In vitro synthesis of structural and nonstructural proteins of Sendai and SV5 viruses

VIROLOGY 100, 22-33 (1980)

In Vitro Synthesis of Structural and Nonstructural Proteins of Sendai and SV5 Viruses POLLY R. ETKIND, 1 RISE K. CROSS, ROBERT A. LAMB, DAVID C. MERZ, AND PURNELL W. CHOPPIN The Rockefeller University, New York, New York 10021 Accepted August 25, 1979 The messenger RNAs of SV5 and two strains of Sendai virus were isolated from infected cells and translated in a wheat germ cell-free system. Comparison of the peptide maps of the polypeptides synthesized in vivo and in vitro established that the nonglycosylated polypeptides P, NP, and M of both SV5 and Sendai virus had been synthesized in vitro. In immunoprecipitation studies of the putative SV5 polypeptides synthesized in vitro, antiserum against whole virions precipitated NP, P, and M, and other polypeptides which did not comigrate with mature virion polypeptides. Monospecific antisera against the HN and F glycoproteins precipitated two of the latter polypeptides with molecular weights of -55,000 and 50,000, respectively, suggesting that the nonglycosylated forms of these polypeptides had been synthesized in vitro. Polypeptide C, previously found in Sendai virus-infected cells and proposed to be a virus-specific nonstructural polypeptide, has been found to exhibit strain-specific differences in migration in polyacrylamide gels. Polypeptides with the appropriate strain-specific migration have been synthesized in vitro with mRNAs from cells infected with the different Sendai virus strains, and shown by peptide mapping to be the same polypeptides as those synthesized in vivo. The results have thus provided further evidence that C is a virus-coded nonstructural protein. Another polypeptide (C'), with migrates slightly slower than C, has been found in infected cells and synthesized in vitro. This polypeptide also exhibits strain-specific differences in migration in polyacrylamide gel electrophoresis, and has been shown by peptide mapping to be similar to C. The explanation for the difference in migration between C and C' is as yet unknown.

1972, 1974; Tozawa et al., 1973), and a glycoprotein involved in cell fusion, hemolysis, and the initiation of infection (F) (Scheid et al., 1972; Homma and Ohuchi, 1973; Scheid and Choppin, 1973, 1977). The F glycoprotein of SV5 and Sendai viruses as well as Newcastle disease virus (NDV), a similar paramyxovirus, consists of two disulfide-bonded subunits (F1 and F2) which are derived by specific proteolytic cleavage of a precursor glycoprotein (Fo) (Homma and Ohuchi, 1973; Scheid and Choppin, 1974, 1976, 1977; Nagai et al., 1976; Nagai and Klenk, 1977). This cleavage by a host protease activates the biological activities of the protein, and the availability of the appropriate protease in the host tissue is thus a major determinant of host range, tissue tropism, and the ability to undergo multiple-cycle replication and cause disease (Scheid and Choppin, 1975,

INTRODUCTION

The proteins of the paramyxoviruses simian virus 5 (SV5) and Sendai, although differing somewhat in molecular weights (MW), are similar in their functions (reviewed by Choppin and Compans, 1975). The virions contain a nucleocapsid protein (NP) (Mountcastle et al., 1971), two other proteins associated with the nucleocapsid (L and P) which are involved in RNA transcriptase activity (Stone et al., 1972; Marx et al., 1974; McSharry et al., 1975; Buetti and Choppin, 1977), a nonglycosylated membrane protein (M) (Mountcastle et al., 1971; McSharry et al., 1975), a glycoprotein associated with hemagglutinating and neuraminidase activities (HN) (Scheid et al., 1 To whom reprint requests should be sent. 0042-6822/80/010022-12502.00/0 Copyright © 1980by AcademicPress, Inc. All rights of reproductionin any formreserved.

