Analyses of the terminal sequences of west nile virus structural proteins and of the in vitro translation of these proteins allow the proposal of a complete scheme of the proteolytic

VIROLOGY 169,365-376 (1989)

Analyses of the Terminal Sequences of West Nile Virus Structural Proteins and of the in Vitro Translation of these Proteins Allow the Proposal of a Complete Scheme

of the Proteolytic Cleavages Involved in Their Synthesis

THOMAS NOWAK, PETRA M. FARBER, GISELA WENGLER, AND GERD WENGLER

lnstitut fiir Virologie, Justus-Liebig-UniversitSt Giessen, D-6300 Giessen, Federal Republic of Germany

Received April 27, 1988; accepted November 23, 1988

The proteolytic processes involved in the synthesis of the structural proteins of the West Nile (WN) flavivirus were analyzed: The carboxy-terminal sequences of the structural proteins were determined and the proteins translated in vitro in the presence of membranes from a mRNA coding for the structural polyprotein were analyzed. The results obtained indicate that the following proteolytic activities are involved in the synthesis and assembly of WN virus structural proteins: The growing peptide chain which contains the sequences of the structural proteins in the order C-pre- M-E is cleaved at three places by cellular signalase(s). This cleavage generates the primary amino acid sequence of the mature structural proteins pre-M and E (and the amino-terminus of the ensuing nonstructural protein NS 1). The amino-terminal part of the polyprotein containing the amino acid residues 1 to 123 is released as a molecule which migrates slightly slower than the mature viral core protein and which presumably is associated to the RER membranes via its carboxy-terminal sequence. This protein is called the anchored C protein. Prior to or during assembly of intracellular virus particles the anchored C protein is converted into mature C protein by removal of the carboxy-terminal hydrophobic segment containing the amino acid residues 106 to 123. Presumably a virus-coded protease which can cleave the polyprotein after two basic amino acid residues is responsible for this cleavage. The cell-associated WN virus particles are constructed from the proteins C, pre-M, and E which contain the amino residues l-l 05, 124-290, and 291-787 of the polyprotein, respectively. Cleavage of the pre-M protein between amino acid residues 215 and 216, presumably by a cellular enzyme located in the Golgi vesicles, and loss of the amino-terminal fragment of this protein are associated with the release of virus from the cells. o t999Academic press, hc.

INTRODUCTION

The proteins specified by flaviviruses have been studied intensively by SDS-PAGE of infected cells, viruses, and subviral particles (see Russell et al., 1980, and Westaway, 1980, for reviews). These experiments have shown that cell-associated flaviviruses consist of a ribonucleoprotein core which contains a core(C) protein of approximately 13 kDa molecular weight which is surrounded by a lipid bilayer containing the two membrane proteins E and pre-M (which was labeled NV 2 in the earlier literature) of about 50 kDa and 22 kDa molecular weight, respectively. Instead of the pre- M protein the extracellular virus contains the shorter protein M (of about 8 kDa molecular weight) which is generated from the pre-M protein by proteolytic cleavage (Shapiro et al., 1972; Castle ef al., 1985).

Recently the complete nucleotide sequences of the genome RNA molecules of the following flaviviruses have been determined: Yellow Fever (YF) virus (Rice et a/., 1985); West Nile (WN) virus (Castle et al., 1985, 1986; Wengler et a/., 1985); Japanese encephalitis (JE) virus (Sumiyoshi et al., 1987); Dengue 2 (Den 2) virus (Hahn et al., 1988); and Kunjin (Kun) virus (Coia et al., 1988). These data have shown that a single long open

reading frame comprising about 10,500 nucleotides exists on these molecules. Amino-terminal sequence analyses of structural proteins of YF virus (Rice et al., 1985) WN virus (Castle et al., 1985; Wengler et al., 1985) St. Louis encephalitis (SLE) virus (Bell et al., 1985) DEN 2 virus (Bell et al., 1985; Biedrzycka et al., 1987) and JE virus (Sumiyoshi et a/., 1987) have led to the conclusion that these proteins are present in the amino-terminal part of the corresponding polyprotein in the order: NH,-C-pre-M-E.

