A comparison of the virus-specific polypeptides of encephalomyocarditis virus, human rhinovirus-1A, and poliovirus

VIROLOGY

66, 439-453 (1973)

A Comparison

of the

carditis

Virus,

Virus-Specific Human BYRON

Polypeptides

Rhinovirus-lA,

and

of EncephalomyoPoliovirus’

E. BUTTERWORTH

Cen.tral Research Department, E. I. du Pont de Nenaours Accepted September

and Company, Wilmington,

Delaware

19898

‘7, 1973

Encephalomyocarditis (EMC) virus, human rhinovirus-IA (HR.V-lA), and poliovirus are each representatives of a different, major subgroup of the picornaviruses. In this comparative study the patterns of biosynthesis of the virus-specific polypeptides of these three viruses were examined in detail. The molecular weight and molar ratio of each polypeptide and the genetic map of each virus were determined, and the kinetics of cleavage of the analogous precursor polypeptides were contrasted. Previous work has shown that there are three main families of EMC virus-specific polypeptides. Translation of the EMC viral ribonucleic acid generates at least three primary products, polypeptides A, F, and C. Polypeptide A undergoes successive cleavages to produce the four major capsid polypeptides. Polypeptide F is stable. Polypeptide C cleaves to form polypeptide D, which in turn cleaves to form polypeptide E. Evolutionary pressures on these viruses have resulted in extensive differences in the antigenicity of the virions. In addition, it was found that each profile had polypeptides that were unique to that particular virus, and the kinetic studies revealed that analogous cleavages proceeded at extensively different rates. However, its judged by these studies with EMC virus, HRV-lA, and poliovirus, the general mechanism of synthesis, size, and genetic order of the three main families of polypeptides remain the same. INTRODUCTION

Experimentation The picornaviruses can be divided into at least four major subgroups: (1) the enteroviruses (e.g., polio-, coxsackie-, and echoviruses), (2) the cardioviruses (e.g., EMC-, mouse-elberfeld (ME)- and mengoviruses), (3) the rhinoviruses (e.g., the commoncold viruses), and (4) the foot-and-mouth disease viruses (Fenner, 1968). Several laboratories have been involved in identifying the virus-specific polypeptides of poliovirus and the post-t.ranslational cleavage mechanism by which they are produced (Summers et al., 1965; Holland and Kiehn, 1968; Jacobson and Baltimore, 1968; Maizel and Summers, 1968; Summers and Maizel, 1968; Jacobson et al., 1970; Koran& I Contribution

No. 2058.

1972). Other detailed studies have revealed profiles of virus-specific polypeptides and post-translational cleavage mechanisms for EMC virus and HRV-1A that are similar to those of poliovirus (Butterworth et al., 1971; Butterworth and Rueckert, 1972b; McLean and Rueckert, 1973). Several lines of evidence suggest that the picornaviral RNA possesses a single initiation site and that, once initiated, each ribosome completes translation of the entire viral RNA (Jacobson and Baltimore, 1968; Kiehn and Holland, 1970; Butterworth and Rueckert, 1972a; oberg and Shatkin, 1972). This property has facilitated the genetic mapping of these viruses by a technique which employs the drug pactamycin. The genetic map has been determined wholly or in part for EMC virus, HRV-lA, and poliovirus (Taber et al., 1971; Summers and Maizel, 1971; 439

Copyright All rights

@ 1973 by Academic Press, of reproduction in any form

Inc. reserved.

440

BUTTERWORTH

Butterworth and Rueckert, 1972a; Rekosh, 1972; McLean and Rueckert, 1973). In order to compare members from different picornavirus subgroups, EMC virus, HRV-lA, and poliovirus were chosen. The genetic map of poliovirus was expanded and compared to those of EMC virus and HRV-lA, the molecular weights of the different viral polypeptides relative to the EMC viral polypeptides were determined, and the rates of cleavage of several analogous precursor polypeptides were contrasted. The results indicate that many common features have been conserved in the evolution of these viruses and point out the areas where change has occurred. Nomenclature and Terminology The virus-specific polypeptides of EMC virus, HRV-lA, and poliovirus were named by different laboratories each using a different nomenclature (Butt.erworth et al., 1971; McLean and Rueckert, 1973; Summers et al., 1965). The EMC noncapsid polypeptides have been designated with capit’al letters and the virion polypeptides have been assigned Greek letters. The HRV-1A polypeptides have been designated by their apparent molecular weight relative to the EMC polypeptides (i.e., polypeptide 92 has an apparent molecular weight of 92,000). The poliovirus polypeptides have been designated by numbers with the prefix NCVP (noncapsid viral polypeptide) or VP (virion polypeptide). In addition, the nomenclature has become further complicated because the discovery of new polypeptides in the EMC- and poliovirus patterns has resulted in their being named with imaginative, confusing new letters or subscript’s. Several new poliovirus-specific polypeptides are reported here. They have been named within the context of the poliovirus nomenclature. However, the NCVP prefix has been dropped. It should be noted that the poliovirus nomenclature for the minor peaks may differ from earlier reports because it is hard to be certain that the different patterns from different laboratories have been labeled in a consistent manner. The “primary products” are defined as those polypeptides released from the poly-

ribosome. The giant uncleaved polypeptide resulting from complete translation of the viral RNA would have a molecular weight of 220,000-260,000 daltons and has been termed “polyprotein.” However, it is possible that the pattern of primary products may change throughout the course of the infection (Kiehn and Holland, 1970). It is not known if the smaller primary products are generated by cleavage of the growing polypeptide chain or by some other release mechanism. Some of the primary products are precursor molecules which undergo proteolytic cleavages to become smaller stable polypeptides. Studies on the kinetics of synthesis of EMC viral-specific polypeptides indicate that in some cases a specific cleavage may occur either on the growing polypeptide or as part of the “maturation” cleavage of a precursor. Thus, a fract,ion of the time a polypeptide may be released by a “nascent cleavage” as a primary product, while the remainder of the time it is generated by a “posttranslational” maturation cleavage. MATERIALS

