VIROLOGY
82, 369-379 (1977)
A Comparison of the Polypeptides of Human and Bovine Respiratory Syncytial Viruses and Murine Pneumonia Virus P. CASH, W . H . WUNNER,' AND C. R. PRINGLE 2 M .R .C . Virology Unit, Institute of Virology, Church Street, Glasgow G11 SJR, Scotland Accepted June 18, 1977 Human and bovine respiratory syncytial (RS) viruses and pneumonia virus of mice (PVM) are morphologically similar viruses and are the only known members of the metamyxovirus (or pneumovirus) group . Comparison of the polypeptides of these viruses by polyacrylamide-gel electrophoresis (PAGE) has shown that the serologically related human and bovine RS viruses have similar polypeptide profiles . However, PVM does not resemble the other two viruses closely . The molecular weights of RS virus polypeptides were established by discontinuous SDS-PAGE in gradient slab gels and were comparable to previous determinations by continuous SDS-PAGE (Wunner and Pringle, 1976), apart from the detection of an additional 10,000-MW polypeptide . Comparison of 11 strains of human RS virus isolated from different localities, or from the same locality at different dates, showed that the relative mobility of VP32 (a nonglycosylated polypeptide of unknown function) was a stable characteristic of each strain . The mobilities of the other viral polypeptides showed little variation . The 11 strains fell into three groups in which the molecular weights of VP32 were estimated to be 31,000, 32,000, and 35,000, but there was no clear correlation with date or place of origin . INTRODUCTION
Human respiratory syncytial (RS) virus was first isolated in 1957 from young children suffering from severe respiratory disease (Chanock et al., 1957) and subsequently has been shown to be an important causative agent of lower respiratory tract disease in young children (Jacobs et al ., 1971) . Bronchiolitis and bronchopneumonia are characteristic features of RS infection in infancy (Holzel et al ., 1963) . Somewhat later a morphologically and antigenically related virus was isolated from cattle with respiratory disease and given the name bovine RS virus (Paccaud and Jacquier, 1970) . Human RS virus is a pleomorphic enveloped virus (Joncas et al ., 1969) and has been reported to contain a single-strand RNA genome with a sedimentation coefficient of approximately 57 S (Zhdanov et al ., 1974) . Melnick (1971) proposed that ' Present address : The Wietar institute, 36th Street at Spruce, Philadelphia, Pennsylvania 19104 . ' Author to whom reprint requests should be addressed.
human and bovine RS viruses and a morphologically similar but antigenically unrelated virus, pneumonia virus of mice (PVM), should be separated from the paramyxovirus group and given separate taxonomic status under the name Metamyxoviridae on the basis of a difference in nucleocapsid diameter and lack of neuraminidase activity . More recently the International Committee on the Taxonomy of Viruses has placed these viruses provisionally in the genus Pneumovirus of the family Paramyxoviridae (Fenner, 1976) . Neither hemagglutination nor hemadsorption activity has been found associated with human RS virus (Richman et al., 1971) or bovine RS virus (Inaba et al., 1970) but both activities have been found with PVM (Compans et al ., 1967) . Since the homogeneity of the group can be questioned we have compared the electrophoretic properties of the polypeptides of bovine RS virus and of PVM with those of human RS virus to substantiate further the inclusion of these viruses within the same taxonomic grouping . 369
Copyright 0 1977 by Academic Press, Inc . All rights of reproduction in any form reserved .
ISSN 0042-6822
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CASH, WUNNER, AND PRINGLE
A minimum of six virus-specified polypeptides have been described for the RSN2 strain of human RS virus, which range from 25,000 to 48,000 in molecular weight (plus a possible additional polypeptide of approximately 200,000 MW), with a single major glycopolypeptide of 48,000 MW and a minor glycopolypeptide of 42,000 MW (Wunner and Pringle, 1976) . However, Levine (1977) has reported seven polypeptides of 20,000-80,000 MW for the Long strain of human RS virus and has resolved three glycopeptides of MW 79,000, 56,000, and 22,000 . It has been necessary, therefore, to compare several different cloned strains of human RS virus, isolated from different geographic localities and also from the same locality at different times, to establish the extent of the variation in the electrophoretic mobilities of the polypeptides of different isolates of human RS virus . MATERIALS AND METHODS
Cells . BHK-21, clone 13, cells, MDBK cells, secondary bovine embryo kidney (BEK) cells, and BS-C-1 cells were maintained in 2-liter roller bottles in medium consisting of Eagle's medium supplemented with twice the normal concentration of amino acids (Glasgow modification) and containing 10% fetal calf serum (virus and mycoplasma screened ; Gibco-Bio-cult, Glasgow) . Human RS virus . Strains of human RS virus were obtained from the sources listed below . The viruses were passaged once or twice in BS-C-1 cells and, as indicated in Table 1, 9 of the 11 strains were cloned by three or four successive isolations from single plaques to produce genetically homogeneous stocks . The RSN-2, RSN-0544, and RSN-1599 strains were obtained from Dr . P. E . Gardner, Royal Victoria Hospital, Newcastle-upon-Tyne . The RSN-2 strain was isolated in 1972 from a child with bronchitis and is the origin of the is mutants described by Faulkner et al . (1976) . RSN-0544 was isolated in December 1974 from lung tissue during autopsy, and RSN-1599 in February 1975 from a child with febrile convulsions . Strains RSS-1 and RSS-2 also originated from
TABLE 1 DESCRIPTION OF STRAINS OF HUMAN
Strain
Cloned
RNS-0544
Yes
RSN-1599
Yes
RSN-2"
Place of isolation
RS
Date
VIRUS
Apparent molecular weight of VP32^
Newcastle, U .K . Newcastle, U .K .
1974 31 x 10'
Yes
Newcastle, U . K.
1972
Long
Yes
1956 35 x 10'
A2`
Yes
RSG-1545
No
RSG-1503
Yes
RSG-1504
Yes
RSG-4988
No
RSS-1
Yes
RSS-2
Yes
Baltimore, U .S .A . Melbourne, Australia Glasgow, U .K . Glasgow, U . K. Glasgow, U . K. Glasgow, U .K . Newcastle, U. K. Newcastle, U . K.
1975 31 x 10'
1961
32 x 10'
35 x 10'
1969 35 x 10' 1972 35 x 10' 1972 35 x 10' 1976 35 x 10' 1976
35 x 10-
1976 35 x 10'
° RSN-2 nomenclature (Wunner and Pringle, 1976) . Wild type and mutants is 2 and is 23 (Faulkner et al ., 1976) . ° Mutants Is Al, is A2, and is A7 (Wright et al ., 1973) .
Newcastle, but were isolated and cloned in human diploid cells (MRC-5) at the M .R .C . Common Cold Research Unit, Salisbury . The following strains were isolated in the Regional Virus Laboratory, Ruchill, Glasgow: RSG-1545 from a child with bronchiolitis in February 1969 ; RSG-1503 from a child with bronchopneumonia in February 1972 ; RSG-1504 from a child with bronchiolitis in February 1972 ; and RSG 4988 from a child with bronchiolitis in March 1976 . The Long strain, isolated in Baltimore in 1956, was obtained from the American Type Culture Collection, and the A2 strain, isolated in Melbourne, Australia, in 1961, was obtained from Dr . R. M .