Analyses of the amino-terminal sequences of the WN virus structural proteins C, pre-M, and E and of the nucleotide sequence coding for these proteins have indicated that these proteins presumably are separated from each other by signalase cleavage of the growing polyprotein chain in the RER (Castle et a/., 1985; Wen- gler et al., 1985). We have now attempted to find direct experimental evidence for the role of signalase in the synthesis of the WN virus structural proteins and to analyze the existence of other proteases which might possibly be involved in their synthesis. Two major types of experiments were made: The carboxy-terminal sequences of the structural proteins were characterized and a mRNA coding for the corresponding polyprotein was translated in vitro in the presence of RER mem-

365 0042.6822189 $3.00 Copynght 0 1989 by Academc Press. Inc. All rights of reproduction I” any form reserved.

366 NOWAK ET AL.

branes and the resulting translation products were analyzed. Together with the earlier experimental data cited above the results obtained allow us to propose a complete scheme of the proteolytic cleavages involved in the generation of the structural proteins of WN virus.

MATERIALS AND METHODS

Materials and reagents

DFP-treated carboxypeptidase A was obtained from Sigma, carboxypeptidase B and endoglycosidase H were purchased from Boehringer-Mannheim. L-

[35S]cysteine and L-[35S]methionine were obtained from NEN. Chemicals were bought from Merck. Cellu- lose thin-layer plates (Polygram CEL 300) were obtained from Macherey-Nagel.

Purification of WN virus

Extracellular virus was purified from the growth medium harvested at about 30 hr p.i. by differential centrifugation followed by equilibrium centrifugation as described previously (Castle et a/., 1985). Intracellular virus was purified from virus-infected cells harvested at about 30 hr p.i. according to the procedure developed by Shapiro et a/. (1972) as previously described (Castle et al., 1985).

Preparation of radioactively labeled virus

In order to prepare virus containing either [35S]methionine or [35S]cysteine the growth medium of 10 mono- layer cultures, each of which contained approximately lo7 cells, was replaced at about 10 hr p.i. by fresh growth medium lacking either methionine or cysteine. The radioactively labeled amino acid was then added to a concentration of 5 &i/ml and the medium and the cells were harvested at about 30 hr p.i. for isolation of extracellular and cell-associated virus, respectively.

Protein fractionation by SDS-PAGE

Analytical and preparative SDS-PAGE was performed according to the procedure of Laemmli (1970) as described (Wengler et a/., 1987).

Determination of carboxy-terminal protein sequences by carboxypeptidase digestion

Carboxy-termini were analyzed by carboxypeptidase treatment as described by Jones (1986). In order to de- termine the carboxy-terminal sequence of the C protein viral cores containing approximately 5 nmol of C protein were solubilized in 40 ~1 of water. After addition of 500 ~1 of 200 m/l/l A/-ethylmorpholine-acetate, pH 8.5, and 10 ~1 of water containing 14 nmol of norleucine,

aliquots containing 2 nmol norleucine were removed either prior to addition of 1.5 pg of carboxypeptidase B (t = 0) or a various times during incubation in the presence of enzyme at 37”. The protein present in these samples was removed by TCA precipitation, the TCA was removed from the supernatant by ether extraction and amino acids released were determined by ion exchange chromatography followed by postcolumn analysis with OPA. The total amount of substrate present in the reaction was determined by amino acid analysis of an acid hydrolyzate of an aliquot which was taken prior to addition of enzyme. The carboxy-terminal sequence of the E protein was determined in a similar manner with the following modifications: The E protein was isolated by preparative SDS-PAGE. Since the resulting denatured protein was not soluble in the buffer described above SDS was added to the buffer to 0.4%. Carboxypeptidase A was present at a relative concentration of 1 fig of enzyme per 150 pg of substrate and the reaction was performed at room temperature. Sam- ples were taken and analyzed as described above. The carboxy-terminal sequence of the M protein which was isolated by preparative SDS-PAGE was determined by treatment with carboxypeptidase A in the presence of 0.4% SDS (1 pg of enzyme per 50 pg of substrate) as described above but since the protein did not precipi- tate in the presence of TCA the total volume of the reaction was reduced to 50 ~1 and the aliquots of the reaction were diluted 20-fold in the pH 2.2 buffer used for amino acid analysis and subjected directly to ion exchange chromatography.