AND

METHODS

Cells and virus. The source and propagation of HeLa cells (“rhino HeLa cells”), HRV-1A (strain 260), and poliovirus type 2 (strain 712-Ch-2ab) have been described (Lonberg-Holm and Koran& 1972). The source of EMC virus has been described (Butterworth et ah, 1971). Materials. The composition of the dialysis buffer, the 10X sodium dodecyl sulfate (SDS) solubilizing solution, medium AL, medium AH, and the scintillation solvent tT21 have been described (Butterworth et al., 1971). Pactamycin was a gift of Upjohn laboratories. A stock solution of 1 X 10B5 M pactamycin in 1 mM acetic acid was stored frozen and used throughout these experiments. The L-3H-amino acid mixture and n-r4C-amino acid mixture each containing 15 amino acids were purchased from New England Nuclear Corporation. Methods. Infection of HeLa cells with picornaviruses in the presence of actinomytin D results in the inhibition of host protein synthesis. The addition of radioactive amino acids at t,he appropriate time allows

PICORNAVIRAL

the incorporation of label almost exclusively into the virus-specific proteins. The entire cell then can be solubilized and the viral polypeptides analyzed by SDS-polyacrylamide gel electrophoresis. The infection procedure has been described (Butterworth et al., 1971) and was used for all three viruses with the exception that the HRV-lAinfected HeLa cell suspension was cultured at 34” instead of 37”. The procedures for pulse-chase and progressive labeling experiments, pactamycin mapping, and SDSpolyacrylamide gel electrophoresis have been described (Butterworth and Rueckert, 1972a, b). Gels were 26 cm long and contained 10% acrylamide and 0.3% (v/v) ethylene diacrylate. The pH of the electrophoresis buffer was 7.0. The gels were fractionated using a Gilson Gel Fractionator (Gilson Medical Electronics, Middleton, Wisconsin). One-millimeter segments of crushed gel in 0.3 ml water were collected into 0.7 ml of 0.1 N NaOH in scintillation vials, followed by incubation at room temperature for at least 0.5 hr. Then 10 ml of scintillation solvent tT21 was added to each. The vials were capped, allowed to stand overnight, and mixed using a Vortex stirrer; 100% f 5 % of the applied radioactivity was recovered from the gels by this procedure. The vials were counted using a liquid scintillation spectrometer equipped with a punched paper tape output. The data were analyzed using a PDP-10 computer (Digital Equipment Corporawere tion) , and the electropherograms plotted using a Calcomp plotter equipped with a liquid ink pen. The gel patterns presented are phot’ographs of the actual computer output. RESULTS

Relative Molecular peptides

Weight of th
Poly-

Figure 1 shows the profile of virusspecific polypeptides produced in EMC virus, HRV-lA, and poliovirus infected cells. In each case the whole cell extracts have been co-electrophoresed with the corresponding differentially labeled virions. These patterns allow the identification of the virion polypeptides within each profile

POLYPEPTIDES

441

and illustrate the similarities in the polypeptide composition of the different viruses. The SDS-polyacrylamide gel technique separates polypeptides on the basis of molecular weight (Dunker and Rueckert, 1969). In order to compare the size of the viral polypeptides, extracts were prepared from cells infected with each of the three different viruses and each differentially labeled extract was coeleetrophoresed with the other. The resulting patterns are shown in Fig. 2. The apparent molecular weights of the EMC viral polypeptides are listed in Table 1. The apparent molecular weights of the HRV-1A polypeptides relative to the EMC viral polypeptides have been calculated (McLean and Rueckert, 1973) and are listed in Table 2. Table 3 shows the apparent molecular weights of the polioviral polypeptides relative to the EMC viral polypeptides. The molecular weights of the corresponding virion polypeptide are similar for each of the three viruses. The sum of the molecular weights of the four EMC virion polypeptides is 96,000 daltons. The sum of the corresponding HRV-1A virion polypeptides (98,000) is slightly larger and the sum of the molecular weights of the four poliovirus virion polypeptides (102,000) is, in turn, slightly larger still. Consistent with the above, the EMC capsid precursor (polypeptide B, MW 90,000) migrates slightly ahead of the HRV-1A capsid precursor (polypeptide 92, MW 92,000), which, in turn, migrates slightly ahead of the poliovirus capsid precursor (polypeptide la, MW 95,000) (Fig. 2). It should be noted that in each case the apparent molecular weight of the capsid precursor is about 6000 daltons below the sum of the appa,rent molecular weights of the four constituent virion polypeptides. Polypeptide Dl of EMC is a proposed intermediate in the formation of the capsid polypeptides. Polypeptides 67 and 3a in the HRV1A and poliovirus extracts have about the same apparent molecular weight as Dl. Polypeptide F is a stable primary product of MW 38,000 which is produced during translation of the EMC viral RNA. The HRV-1A polypeptide 38 (MW 38,000) and

442

BUTTER WORTH

0.751

I

0.50-l

tnn

0.254

II

llY+lI

II II I I c7 --

0.00

II

V”

I

““”

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4.5

C

4.0 r

vrL !I

!