22

PARAMYXOVIRUS TRANSLATION I N VITRO

1976; Choppin and Scheid, 1979; Nagai et al., 1976; Nagai and Klenk, 1977). Biologically inactive Sendai and NDV virions containing the uncleaved Fo glycoprotein are produced by cells which lack an appropriate protease, but such virions have not been found with SV5. However, in SV5-infected cells the glycoprotein precursor (Fo) has been found (Peluso et al., 1977). In addition to the structural polypeptides, the synthesis of another polypeptide (C) (MW -22,000) has been observed in Sendai virus-infected cells (Lamb et al., 1976) and shown to be distinct from the other viral polypeptides (Lamb and Choppin, 1978). An analogous protein (V) has been found in SV5-infected cells (Peluso et al., 1977), and it has been suggested that these are virusspecific nonstructural polypeptides (Lamb et al., 1976; Lamb and Choppin, 1978; Peluso et al., 1977). The genomes of SV5 and Sendai virus are single pieces of - 5 0 S RNA with a MW of - 5 - 6 × 106 (Barry and Bukrinskaya, 1968; Blair and Robinson, 1968; Compans and Choppin, 1968; Kolakofsky, 1974). Sendai and SV5 virions contain a transcriptase which produces viral mRNAs which are complementary to the viral genome (Robinson, 1971; Stone et al., 1971; Buetti and Choppin, 1977). Viral transcripts are found in infected cells in the form of 33, 24, and 18 S segments (Blair and Robinson, 1968; Portner and Kingsbury, 1970; Kingsbury, 1974; Roux and Kolakofsky, 1975). Heating or dimethyl sulfoxide denaturation of these RNAs demonstrated that all of the 24 S RNA and - 6 5 % of the 33 S RNA was aggregated 18 S RNA. Hybridization studies have shown that the 18 S RNA contains transcripts from 60% of the viral genome, and that the 33 S RNA which is not an aggregate is a transcript from the other 40% of the genome (Roux and Kolakofsky, 1975). The 33 and 18 S transcripts appear to serve as monocistronic messenger RNAs for specific viral polypeptides (Blair and Robinson, 1970; Pridgen and Kingsbury, 1972; Kingsbury, 1973; Davies et al., 1976; Glazier et al., 1977; Jones et al., 1978). In this paper we describe the translation in vitro of viral messenger RNAs isolated from Sendal and SV5-infected cells. Evidence

23

is presented for the synthesis of P, NP, and M of both viruses, the nonglycosylated form of HN and F of SV5, and the nonstructural polypeptide C of Sendai virus, providing additional evidence that the latter is virus specific. MATERIALS AND METHODS

Cells. The BHK21-F line of baby hamster kidney cells was grown in reinforced Eagle's medium (REM) with 10% calf serum and 10% tryptose phosphate broth as described previously (Holmes and Choppin, 1966; Choppin, 1969). African green monkey kidney (CV-1). cells were grown in Eagle's medium and 10% fetal calf serum. Primary chick embryo fibroblast (CE F) cultures prepared as described by Borland and Mahy (1968) were grown in medium 199 with 7.5% calf serum and 1.6% chicken serum. Virus. Seed stocks of the RU, Z, MN, Fushimi, and Obayashi strains of Sendai virus were propagated in the allantoic sac of embryonated chicken eggs and [3~S]methionine-labeled virions were propagated in C E F cells and purified as described previously (Lamb et al., 1976; Lamb and Choppin, 1977b). Stocks of the W3 strain of SV5 and [35S]methionine-labeled virions were grown in CV-1 cells and purified as described by Buetti and Choppin (1977). Preparation of radioactively labeled polypeptides from infected cells. Confluent monolayers of C E F cells grown on 35-mm plastic dishes were inoculated with - 5 0 PFU/cell of Sendai virus. After adsorption at 37° for 1 hr, the inoculum was removed and REM added. At 16 hr postinfection (p.i.) the medium was removed and REM deficient in methionine and containing [35S]methionine (20 t~Ci/ml for electrophoresis experiments or 200 t~Ci/ml for peptide mapping experiments) was added for 2 or 4 hr. Samples were prepared for electrophoresis as described previously (Lamb et al., 1976). Preparation of infected cell extracts for viral m R N A isolation. Confluent monolayers of BHK21-F cells were infected with either SV5 or Sendai virus at multiplicities o f - 5 0 - 1 0 0 PFU/cell. After adsorption for 2 hr, the inoculum was removed and REM containing actinomycin D (5 tLg/ml) was added.