PNEUMOVIRUS POLYPEPTIDES
371
Chanock, N .I .H ., Bethesda, Md . The is L-["S]Methionine (5 pzCi/ml, 200-300 Ci/ mutants of this strain, which were used in mmol), L-113H]fucose (10 µCi/ml, 3 .3 Ci/ this study were isolated by Wright et al . mmol) or D-fl 3H]glucosamine (10 µCi/ml, (1973) . 1 Ci/mmol) was then added 2 hr later, as Bovine RS virus . Strain BRS-127 and required . All radiochemicals were purBRS-4642 were obtained from Dr . E . J . chased from the Radiochemical Centre, Stott, A .R . C . Institute for Research on An- Amersham, England . Incubation was conimal Diseases, Compton, and cloned in tinued for a further 24-48 hr at 31° until secondary bovine embryo kidney cells by syncytium formation was extensive and three successive isolations from single the cell monolayer had begun to degenerplaques . Strain BRS-127 was isolated in ate . At this time the cultures were harFebruary 1973, and strain BRS-4642 was vested as described below . isolated in March 1976 from the lungs of Harvesting of cultures and partial puria calf which died from pneumonia . Both fication of virus . The cells were scraped strains were known to be free of mucosal into the medium, chilled, and centrifuged disease virus, a frequent noncytopathic for 30 min at 3000 rpm in an MSE Mistral contaminant in bovine cell cultures and centrifuge at 4° . Polyethylene glycol 6000 fetal calf serum (Thomas et al ., 1976) . (PEG) (AnalaR) was added to the clarified Pneumonia virus of mice . Strain 15 of supernatant to give a final concentration PVM, isolated in 1939, was obtained from of 6% (w/v) PEG . The precipitate obtained the American Type Culture Collection . after 24 hr at 4° was collected by centrifuThe stock used in this study had not been gation for 30 min at 3000 rpm in an MSE cloned. Mistral centrifuge at 4° . The supernatant Infectivity assay . The infectivity titers of was carefully decanted and the surface of the human RS strains were determined by the pellet washed with cold 0 .01 M focal plaque formation in monolayers of HEPES, pH 7 .8 . The PEG pellet from one BS-C-1 cells according to the procedure de- 2-liter roller bottle was then resuspended scribed by Faulkner et al . (1976) . A similar in 0 .3 ml of 0 .01 M HEPES, pH 7 .8 . The procedure has been developed for assay of cell pellet was washed twice with high-salt PVM (Cash and Pringle, in preparation) . buffer (0 .18 M NaCl ; 0.01 M Tris-HCI, pH Bovine RS virus plaqued erratically in BS- 7 .4) and then lysed in 6 ml of 0 .5% NP40 ; C-1 cells, hence infectivity titrations were 0 .01 M Tris-HCl, pH 7 .4 ; 0 .1 mM EDTA on carried out on BEK cells . ice for 10 min before storage at -70° .
Conditions of infection and labeling with radioactive isotopes . Just confluent
Preparation of protein samples for polyacrylamide-gel electrophoresis (PAGE) .
monolayer cultures of BS-C-1, BEK, MDBK, or BHK-21 cells in 2-liter roller bottles were infected with virus at an approximate multiplicity of 0 .02-0 .1 PFU/ cell in 10 ml of a maintenance medium of Eagle's medium with 5% fetal calf serum . A period of 45 min at 31° was allowed for absorption of the inoculum before addition of 90 ml of maintenance medium . The infected and mock-infected cultures were incubated at 31° until viral cytopathic effect was visible in the infected cell cultures (72-96 hr postinoculation) . The culture medium was then replaced with 30 ml of fresh culture medium (Eagle's medium plus 5% fetal calf serum) containing 2 .5 gg/ml of actinomycin D (Cosmegen, Merck, Sharp and Dohme, Rahway, N .J .) .
For continuous SDS-PAGE, the protein from the PEG-precipitated virus was solubilized with an equal volume of 1 M urea, 2% (w/v) SDS, and 2% (v/v) 2-mercaptoethanol in 10 mM sodium phosphate, pH 7 .4, together with sucrose (5%, w/v) and bromophenol blue tracking dye . The mixture was then heated to 95-100° for 90 sec and applied to the tube gels . For discontinuous SDS-PAGE the protein samples from the cell lysate and PEGprecipitated virus were treated with 2% (w/v) SDS and 5% (v/v) 2-mercaptoethanol in 0 .14 M Tris-HCI, pH 6 .7, plus 10% (v/v) glycerol (all final concentrations) and bromophenol blue added as a tracking dye .
SDS polyacrylamide-gel electrophoresis . Tube gels consisting of 10% (w/v)
CASH, WUNNER, AND PRINGLE
372
acrylamide, 5% (w/w)N,N'-methylene bisacrylamide containing 0 .1% SDS and 0 .5 M urea in 10 mM sodium phosphate, pH 7 .4, were prepared for continuous SDSPAGE . Electrophoresis was carried out for 16 hr at a constant current of 4 .5 mA per gel . After electrophoresis the gels were cut into 0 .6-mm slices with a Mickel automatic gel slicer and the amount of radioactivity in each slice was determined as described previously (Wunner and Pringle, 1976) . Discontinuous SDS-PAGE was carried out using gradient slab gels of 6-15% (total) acrylamide containing 0 .1% (w/v) SDS and a constant proportion (5%, w/w) of N,N'-methylene bisacrylamide as described by Marsden et al ., (1976) . Electrophoresis was carried out at 4° for 3-4 hr at a constant current of 40 mA unless stated otherwise . Radioactivity in the gel was detected by the fluorographic method of Bonner and Lasky (1974) as follows : The gel was given two 30-min washes in dimethyl sulfoxide (DMSO) followed by a 3-hr immersion in four times the gel volume of 22 .2% 2,5-diphenyloxazole (PPO) (KochLight Laboratories Ltd ., England) in DMSO . The gel was then washed in a number of changes of water for at least 24 hr to remove all of the DMSO from the gel . The gel was then dried down under vacuum and autoradiography performed with RP Royal X-Omat (Kodak) film at -70° with an exposure of approximately 7 days .
Protein molecular weight determination . Molecular weights on discontinuous SDS slab gels were determined by comparison with nonradioactive standard proteins run on adjacent slots as described by Marsden et al . (1976) . Reagents . All chemicals unless otherwise stated were obtained from BDH Chemicals Ltd ., Poole, Dorset, England . RESULTS
Comparison of Human RS Virus Strains All 11 strains of human RS virus produced the focal plaques described by Faulkner et al . (1976) on BS-C-1 cell monolayers . The Long strain, which has a history of serial passage in cultured cells (KB, Chang L, and Hep-2), grew to signifi-
cantly higher titers than the strains derived more immediately from clinical material ; otherwise there was little to distinguish the strains . Previously we have shown that RS virus can be concentrated from clarified culture supernatant by PEG precipitation, and that virus-specified polypeptides can be clearly resolved in this material by PAGE without resort to further purification by gradient centrifugation (Wunner and Pringle, 1976) . This is advantageous because RS virus is both labile and highly cell-associated which makes rigorous purification extremely difficult . The [ 35 S]methionine-labeled polypeptides of all 11 strains have been compared in parallel tracks in gradient slab gels by the electrophoresis of PEG concentrates from virusinfected and mock-infected cultures . Lysates of virus-infected and mock-infected cells were also included in each comparison . Three strains of human RS virus, RSN-2, RSN-0544, and Long, are compared in Fig. la . All the presumptive virus-specified polypeptides previously described for the RSN-2 strain (Wunner and Pringle, 1976) are resolved in the PEGconcentrated material . In addition a polypeptide with a molecular weight of approximately 10,000 is revealed in the slab gel autoradiography which was not always apparent previously . Intracellular polypeptides corresponding to VP48 (the major viral glycoprotein) and VP27 (a nonglycosylated matrix-like protein) were not as easily detected in some samples, however, as VP41 (nucleocapsid polypeptide) or VP32 . In each strain all the polypeptides, with the exception of VP32, migrated with similar mobilities . The differences in the mobilities of VP32 for the three strains illustrated in Fig . la represents the maximum extent of the variation observed in the 11 human RS virus strains (and is mutants) examined . The data are summarized in Table 1, and three groups are distinguished according to the apparent molecular weight of VP32 . The data also indicate that the electrophoretic mobility of VP32 is a stable characteristic of each strain: No differences were detected between different independently derived is mutants of the A2 and RSN-2 strains .