2.5 4

02

0’2

0‘a

RELAilVE

0’5 DISTANCE

FIG. 1

MIGRATED

PICORNAVIRAL TABLE

1

MOLECULAR

WEIGHT AND MOLAR RATIO EMC VIRAL POLYPEPTIDES~

Polypeptide

A B C D Dl D2 E ; s” L H I 6 A+B+a F C+D+E

OF

Apparent Molar ratio Molar ratio molecular following a following a 6-min pulse lo-min pulse weight 80-min chase 100,000 90,000 84,000 75,000 65,000 59,000 56,000 40,000 38,000 34,000 30,000 23,000 16,000 12,000 11,000 9,000

0.80 0.09 0.29 0.29 0.47 1.00 0.20

1.09 1.00 1.05

0.04 0.72 0.50 1.00 1.00 0.32 1.27 1.06 0.70 1.32 0.27 1.00 1.00 0.76

a All values are from Butterworth et al. (1971) and Butterworth and Rueckert (1972a). the poliovirus polypeptide X (MW 37,000) are about the same size as F. The C, D, E family of EMC polypeptides have molecular weights of 84,000,75,000, and 56,000, respectively. Polypeptides 84,76, and 55 (MW 84,000, 76,000, and 55,000) in the HRV-1A pattern and polypeptides lb, 2, and 4 (MW 85,000,77,000, and 57,000) in the po-

443

POLYPEPTIDES liovirus and E.

pattern

are similar

in size to C, D,

Molar Ratios of the Viral Polypeptides The analytical techniques used here allow the complete recovery and quantitation of each of the radiolabeled viral polypeptides. This information in conjunction with the molecular weights allows the calculation of the relative molar amount of each of the viral polypeptides following any given labeling period. After a brief labeling period most of the radioactivity is found in the large, primary products. Some of these primary products are precursor polypeptides. Their breakdown into smaller, stable

chains can be observed by transferring the infected, labeled cells into medium free of radioactive amino acids and following the change in the profile of the radiolabeled polypeptides. The single initiation site hypothesis requires that each primary product, like the stable chains derived from them, be produced in equimolar amounts. Values very close to the predicted one-to-one ratios were found for the primary and stable EMC viral polypeptides (Table 1). The molar ratios of the primary and stable HRV-1A polypeptides are listed in Table 2. The molar ratios of the primary and stable poliovirus polypeptides are listed in Table 3. Assuming a cleavage pattern for HRV-1A and poliovirus similar to that of EMC, there is roughly an equimolar production of primary and stable polypeptides for both HRV-1A and polio-

FIG. 1. Identification of the virion polypeptides in the complete profiles of virus-specific polypeptides. In this and subsequent experiments, HeLa cells were infected with 200 plaque-forming units of virus per cell and cultured at 4 X lo6 cells/ml at 37’ (the HRV-1A infected cell suspension was cultured at 34”) in medium AL containing 5 rg/ml of actinomycin D as described (Butterworth et al., 1971). (a) At 3 hr 45 min postinfection an EMC virus-infected cell suspension was exposed to 100 &i/ml of a 3Hamino acid mixture. Forty minutes thereafter, a l-ml sample was removed, added to 0.1 ml of 10X solubilizing solution (1070 SDS, 5 M urea, 1% 2-mercaptoethanol), and immediately heated for 5 min in a boiling water bath. After dialysis 20 ~1 of the whole cell, extract was added to 30 ~1 of a suspension of purified EMC virus that had been labeled with r4C-amino acids. The suspension was made 1% in SDS and 0.1% in 2-mercaptoethanol, then incubated at 95” for 1 min. The mixture was subjected to SDS-polyacrylamide gel electrophoresis at 8 ma/tube for 20 hr. In this and subsequent figures the anode is to the right. All gels were 26 cm long. (b) In a similar manner 2 ml of an HRV-lA-infected cell suspension was labeled from 4.0 to 5.0 hr postinfection with 50 &i/ml of a 3H-amino acid mixture. The sample was coelectrophoresed with purified, SDS-disrupted, r4C-labeled HRV-1A virions. (c) Using the same techniques, a poliovirus-infected cell suspension was labeled from 3.5 to 4.0 hr postinfection with 12 rCi/ml of a ‘%-amino acid mixture and coelectrophoresed with purified, SL)S-disrupted, 3H-labeled polio virions.

444

BUTTERWORTH

Y

nb

3c

2

3.0-

POLIO-

x B

2.5-

“= T I J

2.0-

“0

1.5-

/

I--EMC

x

RELATIVE

DISTANCE

FIG. 2

MIGRATED

PICORNAVIRAL TABLE

2

MOLECULAR WEIGHT AND MOLAR RATIO O‘RHRV-1A POLYPEPTIDES

Polypeptide”

92 84 76 67 60 55 47 c + 39 38 a (35,000) B (30,000) Y GWJW 24 14 13 6 (8000) 92 + a 47 + 38 84 + 76 + 55

Molar ratio following an [%min pulse*

Molar ratio following a 20-min pulse SO-min chase

0.88 0.79 0.67

0.04 0.03 0.23 0.03 0.01 0.18 0.48 0.96 0.52 0.77 0.27 0.84 0.24 1.39 0.72 0.20 0.81 1.00 0.44

0.05 0.60 0.40 0.11

0.99 1.00 1.51

= Molecular weights are relative to the EMC viral polypeptides (McLean and Rueckert, 1973). b Values were calculated from the control gel shown in Fig. 3a. The mass of protein was assumed to be proportional to cpm. The molar ratios were calculated by dividing the per cent of total viral radioactivity in each peak by its apparent molecular weight (McLean and Rueckert, 1973) and normalizing with respect to the sum of the 47 + 38 polypeptides. c Each value is the average from two independent experiments that gave similar results. The patterns were as that shown in the control gel in Fig. 3b. The average deviation from the mean values shown was 0.03, and the maximum deviation (polypeptide 7) was 0.15.