24

E T K I N D ET AL.

At 4 hr p.i. [3H]adenosine (5 ~Ci/ml) was added and at - 1 8 - 2 0 hr p.i. the medium was removed, the cells were washed sequentially with cold PBS and cold reticulocyte standard buffer (RBS) (0.01 M KC1, 0.0015 M MgC12, 0.01M Tris-HC1 7.4), and harvested into RSB. Cells were disrupted by homogenization in a Dounce homogenizer to yield a cytoplasmic extract (Krug, 1971, 1972). In some experiments, nuclei were washed with RSB containing the nonionic detergents sodium deoxycholate and Triton X-100 (Penman, 1969; Krug and Etkind, 1973), and the wash was added back to the cytoplasmic extract; however, the presence or absence of the nuclear wash did not affect the quality of the translation products. Isolation of viral messenger R N A . Viral messenger RNA was purified by one of the following methods, all of which gave similar in vitro translation patterns. The procedure used most often involved making the cytoplasmic extract 2% in SDS and layering it over 15-30% sucrose gradients in NETS (0.001 M EDTA, 0.01 M Tris 7.4, 0.1 M NaC1, 0.5% SDS) buffer and centrifuging at 18,000 rpm in a Spinco SW-27 rotor for 16 hr at 20°. [3H]Adenosine-labeled RNAs in the size range of 14-33 S were collected. The RNA was ethanol precipitated at least 2 ×, washed with ethanol, dried, and dissolved in water for in vitro translation work. In some experiments, phenol- chloroform extraction of cytoplasmic extract was done as described previously (Etkind and Krug, 1974, 1975). After extraction, RNA was precipitated with ethanol and further purified on either an oligo(dT)-cellulose column or in S D S sucrose gradients. Another procedure tested was lysis of cells in a cold solubilizing buffer (0.1 M NaC1, 0.01 M Tris pH 8.8, 0.02 M EDTA, 1% Triton N-101, and 0.5% sodium deoxycholate). Once removed from the plate, the lysate was adjusted to 0.4 M NaC1 and 0.5% SDS, and the RNA was isolated by oligo(dT)-cellulose chromatography and concentrated by ethanol precipitation (Collins et al., 1978). Cell-free translation. Wheat germ cellfree extracts were prepared according to the procedure of Roberts and Paterson (1973) and translation carried out as described

previously (Etkind and Krug, 1975, 1977) except that 98 mM potassium acetate was substituted for KC1 (Weber et al., 1977; Lamb et al., 1978). Antisera and immunoprecipitation. Antisera specific for SV5 grown in MDBK cells and Sendai virions grown in BHK21-F cells and monospecific antisera against the HN, F, and M proteins of SV5 were raised in rabbits by repeated injections of high salt, nonionic detergent-disrupted virions (1 mg protein) or purified proteins in an emulsion with complete Freund's adjuvant (Merz, unpublished experiments). Immunoglobulin G (IgG) was isolated from each serum by fractionation on DEAE-cellulose as described by Levy and Sober (1960). The specificity of each antibody was demonstrated by immunodiffusion and immunoprecipitation analyses (Merz, unpublished experiments). For immunoprecipitation, 500 tLg of specific IgG was complexed with immobilized Staphylococcus aureus protein A (protein A-Sepharose CL-4B (pA/CL-4B)). Excess cystine relative to dithiothreitol was added to in vitro translation product mixtures which were then made to 0.025 M Tris-HC1, pH 7.8, 0.25 M NaC1, 1.0% (v/v) Emulphogene BC-720, 0.5% sodium deoxycholate, 0.1% SDS. Aliquots of treated translation mixtures were added to pA/CL-4B immunoadsorbants and incubated for 2 hr on ice with periodic shaking. Excess antigens and extract proteins were washed from immunoadsorbants by six centrifugal washes (250 vol each) with 0.025 M Tris-HC1, pH 7.8, 0.5 M NaC1, 1.0% Emulphogene BC-720, 0.5% sodium deoxycholate, 0.1% SDS, followed by two additional washes with 0.0625 M Tris-HC1, pH 6.8. Antigen-antibody-protein A complexes were disrupted by addition of 4% SDS, 4 M urea in 0.0625 M Tris-HC1, pH 6.8, followed by 2-mercaptoethanol at a final concentration of 2%. After boiling for 3 min, the entire slurry was applied to a gel slot for electrophoretic analysis. Polyacrylamide gel electrophoresis. [35S]Methionine-labeled products of in vitro assays and viral proteins were electrophoresed on either 10 or 13% polyacrylamide gels (Lamb et al., 1976; Lamb and Choppin, 1977a). The acrylamide/Bis ratio of 10% gels was 30.0/0.8, and of 13% gels, 130/1.

