the influence of capsid protein cleavage on the processing of e2 and e1 glycoproteins of rubella...

VIROLOGY 183, 52-60 (1991)

The Influence of Capsid Protein Cleavage on the Processing of E2 and El Glycoproteins of Rubella Virus

HELEN MCDONALD, TOM C. HOBMAN,’ AND SHIRLEY GILLAM’

Department of Pathology, University of British Columbia Research Centre, 950 West 28th Avenue, Vancouver, British Columbia, Canada V5Z 4H4

Received October 11, 1990; accepted February 2 1, 199 1

The structural polyprotein of rubella virus is cotranslationally processed by host cell signal peptidase. Oligonucleo- tide-directed mutagenesis was used to alter the cleavage site between capsid and E2 proteins and to examine the importance of this cleavage for the transport and processing of E2 and El glycoproteins. The in vifro and in vivo expression of the cleavage site mutant revealed that the E2 polypeptide can cross the endoplasmic reticulum membrane without the cleavage of its signal peptide, while the transport of E2 beyond the endoplasmic reticulum requires the cleavage of E2 from capsid. We have shown that capsid protein does not appear to undergo further proteolytic processing after it is cleaved from E2 by signal peptidase. Some of the requirements for the cleavage by signal peptidase between capsid and E2 were examined by the in vitro analysis of wild-type and mutant cDNAs. o ISSI

Academic Press, Inc.

INTRODUCTION

Rubella virus (RV), a member of the Togavirus family (Matthews, 1982) gives rise to two mRNA species in infected cells, a genomic 40 S mRNA and a subgenomic 24 S mRNA (Oker-Blom et a/., 1984). The RV structural proteins are coordinately translated from the 24 S subgenomic RNA species as a 1 1 0-kDa polyprotein precursor (Oker-Blom, 1984) and are processed by endoproteolytic cleavage by host signal peptidase (Clarke et a/., 1987, 1988) to yield three structural proteins, in the order NH2-C-E2-El -COOH (Oker-Blom, 1984). C is a capsid protein, whereas E2 and El are transmembrane glycoproteins which traverse the host cell’s exocytic pathway. The cleavage sites between C/E2 and E2/El proteins have been determined from the N-terminal amino acid analysis of the E2 and El proteins (Kalkkinen et a/., 1984). Analysis of the amino acid sequences which surround the two cleavage sites suggests the presence of signal peptide sequences preceding E2 and El proteins within the polyprotein (Clarke et a/., 1987). The hydrophobic signal sequences have been shown to be necessary for the translocation of E2 and El across the membrane of the rough endoplasmic reticulum (ER) (Hobman and Gil- lam, 1989; Hobman et a/., 1988) and are likely cleaved

’ Present address: Division of Cellular and Molecular Medicine, University of California, San Diego, M-051, La Jolla, CA 92093.

’ To whom correspondence and reprint requests should be ad- dressed.

0042-6822/91 $3.00 Copyright 0 1991 by Academic Press. Inc

by signal peptidase on the luminal side of the ER membrane (Blobel and Dobberstein, 1975).

Although not closely related, the alphaviruses and RV share a similar strategy for the organization and expression of their structural genes (Oker-Blom et al., 1984; Soderlund et al., 1985). However, the alphavirus capsid protein differs from the RV capsid with respect to the mechanism of its cleavage from the polyprotein. The alphavirus capsid protein is an autoprotease and quickly releases itself from rest of the polyprotein precursor within the cytoplasm (Simmons and Strauss, 1974; Aliperti and Schlesinger, 1978; Boege et al., 1981; Hahn et a/., 1985; Melancon and Garoff, 1987; Hahn and Strauss, 1990) whereas RV capsid protein requires the presence of microsomal membranes for its cleavage from the polyprotein precursor in vitro (Clarke et al., 1988). Site-directed mutagenesis of the putative active site of the capsid protein of two alphaviruses, Semliki Forest virus (SFV) (Melancon and Gar- off, 1987) and Sindbis virus (SV) (Hahn and Strauss, 1990) has demonstrated the importance of a con- served tetrapeptide sequence (Gly-Asp-Ser-Gly) containing the active serine residue for the protease function. Although the RV capsid protein shares little amino acid sequence homology with the alphavirus capsid protein (Frey and Marr, 1988) it has a peptide sequence at its C-terminus that resembles the active site of serine proteases.

We are interested in the processing of RV structural proteins from the polyprotein precursor with respect to the cleavage events that are required to separate the

52

All rights of reproduction in any form reserved

CAPSID CLEAVAGE INFLUENCE ON E2 AND El PROCESSING 53

individual proteins from the polyprotein precursor. Al- though the role of signal peptidase in the cotransla- tional cleavage of the C/E2 polyprotein has been demonstrated (Clarke et al., 1988) the nature of the C/E2 cleavage and its effect on the processing of E2 and El proteins have not been reported. Some important fea- tures of the C/E2 cleavage are that the capsid protein is cleaved by a signal peptidase after the E2 signal sequence (Kalkkinen et al., 1984; Hobman and Gillam, 1989) and that the E2 signal sequence appears to re- main attached to the C-terminus of the capsid protein (Suomalainen et al., 1990; Lee et al., 1991).