445

POLYPEPTIDES

virus. Deviations from the one-to-one ratios, along with possible explanations will be discussed later. Pactamycin Mapping The theory and procedures for pactamytin mapping have been discussed in detail (Butterworth and Rueckert, 1972a). The results of pactamycin mapping with EMC virus-infected cells indicate that the primary products are ordered on the viral RNA 5’ + 3’ A-F-C and that the stable products are ordered S-/?-y-a-G-1-F-H-E. The intermediate chains B and E map in the capsid region and the intermediate chain D maps in the E region (Butterworth and Rueckert, 1972a). A similar order has been obtained for the HRV-1A polypeptides (McLean and Rueckert, 1973). Figure 3 shows that the normal patterns of both the primary and stable, radiolabeled HRV-1A polypeptides are dramatically changed if the polypeptides are labeled during the runoff period. The appropriate calculations yield the complete pactamycin map for HRV-1A (Fig. 4). With the exception of polypeptide 55, this confirms the order found by McLean and Rueckert. The values obtained indicate the order of the primary products on the HRV-1A RNA to be 5’ + 3’ 92-47-38-84. The order obtained for the stable polypeptides is 6-@-r-a-24-14-38-47-55. The intermediate polypeptide 76 mapped near the 3’ end with polypeptide 84. The relatively large molar amount of e indicates the presence of a comigrating polypeptide (polypeptide 39). The map position of this peak is, therefore,

an average

of the two.

Similar experiments in which there was a

FIG. 2. Comparison of the profiles of virus-specific polypeptides from EMC virus, HRV-lA, and poliovirus-infected cells. The whole cell extracts were labeled, prepared, dialyzed, and electrophoresed as described in Materials and Methods and in legend to Fig. 1. (a) EMC virus-infected cell suspension labeled with a 3H-amino acid mixture for 40.0 min beginning 3 hr 45 min postinfection (solid line), coelectrophoresed with an HRV-lA-infected cell suspension labeled with a ‘%-amino acid mixture for 60.0 min beginning 4 hr 5 min postinfection (dashed line). (b) HRV-lA-infected cell suspension labeled with a ‘%-amino acid mixture for 60.0 min beginning 4 hr 5 min postinfection (solid line), coelectrophoresed with a poliovirus-infected cell suspension labeled with a 3H amino acid mixture for 20.0 min beginning 3 hr 30 min postinfection (dashed line). (c) Poliovirus-infected cell suspension labeled with a 1% amino acid mixture for 20.0 min beginning 3 hr 30 min postinfection (solid line), coelectrophoresed with an EMC virus-infected cell suspension labeled with a 3H-amino acid mixture for 40.0 min beginning 3 hr 45 min postinfection (dashed line).

BUTTEBWORTH

446 TABLE MOLECULAR WEIGHT OF POLIOVIRUS

Polypeptide la lb 2 3a 3b 4 5a 5b VP0 x + VP1 6b VP2 VP3 7a 7b 7c 7d 8 9a 9b 10 VP4 la + 3a x f VP1 lb+2+4

Molecular weight” 95,000 85,000 77,000 70,000 65,000 57,000 50,000 47,000 42,000 37,000 34,000 31,000 26,000 24,000 23,000 22,000 16,000 14,000 13,000 11,000 10,000 8,000

3

AND MOLAR POLYPEPTIDES

Il.4~10

8-min 20-min pulse pulseh SO-minchase” 0.34 0.42 0.73 0.26 0.29 0.16

1.40

0.04 0.16 0.15 0.73 0.12 0.64 2.00 0.32 0.12 1.01 0.19 0.86 0.18 0.78 0.83 0.75 1.60

0.60 1.40 1.31

0.15 2.00 0.93

a The apparent molecular weight of each polypeptide was calculated from its mobility on SDSpolyacrylamide gels relative to the EMC virusspecific polypeptides (Butterworth el al., 1971). Differentially labeled EMC virus and poliovirus extracts were coelectrophoresed and produced the pattern shown in Fig. 2c. A straight line was fitted by regression analysis to a plot of log molecular weight vs mobility. The values shown are the average of 5 different experiments which gave practically identical results. b Values were calculated from the control gel shown in Fig. 5a, as described in Table 2. The molar ratios were normalized to 2.00 for the sum of X + VPl. c Each value is the average from two independent experiments that gave similar results. The patterns were as that shown in the control gel in Fig. 5b. The average deviation from the mean values shown was 0.03 and the maximum deviation (polypeptide 10) was 0.12.

delay between the addition of pactamycin and the addition of the radioactive amino acids confirmed that polypeptide 92 maps nearest the initiat’ion site, polypeptides