PARAMYXOVIRUS TRANSLATION I N V I T R O

Autoradiography and fluorography. Gels which underwent fluorography were treated with dimethyl sulfoxide and 2,5-diphenyloxazole prior to drying down, as described by Bonner and Laskey (1974), and dried gels were exposed to Kodak RP-Royal "X-omat" film at -70 °. Nonfluorographed gels were exposed to Dupont Cronex 2DC X-ray film. Peptide mapping by limited proteolysis in SDS and analysis by gel electrophoresis was done as described by Cleveland et al. (1977) with minor modifications (Lamb and Choppin, 1977b). Peptide mapping by trypsin digestion and analysis on thin-layer plates. [35S]Methionine-labeled in vitro products and infected cell lysates were subjected to electrophoresis on either 10 or 13% gels. The relevant polypeptide gel bands were removed, digested, and analyzed as described previously in detail (Lamb et al., 1978). U

MN

Z

FU

OB

RU

P HN

Fo

25

Chemicals, enzymes, and isotopes. Sucrose-ultrapure (density gradient grade) was obtained from Schwarz/Mann (Orangeburg, N. Y.); SDS (BDH specially pure), from Gallard-Schlesinger Chemical Manufacturing Corporation (Carle Place, N. Y.); phenol (liquified) and chloroform, from Mallinkrodt (St. Louis, Mo.); oligo(dT)-cellulose from Collaborative Research (Waltham, Mass.); Cleland's reagent and glycine (ammonium free), from Calbiochem, (La Jolla, Calif.); spermidine trihydrochloride, from ICN Pharmaceuticals (Cleveland, Ohio); DEAEcellulose (DE-52), from Whatman (Clifton, N. J.); S. aureus protein A-Sepharose CL-4B, from Pharmacia Fine Chemicals (Piscataway, N. J.); Emulphogene BC-72, from GAF Corporation (New York); Canalco acrylamide (ultrapure) and Canlco bisacrylamide (ultrapure), from Miles Labs, (Elkhart, Ind.); TPCK-treated trypsin and chymotrypsin, from Worthington Biochemical Corporation (Freehold, N. J.); Triton X-100, Triton N-101, sodium deoxycholate, actinomycin D, ATP, GTP, creatine phosphate, creatine phosphokinase, and HEPES buffer, from Sigma Chemical Company (St. Louis, Mo.); complete Freund's adjuvant, from Difco (Detroit, Mich.); [3H]adenosine, from New England Nuclear Corporation (Boston, Mass.); and [35S]methionine, from Amersham Corporation (Arlington Heights, Ill.).

NP

RESULTS B M

CI

C

FIG. 1. Polyacrylamide gel electrophoresis of [3~S]methionine-labeled polypeptides in cytoplasmic extracts of uninfected CEF cells (U) and cells infected with Sendai virus strains MN, Z, Fushimi (FU), Obayashi (OB), and RU. In this and all subsequent gels the positions indicated for the marker polypeptide species in the infected cell lysates were determined by the migration of the polypeptides of the appropriate virus strain run in another lane of the same slab gel.