C
MI
(a)
P C P C P C P
RSN LONG RSN2 0544
ACT N-,,~~
(b)
RSN2 RSN2
LONG A2
Fio. 1 . (a) Autoradiograph of ["S]methionine-labeled polypeptides of the RSN-0544, RSN-2, and Long strains of RS virus . (C) Cell pellet fraction ; (P) polyethylene glycol precipitate of culture supernatant ; (MI) mock infection . (b) Autoradiograph of [ 35 S]methionine-labeled polypeptides of the Long, A2, and RSN-2 strains of RS virus, coelectrophoresed in the same gel . (MI) mock infection .
w
CASH, WUNNER, AND PRINGLE
374
ACTIN-
Fic . 2 . Autoradiograph of PsS]methionine-labeled polypeptides of RS virus (strain RSN-2) grown in BSC-1 cells and BEK cells and of BRS (strain BRS-127) grown in BEK cells . (C) Cell pellet fraction ; (P) polyethylene glycol precipitate of culture supernatant ; (MI) mock infection .
There appears to be no clear correlation between VP32 mobility and place or data of original isolation except that the exceptional strains were all isolated in Newcastle prior to 1976 . In Fig . lb the [asS]methionine-labeled polypeptides from the PEG precipitates of the RSN-2 and Long strains have been compared by coelectrophoresis in the same slab gel track in Fig . 2 to show by direct comparison that the difference in mobility of VP32 is not a gel artifact . This autoradiograph was underexposed to resolve the twin VP32 bands ; consequently the band corresponding to the 10,000-molecular weight poly-
peptide does not appear . In this gel the major viral glycoprotein (VP48) was clearly resolved . By extension of the electrophoresis time a difference was observed between the electrophoretic mobility of VP32 in the PEG virus concentrate and the corresponding intracellular polypeptide (data not shown) . The intracellular VP32 migrated faster and therefore appeared to be of lower molecular weight in the SDSpolyacrylamide gel . This suggests that a form of polypeptide processing may occur during virus maturation . The function of VP32 is unknown, but it does not appear to
PNEUMOVIRUS POLYPEPTIDES
375
be glycosylated (Wunner and Pringle, 1976) or sulfated (unpublished observation) . Some other minor variations in electrophoretic mobility of virus-specific polypeptides were observed . For instance, a slight difference in mobility of the VP27 of the RSN and Long strains could be resolved by coelectrophoresis . There may also be differences in the relative mobilities of VP48, but the diffuseness and spread of the glycoprotein band make discrimination of differences by autoradiography uncertain .
Comparison of Human RS Virus and Bovine RS Virus Human RS virus and bovine RS virus differ in their in vitro host range (Matumoto et al ., 1974) . Both viruses grow in cells of bovine origin, but only human RS virus grows well in primate cells . Consequently, bovine RS virus was grown in BEK cells and compared with human RS virus grown in both BEK and BS-C-1 cells . Figure 2 shows that there was little difference in the characteristic viral polypeptides obtained after infection of either BEK or BS-C-1 cells with the RSN-2 strain of human RS virus . In Fig . 2 bovine RS virus (strain BRS-127) is compared directly with the RSN-2 strain of human RS virus . The four major [PS]methionine-labeled bovine RS virus polypeptides (VP48, VP41, VP33, and VP27) appeared to have molecular weights identical to, or very close to, RSN-2 virus polypeptides . Two of these (VP41 and VP27) were nonglycosylated and had molecular weights identical with those of the corresponding nonglycosylated polypeptides of RSN-2 . A third nonglycosylated virus-specific polypeptide with a molecular weight of 33,000 corresponded to VP32 of human RS virus, and the single major glycosylated polypeptide of bovine RS virus had a molecular weight of 48,000 as in human RS virus (Fig. 3) . One noticeable difference between the human and bovine viruses was the absence of VP38 in BEK cells infected with bovine RS virus and in PEG-concentrated virus from these cells . A minor 38,000-molecular weight virus-specific polypeptide was de-
0
0 20 30 40 50 60 70 80 Fraction No . Fin . 3 . Electrophoretic profile of polypeptides of the BRS-127 strain of BRS virus . The virus was grown in BEK cells and double-labeled with [USlmethionine (0-0) and 13H]glucosamine (0 0) . (a) Polyethylene glycol precipitate of the supernatant from a virus-infected culture ; (b) polyethylene glycol precipitate of the supernatant of an uninfected culture .
tected occasionally, however, especially when bovine RS virus was grown in MDBK cells (unpublished observations) . A minor polypeptide with a molecular weight of 10,000 was also detected by SDSdiscontinuous PAGE in slab gels which appeared to be identical to a similar polypeptide of human RS virus . A second minor polypeptide of 25,000 molecular weight was also common to both viruses . The second strain (BRS-4642) of bovine RS virus gave an electrophoretic profile identical to that of BRS-127 .
Comparison of Human RS Virus and PVM According to Berthiaume et al . (1974) PVM grows well in BHK-21 cells, but not in African green monkey kidney cells (Vero) . In our hands PVM can grow and even form plaques in BS-C-1 cells under certain conditions, which makes it possible to compare the polypeptides of PVM grown in both BHK-21 cells and BS-C-1 cells: No difference was apparent . Consequently
376
CASH, WUNNER, AND PRINGLE
PVM grown in BHK-21 cells can be compared directly with human and bovine RS virus grown in BS-C-1 cells (data not shown) . Figure 4a compares PVM with the RSN-2 strain of human RS virus . Three PVM-specified polypeptides (labeled IV, V, and VI in Fig . 4a) in infected BHK-21 cells and in the PEG concentrate of virus released from these cells, had electrophoretic mobilities which correspond to viral polypeptides of human RS virus (RSN-2 strain) . Their molecular weights were approximately 43,000, 40,000, and 28,000 .
The 43,000-molecular weight polypeptide has been identified as the nucleocapsid polypeptide by ['H]uridine-labeling and banding in metrizamide (Cash and Pringle, in preparation) . Three polypeptides (II, III, and VII in Fig . 4a) in the PEG concentrate of PVM with molecular weights of approximately 81,000, 63,000, and 26,000, respectively, appeared as broad diffuse bands suggestive of glycopolypeptides . Labeling with [3 H]fucose has shown that II and III, but not VII, are glycosylated (Fig . 4b) . No gly-
ACTIN
(a)
(b)
Fm. 4 . (a) Autoradiograph of ["S]methionine-labeled polypeptides of PVM grown in BHK-21 cells and of RS virus (strain RSN-2) grown in BS-C-1 cells . The PVM polypeptides are labeled I-VIII . (C) Cell pellet fraction ; (P) polyethylene glycol precipitate of culture supernatant ; (MI) mock infection . (b) Autoradiograph of ["Slmethionine- and ['H]fucose-labeled polypeptides of PVM . (C) Cell pellet fraction; (P) polyethylene glycol precipitate of culture supernatant, The left and center tracks are ['H]fucose-labeled and the right track ["S]methionine-labeled .
377
PNEUMOVIRUS POLYPEPTIDES TABLE
2
AVERAGE MOLECULAR WEIGHTS OF PNEUMOVIRUS POLYPEPTIDES °
Virus
Glycosylated VP48
ma
BRS
PVM
Nucleocapsid VP41
Others VP200
46 .1x1 .2 (4)
41 .5x1 .1 200 (14) (2)
47 .5x0 .7 (2)
41 .8x1 .4 (3)
II
III
N .D.
IV
VP38
VP32
VP27
40 .5x1 .1 (14)
33 .711 .8 (14)
26 .2±1 .1 (14)
N .D .