In this report we have examined the nature of the C/E2 cleavage by analysis of both the wild-type precursor polyprotein and a mutant that had its consensus signal peptidase cleavage site altered by oligonucleotide-directed mutagenesis. The effect of C/E2 cleavage on the transport of E2 and El proteins was studied by in vitro and in vivo expression of the wild-type and mutant polyproteins. Indirect immunofluorescence was used to determine the subcellular location of RV structural proteins in transfected COS cells. Our results suggest that sequences beyond the C/E2 cleavage site affect the C/E2 cleavage and signal peptide function. The introduction of a mutation at the C/E2 cleavage site causes a decrease in the efficiency of the processing of the C/E2 polyprotein and has a profound effect on the transport of the E2 protein. However, this mutation does not appear to alter the processing of El in the polyprotein containing the three structural proteins.

MATERIALS AND METHODS

Materials

Restriction endonucleases and DNA modifying en- zymes were purchased from commercial suppliers and used according to manufacturers’ specifications. Esch- erichia co/i strains DH~Lu and DH5aF’ from Bethesda Research Laboratories, and E. coliCJ236 from Bio-Rad were used for the propagation of recombinant clones. The [T-~‘P]ATP (3000 Ci/mmol), [a-32P]dATP (3000 Ci/ mmol), and L-[35S]methionine (600-800 Ci/mmol) were from DuPont. The vectors used in expression studies were pSPTl9 (Pharmacia) and pCMV5 (Dr. David Rus- sell, Department of Molecular Genetics, University of Texas, Dallas). The rabbit reticulocyte lysate, microsomes, and phage SP6 RNA polymerase used for in vitro translations were purchased from Promega Bio- tech. The synthetic deoxyribonucleotides were ob- tained from the laboratory of M. Smith (University of British Columbia). Fluorescein-conjugated goat anti- human or anti-mouse IgG was from Kirkegaard and Perry Laboratories or Tago, Inc. Tetramethylrhoda-

mine isothiocyanate (TRITC)-conjugated wheat germ agglutinin (WGA) and TRITC-conjugated concanavalin A (Con A) were from Sigma.

Mutagenesis

The method of oligonucleotide-directed mutagenesis described by Kunkel (1985) was used to alter sequences in the structural genes of rubella virus. The synthetic deoxyribonucleotide used to alter the cleavage site between capsid and E2 was S’CTGGAGCCC- GGGGCGCGCGGY (the altered nucleotide residue is underlined). Mutants were selected by screening of transformants with the mutagenic oligonucleotide. The presence of mutations was confirmed by dideoxy se- quencing (Sanger et al., 1977) of the cDNAs according to the Sequenase protocol (U.S. Biochemicals).

Plasmid constructs

pCE2 was derived from p24S, which codes for the three structural proteins (Hobman et al., 1990), by ex- cision of the Pstl fragment containing the coding region of the El gene, followed by religation. pC5’E2, a truncated cDNA coding for capsid and the N-terminal 124 amino acids of E2, was constructed as described previously (Clarke et al., 1987). pCP5’E2, the mutant plasmid, encodes a proline (P) residue at the (-1) position of the cleavage site rather than an alanine residue as in pCE2 (Fig. 3a). pCE2X codes for capsid and 86 amino acids of E2 and was constructed by cutting pC5’E2 at the BstXl site of E2, removing the 3’ over- hang with T4 DNA polymerase, and religating to the Smal site of the vector (Fig. la). The mutant cDNA, pCE2X-G1/2 (Fig. 2a), was derived from an E2 cDNA, clone which had the first two N-linked glycosylation sites altered (Hobman et a/., 1991). It was constructed by isolation of the BstXl fragment from the E2 mutant cDNA, which was then blunt-ended with T4 DNA polymerase and cut with BstEll. The digested E2 fragment was then ligated into the BstEll- and Seal-digested pC5’E2 plasmid (Fig. 1 a). For in vitro and in viva analysis of the endoproteolytic cleavage between capsid and E2, the respective cDNAs were subcloned into the EcoRl and HindIll sites of the vectors pSPT19 and pCMV5, respectively. pCMV5 is an in viva transient expression vector containing the human cytomegalovi- rus major immediate early gene promoter (Andersson et a/., 1989).

In vitro transcription and translation

Plasmid DNA was linearized at either the HindIll site of the vector or, for the expression of truncated peptides, at restriction sites within the cDNA coding sequence. The transcription by SP6 RNA polymerase and

54 MCDONALD, HOBMAN, AND GILLAM

in vitro translations using a rabbit reticulocyte lysate system (in some cases supplemented with microsomes) were carried out as previously described (Hob- man et a/., 1988).