Viral Polypeptide Patterns in Cells Infected by Various Strains of Sendai Virus. Figure 1 shows the [35S]methionine-labeled polypeptides in cytoplasmic extracts of uninfected CEF cells and cells infected with Sendal virus strains MN, Z, Fushimi, Obayashi, and RU. There are differences in the mobility of certain polypeptides from these different strains. The NP polypeptide of the Z strain is smaller than that of the other strains, and the B and M polypeptides of the Z strain are larger. B is the phosphorylated form of M (Lamb and Choppin, 1977b). The P, HN, Fo polypeptides migrate similarly in the various strains. Of particular interest is the fact that the mobilities of the C polypeptides of the different strains vary, e.g.,

26

ETKIND ET AL.

in the Z strain it migrates more rapidly than in the other strains. These strain-specific differences in migration of the C polypeptide synthesized in the same host cell provide additional evidence supporting the conclusion that C is a virus-specific polypeptide (Lamb et al., 1977a; Lamb and Choppin, 1978). Figure 1 also shows that there is a faint band (C') just above C which also shows strain specificity in its migration pattern. The Z and RU strains of Sendai virus were selected for subsequent experiments because the strain-specific mobility differences of certain of their polypeptides facilitated the preliminary identification of these polypeptides synthesized in vitro.

RU

Z

NP--_ D --NP-~

M___

Comparison of Polypeptides of the Z and R U Strains Synthesized in Vitro C~ Figure 2 shows the [3~S]methioninelabeled products synthesized in a wheat germ cell-free extract from mRNA isolated from BHK21-F cells infected with the Z or RU strains of Sendai virus. [3H]AdenosineFIG. 2. Polyacrylamide gel electrophoresis of [35S]labeled mRNA was isolated from infected methionine-labeled polypeptides synthesized in vitro cell extracts using sucrose density centrifu- in a wheat germ extract programmed with RNA from gation as described under Materials and cells infected with the Z and RU strains of Sendai Methods. As expected from the migration virus. Two lanes (RU and Z) of left panel are from of the authentic viral polypeptides synthe- the same experiment and slab gel electrophoresis while sized in vivo (see Fig. 1), the putative P right single panel (Z) is from a different experiment polypeptides of the two strains synthesized and gel. in vitro comigrated, whereas the putative NP of the Z strain RNA was smaller than comigrated with the marker polypeptides that of the RU and the M of the Z strain is P, NP, M, C, and C'. The authenticity of larger than that of the RU strain. The puta- these as viral polypeptides is demonstrated tive C polypeptides synthesized in vitro also below. Polypeptide bands are also seen just showed the expected strain-specific migra- above NP and between NP and M. These tion, and a polypeptide was synthesized in are thought to be virus specific because they vitro which corresponds to C' seen in Fig. 1 were precipitated with antisera directed and showed strain specificity in its migra- against whole Sendai virions (not shown). tion. The right lane of Fig. 2 shows another However, because of the small amounts of example of the products programmed with these polypeptides normally synthesized in the mRNA from cells infected with the Z vitro, we have not yet been able to obtain strain. There was better definition of the enough material for further analysis. Peptide mapping by limited proteolysis individual polypeptides, particularly C and C', in this gel. None of the putative viral of the M polypeptide synthesized in vitro. polypeptides was synthesized using mRNA Figure 3 shows the comparison of the limited protease digests of M polypeptides from mock-infected cells. obtained from virions and those synthesized Characterization of Sendai Virus Polypep- in vitro. The [35S]methionine-labeled M tides of Z Strain Synthesized in Vitro bands of the polypeptides were removed As shown in the right lane of Fig. 2, poly- from gels of virions or the in vitro products, peptides were synthesized in vitro which inserted into the sample well of another gel,

C!

---C !~ ~ i'

PARAMYXOVIRUSTRANSLATIONIN VITRO M V.