47 and 38 map together near the center of the RNA and polypeptides 84, 76, and 55 map together nearest the 3’ end of the RNA. There are several peaks in the HRV-1A pattern that are missing in the EMC profile (Fig. 1). Polypeptide 129 probably represents a cleavage intermediate between polyprotein and the 92, 47, 84 level. The HRV-IA profile is similar to the EMC virus profile in that there arc only 3 or 4 peaks present that are smaller than y. The pactamycin map for t’he primary and intermediate poliovirus polypeptides has been shown to be 5’ + 3’ la-X-2 (Taber et al., 1971; Summers and Maizel, 1971). The order of the capsid polypeptides within the capsid precursor is VP4-VP2VP3-VP1 (Rekosh, 1972). This order was confirmed and the remaining polypeptides were mapped in the experiment described in Fig. 5. The normal distributions of radioactivity in both the primary and stable profiles of poliovirus-specific polypeptides are changed if the polypeptides are labeled during the runoff period (Fig. 5). The complete poliovirus pactamycin map calculated from Fig. 5 is shown in Fig. 6. Once again, several features of the poliovirus pattern were found to be analogous to those for EMC virus and HRV-1A. The order of the primary products on the polioviral RNA is 5’ -+ 3’ la-X-lb. The capsid polypeptides along with the intermediate polypeptides 3a and VP0 all map together with la nearest the initiation site. The intermediate polypeptide 2 and the stable polypeptide 4 map together with lb nearest the 3’ end of the RNA. Kinetics of Cleavage Detailed studies on the rates of cleavage of the EMC viral precursor polypeptides have helped establish precursor-product relationships and have provided information as to the mechanism and kinetics of synthesis of the viral polypeptides (Butterworth and Rueckert, 197213). Similar kinetics studies have been carried out with HRV-IA (McLean and Rueckert, in preparation). Figure 7 illustrat’es the change in the profile of poliovirus-specific polypeptides in a pulse-chase experiment as the pre-

PICORNAVIRAL

4 17

POLYPEPTIDES

700. lb

“0ti

0:2

013 RELATIVE

0:4

015

DISTANCE

016

0:7

018

0:9

I :3

MIGRATED

FIG. 3. Elfect of pact.amycin on the distribubion of radioactivity incorporated into the virus-specific polypeptides of HRV-1A. (a) At 4 hr 2 min postinfection, 2 ml of an infected cell suspension was removed Eight minutes thereafter the suspension was and exposed to 12 pCi/ml of a i4C amino acid mixture. solubilized in hot 1% SDS as described for Fig. 1. At 4 hr 3 min postinfection, an equivalent sample from the same suspension was exposed to 2 X lo-’ M pactamycin for 4 min, labeled for 8 min with 12 &i/ml of the same W-amino acid mixture, and solubilized as the control. Both samples were dialyzed overnight, concentrated about 2X by dialysis against Ficoll, and electrophoresed as described in Materials and Methods. The two extracts were electrophoresed separately, and the electropherograms have been superimposed for comparison. Control, 8 min pulse (solid line) ; pactamycin, 4 min delay, 8 min pulse (dashed line). (b) At 4 hr postinfection, a 2-ml sample from an HRV-1A cell suspension was removed and exposed simultaneously to 2 X 10e7 M pactamycin and 12 &i/ml of a ‘%-amino acid mixture. After a 20.0-min incubation, 50 ml of medium AH containing 2 X 1W7 M pactamycin was added to the suspension, and the cells were immediately sedimented and suspended in 8 ml of fresh, prewarmed medium AH free of drug and isotope. The total incubation in medium AH was continued for 80 min at 34”. The cells were sedimented, suspended in 0.5 ml water, solubilized, and dialyzed as described for Fig. 1. An identical control sample was taken simultaneously from the original infected cell suspension and treated in the same way, except that all solutions were free of pactamycin. The two samples were electrophoresed separately, and the electropherograms have been superimposed here for comparison. Control, 20 min pulse, 80 min chase (solid line) ; pactamycin, 20 min pulse, 80 min chase (dashed line).

448

BUTTERWORTH

c+39 -6 as gg c, 13 24/m7 5 S+a~ se y 300 [ I I . IS’%+0 aNm 00 I.0 ,‘,‘_\ ;-i? -, 3 PACTAMYCIN ,’ CONTROL ’ FIG. 4. Gene sequence of HRV-1A polypeptides as determined by the pactamycin mapping technique. The amount of radioactivity in each peak in Fig. 3 was calculated as the percent relative to the total radioactivity recovered from all viral peaks on a gel. The ratio of this percentage in the pactamycin-treated extract relative to the value of its corresponding peak from the control extract was calculated. This pactamycin-control ratio is a reflection of the relative position of the polypeptide on t,he viral mRNA. In the above graph, the viral polypeptides have been ordered from left to right according to increasing values of the pactamycin-cont,rol ratio so that the 5’ end of the RNA is to the left. Polypeptides 60 and 67 each contained less than 3y0 of the total viral cpm, and their positions should be considered tentative. Accurate measurements of the amount of polypeptide 6 present could not be made in this experiment; its position to the far left of the map is based on other observations (McLean and Rueckert, 1973).