COS cell transfection and metabolic labeling

The transfection of COS cells and labeling of intracellular proteins were performed as described previously (Hobman and Gillam, 1989) with the follow- ing modifications. Transfected cells grown in 35 mm wells were labeled for 30 min with medium containing 100 &i L-[35S]methionine. Then cells were incubated with a medium containing 100 pg/ml cycloheximide and 2 mM methionine for 2 hr for viral protein processing studies. After labeling, cells were washed with Tris-saline (25 mM Tris/CI, pH 7.4; 140 mM NaCI; 0.3 mM KCI; 1 mM CaCI,; 0.5 mM MgCI,; 0.9 mM Na,HPO,) and lysed in 400 ~1 of cold lysis buffer (1% Triton X-l 00; 10 mM EDTA; 50 mMTris/CI, pH 7.5; 1% sodium deoxycholate; 0.15 NI NaCI; 0.1% SDS). Fol- lowing centrifugation of the cellular lysates, the super- natants were removed for immunoprecipitation.

Immunoprecipitation, endo-P-A/-acetyl-o- glucosaminidase H (endo H) digestion, and indirect immunofluorescence

lmmunochemical methods and digestion of immunoprecipitated products with endo H glycosidase (Tar- entino and Maley, 1974) were as described previously (Hobman and Gillam, 1989). Human anti-RV serum (a gift from Dr. A. Tingle, Department of Pediatrics, Univer- sity of British Columbia) was used for the immunoprecipitation of intracellular-labeled proteins. The immunoprecipitated products were analyzed by SDS-PAGE analysis. Mouse monoclonal antibody to capsid (Dr. John Safford, Abbot Laboratories) and to E2 (a gift from Dr. Jerry Wolinsky, Department of Neurology, Univer- sity of Texas) was used for the indirect immunofluorescence studies of permeabilized cells.

RESULTS

Effect of translocated E2 on C/E2 cleavage

There is evidence that some preproteins may require peptide domains beyond the signal peptide region for the efficient translocation and cleavage of their signal peptides (Andrews et a/., 1988). Cleavage between capsid and E2 occurs cotranslationally as a result of insertion of the signal sequence for E2 into the ER membrane (Hobman and Gillam, 1989). The influence of translocated E2 peptide sequences on the cleavage between capsid and E2 proteins by signal peptidase was studied by in vitro expression of run-off transcripts

from progressively 3’ truncated cDNAs coding for full- length capsid and E2 of different lengths (Fig. 1 a). Fig- ure 1 b shows the in vitro translation products of the phage SP6 transcripts translated in the presence and absence of microsomes and separated by SDS-gel electrophoresis. This preparation of microsomes was inefficient in processing the synthesized peptides and some unprocessed precursor polypeptides were present in all of the translation products. In the presence of microsomes cleavage of capsid was observed in the translation products from transcripts encoding C/E2 precursors that contained full-length E2 (Fig. lb-A), the N-terminal 124 (Fig. 1 b-B) or 86 residues (Fig. 1 b-C) of E2. There was no cleavage of capsid by signal peptidase of the microsomes when the C/E2 precursor contained only the N-terminal 25 residues of E2 (Fig. 1 b-D). These results suggest that there is a minimum length requirement for the translocation and signal peptidase cleavage of RV E2 glycoprotein, which is in between 25 and 86 amino acid residues of E2. The influence of E2 is not restricted to the in vitro processing of the C/E2 precursor by membranes. A similar result has also been observed in vivo for a vaccinia virus recombinant which also codes for the same precursor protein (Gil- lam et al., 1991).

Influence of glycosylation on the cleavage between capsid and E2

In this region between the N-terminal 25 and 86 residues of E2 are two asparagine-linked glycosylation sites (Clarke et al., 1987). The influence of carbohy- drate moieties at these sites on the C/E2 cleavage was studied using a glycosylation mutant. The glycosylation sites at position 53 (Gl) and 71 (G2) were altered by oligonucleotide-directed mutagenesis (Hobman et al., 1991). The asparagine residue at the Gl site was changed to glutamine and the serine residue at the G2 site to glycine (Fig. 2a). The mutated cDNA was subcloned into the pSPT19 vector, creating pCE2X-G1/2, which codes for full-length capsid and the N-terminal 86 residues of E2 containing two mutated glycosylation sites. The mutant and wild-type cDNAs were linearized at the Hindlll site of the vector and used as templates for in vitro synthesis of synthetic RNA,transcripts. In vitro translation of the transcripts in the presence and absence of microsomes showed that there was no apparent difference in the efficiency of C/E2 cleavage observed in the glycosylated and the ungly- cosylated C/E2 polypeptide precursors (Fig. 2b). SDS- PAGE showed only the cleaved capsid as the excised 86 residues of E2 ran off the gel under these condi- tions. This indicates that glycosylation is not important for the in vitro cleavage of this polyprotein precursor by signal peptidase.


a

5’ ATG

C E2 B X

spl P 3’