I.V.

|

ii FIG. 3. A comparison of the peptides obtained by limited proteolysisof the M polypeptideof the Z strain Sendal virions (V) with the putative M polypeptide synthesized in vitro (I.V.). The [35S]methionine-labeled M polypeptides were isolated from 13% gels and digested with ehymotrypsinas describedunder Materials and Methods.

overlaid with chymotrypsin, and subjected to electrophoresis as described under Materials and Methods. The two polypeptides show a series of similar peptides after limited protease digestion, indicating a similarity between the M polypeptides synthesized in vivo and in vitro. Peptide mapping of the P, NP, C, and C' polypeptides. [3~S]Methionine-labeled polypeptides P, NP, C, and C' were isolated from infected cells, and the putative P, NP, C, and C', also [35S]methionine-labeled, were isolated from gels of the products synthesized in vitro. The polypeptides were removed from gels and digested with trypsin, and the tryptic peptides were analyzed on cellulose plates by high voltage electrophoresis in the first dimension and chromatography in the second as described under Materials and Methods. Figure 4 (top) depicts the peptide map of the P polypeptides synthesized in vivo (Pc) and in vitro (P,.v.), and the mixture of the two. The peptide maps are nearly identical.

27

Figure 4 (bottom) shows the peptide maps of NP synthesized in vivo (NPc) and in vitro (NP,.v.). There are two or three extra peptides in the NP made in vitro, but on the whole the patterns are very similar. The similarity of these peptide patterns indicates the authenticity of the P and NP polypeptide made in vitro. The peptide maps depicted in the top row of Fig. 5 show that the C polypeptides from Z and RU strain-infected cells have similar peptide maps, and that C made in vitro (Ci.v.) has almost the same tryptic peptide pattern as the authentic C polypeptide from infected cells. The bottom panels in Fig. 5 show the similarity between the C' and C polypeptides from infected cells, and between the C' polypeptides synthesized in vivo and in vitro. Thus, the C and C' polypeptides synthesized in vitro are authentic, and C and C' are similar polypeptides with different electrophoretic mobilities. Translation of SV5 Polypeptides In Vitro The products synthesized in a wheat germ cell-free extract programmed by mRNA isolated from BHK21-F or CV-1 cells infected with SV5 were also examined. Figure 6 shows that polypeptides which comigrated with the NP, P, and M polypeptides from virions were synthesized in vitro. Also present are additional polypeptides which did not comigrate with virion polypeptides. Some of these bands are faint, e.g., two bands between NP and F,, but can be seen much more easily upon exposure of the gel, as is shown in the third panel. Some bands are obscured by others, but can be identified by immunoprecipitation as discussed below. Peptide mapping of SV5 polypeptides synthesized in vitro. [35S]Methionine-labeled polypeptides HN, NP, F, P, and M obtained from virions and the major polypeptide bands synthesized in vitro were mapped by limited protease digestion in SDS. As shown in Fig. 7, the three in vitro products which comigrated with P, NP, and M contain essentially the same peptides as their in vivo counterparts, indicating that these polypeptides synthesized in vitro are authentic viral polypeptides. Those polypeptides made in vitro which ran below F1 and above M (see

28

ETKIND ET AL.

Pc

P=

pc" p,.,,

e

9

e

e

9

j

NP c

.

o

|

.

NP..~

t

,

N P c * NPLv "

;

B

!

B

O~ a,

•

~

i ~m

Q

FIG. 4. Two-dimensionalpeptide maps of trypsin digests of [35S]methionine-labeledpolypeptidesof the Z strain of Sendaivirus by chromatographyand high voltageelectrophoresis. P polypeptidefrom infected cells (Pc); P made in vitro (P~.v.); a mixture of P from infected cells and P made in vitro (Pc + Pz.v.);NP frominfectedcells (NPc); NP madein vitro (NP,.v.); a mixture of NP frominfected cells and NP madein vitro (NPc + NP~.v.). Fig. 6) and contained enough material for analysis could not be related by mapping to any of the viral polypeptides, although they did appear to share some peptides with NP and thus contain NP fragments. Immunoprecipitation of SV5 polypeptides synthesized in vitro. [3~S]Methionine-labeled products in vitro were precipitated with either antisera directed against whole SV5 virions, or monospecific antisera directed against either the HN, F, or M polypeptides of the virus. As shown in Fig. 8, antisera against whole virions precipitated NP, P, and M, as well as two faint bands below NP, two heavier bands flanking P, faint bands below M, and some rapidly migrating bands in the 18,000-26,000 MW range. Antisera against the HN polypeptide precipitated from the in vitro products a polypeptide with a molecular weight of 55,000 which appears to co-run with the faint polypeptide band seen below NP in nonprecipitated extracts. Apparent contamination with other viral polypeptides NP, P, and M occurs in these immunopre-