cursor polypeptides cleave to generate the smaller stable chains. In this system t’here is an extremely rapid cleavage of the capsid precursor polypeptide la. After a 15-min pulse followed by a 15-min chase the radiolabeled la is completely gone (Fig. 7b). This rapid cleavage is not reflected by a corresponding rapid production of the capsid polypeptides. Instead, radioactivity piles up in intermediate polypeptides such as 3a (polypeptide 3a is the size and has a map position of a VPO-VP3 intermediate). Polypeptide 2 cleaves relatively slowly, and there is a corresponding slow increase in the production of polypeptide 4. The poliovirus profile differs from the other two in that it is very complicated in the lO,OOO-20,000 dalton molecular weight range. Polypeptides 5a, 5b, 610, 7a,

S, Ya, and 10 are all present’ following a IO-min pulse (Fig. 7). After a 15.min pulse followed by a 15-min chase, it can be seen that the amounts of 5a and 7a arc decrtasing. Polypeptides 7c and 9b appear, and the 6b and 10 peaks continue to grow. By 60 min of chase, 5a and 7a are practically gone and 7d has appeared. The 20-min pulse 80-min chase pattern shoxs the presence of polypept,ide 7b (Fig. 5). Such activity is either lacking in EMC virus and HRV-lA-infected cells or it is taking place very rapidly and degradation continues on to small fragments. In all three virus patterns, various amounts of low molecular weight, nondialyzable material is observed migrating near the front. This is especially pronounced in HRV-1A extracts after a long chase and may be the result of extensive degradation (Fig. 3). DISCUSSION

Caps&Related Polypeptides The native virions of EMC virus, HRVIA, and poliovirus are not antigenically relat’ed. However, architectural restrictions imposed during their evolution have resulted in the retention of several common features, including the number, approximate size, and order of the capsid polypeptides within the capsid precursor. The analogous virion polypeptides of EMC virus, HRV-lA, and poliovirus are shown in Fig. 8. Observations on the biosynthesis of the virion polypeptides of the three viruses studied here indicate that the overall mechanism of capsid precursor synthesis, cleavage, and assembly also has been conserved in their evolution. In each case t.here exists a capsid precursor whose gene locus is locat,ed nearest the single, ribosomal initiation site on the viral RNA. There is some evidence that there may be a lead-in peptide sequence before the coding of the actual capsid pol_ypeptide begins (Oberg and Shatkin, 1972). In the case of EMC virus the capsid precursor, polypeptide A, may contain a short noncapsid sequence (possibly the lead-in sequence). It has been proposed that polypeptide A cleaves via an intermediate (B) which is in turn rapidly cleaved to generate the capsid polypeptides

PICORNAVIRAL

449

POLYPEPTIDES

b

P x 4I

3.0

%

2.5-j

:

i I

XJLVPI

II

II t 1

F

VP3

t

0.5

RELATIVE

DISTANCE

MIGRATED

5. Effect of pactamycin on the distribution of radioactivity incorporated into the virus-specific polypeptides of poliovirus. The procedure used was as described in Fig. 3 except that the labeling of the poliovirus-infected suspension was initiated at 3 hr 30 min postinfection with 90 &X/ml of a 3H-amino acid mixture and the pactamycin concentration used was lo-’ M. In addition, the pulse-chase samples were loaded directly on the gels without dialysis. (a) Control, 8 min pulse (solid line); pactamycin, 4 min delay, 8 min pulse (dashed line). (b) Control, 20 min pulse, 80 min chase (solid line) ; pactamycin, 20 min pulse, 80 min chase (dashed line). FIG.

(Butterworth and Rueckert, 197213). The poliovirus capsid precursor (la) and the analogous HRV-1A polypeptide (92) migrate faster on SDS gels than the EMC capsid precursor (A). This was unexpected because the sum of the molecular weights of the four individual poliovirus and HRV1A capsid polypeptides exceeds those of EMC virus. Thus, on the basis of molec-

ular weight it appears that B, 92, and la are the analogous capsid precursor polypeptides for EMC virus, HRV-IA, and poliovirus, respectively. Traces of polypeptides about the size of polypeptide A can be found in the HRV-1A and poliovirus patterns. It is not known if these are shortlived precursors comparable to the EMC viral polypeptide A.

450

BUTTERWOIITH

1 0.0

5'

I IO

PACTAMYCIN/CONTROL

I

29

3

FIG. 6. Gene sequence of poliovirus polypeptides as determined by the pactamycin mapping technique. The relative position of each viral polypeptide on the viral RNA was calculated from the gels in Fig. 5 as described in Fig. 4. Each value in the pulse-chase section is the average from two independent experiments that gave similar results. The average deviation from t,he mean values shown above is 0.04. The maximum deviation (polypeptide 2) is 0.16. Polypeptides VP2, 5a, 5b, 7a, 7b, and 7d each contained less than 37, of the total viral cpm, and their positions should be considered t,entative. Accurate measurements of the amount of polypeptide VP4 could not be made in t,his experiment, and its position to the far left of the map is based on other observations (Rekosh, 1972).