TixF b

A B C D I I I

I--+ mic - +I- +t- +I- +

I I I

P A : I

E Et I p&2

E C I

T pCE2X

E 8 D

I 1

FIG. 1. ln vitro analysis of a minimum length requirement for signal peptidase’s cleavage. (a) A representation of the cDNAs coding for capsid and E2 of different lengths. The restriction sites BstEll (B), BsfXl (X), Psfl (P), EcoRl (E), and Hindlll (H), and signal peptide (SP) and transmembrane (TM) domains are indicated. A-D represent linearized cDNAs cloned into pSPTl9 which were used as templates for transcription. A codes for full length capsid and E2, linearized at the Pstl site at the 3’terminus of E2. B, pC5’E2 encodes capsid and 124 amino acids of E2, linearized at the Hindlll site of the vector. C, pCE2X codes for capsid and 86 amino acids of E2, linearized at the Hindlll site of the vector. D codes for capsid protein and 25 amino acids of E2 and was derived from pC5’E2 by linearization at the BstEll site at the 5’ terminus of E2. (b) The translation products from SP6 polymerase transcripts separated by SDS-PAGE. Translations were in the presence and absence of microsomes (mic). Molecular weight markers are indicated (in kDa) and the capsid protein cleavage product (C) is shown.

Mutation of the signal peptidase cleavage site between capsid and E2

Usually the specificity of the cleavage reaction by signal peptidase is very high in that the region around the cleavage site shows strong preferences for particu- lar amino acids in specific positions. Small, neutral residues are found in positions -1 and -3 (counting from the cleavage site between positions -1 and +l), but are rare in -2 (von Heijne, 1984). The signal peptidase cleavage site between C and E2 was altered by oligo-

nucleotide-directed mutagenesis in a cDNA encoding the entire capsid protein and the N-terminal 124 residues of E2. In this constructed mutant (pCP5’E2) the (- 1) residue at the cleavage site was changed from an alanine to a proline (P) residue (Fig. 3a). ln vitro expression of the wild-type and cleavage mutant cDNAs showed that the change at the cleavage site did not completely block the cleavage of the C/E2 polyprotein, but caused a reduction in the efficiency of cleavage as compared to wild-type (Fig. 3b). The appearance of a translation product of higher molecularweight (Fig. 3b,

b I pCE2X ; pCE2X-G112

mic - +‘- +

a C E2

1 2

pCE2X wt NAS wt NAS

pCE2 X-G1/2 Gl QAS G2 NAG

FIG. 2. In vitro analysis of the signal peptidase cleavage of an E2 glycosylation-deficient mutant. (a) The wild-type cDNA, pCE2X (C in Fig. l), is represented and the two sites in this construct which encode the sequence for N-linked glycosylation are indicated by arrows. The mutant cDNA, pCE2X-G1/2, was derived from an E2 cDNA clone previously constructed in this laboratory which had been altered at the first two N-linked glycosylation sites of E2 (see Materials and Methods). E, B, X, and H denote the EcoRI, BstEll, BsfXI, and Hindlll restriction sites, respectively. (b) The translation products from transcripts derived from the HindIll-linearized cDNAs, pCE2X and pCE2X-G1/2. Capsid protein(C) is the cleavage product shown which appears when microsomes (mic) are added to the translation mixture. Protein molecular weight standards are indicated (in kDa).


b 1 2 314 5 6 I

endoH - - f 1 - - •f-

mic - t -1 I - + +

a C E2 68,

5 /E

3’ 43,

-7 29, W,

PCS’EZ

Gs “c GCC GAT G&C ACC GCG CCC CCC Gs

AVAVGTAR;;G .5’E2

1 184, mutan, pCP5’EZ

ccc P 14, *

FIG. 3. ln vitro analysis of the C/E2 signal peptidase cleavage site mutant. (a) Mutation of the signal peptidase cleavage site between capsid (C) and E2. The cDNA, pC5’E2 (B in Fig. l), was used as a template for the mutagenesis of the cleavage site between C and E2. The alanine residue was altered to proline. E and H denote the EcoRl and Hindlll restriction sites flanking the 5’and B’ends of the construct. (b) The cDNA constructs, pC5’E2 and pCP5’E2, were linearized with Hindlll and transcribed by SP6 polymerase. SP6 transcripts were translated in the presence and absence of microsomes (mic) and the translation products were immunoprecipitated with human anti-RV serum and then digested with endo H and analyzed by SDS-PAGE. The cleavage products from the translations with microsomes (mic) added are capsid (C) and a truncated E2 from the 5’coding region (5’EZ). The uncleaved C/E2 precursor is approximately45 kDa. Aglycosylated form of this precursor is seen in lane 5, above the 45-kDa peptide (shown by arrowhead). Lanes l-3: pWE2 translation products. Lanes 4-6: pCP5’E2 translation products. Protein molecular weight standards are indicated (in kDa).

shown by arrow) than that of the uncleaved precursor in the translation products of the mutant suggests that some mutant precursor is translocated and core-glycosylated, but not cleaved, by signal peptidase. This was confirmed by treatment with endo H, an enzyme that removes N-linked high mannose sugar residues (Tarentino and Maley, 1974) which resulted in a loss of the higher molecularweight band corresponding to the increase in the amount of deglycosylated precursor (Fig. 3B, lanes 5 and 6). This result indicates that the change at the cleavage site affected only the cleavage and not the translocation or glycosylation of the precursor C/E2 peptide.