cipitation experiments using monospecific antisera and varies in amount in different experiments. This 55K polypeptide, however, is precipitated only with anti-HN serum or anti-whole SV5 virus serum, and not by anti-M or anti-F sera. These results suggest that this polypeptide is the nonglycosylated form of the HN protein. Antisera directed against the F polypeptide of SV5 precipitated a faint band in the range of 50K. This apparently represents the nonglycosylated form of Fo, the uncleaved precursor to the polypeptides F1 and F2. (The glycosylated form of the Fo polypeptide runs under the NP.) Antisera directed against the viral M polypeptide specifically precipitated the M protein made in vitro. DISCUSSION Although there have been no previous reports of the synthesis of SV5 polypeptides in vitro, with Sendai virus evidence has been obtained for the synthesis in vitro of

8

j.

4)

!

o #

O

O

Q

*

¢IIIUL CELL

C,u÷¢'lu

o

CELL

C~

~ e

e

*

I

,.v.!

C z I.V.

$

!

e

CELL

I.V.

Cz + C z

0

IV.

Cz~C z C|LL

FIG. 5. Tryptic peptide maps of the following polypeptides: C polypeptides from cells infected with the Z strain (CzecH) and RU strain of Sendai virus (CRu¢~,~); C made in vitro from RNA isolated from Z strain-infected cells (CzLv.); a mixture of C polypeptides from Z strain-infected cells and C made in vitro (Cz,.ol] + Cz,.v.); C' from RU strain-infected cells (C'aucel]); a mixture of C from RU strain-infected cells and C' from R U strain-infected cells (Cat~H + C'Ru~,~); C' made in vitro by RNA isolated from Z strain-infected cells (C'z~.v.); and a mixture of C from Z strain-infected cells and C' made in vitro from RNA isolated from Z strain-infected cells (Cz¢~l, + C'z[.v.).

e

O

¢z CuLL

e~

GO

GO

o

30

ETKIND ET AL.

three polypeptides: P, NP, and M (Kingsbury, 1973; Davies et al., 1976; Jones et al., 1978). With Newcastle disease virus, polypeptides corresponding to viral polypeptides L, MN, Fo, 47K, and M have been synthesized in vitro (Morrison et al., 1975; Clinkscales et al., 1977; Collins et al., 1978). No putative nonstructural polypeptide has been reported for NDV thus far. In the present studies with SV5 and Sendai virus, although we have not detected the in vitro synthesis of all of the polypeptides of each virus, between these two paramyxoviruses we have obtained evidence for the synthesis of the nonstructural protein (C) and each of the virion proteins except L. The evidence for the authenticity of these polypeptides synthesized in vitro included strain-specific differences in electrophoretic mobility, peptide mapping, and, in the case of the SV5 glycoproteins, HN and Fo, precipitation of a polypeptide with specific antisera to individual viral polypeptides. In the case of Sendai virus, polypeptides were V.

I.V.

I,V.

b . . . .

HN

. . . .

NP . . . . . F1 A

..... -

M----

"

qlP ~

qlmlm

~mD

qllD

m

FIG. 6. Polyacrylamide gel electrophoresis of the [3~S]methionine-labeled polypeptides synthesized in vitro in wheat germ extracts programmed with RNA isolated from SV5-infected cells. The left lane (V) shows [3~S}methionine-labeled polypeptides from SV5 virions run as markers to localize the polypeptides synthesized in vitro. (A = cellular actin.) The middle lane shows the polypeptides synthesized in a cell-free extract programmed by SV5 RNA, and the right lane shows the same polypeptides overexposed to allow better visualization of the two bands running below NP.

