.2 2.9 5.0
2.1 4.0 5.0 5.3
2.7 6.0 5.8 1.4
N
4.5 x 10’
x x x x
106 104 104 104
x x x x
106 104 103 105
2.5 x 10’
SDS treatment
714 9 68
381 7 294
lo3 lo3 103 103
1097 9 5 36
781 7 1 380
5.2 x lo4
861
486
x x x x
a Specific infectivity = PFU/cpm b Sodium dodecyl sulfate was added to a final concentration of 0.5%, the reaction was held temperature for 10 to 15 min and then diluted for assay. r An aliquot of the original virus-ME reaction mixture was held at 0” for 3 hr and then assayed having been subjected to centrifugation.
at room without
UNCOATING
8-
OF
POLIOVIRUS
iY R&),H
IN
VITRO.
559
II
r; ‘;
-4.2 0 1.5
6
8-
8-
FRACTION
NUMBER
FIG. 2. Characterization of type I poliovirus modified by membrane extract. 32P-labeled virus was treated with membrane extract for 1 hr at 36”. The reaction mixture was subdivided and treated with various reagents for 20 min at 23”. The percentage distribution in each gradient is shown in A after secondary treatment with water (0) or with 0.5 mglml chymotrypsin (0); in B after secondary treatment with chymotrypsin as above, followed by 10 @g/ml RNase (01, or with RNase alone (0); in C after secondary treatment with 1% sodium dodecyl sulfate (01, or with sodium dodecyl sulfate followed by RNase (01.
The results in Fig. 2 show that treatment with ME (panel A) produces a population of M particles which sediment slower than N particles. Secondary treatment with chymotrypsin (panel A) does not visibly affect N particles but converts M particles to the more slowly sedimenting C particle. Treatment with RNase after chymotrypsin (panel B) degrades the RNA of the C particles and also the RNA of some of the N particles. SDS causes the release of viral RNA from M particles, and the RNA is seen in its conserved (i.e., 35 S)
state and is degradable by RNase (panel C). A puzzling result is seen when RNase is used directly after ME treatment (panel B). A class of particles appears intermediate in S value between N and M, probably derived from the N population. It is possible that some particles considered to be N have been minimally modified without change in S value but with induced sensitivity to various reagents. The decrease in the proportion of particles in the N peak after chymotrypsin-RNase treatment and SDS treatment suggests this possibility.
560
DE
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AND
The peak appearing after RNase treatment may represent a population of pseudo-N particles. Such particles may therefore represent the earliest stage in modification which could be a simple rearrangement of the quaternary structure of the capsid. Effect
of Membrane Extract Serotypes II and III
on Poliovirus
In previous studies (De Sena and Mandel, 1976) it was reported that, unlike type I, poliovirus types II and III were insensi-
MANDEL
tive to the action of membrane extract. This conclusion was based on biochemical analysis which detects an induced state of sensitivity to chymotrypsin. Inasmuch as modification is also recognizable by a reduction in sedimentation rate, the effect of ME on serotypes II and III was reexamined for a possible change in this parameter. 32P-labeled virus of each type was treated either with water or ME for 1 hr at 36”. Aliquots were taken for biochemical analysis and gradient centrifugation analysis. Data depicted in Fig. 3 show that M
60
FRACTION
NUMBER
FIG. 3. Production of M particles by treatment of poliovirus serotypes extract. Each $*P-labeled virus was mixed either with water or membrane then centrifuged through sucrose density gradients. Percentage distribution (0) and virus-extract (0) mixtures is shown for types I, II, and III.
I, II, and III with membrane extract, held for 1 hr at 36”, and of radioactivity of virus-water
UNCOATING
OF
POLIOVIRUS
particles are produced by ME treatment of all three types. Although type III is more resistant to modification, the M particles of all three appear to be alike in respect to S value. Of interest is the observation that in the absence of ME no M particles are produced indicating that the formation of M particles is not a spontaneous occurrence that is accelerated by ME. However, in view of the appearance of viral oligonucleotides in the absence of ME, there is the possibility that M particles are produced, but under these conditions they are unstable and are rapidly degraded. Biochemical data in Table 3 show that all three types, whether exposed to water or ME but held at O”, are insensitive to sequential treatment with chymotrypsin and RNase. At 36” spontaneous degradation of viral RNA to an acid-soluble state occurs but to a lesser extent in the presence than in the absence of ME. Treatment with RNase reveals in each case accessibility of viral RNA, which, only in the case of type I, is enhanced by prior treatment with chymotrypsin. The previous conclusion that types II and III viruses do not undergo modification (De Sena and Mandel, 1976) was TABLE BIOCHEMICAL
Specimen
ANALYSIS
FOR MODIFICATION
Incubated at
BASED
1 hr
VITRO.