VP25
40 .6 , 1 .3 (3)
33 .7-0 .8 (3)
26 .5x0 .9 (3)
24 .0 (2)
V
VI
VII
VIII
VP10 10 .5x0 .9 (6)
80 .5±8 .1 63 .2x4 .2 42 .7x1 .6 191 .4±13 .9 40 .4±1 .2 27 .9±2 .4 25 .5x1 .0 16 .9x0 .6 (5) (5) (5) (5) (5) (5) (3) (4)
9.4 (1)
-
° Molecular weights (x 10') were determined by reference to a set of standard proteins as described by Marsden et at . and Wunner and Pringle (1976) . The number of separate determinations on which the average value was derived is indicated in parentheses . Pooled data from different strains ; hence the value for VP32 does not distinguish the three types of Table 1 . (1976)
copolypeptide corresponding to the glycosylated VP48 of human and bovine RS viruses was observed. A large polypeptide of approximately 200,000 molecular weight is also apparent in both the cell lysate and PEG concentrate, but there is no PVM-specified polypeptide corresponding to the VP32 of RS virus . The major radioactive band in the PEG precipitate from PVM-infected cultures was not the nucleocapsid polypeptide, as in the case of human and bovine RS virus, but rather the nonglycosylated polypeptide VP27 . However, the nucleocapsid polypeptide (VP41 or band IV) was the most prominent band intracellularly with all three viruses . It is not known whether the rapidly migrating bands beyond band VII in the PVM tracks are present consistently . The results of the comparisons of the electrophoretic properties of the polypeptides of PVM and bovine and human RS viruses are summarized in Table 2 . It is obvious that PVM is distinct from the other two viruses .
much significance in human disease (Coates et al ., 1966) . In explanation of these observations it has been suggested that a number of antigenic variants of human RS virus may be in circulation in the population, causing frequent reinfection and a consequent broadening of the antibody spectrum of human serum . Our comparison of electrophoretic mobilities of the polypeptides of human RS virus strains provides some indirect support for this hypothesis, since differences were detected in the mobility of at least one (VP32) of the six (or seven) virion polypeptides, confirming the existence of interstrain variation . The mobility characteristic of VP32 appeared to be a stable genetic property since independently isolated is mutants resembled one another and the wild-type parent . The close serological resemblance of human and bovine RS virus is reflected in the similarity of their polypeptide profiles (Table 2 and Fig. 3) . It is likely that variation between strains of bovine RS virus is no greater than that observed between human RS virus strains, since the polypeptide profiles of the two strains of bovine DISCUSSION RS virus isolated at Compton 3 years apart Characteristically, human RS virus were essentially identical . The number and estimated molecular shows little antigenic variation . Serological differences between different isolates of weights of the RS virus polypeptides rehuman RS virus can be detected if individ- solved by discontinuous SDS-PAGE in ual antisera for each isolate are prepared gradient slab gels are very similar to those in ferrets . These differences cannot be re- previously established by continuous SDSsolved, however, when human convales- PAGE in tube gels (Wunner and Pringle, cent antiserum is used, and it is unlikely 1976) . One additional polypeptide of 10,000 that this minor serological variation has molecular weight was detected by discon-
378
CASH, WUNNER, AND PRINGLE
tinuous SDS-PAGE which had not been observed previously . The estimates differ, however, from determinations of the molecular weights of human RS virus polypeptides reported recently by Levine (1977), using the Long strain grown in HeLa cells . Levine resolved seven polypeptides with molecular weights of 79,000, 56,000, 44,000, 32,000, 28,000, 25,000, and 22,000 and showed that three (the 79,000, 56,000, and 22,000 polypeptides) were glycosylated . The presence of the two high molecular weight glycosylated polypeptides is the major discrepancy between these results and those reported here . The conditions of infection and radiolabeling were different, notably multiplicity, isotope, and presence of actinomycin D, which may in part account for the discrepancy . It is conceivable that the large glycopeptides might lack methionine and remain undetected in our experiments . However, the PAGE profiles were not altered when [14C]leucine (Wunner and Pringle, 1976) or protein hydrolysate (Cash, unpublished) was used in place of [35 S]methionine . The presence of actinomycin D in our experiments might have an effect on viral protein synthesis since this inhibitor reduced the yield of infectious virus (Faulkner et al ., 1976) . Omission of actinomycin D increased the incorporation of isotope into the background of host proteins, but did not reveal any additional presumptive viral proteins (unpublished data). Previously we have considered high molecular weight glycoproteins to be of host origin, and the consistency of the present findings supports this interpretation . RS virus infectivity is predominantly cell-associated and rigorous purification of RS virus cannot be achieved . For the present our criteria for identity of RS virus proteins remain synthesis in the presence of actinomycin D and abolition of synthesis when certain is mutants of RS virus are incubated at restrictive temperature (Wunner and Pringle, 1976 ; Gimenez and Pringle, in preparation) . The polypeptide profile of PVM is distinct from that of both human and bovine RS virus . In particular a polypeptide equivalent to the major glycopolypeptide
of RS virus is missing (Fig . 4b) . Results of analyses of polypeptides from cloned and gradient-purified PVM have reinforced this observation (Cash and Pringle, in preparation) . However, since is mutants of PVM have not been isolated, confirmation that these polypeptides are products of the viral genome is lacking . The distinctive polypeptide profile, the absence of cross-neutralization, and the presence of hemagglutinin all suggest that PVM is probably not closely related to either human or bovine RS virus . ACKNOWLEDGMENTS We are indebted to Professor J . H . Subak-Sharpe for critical appraisal of the manuscript . One of us (P .C .) is the holder of a Medical Research Council Award for Training in Research Methods .
REFERENCES BERTHIAUME, L ., JONCAS, J ., and PAVxwNI5, V . (1974) . Comparative structure, morphogenesis and biological characteristics of respiratory syncytial (RS) virus and pneumonia virus of mice (PVM) . Arch . Gesamte Virusforsch . 45, 39-51, BONNER, W . M ., and LASKEY, R . A . (1974) . A film detection method for tritium-labeled proteins and nucleic acids in polyacrylamide gels . Ear . J : Bio-
chem . 46, 83-88 . CHANOCK, R ., ROIZMAN, B ., and MOYERS, R . (1957) . Recovery from infants with respiratory illnesses of a virus related to chimpanzee coryza agent (CCA) . 1 . Isolation, properties and characterisation . Amer . J . Hyg . 66, 281-290 . COATES, H . V ., PILLING, D . W ., and CHANOCK, R . M . (1966) . An antigenic analysis of respiratory syncytial virus isolates by a plaque reduction neutralisation test . Amer . J . Epidem . 83, 299-313 . COMPANS, R . W ., HARmR, D . H ., and CHOenIN, P . W . (1967). Studies on pneumonia virus of mice (PVM) in cell culture . J . Exp . Med . 126, 267-276 . FAULKNER, G. P ., SHIRODARIA, P . V ., FOLLETT, E . A . C ., and PRINGLE, C . R. (1976) . Respiratory syncytial virus is mutants and nuclear immunofluorescence . J . Virol . 20, 487-500 . FENNER, F. (1976) . Classification and nomenclature of viruses . Second report of the International Committee on Taxonomy of Viruses . Interuirology 7, 1-102 . HOLZEL, A ., PARKER, L ., PATTERSON, W. H ., WHITE, L . R ., THOMaoN, K . M ., and TOBIN, J . O'M . (1963) . The isolation of respiratory syncytial virus from children with acute respiratory disease . Lancet 1, 295-298 .