NP

V,

M

I.V.

VT

Q

p

V.

I.V.

o

I.V.

)

FIG. 7. A comparison of the peptides obtained by limited proteolysis of the NP, M, and P polypeptide from SV5 virions (V), and the respective putative polypeptides made i,n vitro (I.V.). All polypeptides were [35S]methionine-labeled and were isolated from 13% gels and digested with chymotrypsin as described under Materials and Methods.

synthesized which were precipitated by antibody to the virion and which migrated just above NP and between NP and M. Although these could represent nonglycosylated viral glycoproteins, monospecific antisera were not available to these Sendai polypeptides, in contrast to SV5, and the lack of sufficient material has hindered identification by peptide mapping. Of particular interest in these studies was the synthesis of the putative nonstructural polypeptide (C). Previous studies showed that C was synthesized in several different types of cells infected with the same strain of Sendai virus but not in uninfected cells, and C was shown to be unrelated to any of the virion polypeptides by peptide mapping (Lamb et al., 1976; Lamb and Choppin, 1978). These studies provided strong evidence that C was a virus-coded nonstructural protein. The present studies have provided additional evidence supporting this conclu-

PARAMYXOVIRUS TRANSLATION I N VITRO

NpHN--- I

::::::

;

!

_HN (~.v.)7 -

31

I O --E

_F(I.V.)Ijt

--PA I_M

m O

SV5 aM etHN <~F


FIG. 8. Polyacrylamide gel electrophoresis of the [35S]methionine-labeled SV5 polypeptides synthesized in vitro and precipitated by antisera directed against the M polypeptide (aM), the HN protein (aHN), the F protein (aF), or whole SV5 virions (aSV5). [35S]Methionine-labeled SV5 virion polypeptides run as markers are shown in the two outermost lanes. The gel used in the left panel contained 10% acrylamide and 4 M urea, and that in the right panel, 13% acrylamide.

sion. Strain-specific differences in electrophoretic mobility were found in the C polypeptide synthesized in the same host cell infected with different strains of Sendai virus, and polypeptides with these strainspecific differences were synthesized in vitro using mRNA from cells infected with the respective strains but not m R N A from mock-infected cells. Peptide mapping showed that the C polypeptides made in vitro were the same as those made in vivo, and the maps of C polypeptides from different strains were similar. An additional finding in these studies was the synthesis in vivo and in vitro of a polypeptide (C') migrating slightly lower than C. This was shown by peptide mapping to be similar to C, and is thus clearly another form of C. Whether it is a slightly larger form or is the result of some other modification is not clear. Although extra [35S]methionine-labeled peptides were not identified in C' (Fig. 5) an additional undetected peptide(s) could be present. Short pulse-label experiments have provided no evidence that it is a precursor of C, and it is not a phosphorylated form of C (Lamb, unpublished experiments).

Polypeptides analogous to the C polypeptide of Sendai virus have now been found in several paramyxoviruses, including not only SV5 (Peluso et al., 1977) but also canine distemper and measles viruses (Hall et al., 1979; and unpublished experiments) and the shipping fever strain of bovine parainfluenza virus 3 (Merz, unpublished experiments). The function of this polypeptide in infected cells is unknown. An attractive possibility is that it could be involved in viral RNA replication but there is no evidence for this at the present time. These and previous studies have shown that in vitro synthesis of paramyxovirus proteins is feasible, and this should make possible the investigation of a number of remaining questions concerning paramyxovirus proteins, e.g., the existence of a signal sequence on the viral glycoproteins, the effect of the absence of carbohydrate on proteolytic cleavage of the glycoproteins, and the possibility of a precursor to the HN glycoprotein which has thus far been found in vivo only with two strains ofNDV (Nagai et al., 1976; Nagai and Klenk, 1977) but not with other NDV strains or other paramyxoviruses.

32

ETKIND ET AL. ACKNOWLEDGMENTS

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