The mutant precursor was cleaved at a reduced rate compared to the wild-type precursor. Since alternate processing by signal peptidase can occur when a bulky residue replaces the normal (-1) residue of a signal peptide (Hot-tin and Boime, 1981), an alternate cleavage site may be chosen by signal peptidase when the proline residue is introduced into the C/E2 cleavage site. The proline may inhibit cleavage at the normal site through steric hindrance and in addition it may create a new cleavage site recognized by signal peptidase. The (-7) residue from the N-terminus of E2 is a potential candidate for an alternate cleavage site (Fig. 3A) since it is another alanine residue surrounded by residues characteristic of a signal peptidase cleavage site (Ala-X-Ala) (von Heijne, 1986).

The mutation at the cleavage site introduced a new Smal recognition sequence at the cleavage site between capsid and E2. This cleavage site was used to linearize the C/E2 template at the 3’ end of the coding

region of the capsid gene for in vitro transcription. The SP6 RNA polymerase transcript was translated in vitro and the product was compared by SDS-PAGE analysis with the capsid protein extracted from RV virions. There is no apparent difference in mobility between the full-length capsid translation product and the mature capsid protein of virions (data not shown). This suggests that the signal peptide of E2 remains at the carboxy terminus of capsid and is not cleaved by other endoproteolytic activity. This result confirms the re- ports of Suomalainen eta/. (1990)and Leeetal. (1991).

In order to study the effect of C/E2 cleavage on the processing of E2 and El glycoproteins in viva, the cleavage site mutants, pCPE2 and p24S-CPE2, were generated by replacing the f3stEIIIEcoRI fragment of the wild-type cDNAs in pCE2 and p24S with the corresponding fragment from the mutant, pCP5’E2. The C/ E2 construct encodes capsid protein and E2, and the 24s construct encodes the three RV structural proteins, capsid, E2, and El. Transfected COS cells were labeled with [35S]methionine for a period of 30 min, and chased with nonradioactive methionine for a period of 2 hr. Labeled proteins were immunoprecipitated with human anti-RV serum and analyzed by SDS-PAGE. Analysis of the immunoprecipitated products from the C/E2-transfected cell lysates (Fig. 4) showed that after a chase period the majority of the wild-type C/E2 precursor was cleaved to a 35-kDa capsid protein and a 39-kDa core-glycosylated form of the E2 protein, while some of the mutant C/E2 precursor remained uncleaved. The presence of the mutation at the C/E2 cleavage site in the polyprotein containing the three


pCE2 ; pCPE2 j p24S j p24S-CPE2 1 I 1 I

I endo H - -f- I - -f- 1 - fj- +

I I I I

FIG. 4. In viva pulse-chase study of the C/E2 signal peptidase cleavage site mutant. COS cells were transfected with the pCMV5 cDNAs (mutant and wild-type). Transfected cells were labeled with [36S]methionine for a pulse period of 30 min and chased for a period of 2 hr. Labeled lysates were immunoprecipitated with human anti- RV serum, and aliquots of the immunoprecipitated products were digested with endo H. SDS-PAGE analysis was performed to separate the various immunoprecipitated products. The endo H-sensitive (core-glycosylated) forms of the C/E2 precursor (C/E2), El, and E2 (39 kDa) are indicated by arrowheads. Protein molecular weight standards are shown (in kDa). Wild-type (pCE2, ~24s). Cleavage site mutant (pCPE2, p24S-CPE2).

structural proteins did not affect the topology or the processing of the signal peptide for El. El cleavage product was equally abundant in wild-type- and mutant-transfected cells (Fig. 4). An incompletely cleaved C/E2 precursor was also seen in the transfected cell products of the mutant 24s cDNA, p24S-CPE2 (Fig. 4). These results indicate that the translocation and cleavage of the signal peptide for El is independent of the cleavage of the E2 protein from capsid.

Endo H glycosidase digestion of the 2-hr chase products showed that the oligosaccharide structures on the mutant C/E2 precursor were sensitive to glycosidase, as seen by the shift in mobility of the polypeptide from 74 kDa to approximately 65 kDa (Fig. 4). This suggests that most of the mutant precursor was translocated, since it is N-glycosylated, and that it was re- tained within the ER or cis-Golgi vesicles, where oligosaccharide structures show sensitivity to endo H glycosidase (Dunphy and Rothman, 1985). Endo H digestion reduced the 57-kDa El and 39-kDa E2 glycoproteins to 5 1 and 3 1 kDa, respectively. The mobility of capsid protein was not affected by endo H treatment (Fig. 4).

lmmunofluorescence studies

Transfected COS cells were processed for indirect immunofluorescence studies on the subcellular local-

ization of the C/E2 mutant and wild-type precursor polypeptides and their cleavage products. Figures 5 and 6 show the distribution of C and E2 in cells transfected with pCE2 (wild-type) and pCPE2 (mutant) plasmids. Monoclonal antibodies against structural proteins