PNEUMOVIRUS POLYPEPTIDES Y ., TANKA, Y ., OMoiu, T ., and MATUMOTO, M . (1970) . Isolation of bovine respiratory syncytial virus . Japan . J . Exp . Med . 40, 473-474. JACOB, J . W ., PEACOCK, D . B ., CORNER, B . D ., CAUL, E . 0 ., and CLARKE, S . K . K . (1971) . Respiratory syncytial and other viruses associated with respiratory disease in infants. Lancet, 1, 871-876. JONCAS, J ., BERTHIAUME, L ., and PAVILANIS, V. (1969) . The structure of the respiratory syncytial virus . Virology 38, 493-496 . LEVINE, S . (1977) . Polypeptides of respiratory syncytial virus . J . Viral, 21, 427-431 . MARSDEN, H . S., CROMBIE, I . K ., and SUEAxSHARPE, J. H . (1976) . Control of protein synthesis in berpesvirus-infected cells : Analysis of the polypeptides induced by wild type and sixteen temperature-sensitive mutants of HSV strain 17 . J . Gen . Virol . 31, 347-372. MATUMOTO, M ., INABA, Y ., KURooi, H ., SATs, K ., OMORI, T ., GOTO, Y ., and HIROSE, 0 . (1974) . Bovine respiratory syncytial virus : Host range in laboratory animals and cell cultures . Arch . Gesamte Virusforsch . 44, 280-290 . MELNICK, J. L . (1971) . Classification and nomenclaINABA,
379
Lure of animal viruses . Progr . Med . Virol . 13, 462484 . PACCAUD, M . F., and JACQUIER, C . (1970) . A respiratory syncytial virus of bovine origin . Arch . Gesamte Virusforsch . 30, 327-342 . RICHMAN, A . V ., PEDREIRA, F. A ., and TAURASO, N . M . (1971) . Attempts to demonstrate haemagglutination and haemadsorption by respiratory syncytial virus . Appl . Microbiol . 21, 1099-1100. THOMAS, L. H ., STOTT, E . J ., JEBBETT, J ., and HAMILTON, S . (1976) . The growth of respiratory syncytial virus in organ cultures of bovine fetal trachea . Arch . Viral . 52, 251-258. WRIGHT, P . F ., CHARPURE, M . A., HODES, D . S ., and CHANOCK, R . M . (1973) . Genetic studies of respiratory syncytial virus temperature-sensitive mutants. Arch . Gesamte Virusforsch . 41, 238-247. WUNNER, W . H ., and PRINGLE, C . (1976) . Respiratory syncytial virus proteins. Virology 73, 228243 . ZHDANOV, V . M., DREIZIN, S ., YANKEVICI, 0 . D ., and A5TARHOVA, A . K . (1974) . Biophysical characteristics of respiratory syncytial virus . Rev . Roum . Viral . 25, 277-280 .
82, 369-379 (1977)
A Comparison of the Polypeptides of Human and Bovine Respiratory Syncytial Viruses and Murine Pneumonia Virus P. CASH, W . H . WUNNER,' AND C. R. PRINGLE 2 M .R .C . Virology Unit, Institute of Virology, Church Street, Glasgow G11 SJR, Scotland Accepted June 18, 1977 Human and bovine respiratory syncytial (RS) viruses and pneumonia virus of mice (PVM) are morphologically similar viruses and are the only known members of the metamyxovirus (or pneumovirus) group . Comparison of the polypeptides of these viruses by polyacrylamide-gel electrophoresis (PAGE) has shown that the serologically related human and bovine RS viruses have similar polypeptide profiles . However, PVM does not resemble the other two viruses closely . The molecular weights of RS virus polypeptides were established by discontinuous SDS-PAGE in gradient slab gels and were comparable to previous determinations by continuous SDS-PAGE (Wunner and Pringle, 1976), apart from the detection of an additional 10,000-MW polypeptide . Comparison of 11 strains of human RS virus isolated from different localities, or from the same locality at different dates, showed that the relative mobility of VP32 (a nonglycosylated polypeptide of unknown function) was a stable characteristic of each strain . The mobilities of the other viral polypeptides showed little variation . The 11 strains fell into three groups in which the molecular weights of VP32 were estimated to be 31,000, 32,000, and 35,000, but there was no clear correlation with date or place of origin . INTRODUCTION
Human respiratory syncytial (RS) virus was first isolated in 1957 from young children suffering from severe respiratory disease (Chanock et al., 1957) and subsequently has been shown to be an important causative agent of lower respiratory tract disease in young children (Jacobs et al ., 1971) . Bronchiolitis and bronchopneumonia are characteristic features of RS infection in infancy (Holzel et al ., 1963) . Somewhat later a morphologically and antigenically related virus was isolated from cattle with respiratory disease and given the name bovine RS virus (Paccaud and Jacquier, 1970) . Human RS virus is a pleomorphic enveloped virus (Joncas et al ., 1969) and has been reported to contain a single-strand RNA genome with a sedimentation coefficient of approximately 57 S (Zhdanov et al ., 1974) . Melnick (1971) proposed that ' Present address : The Wietar institute, 36th Street at Spruce, Philadelphia, Pennsylvania 19104 . ' Author to whom reprint requests should be addressed.
human and bovine RS viruses and a morphologically similar but antigenically unrelated virus, pneumonia virus of mice (PVM), should be separated from the paramyxovirus group and given separate taxonomic status under the name Metamyxoviridae on the basis of a difference in nucleocapsid diameter and lack of neuraminidase activity . More recently the International Committee on the Taxonomy of Viruses has placed these viruses provisionally in the genus Pneumovirus of the family Paramyxoviridae (Fenner, 1976) . Neither hemagglutination nor hemadsorption activity has been found associated with human RS virus (Richman et al., 1971) or bovine RS virus (Inaba et al., 1970) but both activities have been found with PVM (Compans et al ., 1967) . Since the homogeneity of the group can be questioned we have compared the electrophoretic properties of the polypeptides of bovine RS virus and of PVM with those of human RS virus to substantiate further the inclusion of these viruses within the same taxonomic grouping . 369
Copyright 0 1977 by Academic Press, Inc . All rights of reproduction in any form reserved .
ISSN 0042-6822
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CASH, WUNNER, AND PRINGLE
A minimum of six virus-specified polypeptides have been described for the RSN2 strain of human RS virus, which range from 25,000 to 48,000 in molecular weight (plus a possible additional polypeptide of approximately 200,000 MW), with a single major glycopolypeptide of 48,000 MW and a minor glycopolypeptide of 42,000 MW (Wunner and Pringle, 1976) . However, Levine (1977) has reported seven polypeptides of 20,000-80,000 MW for the Long strain of human RS virus and has resolved three glycopeptides of MW 79,000, 56,000, and 22,000 . It has been necessary, therefore, to compare several different cloned strains of human RS virus, isolated from different geographic localities and also from the same locality at different times, to establish the extent of the variation in the electrophoretic mobilities of the polypeptides of different isolates of human RS virus . MATERIALS AND METHODS
Cells . BHK-21, clone 13, cells, MDBK cells, secondary bovine embryo kidney (BEK) cells, and BS-C-1 cells were maintained in 2-liter roller bottles in medium consisting of Eagle's medium supplemented with twice the normal concentration of amino acids (Glasgow modification) and containing 10% fetal calf serum (virus and mycoplasma screened ; Gibco-Bio-cult, Glasgow) . Human RS virus . Strains of human RS virus were obtained from the sources listed below . The viruses were passaged once or twice in BS-C-1 cells and, as indicated in Table 1, 9 of the 11 strains were cloned by three or four successive isolations from single plaques to produce genetically homogeneous stocks . The RSN-2, RSN-0544, and RSN-1599 strains were obtained from Dr . P. E . Gardner, Royal Victoria Hospital, Newcastle-upon-Tyne . The RSN-2 strain was isolated in 1972 from a child with bronchitis and is the origin of the is mutants described by Faulkner et al . (1976) . RSN-0544 was isolated in December 1974 from lung tissue during autopsy, and RSN-1599 in February 1975 from a child with febrile convulsions . Strains RSS-1 and RSS-2 also originated from
TABLE 1 DESCRIPTION OF STRAINS OF HUMAN
Strain
Cloned
RNS-0544
Yes
RSN-1599
Yes
RSN-2"
Place of isolation
RS
Date
VIRUS
Apparent molecular weight of VP32^
Newcastle, U .K . Newcastle, U .K .