The EMC virus polypept’ide Dl is about the size of an c-y polypeptide and may represent this intermediate in the cleavage of the capsid precursor. ,4 peak of about this same size can be detected in extracts from HRV-IA (polypeptide 67) and poliovirus (polypeptide 3a) infected cells. In the case of poliovirus relatively large amounts of this unstable polypeptide are generated. The results of pactamycin mapping indicate that 3a does map with la nearest the initiation site (Fig. 6). This

is consistc>nt with its assignment as thr VPO-VP3 intermediate. In a similar way, the D2, 60 and 3b polyprpt,ide of E1\IC virus, HRV-IA, and poliovirus may crnwspond to a y-or (VP3-VPl) cleavage intermediate. However, t’he mapping results are not as clear in this case. Polypeptide cleavage rates can be measured by following the disappearance of a polypeptide in a pulse-chase experiment or by measuring its steady-state concentration in a progressive-labeling experiment (Buttarworth and Rueckert, 197213). The individual rates of cleavage differ greatly for the different capsid precursors. The rapid disappearance of the polio precursor la in a pulse-chase experiment (Fig. 7), and its relative low level following a 20-min pulse (Fig. 2) indicates that under these conditions of infection it is cleaving much more rapidly hhan the capsid precursor of EMC virus (polypepbide A), which in turn is cleaved more rapidly than polypeptide !3L of HRV-IA. Similar analysis of all the various capsid precursors shows that the rate-limiting step in the generation of the capsid polypeptides to the level of E, y, a! is at the cleavage of A, 92, and 3a for EAIC virus, HRV-IA, and poliovirus, rrspectively. The final maturation cleavage (E -+ 6 + /3, VP0 + VP4 + VP2) is common to the assembly of all three viruses. Polypeptides An,alogous to F Polypeptide F is one of the primary products generated by translation of the EMC viral RNA. It is a stable polypeptide of molecular weight 37,000 daltons whose gene locus is near the middle of the genome.

FIG. 7. Change in the profile of radiolabeled poliovirus-specific polypeptides during a pulse-chase experiment. (a) At 3 hr 30 min postinfection a HeLa cell suspension was exposed to 12 &i/ml of a lnCamino acid mixture. Ten minutes thereafter 0.9 ml was withdrawn, added to 0.1 ml 10X solubilizing solution, immediately heated for 5 min in a boiling water bath, dialyzed, and electrophoresed as described (10 min pulse). (b) At 3 hr 41 min postinfection, 5 ml of the original, infected cell suspension containing the %-amino acids was sedimented for 3 min at 1500 rpm in a clinical centrifuge. The chase period was initiated at 3 hr 45 min postinfection by suspending the cells in 20 ml of medium AH which had been prewarmed to 37”. At 3 hr 55 min postinfection, 6 ml of the infected, labeled suspension was sedimented and suspended in 0.5 ml water, and at 4 hr postinfection it was solubilized with 10X solubilizing solution as described (15 min pulse, 15 min chase). (c) At 4 hr 40 min postinfection an additional 6 ml was removed and t,reated as the previous sample (15 min pulse, 60 min chase).

PICORNAVIRAL

:Q

POLYPEPTIDES

0.75

x k 0

0.50

t

0.00 2.25 2.00 I .75 1.50 b M

I .25

z

I.BB

I

0 0.75 0.50 0.25 0.00 2.0G

I .75

1.50

n

I .K

b s I.00 it 0 f

8.75

8.50

8.25

0.e0

RELATIVE

DISTANCE

FIG. 7

MIGRATED

451

BUTTERWORTH

452 EMCVIRUS A

92 Iik

-CL aa

~'-.Yk

HRV-IA 47 3884

67L

E

L i%

FIG. 8. Common features in the biosynthesis of lA, and poliovirus. On the basis of molecular weight of the patterns of viral protein biosynthesis were peptides are shown in the same relative positions. of the corresponding gene locus on the viral RNA position represents precursor-product relationships proportional to molecular weight.

Polypeptide 38 in the HRV-IA profile is about the same size, maps in the same region of the genome, and seems to be analogous to F. Consistent with the single initiation site hypothesis, it can be shown that the primary products generated by translation of the EMC viral RNA are produced in approximately equimolar amounts (after correcting for post-translational cleavage) (Table 1). However, after a short pulse, t’he amount of 38 present is lower than the corrected amount of 92 (92 + a! = 0.99; 38 = 0.40) (Table 2). Polypeptide 47, which is also prominent in the HRV-1A profile, is missing or present only in small amounts in both the EMC virus and poliovirus patterns (Fig. 2). Several lines of evidence suggest that 47 may be an alternative cleavage form of 38: (1) after a brief pulse the molar amount of the capsid precursor and its cleavage products does equal the sum of 47 and 38 (92 + (Y = 0.99; 47 + 38 = 1.00) (Table 2), and (2) polypeptides 47 and 38 always map together (Fig. 4). The poliovirus polypeptide X is about the same size and maps in the same posit,ion as the EMC polypeptide F (Summers and Maizel, 1971; Taber et al., 1971; Fig. 6). Although measurements involving X are complicated because it remains unresolved from VPl, it appears that X and F are comparable. Polypepticles Analogous to C, D, ancl E The EMC viral polypept.ide C maps nearest the 3’ end of the viral RNA. C cleaves to produce D which in turn cleaves to produce E. Kinetic studies indicate that although C can be kanslated in the intact