561
II
based on two observations: (1) that chymotrypsin did not enhance sensitivity to RNase, and (2) that there was no difference in degree of sensitivity to RNase between virus incubated in the presence of ME or water. Although this conclusion is supported by the data in Table 3, it is clear from the centrifugation results in Fig. 3 that all three types yield M particles when treated with ME. On the other hand, they differ in that the M particles of type I are sensitive to chymotrypsin unlike the M particles of types II and III (Table 3). The Effect of Chymotrypsin and Sodium Dodecyl Sulfate on Modified (M) Particles of Types II and III Viruses Treatment of type I M particles with chymotrypsin produces an additionally modified (C) particle, and treatment of M particles with SDS results in release of 35 S viral RNA. The effect of these reagents on types II and III M particles was examined. The data in Fig. 4 show that the M particles produced from types II and III viruses (panel A) are degraded by chymotrypsin with the release of viral oligonucleotides (panel B), whereas SDS promotes 3 ON INDUCED
Percentage Water
Type I virus + ME + H,O Type II virus + ME + H,O Type III virus i ME + H,O Type I virus + ME + H,O Type II virus + ME +H,O Type III virus + ME + H,O
IN
SENSITIVITY
acid-soluble
Chymotrypsin
TO CHYMOTRYPSIN
RNA RNase
after Chymotrypsin and RNase
36” 5.4 21.6
14.6 17.3
53.0 38.1
77.3 49.3
6.9
4.3 6.5
21.0 38.1
23.2 35.6
4.4 7.0
2.0 4.6
14.5 16.8
15.1 20.0
2.3 3.9
-
-
3.3 3.6
3.5 2.6
-
-
3.6 3.8
3.6 3.2
-
-
3.8 3.3
36 11.0
36”
0”
0”
0”
562
DE
6
SENA
AND
MANDEL
I
R(%)
Q
030.0 6.0
t
8
0
40
FRACTION
50
60
70
NUMBER
FIG. 4. The effect of chymotrypsin and sodium dodecyl sulfate on M particles of poliovirus types II and III. After reacting with membrane extract for 1 hr at 36”, aliquots of each reaction were treated either with chymotrypsin (0.5 mg/ml) or with SDS (1%) for 20 min at room temperature. All specimens, including an untreated control, were centrifuged through density gradients. The results for types II (0) and III (0) viruses are shown without secondary treatment (A), after chymotrypsin (B), and after SDS treatment (0.
the release of 35 S viral RNA (panel C!). In respect to chymotrypsin, types II and III M particles differ from type I, while all three are affected similarly by SDS. Effect of Trypsin on M Particles of Types I, II, and III Poliovirus In view of the different effect of chymotrypsin on the M particles of the three serotypes, an examination of the effect of trypsin was undertaken. 32P-labeled virus of each type was held for 1 hr at 36” with ME. Each reaction was subdivided and
either held as is or treated with trypsin (0.5 mg/ml) for 20 min at room temperature, then centrifuged as usual. The results are depicted in Fig. 5. Unlike the effect of chymotrypsin, no particles corresponding to C particles are generated when type I is treated with trypsin. A portion of the M particles are digested with the formation of broadly heterogeneous material. As with type I, types II and III M particles are degraded by trypsin. Interpretation of the results of trypsin treatment of types II and III is made difficult by
UNCOATING
OF
IN VITRO.
POLIOVIRUS
77
7
II
563
‘;
L
8-
FRACTION FIG. held for trypsin sucrose (0) and
NUMBER
5. Effect of trypsin on M particles of types I, II, and III poliovirus. 32P-labeled virus of each type was 1 hr at 36” with membrane extract. Each reaction was subdivided and either held as is, or with (0.5 mg/ml) for 20 min at room temperature. Each reaction was subjected to centrifugation through a density gradient. The percentage distribution of radioactivity is shown for each virus type without with trypsin treatment (0).
the large reduction in the fraction of residual material. Possibly in the case of type II the increase in the proportion of N particles after trypsinization reflects the reduction in residual material. An alternative explanation is that some of the M particles are not in fact M particles but are N particles bound to host material and they are restored to their true sedimentation behavior after treatment. Such an effect has been described after detergent treatment of virus recovered from infected cells (Lonberg-Holm et al., 1975).
A Comparative Study of M Particle Formation, and the Effect of Chymotrypsin and Ribonuclease Treatment, Based on the Use of Viral RNA and Protein Tracers Since all the studies thus far were based on the use of a viral RNA label, the basic observations were repeated using a viral protein label. Singly-labeled 32P and 3H stocks were treated with ME and then with either chymotrypsin or RNase following the usual
564
DE
SENA
AND
MANDEL
-6
-6
FRACTION FIG. 6. Characterization of the products of the of RNA V*P) and protein c3H) labeled viruses. membrane extract, then subdivided and treated then centrifuged through sucrose density gradients. shown after reacting with membrane extract (A) with RNase (Cl.