1974 31 x 10'
Yes
Newcastle, U . K.
1972
Long
Yes
1956 35 x 10'
A2`
Yes
RSG-1545
No
RSG-1503
Yes
RSG-1504
Yes
RSG-4988
No
RSS-1
Yes
RSS-2
Yes
Baltimore, U .S .A . Melbourne, Australia Glasgow, U .K . Glasgow, U . K. Glasgow, U . K. Glasgow, U .K . Newcastle, U. K. Newcastle, U . K.
1975 31 x 10'
1961
32 x 10'
35 x 10'
1969 35 x 10' 1972 35 x 10' 1972 35 x 10' 1976 35 x 10' 1976
35 x 10-
1976 35 x 10'
° RSN-2 nomenclature (Wunner and Pringle, 1976) . Wild type and mutants is 2 and is 23 (Faulkner et al ., 1976) . ° Mutants Is Al, is A2, and is A7 (Wright et al ., 1973) .
Newcastle, but were isolated and cloned in human diploid cells (MRC-5) at the M .R .C . Common Cold Research Unit, Salisbury . The following strains were isolated in the Regional Virus Laboratory, Ruchill, Glasgow: RSG-1545 from a child with bronchiolitis in February 1969 ; RSG-1503 from a child with bronchopneumonia in February 1972 ; RSG-1504 from a child with bronchiolitis in February 1972 ; and RSG 4988 from a child with bronchiolitis in March 1976 . The Long strain, isolated in Baltimore in 1956, was obtained from the American Type Culture Collection, and the A2 strain, isolated in Melbourne, Australia, in 1961, was obtained from Dr . R. M .
PNEUMOVIRUS POLYPEPTIDES
371
Chanock, N .I .H ., Bethesda, Md . The is L-["S]Methionine (5 pzCi/ml, 200-300 Ci/ mutants of this strain, which were used in mmol), L-113H]fucose (10 µCi/ml, 3 .3 Ci/ this study were isolated by Wright et al . mmol) or D-fl 3H]glucosamine (10 µCi/ml, (1973) . 1 Ci/mmol) was then added 2 hr later, as Bovine RS virus . Strain BRS-127 and required . All radiochemicals were purBRS-4642 were obtained from Dr . E . J . chased from the Radiochemical Centre, Stott, A .R . C . Institute for Research on An- Amersham, England . Incubation was conimal Diseases, Compton, and cloned in tinued for a further 24-48 hr at 31° until secondary bovine embryo kidney cells by syncytium formation was extensive and three successive isolations from single the cell monolayer had begun to degenerplaques . Strain BRS-127 was isolated in ate . At this time the cultures were harFebruary 1973, and strain BRS-4642 was vested as described below . isolated in March 1976 from the lungs of Harvesting of cultures and partial puria calf which died from pneumonia . Both fication of virus . The cells were scraped strains were known to be free of mucosal into the medium, chilled, and centrifuged disease virus, a frequent noncytopathic for 30 min at 3000 rpm in an MSE Mistral contaminant in bovine cell cultures and centrifuge at 4° . Polyethylene glycol 6000 fetal calf serum (Thomas et al ., 1976) . (PEG) (AnalaR) was added to the clarified Pneumonia virus of mice . Strain 15 of supernatant to give a final concentration PVM, isolated in 1939, was obtained from of 6% (w/v) PEG . The precipitate obtained the American Type Culture Collection . after 24 hr at 4° was collected by centrifuThe stock used in this study had not been gation for 30 min at 3000 rpm in an MSE cloned. Mistral centrifuge at 4° . The supernatant Infectivity assay . The infectivity titers of was carefully decanted and the surface of the human RS strains were determined by the pellet washed with cold 0 .01 M focal plaque formation in monolayers of HEPES, pH 7 .8 . The PEG pellet from one BS-C-1 cells according to the procedure de- 2-liter roller bottle was then resuspended scribed by Faulkner et al . (1976) . A similar in 0 .3 ml of 0 .01 M HEPES, pH 7 .8 . The procedure has been developed for assay of cell pellet was washed twice with high-salt PVM (Cash and Pringle, in preparation) . buffer (0 .18 M NaCl ; 0.01 M Tris-HCI, pH Bovine RS virus plaqued erratically in BS- 7 .4) and then lysed in 6 ml of 0 .5% NP40 ; C-1 cells, hence infectivity titrations were 0 .01 M Tris-HCl, pH 7 .4 ; 0 .1 mM EDTA on carried out on BEK cells . ice for 10 min before storage at -70° .
Conditions of infection and labeling with radioactive isotopes . Just confluent
Preparation of protein samples for polyacrylamide-gel electrophoresis (PAGE) .
monolayer cultures of BS-C-1, BEK, MDBK, or BHK-21 cells in 2-liter roller bottles were infected with virus at an approximate multiplicity of 0 .02-0 .1 PFU/ cell in 10 ml of a maintenance medium of Eagle's medium with 5% fetal calf serum . A period of 45 min at 31° was allowed for absorption of the inoculum before addition of 90 ml of maintenance medium . The infected and mock-infected cultures were incubated at 31° until viral cytopathic effect was visible in the infected cell cultures (72-96 hr postinoculation) . The culture medium was then replaced with 30 ml of fresh culture medium (Eagle's medium plus 5% fetal calf serum) containing 2 .5 gg/ml of actinomycin D (Cosmegen, Merck, Sharp and Dohme, Rahway, N .J .) .
For continuous SDS-PAGE, the protein from the PEG-precipitated virus was solubilized with an equal volume of 1 M urea, 2% (w/v) SDS, and 2% (v/v) 2-mercaptoethanol in 10 mM sodium phosphate, pH 7 .4, together with sucrose (5%, w/v) and bromophenol blue tracking dye . The mixture was then heated to 95-100° for 90 sec and applied to the tube gels . For discontinuous SDS-PAGE the protein samples from the cell lysate and PEGprecipitated virus were treated with 2% (w/v) SDS and 5% (v/v) 2-mercaptoethanol in 0 .14 M Tris-HCI, pH 6 .7, plus 10% (v/v) glycerol (all final concentrations) and bromophenol blue added as a tracking dye .
SDS polyacrylamide-gel electrophoresis . Tube gels consisting of 10% (w/v)
CASH, WUNNER, AND PRINGLE
372
acrylamide, 5% (w/w)N,N'-methylene bisacrylamide containing 0 .1% SDS and 0 .5 M urea in 10 mM sodium phosphate, pH 7 .4, were prepared for continuous SDSPAGE . Electrophoresis was carried out for 16 hr at a constant current of 4 .5 mA per gel . After electrophoresis the gels were cut into 0 .6-mm slices with a Mickel automatic gel slicer and the amount of radioactivity in each slice was determined as described previously (Wunner and Pringle, 1976) . Discontinuous SDS-PAGE was carried out using gradient slab gels of 6-15% (total) acrylamide containing 0 .1% (w/v) SDS and a constant proportion (5%, w/w) of N,N'-methylene bisacrylamide as described by Marsden et al ., (1976) . Electrophoresis was carried out at 4° for 3-4 hr at a constant current of 40 mA unless stated otherwise . Radioactivity in the gel was detected by the fluorographic method of Bonner and Lasky (1974) as follows : The gel was given two 30-min washes in dimethyl sulfoxide (DMSO) followed by a 3-hr immersion in four times the gel volume of 22 .2% 2,5-diphenyloxazole (PPO) (KochLight Laboratories Ltd ., England) in DMSO . The gel was then washed in a number of changes of water for at least 24 hr to remove all of the DMSO from the gel . The gel was then dried down under vacuum and autoradiography performed with RP Royal X-Omat (Kodak) film at -70° with an exposure of approximately 7 days .