POLIWIRUS

76

As-

lo Al!PL

XQVE VW!&?2

X

lb

2 4

the virus-specific polypeptides of EMC virus, HRVand position on the genetic map, the above portions the same for these three viruses. Analogous polyThe lateral position represents the relative location (the 5’ end of the RNA is to the left). The vertical or alternative cleavage forms. Line lengths are

form, most of the time nascent cleavages occur generating D or E as primary products. The HRV-1A polypeptides 84, 76, and 55 map together near the 3’ end of the RNA, are similar in molecular w-eight and, thus, appear to be analogous to the EMC polypeptides C, D, and E. After a short pulse the molar amount of 84 + 76 + 55 (l.cil) exceeds the amount of 92 and its cleavage products (0.99) (Table 2). However, after post-translational cleavages the molar amount of 84 + 76 + 5.5 (0.44) is lower than the amount of the capsid family (0.81) (Table 2). This indicates the possible presence of an unstable comigrating polypeptide and further unrecognized cleavages. The poliovirus polypeptidrs lb, 2, and 4 also map together near the 3’ end of the RNA and have molecular weights similar to the EMC virus polypept’ides C, D, and E (Fig. 6). Furthermore, the relat’ive slow cleavage of 2 and corresponding slow appearance of 4 is reminiscent of the slow D + E conversion in EMC virus-infected cells. As was the case with HRV-lA, following a short pulse the relative molar amount of lb + 2 + 4 is higher than would be predicted (capsid + X = 2.00, lb + 2 + 4 = 1.31) (Table 3). However, the molar ratio of the stable polypeptides is close to Ohe one-to-one ratio predicted (X + VP1 = 2.00; lb + 2 + 4 = 0.93). Again, it seems as though there is an unstable comigrating polypeptide present. Conclusion

Evolutionary pressures on the picornaviruses have resulted in extensive differences in the antigenicity of the virions and in the size and number of many of the

PICORNAVIRAL

smaller viral polypeptides. However, as judged by these studies with EMC virus, HRV-lA, and poliovirus, the general mechanism of synthesis, size, and genetic order of the three main families of polypeptides remain the same (Fig. 8). ACKNOWLEDGMENTS I would like to thank C. McLean and R. R. Rueckert for helpful discussions, R. Z. Lockart, Jr. for support and valued advice, K. K. LonbergHolm for supplying radiolabeled virus, and Sadie Massado for excellent technical assistance. REFERENCES BUTTERWORTH, B. E., and RUECKERT, R. R. (1972a). Gene order of encephalomyocarditis virus as determined by studies with pactamycin. J. Viral. 9, 523-828. BUTTERIVORTH, B. E., and RUECKERT, R. R. (1972b). Kinetics of synthesis and cleavage of encephalomyocarditis virus-specific proteins. virozogy 50, 535-549. BUTTERWORTH, B. E., HALL, L., STOLTZFUS, C. M., and RUECKERT, R. R. (1971). Virusspecific proteins synthesized in encephalomyocarditis virus-infected HeLa cells. Proc. Nat. Acad. Sci. U.S. 68, 30833087. DUNKER, A. K., and RUECKERT, R. R. (1969). Observations on molecular weight determinations on polyacrylamide gels. J. Biol. Chem. 244, 50745080. FENNER, J. (1968). The classification and nomenclature of animal viruses. In “The Biology of Animal Viruses,” pp. l-33. Academic Press, New York. HOLLAND, J. J., and KIEHN, E. D. (1968). Specific cleavage of viral proteins as steps in the synthesis and maturation of enteroviruses. Proc. Nat. Acad. Sci. U.S. 60, 1015-1022. JACOBSON, M. F., and BALTIMORE, D. (1968). Polypeptide cleavages in the formation of poliovirus proteins. Proc. IVat. Acad. Sci. U.S. 61, 77-84.

POLYPEPTIDES

453

JACOBSON, M. F., ASSO, J., and BALTIMORE, D. (1970). Further evidence on the formation of poliovirus proteins. J. Mol. Biol. 49, 657-669. KIEHN, E. D., and HOLLAND, J. J. (1970). Synthesis and cleavage of enterovirus polypeptides in mammalian cells. J. Viral. 5, 358-367. KORANT, B. D. (1972). Cleavage of viral precursor proteins in vivo and in vitro. J. Viral. 10, 751-759. LONBERG-HOLM, K., and KORANT, B. D. (1972). Early interaction of rhinoviruses with host cells. J. Viral. 9, 29-40. MCLEAN, C., s.nd RUECKERT, R. R. (1973). Picornaviral gene order: comparison of a rhinovirus with a cardiovirus. J. Viral. 11, 341-344. MAIZEL, J. V., JR., and SUMMERS, D. F. (1968). Evidence for differences in size and composition of the poliovirus-specific polypeptides in infected HeLa cells. Virology 36, 48-54. ~BERG, B. F., and SHATKIN, A. J. (1972). Initiation of picornavirus protein synthesis in ascites cell extracts. Proc. Nat. Acad. Sci. U.S. 69, 3589-3593. REKOSH, D. (1972). Gene order of the poliovirus capsid proteins. J. Vi~ol. 9, 479487. RUECKERT, R. R. (1971). Picornaviral architecture. In “Comparative Virology” (K. Maramorosch and E. Kurstak, eds.), pp. 255306. Academic Press, New York. SUMMERS, D. F., and MAIZEL, J. V., JR. (1968). Evidence for large precursor proteins in poliovirus synthesis. Proc. Nat. Acad. Sci. U.S. 59, 966-971. SUMMERS, D. F., and MAIZEL, J. V., JR. (1971). Determination of the gene sequence of poliovirus with pactamycin. Proc. Nat. Acad. Sci. U.S. 68, 2852-2856. SUMMERS, D. F., MAIZEL, J. V., JR., and DARNELL, J. E., JR. (1965). Evidence for virus-specific noncapsid proteins in poliovirus-infected HeLa cells. Proc. Nat. Acad. Sci. U.S. 5A. 505513. TABER, R., REKOSH, D., and BALTIMORE, D. (1971). Effect of pactamycin on synthesis of poliovirus proteins : a method for genetic mapping. J. Viral. 8, 395401.