FIG. 5. Indirect immunofluorescence of pCE2- and pCPE2-transfected COS cells: E2 localization. Cells were permeabilized with 0.05% Nonidet P-40 for the detection of intracellular antigens and treated with anti-E2 serum. (A) pCE2-transfected cell, anti-E2; (E?) same cell, TRITC-WGA; (C) pCE2-transfected cell, anti-E2; (D) same cell, TRITC-Con A; (E) pCPEZ-transfected cell, anti-E2; (F) same cell, TRITC-WGA; (G) pCPE2-transfected cell, anti-E2 (H) same cell, TRITC-Con A.


were used to localize these antigens. The same cells were also treated with rhodamine-conjugated WGA to identify Golgi and post-Golgi structures (Virtanen et al., 1980) or with Con A to identify the ER. The E2 expressed in pCE2-transfected cells was found distrib- uted intracellularly throughout the ER and Golgi-like re- gions (Figs. 5A-5D). In contrast, E2 expressed from pCPE2 (Fig. 5E) showed a predominantly reticular staining pattern and does not appear to concentrate in the Golgi region of the cell (Fig. 5F). Figure 5G shows another cell transfected with pCPE2, with a reticular staining pattern that is similar to the staining pattern of the ER (Fig. 5H). Both wild-type- and mutant-transfected cells exhibited cell surface staining of E2 anti- gen (data not shown).

FIG. 6. Indirect immunofluorescence of pCE2- and pCPE2-transfected COS cells: capsid localization. Cells were permeabilized and treated with anti-capsid (C) serum. (A) pCE2-transfected cells, anti- C; (B) same cells, TRITC-WGA; (C) pCE2-transfected cells, anti-C; (D) same cells. TRITC-Con A; (E) pCPE2-transfected cells, anti-C; (F) same cells, TRITC-Con A.

FIG. 7. lndrrect rmmunofluorescence of p24S and p24SCPE2- transfected COS cells: E2 and capsid localization. (A-D) Cells were permeabiltzed and treated with ant+capsid (C) serum. (A) p24S transfected cell, anti-C; (B) same cell, TRITC-WGA; (C) p24SCPE2- transfected cells, anti-C; (D) same cells, TRITC-Con A. (E and F) Cells were permeabilized and treated with anti-E2 serum. (E) p24S CPE2-transfected cells, anti-E2; (F) same cells, TRITC-Con A.

Wild-type C expressed from pCE2 is seen diffused throughout the cell cytoplasm, rather than concen- trated in the ER or Golgi region, (Figs. 6A-6D). The staining pattern of pCPE2-derived C is very different from that of the wild-type C (Figs. 6E and 6F), suggest- ing a reticular location of this protein, probably corresponding to the ER.

Figure 7 shows the subcellular distribution of C and E2 expressed from the 24s cDNA constructs, p24S and p24S-CPE2. When wild-type El and C were coex- pressed, a strong juxtanuclear staining pattern that corresponds to the Golgi region was observed for C (Figs. 7A and 7B), in contrast to the diffuse cytoplasmic distribution observed when C was expressed in the absence of El (Fig. 6A). It is likely that in COS cells C concentrates at the Golgi membrane in association with the cytoplasmic tail of El (Hobman et al., 1990). Capsid protein expressed from p24S-CPE2 is less con- centrated in the juxtanuclear region and appears to reside in the reticular network of the ER (Figs. 7C and


70). The staining of E.2 protein expressed from the wild-type construct, p24S was found to be similar to that of the E2 expressed in pCE2-transfected cells (Figs. 5A and 5B). It was predominantly in a juxtanuclear position which indicates that it has been transported to the Golgi apparatus. However, E2 expressed from p24S-CPE2 shows a more diffuse staining pattern, similar to the pattern of Con A staining which is characteristic of the ER network (Figs. 7E and 7F).

DISCUSSION

We have examined the cleavage of a wild-type and a cleavage mutant C/E2 precursor to RV capsid and E2 proteins in vitro and the processing of precursor polyproteins both in vitro and in viva. We have shown that there is a minimum length requirement for the translocation and cleavage of this precursor. More than 25 amino acid residues of E2 must be translocated and folded for this processing to occur. The E2 peptide may be important for the stabilization of the conforma- tional determinants for the signal peptide function. An- drews et al. (1988) have shown that the protein domain being translocated can have a profound impact on the efficiency of the translocation. Deletions in the mature prolactin “passenger” domain, beyond the signal cleavage site, reduce the efficiency of signal function.

In vitro analysis has shown that N-linked glycosylation occurs cotranslationally as the nascent peptide is inserted into the lumen of the ER (Rothman and Lodish, 1977). Glycosylation is known to influence protein transport in some cases by its effect on protein confor- mation (reviewed in Strauss and Strauss, 1985) and it can also influence the cleavability of a peptide. A mutation in the influenza virus hemagglutinin protein that prevents glycosylation can result in enhanced cleavability of the protein (Kawaoka and Webster, 1989). From our in vitro studies (Fig. 2) it appears that the glycosylation is not essential for the cleavage of C/E2 precursor polyprotein. This is consistent with the find- ing that RV E2 is similar to the SFV envelope proteins, which are translocated and cleaved in tunicamycin- treated cells (Garoff and Schwarz, 1978; Clarke et al., 1987).