NUMBER
reaction of virus with membrane extract based on the use Singly-labeled viruses were held for 1 hr at 36” with with either chymotrypsin or RNase. The specimens were The percentage distributions of 32P (@) and 3H (0) are and after treatment with chymotrypsin (B) or treatment
protocol, then analyzed by the usual centrifugation procedure. As seen in Fig. 6, the azPtracer showed the expected results, namely, the formation of M particles which were converted to C particles by chymotrypsin. RNase had no effect, unlike the results shown in Fig. 2. As mentioned previously, after treatment of virus with ME, the resultant particles showed a variable sensitivity to RNase, probably reflecting different degrees of modification. Basically, the results seen via the protein tracer show that the sedimentation
rate of native virions is reduced by ME and that the resulting M particle is sensitive to chymotrypsin. The ratio of protein to RNA decreases when M particles are converted to C particles, indicating the possibility of some loss of capsid material which is consistent with the lowered sedimentation rate. DISCUSSION
Recent studies have implicated a modification in the viral capsid as the forerunner stage in the uncoating of picornaviruses
UNCOATING
OF
POLIOVIRUS
(Crowell and Philipson, 1971; LonbergHolm and Korant, 1972; Fenwick and Wall, 1973; Lonberg-Holm et al., 1975). The modification very likely is the result of virus interacting with the plasma membrane of the intact cell (Lonberg-Holm, 1975; Lonberg-Holm and Whiteley, 1976), or with isolated membranous material derived from susceptible cells (Holland and Hoyer, 1962; Chan and Black, 1970; Roesing et al., 1975; De Sena and Mandel, 1976). The modification in the capsid seems to be the same whether the particle (1) is found intracellularly, (2) has eluted from the surface of cells, (3) is produced in vitro by extracted membranes, or (4) is produced in vitro by physicochemical stress such as heat or acid. The principal characteristics of the modified particle are loss of VP, polypeptide, lo-20% loss in S value, loss of infectivity, retention of functional genome in a RNase-resistant state, and sensitivity to proteolytic enzymes and detergents. Little is known of the events that lead from modification to uncoating. Uncoating, for the purpose of this report, is taken to mean release of the viral genome from the enveloping capsid. In a study of the early stages of infection of cells with poliovirus, Fenwick and Wall (1973) showed that three forms of the parental virus can be recovered from disrupted cells: native and modified particles, and viral RNA associated with endoplasmic reticulum. Similarly, in studies of polio- and rhinoviruses (Lonberg-Holm and Korant, 1972; Lonberg-Holm and Noble-Harvey, 1973; Lonberg-Holm et al., 1975) only one type of modified particle was found intracellularly early in infection. Lonberg-Holm and Whiteley (1976) have proposed that the uncoating process involves early modification, followed by intercalation in the plasma membrane, followed by final uncoating. However, failure to find particles modified beyond the stage of early modification, but not yet uncoated, does not rule out the occurrence of transitory unstable intermediates between early modification and final uncoating. The inaccessibility of viral RNA to RNase, and the sensitivity to pro-
IN VITRO.