Protein molecular weight determination . Molecular weights on discontinuous SDS slab gels were determined by comparison with nonradioactive standard proteins run on adjacent slots as described by Marsden et al . (1976) . Reagents . All chemicals unless otherwise stated were obtained from BDH Chemicals Ltd ., Poole, Dorset, England . RESULTS
Comparison of Human RS Virus Strains All 11 strains of human RS virus produced the focal plaques described by Faulkner et al . (1976) on BS-C-1 cell monolayers . The Long strain, which has a history of serial passage in cultured cells (KB, Chang L, and Hep-2), grew to signifi-
cantly higher titers than the strains derived more immediately from clinical material ; otherwise there was little to distinguish the strains . Previously we have shown that RS virus can be concentrated from clarified culture supernatant by PEG precipitation, and that virus-specified polypeptides can be clearly resolved in this material by PAGE without resort to further purification by gradient centrifugation (Wunner and Pringle, 1976) . This is advantageous because RS virus is both labile and highly cell-associated which makes rigorous purification extremely difficult . The [ 35 S]methionine-labeled polypeptides of all 11 strains have been compared in parallel tracks in gradient slab gels by the electrophoresis of PEG concentrates from virusinfected and mock-infected cultures . Lysates of virus-infected and mock-infected cells were also included in each comparison . Three strains of human RS virus, RSN-2, RSN-0544, and Long, are compared in Fig. la . All the presumptive virus-specified polypeptides previously described for the RSN-2 strain (Wunner and Pringle, 1976) are resolved in the PEGconcentrated material . In addition a polypeptide with a molecular weight of approximately 10,000 is revealed in the slab gel autoradiography which was not always apparent previously . Intracellular polypeptides corresponding to VP48 (the major viral glycoprotein) and VP27 (a nonglycosylated matrix-like protein) were not as easily detected in some samples, however, as VP41 (nucleocapsid polypeptide) or VP32 . In each strain all the polypeptides, with the exception of VP32, migrated with similar mobilities . The differences in the mobilities of VP32 for the three strains illustrated in Fig . la represents the maximum extent of the variation observed in the 11 human RS virus strains (and is mutants) examined . The data are summarized in Table 1, and three groups are distinguished according to the apparent molecular weight of VP32 . The data also indicate that the electrophoretic mobility of VP32 is a stable characteristic of each strain: No differences were detected between different independently derived is mutants of the A2 and RSN-2 strains .
C
MI
(a)
P C P C P C P
RSN LONG RSN2 0544
ACT N-,,~~
(b)
RSN2 RSN2
LONG A2
Fio. 1 . (a) Autoradiograph of ["S]methionine-labeled polypeptides of the RSN-0544, RSN-2, and Long strains of RS virus . (C) Cell pellet fraction ; (P) polyethylene glycol precipitate of culture supernatant ; (MI) mock infection . (b) Autoradiograph of [ 35 S]methionine-labeled polypeptides of the Long, A2, and RSN-2 strains of RS virus, coelectrophoresed in the same gel . (MI) mock infection .
w
CASH, WUNNER, AND PRINGLE
374
ACTIN-
Fic . 2 . Autoradiograph of PsS]methionine-labeled polypeptides of RS virus (strain RSN-2) grown in BSC-1 cells and BEK cells and of BRS (strain BRS-127) grown in BEK cells . (C) Cell pellet fraction ; (P) polyethylene glycol precipitate of culture supernatant ; (MI) mock infection .
There appears to be no clear correlation between VP32 mobility and place or data of original isolation except that the exceptional strains were all isolated in Newcastle prior to 1976 . In Fig . lb the [asS]methionine-labeled polypeptides from the PEG precipitates of the RSN-2 and Long strains have been compared by coelectrophoresis in the same slab gel track in Fig . 2 to show by direct comparison that the difference in mobility of VP32 is not a gel artifact . This autoradiograph was underexposed to resolve the twin VP32 bands ; consequently the band corresponding to the 10,000-molecular weight poly-
peptide does not appear . In this gel the major viral glycoprotein (VP48) was clearly resolved . By extension of the electrophoresis time a difference was observed between the electrophoretic mobility of VP32 in the PEG virus concentrate and the corresponding intracellular polypeptide (data not shown) . The intracellular VP32 migrated faster and therefore appeared to be of lower molecular weight in the SDSpolyacrylamide gel . This suggests that a form of polypeptide processing may occur during virus maturation . The function of VP32 is unknown, but it does not appear to
PNEUMOVIRUS POLYPEPTIDES
375
be glycosylated (Wunner and Pringle, 1976) or sulfated (unpublished observation) . Some other minor variations in electrophoretic mobility of virus-specific polypeptides were observed . For instance, a slight difference in mobility of the VP27 of the RSN and Long strains could be resolved by coelectrophoresis . There may also be differences in the relative mobilities of VP48, but the diffuseness and spread of the glycoprotein band make discrimination of differences by autoradiography uncertain .
Comparison of Human RS Virus and Bovine RS Virus Human RS virus and bovine RS virus differ in their in vitro host range (Matumoto et al ., 1974) . Both viruses grow in cells of bovine origin, but only human RS virus grows well in primate cells . Consequently, bovine RS virus was grown in BEK cells and compared with human RS virus grown in both BEK and BS-C-1 cells . Figure 2 shows that there was little difference in the characteristic viral polypeptides obtained after infection of either BEK or BS-C-1 cells with the RSN-2 strain of human RS virus . In Fig . 2 bovine RS virus (strain BRS-127) is compared directly with the RSN-2 strain of human RS virus . The four major [PS]methionine-labeled bovine RS virus polypeptides (VP48, VP41, VP33, and VP27) appeared to have molecular weights identical to, or very close to, RSN-2 virus polypeptides . Two of these (VP41 and VP27) were nonglycosylated and had molecular weights identical with those of the corresponding nonglycosylated polypeptides of RSN-2 . A third nonglycosylated virus-specific polypeptide with a molecular weight of 33,000 corresponded to VP32 of human RS virus, and the single major glycosylated polypeptide of bovine RS virus had a molecular weight of 48,000 as in human RS virus (Fig. 3) . One noticeable difference between the human and bovine viruses was the absence of VP38 in BEK cells infected with bovine RS virus and in PEG-concentrated virus from these cells . A minor 38,000-molecular weight virus-specific polypeptide was de-
0
0 20 30 40 50 60 70 80 Fraction No . Fin . 3 . Electrophoretic profile of polypeptides of the BRS-127 strain of BRS virus . The virus was grown in BEK cells and double-labeled with [USlmethionine (0-0) and 13H]glucosamine (0 0) . (a) Polyethylene glycol precipitate of the supernatant from a virus-infected culture ; (b) polyethylene glycol precipitate of the supernatant of an uninfected culture .
tected occasionally, however, especially when bovine RS virus was grown in MDBK cells (unpublished observations) . A minor polypeptide with a molecular weight of 10,000 was also detected by SDSdiscontinuous PAGE in slab gels which appeared to be identical to a similar polypeptide of human RS virus . A second minor polypeptide of 25,000 molecular weight was also common to both viruses . The second strain (BRS-4642) of bovine RS virus gave an electrophoretic profile identical to that of BRS-127 .
Comparison of Human RS Virus and PVM According to Berthiaume et al . (1974) PVM grows well in BHK-21 cells, but not in African green monkey kidney cells (Vero) . In our hands PVM can grow and even form plaques in BS-C-1 cells under certain conditions, which makes it possible to compare the polypeptides of PVM grown in both BHK-21 cells and BS-C-1 cells: No difference was apparent . Consequently
376
CASH, WUNNER, AND PRINGLE
PVM grown in BHK-21 cells can be compared directly with human and bovine RS virus grown in BS-C-1 cells (data not shown) . Figure 4a compares PVM with the RSN-2 strain of human RS virus . Three PVM-specified polypeptides (labeled IV, V, and VI in Fig . 4a) in infected BHK-21 cells and in the PEG concentrate of virus released from these cells, had electrophoretic mobilities which correspond to viral polypeptides of human RS virus (RSN-2 strain) . Their molecular weights were approximately 43,000, 40,000, and 28,000 .