The importance of the signal peptidase cleavage site for the cleavage of capsid and E2 has been demonstrated by the analysis of a mutant at the cleavage site. Changing the alanine residue to a proline residue at this site causes the precursor to be only partially cleaved by signal peptidase both in vitro and in vivo (Figs. 3 and 4). The introduction of a mutation at the cleavage site between capsid and E2 causes a decrease in the efficiency of the processing of the C/E2

precursor polyprotein and also has a profound effect on the transport of the E2 protein. An alternate cleavage site may be chosen by the signal peptidase, which may have an effect on the rate and efficiency of transport of E2 through the host cell secretory pathway.

lmmunofluorescence studies indicated that the uncleaved precursor remains predominantly within the ER (Figs. 5-7). Some viral envelope proteins must be correctly folded as a requirement for transport from the ER (Gething and Sambrook, 1986). Since unprocessed E2 will be held within the membrane at its N-terminus by its signal peptide, it may be prevented from exiting the ER due to incorrect folding. Some E2 expressed from the mutant cDNA is also expressed at the cell surface. This may be explained by the partial cleavage of the mutant precursor, observed by SDS-PAGE analysis. However, it is possible that some of the uncleaved precursor was also transported to the cell surface. Further studies are required to identify the proteins present at the cell surface.

By the in vitro analysis of capsid protein expressed from the cleavage site mutant, it was demonstrated that the signal peptide for E2 remains at the carboxy terminus of capsid. Using an antiserum directed against the E2 signal sequence, Suomalainen et al. (1990) have demonstrated that this signal peptide remains attached to both the in vitro synthesized capsid protein and that in mature virus. Therefore, the processing of RV capsid protein is quite different from that of the closely related alphaviruses. The alphavirus C protein autoproteolytically cleaves itself from the polyprotein and the signal sequence of the p62 membrane protein remains part of that protein (Melancon and Gar- off, 1987). The failure of the RV capsid protein to be cleaved from the C/E2 polypeptide may reduce its abil- ity to function in nucleocapsid assembly and virus bud- ding, although there is evidence for a membrane association of the capsid protein, the significance of which remains to be defined (Suomalainen et al., 1990). How- ever, the separation of the capsid protein from the E2 protein is clearly important for the correct intracellular targeting of the E2 glycoprotein.

ACKNOWLEDGMENTS

This work was supported jointly by grants from the Medical Re- search Council of Canada and British Columbia Health Care Re- search Foundation. T. Hobman was a predoctoral trainee receiving support from the Medical Research Council of Canada in the biotech- nology training program.

REFERENCES

ALIPERTI, G., and SCHLESINGER, M. J. (1978). Evidence for an autoprotease activity of Sindbis virus capsid protein. Wology 90, 366- 369.


ANDERSSON, S., DAVIS, D. L., DAHLBACK, H., JORNVALL, H., and Rus- SELL, D. W. (1989). Cloning, structure, and expression of the mito- chondrial cytochrome P-450 sterol26-hydroxylase, a bile acid bio- synthetic enzyme. 1. Biol. Chem. 264, 8222-8229.

ANDREWS, D., PERARA, E., LESSER, C., and LINGAPPA. V. (1988). Se- quences beyond the cleavage site influence signal peptide function. J. Biol. Chem. 263, 15,791-15,798.

BLOBEL, G., and DOBBERSTEIN, 6. (1975). Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane- bound ribosomes of murine myeloma. J. Cell Biol. 67, 852-862.

BOEGE, U., WENGLER, G., WENGLER, G., and WITMANN-LIEBOLD, B. (1981). Primary structure of the core proteins of the alphaviruses Semliki Forest virus and Sindbis virus. Virology 113, 293-303.

CLARKE, D. M., Loo, T. W., HUI, I., CHONG, P., and GILLAM, S. (1987). Nucleotide sequence and in vitro expression of rubella virus 24s subgenomic mRNA encoding the structural proteins El, E2, and C. Nucleic Acids Res. 15, 3041-3057.

CLARKE, D. M., Loo, T., MCDONALD, H., and GILLAM, S. (1988). Ex- pression of rubella virus cDNA coding for the structural proteins. Gene 65,23-30.

DUNPHY, W. G., and ROTHMAN, J. E. (1985). Compartmental organization of the Golgi stack. Ce// 42, 13-21.

FREY, T. K., and MARR, I. D. (1988). Sequence of the region coding for virion proteins C and E2 and the carboxy terminus of the nonstruc- tural proteins of rubella virus: Comparison to alphaviruses. Gene 62, 85-99.

GAROFF, H.. and SCHWARZ, R. T. (1978). Glycosylation is not necessary for membrane insertion and cleavage of Semliki Forest virus membrane proteins. Nature (London) 274, 487-489.

GETHING, M. J., and SAMBROOK, J. (1986). Expression of wild-type and mutant forms of influenza hemagglutinin: The role of folding in intracellular transport. Cell 46, 939-950.

GILLAM, S., HOBMAN, T., and MCDONALD, H. (1991). Antigenic properties and cellular localization of rubella virus structural proteins expressed via recombinant vaccinia virus. Submitted for publication.