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tease of the early modified particle indicate that although the capsid has been modified it is still fairly intact, but now is vulnerable to attack by intracellular proteases. The possibility that proteolytic attack represents the event following early modification has been investigated in the present study utilizing the in vitro system previously described (De Sena and Mandel, 1976) to produce modified particles. The results have shown that chymotrypsin treatment of early modified (i.e., M) particles produces a further modified (i.e., C) particle characterized by a decreased S value and RNase sensitivity. One puzzling aspect of the C particle is that its S value is about the same as that of empties. It has, however, been shown (Breindl, 1971) that it is possible for ribonucleoprotein particles of poliovirus to occur with an S value of about 80. The low S value of the C particle may be a composite result of loss of capsid material as well as rearrangement of the residual structure. The present studies have also shown that treatment of M particles with a detergent (sodium dodecyl sulfate) resulted in the release of 35 S viral RNA. It is therefore possible to envision two alternate pathways for uncoating: (1) M particles are uncoated directly by a detergent-like cellular constituent, or (2) M particles are further modified by an intracellular protease, prior to uncoating. Roesing et al. (1975) have reported that a distinction can be made between plasma membrane receptors that induce modification, and receptors that induce uncoating of coxsackievirus B3. The criterion for uncoating in these studies was sensitivity to RNase. These results raise the possibility that the “uncoated” virus corresponds to the C particles described in the present report. There is the further intriguing possibility that the two receptors described by Roesing et al. (1975) are responsible for the successive production of M and C particles. In a previous report (De Sena and Mandel, 1976) it was stated that types II and III poliovirus were unlike type I since they resisted in vitro modification. The present study shows that all three types are in fact modified, if the reaction is analyzed by
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gradient centrifugation. However, types II and III M particles are sensitive to RNase which obviates the potentiating effect of chymotrypsin on RNA sensitivity to RNase. These types also differ in that chymotrypsin does not produce C particles but rather causes extensive degradation. A constant source of uncertainty in the interpretation of results of studies on picornaviruses is the low efficiency of infection and the occurrence of extensive degradation of intracellular uncoated viral RNA (Joklik and Darnell, 1961). Against this background, the present study may indicate two distinct pathways open to incoming parental genomes, one leading to productive, the other to abortive infection. If this consideration is valid, it is not possible at present to relate which of the two proposed possible pathways terminates in productive or abortive infection. ACKNOWLEDGMENTS These studies were supported in part by Grant No. AI-10429 from the National Institute of Allergy and Infectious Diseases, U. S. Public Health Service. The authors wish to acknowledge the excellent technical assistance of Regina Krivickas. REFERENCES BREINDL, M. (1971). The structure of heated poliovirus particles. J. Gen. Virol. 11, 147-156. BRUNETTE, D. M., and TILL, J. E. (1971). A rapid method for the isolation of L-cell surface membranes using an aqueous two-phase polymer system. J. Membrane Biol. 5, 215-224. CHAN, V. F., and BLACK, F. L. (1970). Uncoating of poliovirus by isolated membranes. J. Virol. 5, 309-312. CROWELL, R. L., and PHILIPSON, L. (1971). Specific alterations of coxsackievirus B3 eluted from HeLa cells. J. Virol. 8, 509-515. DE SENA, J., and MANDEL, B. (1976). Studies on the in vitro uncoating of poliovirus. I. Characterization of the modifying factor and the modifying reaction. Virology 70, 470-483.
MANDEL FENWICK, M. L., and WALL, M. J. (19731. Factors determining the site of synthesis of poliovirus proteins: The early attachment of virus particles to endoplasmic membranes. J. Cell Sci. 13, 403-413. HOLLAND, J. J., and HOYER, B. H. (1962). Early stages of enterovirus infection. Cold Spring Harbor Symp. Quant. Biol. 27, 101-111. JOKLIK, W. K., and DARNELL, J. E., JR. (1961). The adsorption and early fate of purified poliovirus in HeLa cells. Virology 13, 439-447. LONBERG-HOLM, K. (1975). The effects of concanavalin A on the early events of infection by rhinovirus type 2 and poliovirus type 2. J. Gen. Viral. 28, 313-327. LONBERG-HOLM, K., and KORANT, B. D. (1972). Early interaction of rhinovirus with host cells. J. Virol. 9, 29-40. LONBERG-HOLM, K., and NOBLE-HARVEY, J. (1973). Comparison of in vitro and cell-mediated alteration of a human rhinovirus and its inhibition by sodium dodecyl sulfate. J. Virol. 12, 819-826. LONBERG-HOLM, K., and WHITELEY, N. M. (1976). Physical and metabolic requirements for early interaction of poliovirus and human rhinovirus with HeLa cells. J. Virol. 19, 857-870. LONBERG-HOLM, K., GOSSER, L. B., and KAUER, J. C. (1975). Early alteration of poliovirus in infected cells and its specific inhibition. J. Gen. Virol. 27, 329-342. LoNaxaG-Holm, K., GOSSER, L. B., and SHIMSHICK, E. J. (1976). Interaction of liposomes with subviral particles of poliovirus type 2 and rhinovirus type 2. J. Virol. 19, 746-749. MANDEL, B. (1961). Reversibility of the reaction between poliovirus and neutralizing antibody. Virology 14, 316-328. MANDEL, B. (1962). Early stages of virus-cell interaction as studied by using antibody. Cold Spring Harbor Symp. Quant. Biol. 27, 123-131. MANDEL, B. (1967). The interaction of neutralized poliovirus with HeLa cells. I. Adsorption. Virology 31, 238-247. ROESING, T. G., TOSELLI, P. A., and CROWELL, R. L. (1975). Elution and uncoating of coxsackievirus B3 by isolated HeLa cell plasma membranes. J. Virol. 15, 654-667. WEINTRAUB, S., and DALES, S. (1974). Biogenesis of poxviruses: Genetically controlled modifications of structural and functional components of the plasma membrane. Virology 60, 96-127.