The 43,000-molecular weight polypeptide has been identified as the nucleocapsid polypeptide by ['H]uridine-labeling and banding in metrizamide (Cash and Pringle, in preparation) . Three polypeptides (II, III, and VII in Fig . 4a) in the PEG concentrate of PVM with molecular weights of approximately 81,000, 63,000, and 26,000, respectively, appeared as broad diffuse bands suggestive of glycopolypeptides . Labeling with [3 H]fucose has shown that II and III, but not VII, are glycosylated (Fig . 4b) . No gly-
ACTIN
(a)
(b)
Fm. 4 . (a) Autoradiograph of ["S]methionine-labeled polypeptides of PVM grown in BHK-21 cells and of RS virus (strain RSN-2) grown in BS-C-1 cells . The PVM polypeptides are labeled I-VIII . (C) Cell pellet fraction ; (P) polyethylene glycol precipitate of culture supernatant ; (MI) mock infection . (b) Autoradiograph of ["Slmethionine- and ['H]fucose-labeled polypeptides of PVM . (C) Cell pellet fraction; (P) polyethylene glycol precipitate of culture supernatant, The left and center tracks are ['H]fucose-labeled and the right track ["S]methionine-labeled .
377
PNEUMOVIRUS POLYPEPTIDES TABLE
2
AVERAGE MOLECULAR WEIGHTS OF PNEUMOVIRUS POLYPEPTIDES °
Virus
Glycosylated VP48
ma
BRS
PVM
Nucleocapsid VP41
Others VP200
46 .1x1 .2 (4)
41 .5x1 .1 200 (14) (2)
47 .5x0 .7 (2)
41 .8x1 .4 (3)
II
III
N .D.
IV
VP38
VP32
VP27
40 .5x1 .1 (14)
33 .711 .8 (14)
26 .2±1 .1 (14)
N .D .
VP25
40 .6 , 1 .3 (3)
33 .7-0 .8 (3)
26 .5x0 .9 (3)
24 .0 (2)
V
VI
VII
VIII
VP10 10 .5x0 .9 (6)
80 .5±8 .1 63 .2x4 .2 42 .7x1 .6 191 .4±13 .9 40 .4±1 .2 27 .9±2 .4 25 .5x1 .0 16 .9x0 .6 (5) (5) (5) (5) (5) (5) (3) (4)
9.4 (1)
-
° Molecular weights (x 10') were determined by reference to a set of standard proteins as described by Marsden et at . and Wunner and Pringle (1976) . The number of separate determinations on which the average value was derived is indicated in parentheses . Pooled data from different strains ; hence the value for VP32 does not distinguish the three types of Table 1 . (1976)
copolypeptide corresponding to the glycosylated VP48 of human and bovine RS viruses was observed. A large polypeptide of approximately 200,000 molecular weight is also apparent in both the cell lysate and PEG concentrate, but there is no PVM-specified polypeptide corresponding to the VP32 of RS virus . The major radioactive band in the PEG precipitate from PVM-infected cultures was not the nucleocapsid polypeptide, as in the case of human and bovine RS virus, but rather the nonglycosylated polypeptide VP27 . However, the nucleocapsid polypeptide (VP41 or band IV) was the most prominent band intracellularly with all three viruses . It is not known whether the rapidly migrating bands beyond band VII in the PVM tracks are present consistently . The results of the comparisons of the electrophoretic properties of the polypeptides of PVM and bovine and human RS viruses are summarized in Table 2 . It is obvious that PVM is distinct from the other two viruses .
much significance in human disease (Coates et al ., 1966) . In explanation of these observations it has been suggested that a number of antigenic variants of human RS virus may be in circulation in the population, causing frequent reinfection and a consequent broadening of the antibody spectrum of human serum . Our comparison of electrophoretic mobilities of the polypeptides of human RS virus strains provides some indirect support for this hypothesis, since differences were detected in the mobility of at least one (VP32) of the six (or seven) virion polypeptides, confirming the existence of interstrain variation . The mobility characteristic of VP32 appeared to be a stable genetic property since independently isolated is mutants resembled one another and the wild-type parent . The close serological resemblance of human and bovine RS virus is reflected in the similarity of their polypeptide profiles (Table 2 and Fig. 3) . It is likely that variation between strains of bovine RS virus is no greater than that observed between human RS virus strains, since the polypeptide profiles of the two strains of bovine DISCUSSION RS virus isolated at Compton 3 years apart Characteristically, human RS virus were essentially identical . The number and estimated molecular shows little antigenic variation . Serological differences between different isolates of weights of the RS virus polypeptides rehuman RS virus can be detected if individ- solved by discontinuous SDS-PAGE in ual antisera for each isolate are prepared gradient slab gels are very similar to those in ferrets . These differences cannot be re- previously established by continuous SDSsolved, however, when human convales- PAGE in tube gels (Wunner and Pringle, cent antiserum is used, and it is unlikely 1976) . One additional polypeptide of 10,000 that this minor serological variation has molecular weight was detected by discon-
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tinuous SDS-PAGE which had not been observed previously . The estimates differ, however, from determinations of the molecular weights of human RS virus polypeptides reported recently by Levine (1977), using the Long strain grown in HeLa cells . Levine resolved seven polypeptides with molecular weights of 79,000, 56,000, 44,000, 32,000, 28,000, 25,000, and 22,000 and showed that three (the 79,000, 56,000, and 22,000 polypeptides) were glycosylated . The presence of the two high molecular weight glycosylated polypeptides is the major discrepancy between these results and those reported here . The conditions of infection and radiolabeling were different, notably multiplicity, isotope, and presence of actinomycin D, which may in part account for the discrepancy . It is conceivable that the large glycopeptides might lack methionine and remain undetected in our experiments . However, the PAGE profiles were not altered when [14C]leucine (Wunner and Pringle, 1976) or protein hydrolysate (Cash, unpublished) was used in place of [35 S]methionine . The presence of actinomycin D in our experiments might have an effect on viral protein synthesis since this inhibitor reduced the yield of infectious virus (Faulkner et al ., 1976) . Omission of actinomycin D increased the incorporation of isotope into the background of host proteins, but did not reveal any additional presumptive viral proteins (unpublished data). Previously we have considered high molecular weight glycoproteins to be of host origin, and the consistency of the present findings supports this interpretation . RS virus infectivity is predominantly cell-associated and rigorous purification of RS virus cannot be achieved . For the present our criteria for identity of RS virus proteins remain synthesis in the presence of actinomycin D and abolition of synthesis when certain is mutants of RS virus are incubated at restrictive temperature (Wunner and Pringle, 1976 ; Gimenez and Pringle, in preparation) . The polypeptide profile of PVM is distinct from that of both human and bovine RS virus . In particular a polypeptide equivalent to the major glycopolypeptide
of RS virus is missing (Fig . 4b) . Results of analyses of polypeptides from cloned and gradient-purified PVM have reinforced this observation (Cash and Pringle, in preparation) . However, since is mutants of PVM have not been isolated, confirmation that these polypeptides are products of the viral genome is lacking . The distinctive polypeptide profile, the absence of cross-neutralization, and the presence of hemagglutinin all suggest that PVM is probably not closely related to either human or bovine RS virus . ACKNOWLEDGMENTS We are indebted to Professor J . H . Subak-Sharpe for critical appraisal of the manuscript . One of us (P .C .) is the holder of a Medical Research Council Award for Training in Research Methods .
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