HAHN, C. S., and STRAUSS, J. H. (1990). Site-directed mutagenesis of the proposed catalytic amino acids of the Sindbis virus capsid protein autoprotease. J. viral. 64, 3069-3073.

HAHN, C. S., STRAUSS, E. G., and STRAUSS, J. H. (1985). Sequence analysis of three Sindbis virus mutants temperature-sensitive in the capsid autoprotease. Proc. Nat/. Acad. Sci. USA 82, 4648- 4652.

HOBMAN, T. C.. and GILLAM, S. (1989). /n vitro and in viva expression of rubella virus glycoprotein E2: The signal peptide is contained in the C-terminal region of capsid protein. Virology 173, 241-250.

HOBMAN, T. C., LUNDSTROM, M. L., and GILLAM, S. (1990). Processing and intracellular transport of Rubella virus structural proteins in COS cells. Virology 178, 122-l 33.

HOBMAN, T., QIU, Z.. MCDONALD, H., and GILLAM, S. (1991). The role of asparagine-linked oligosaccharides in processing and cell surface expression of E2 glycoprotein of rubella virus. Submitted for publication.

HOEMAN, T. C., SHUKIN. R., and GILLAM, S. (1988). Translocation of rubella virus glycoprotein El into the endoplasmic reticulum. J. Viral. 62, 4259-4264.

HORTIN, G., and BOIME, I. (1981). Miscleavage at the presequence of rat preprolactin synthesized in pituitary cells incubated with a thre- onine analog. Cell 24, 453-46 1.

KALKKINEN, N., OKER-BLOM, C., and PE-~~ERSSON, R. F. (1984). Three genes code for rubella virus structural proteins, El, E2a, E2b, and C. 1. Gen. Viral. 65, 1549-l 557.

KAWAOKA, Y., and WEBSTER, R. G. (1989). Interplay between the car- bohydrate in the stalk and the length of the connecting peptide determines the cleavability of influenza virus hemagglutinin. J. Viral. 63, 3996-3400.

KUNKEL, T. A. (1985). Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc. Nat/. Acad. Sci. USA 82, 4753-4757.

LEE, D. M., SANCHEZ, A., and FREY, T. K. (1991). Efficient in vitro translation and processing of the Rubella virus structural proteins in the presence of microsomes. Virology 180, 400-405.

MATTHEWS, R. E. F. (1982). Classification and nomenclature of viruses. Third report of the international committee on taxonomy of viruses. Intervirology 17, l-l 99.

MELANCON, P., and GAROFF, H. (1987). Processing of the Semliki Forest virus structural polyprotein: Role of the capsid protease. J. Viral. 61, 1301-1309.

OKER-BLOM, C. (1984). The gene order for rubella virus structural proteins is NH2-C-E2-El-COOH. J. l&o/. 51, 354-358.

OKER-BLOM, C., ULMANEN, I., KAARIAINEN, L., and PEITERSSON, R. F. (1984). Rubella virus 40s RNA specifies a 24s subgenomic RNA that codes for a precursor to structural proteins. J. Viral. 49,403- 408.

ROTHMAN, J. E., and LODISH, H. F. (1977). Synchronized transmembrane insertion and glycosylation of a nascent membrane protein. Nature (London) 269, 775-780.

SANGER. F., NICKLEN, S., and COULSON, A. R. (1977). DNA sequenc- ing with chain terminating inhibitors. Proc. Nat/. Acad. Sci. USA 74, 5463-5467.

SIMMONS, D. T., and STRAUSS, J. H. (1974). Translation of Sindbis virus 26s RNA and 49s RNA in lysates of rabbit reticulocytes. J. Mol. Biol. 86, 397-409.

SODERLUND. H., TAKKINEN, K., JALANKO, A., and KALKKINEN, N. (1985). The expression and organization of the alphavirus genome. In “Viral Messenger RNA” (Y. Becker, Ed.), pp. 323-337. Martinus Nijhoff, Boston.

STRAUSS, E. G., and STRAUSS, J. H. (1985). Virus structure and assembly. /n “Assembly of Enveloped Viruses” (S. Casjens, Ed.), pp. 206-234. Jones and Bartlett, Boston.

SUOMALAINEN, M., GAROFF, H., and BARON, M. (1990). The E2 signal sequence of Rubella virus remains part of the capsid protein and confers membrane association in vitro. J, Viral. 64, 5500-5509.

TARENTINO, A. L., and MALEY, F. (1974). Purification and properties of an endo-fi-N-acetylglucosaminidase from Streptomyces griseus. J. Biol. Chem. 249, 81 l-81 7.

VIRTANEN, I., EKBLOM. P., and LAURILA, P. (1980). Subcellular com- partmentalization of saccharide moieties in cultured normal and malignant cells. J. Ce// Biol. 85, 429-434.

VON HEIJNE, G. (1984). How signal sequences maintain cleavage specificity. J. Mol. Biol. 173, 243-251.

VON HEIJNE, G. (1986). A new method for predicting signal sequence cleavage sites. Nucleic Acids Res. 14, 4683-4691.

the influence of capsid protein cleavage on the processing of e2 and e1 glycoproteins of rubella...

Documents