Preparation of mRNA coding for WN virus structural proteins

The mRNA used for in vitro translation of the structural polyprotein was generated by in vitro transcription of cloned cDNA. DNA complementary to the 5’-terminal region of the WN genome was synthesized in vitro using a primer for second strand synthesis which contains a C/al recognition sequence in front of the 5’-terminal sequence of the viral genome. The resulting ds- DNA was cleaved with C/al (the recognition sequence of which is not present in WN virus cDNA) and with Sall, which cleaves the cDNA behind position 2707 of the WN cDNA sequence, 246 nucleotides downstream of the nucleotide triplet GCT which codes for the carboxy- terminal alanine residue of the viral E protein. The cDNA was then cloned into the pGEM-4 plasmid (Pro- mega-Biotec) which had been modified by insertion of a C/al recognition sequence into the polylinker region. The resulting cloned plasmid was linearized by cleavage at the unique SalI cleavage site and transcribed by SP 6 polymerase in the presence of m’GpppG. The

SYNTHESIS OF WN VIRUS STRUCTURAL PROTEINS 367

resulting RNA which has a total length of 2732 nucleotides contains the 5’-terminal sequence m’GpppGAA- TACACGGAATXATCGATA (22). The A residue in position 22 represents the 5’-terminal residue of the WN viral genome (Castle and Wengler, 1987). Transcription was performed according to the procedures given by Promega-Biotec. Transcribed RNA was purified by chromatography on a lo-ml column of Sepharose 6B (Maniatis et al., 1982). Details of the construction and analysis of this cDNA clone will be described else- where in the context of the preparation of a complete cDNA copy of the WN virus genome.

In vitro translation of mRNA

Nuclease-treated rabbit reticulocyte lysate (code N 90) and canine pancreatic microsomal membranes (code N 270) were obtained from Amersham and assembled for in vitro translation as suggested by the manufacturer. The reaction contained, in a final volume of 10 ~1, 4.5 ~1 of lysate, 3.3 ~1 of membranes, 0.2 ~1 of water containing 40 ng mRNA, and 2 ~1 (20 &i) of either [35S]methionine or [35S]cysteine. Further addition of buffers or salts did not improve the quality or efficiency of translation or processing. Translations were generally performed for 90 min at 30” followed by a 15- min centrifugation of the reaction mixture at 15,000 g. Only the material present in the resulting pellet was used for further analyses. More than 90% of the incor- porated radioactivity was present in this fraction.

Fingerprint analyses of proteins

Proteins were digested by tr-ypsin or chymotrypsin in the presence of bovine serum albumin as carrier for 3 hr at 37” with 5 pg of enzyme per 50 pg of substrate in 100 mM N-ethylmorpholine-acetate, pH 8.5. After removal of insoluble material by centrifugation the frag- ments were loaded onto cellulose thin-layer plates and subjected to electrophoresis (pH 4.4) followed by chromatography, as described (Boege et a/., 1980).

Amino-terminal sequencing of proteins

Labeled proteins were sequenced manually according to Chang et al. (1978) as described in detail else- where (Boege et al., 1980). During each sequencing cycle the solvent containing the released amino acid was subjected to radioactivity determination by liquid scintillation counting.

Treatment of proteins with endoglycosidase H

Radioactively labeled proteins were purified by preparative SDS-PAGE. Purified proteins were digested with endoglycosidase H (1 mU per reaction volume of

30 ~1) in buffer containing 66 mM Na-acetate, pH 5.2, 0.02% SDS, and 2 mM PMSF for 3 hr at 37”. Proteins were then analyzed directly by SDS-PAGE in the presence of mercaptoethanol followed by autoradiography.

Chromatography of proteins on organomercurial agarose

[35S]Methionine-labeled authentic core protein or [35S]methionine-labeled in vitro synthesized p 14 protein was isolated by preparative SDS-PAGE. The purified protein was subjected to covalent chromatography on organomercurial agarose using an Affi-Gel 501 (Bio-Rad) column of 750 ~1 volume. Proteins were loaded in 150 ~1 buffer containing 50 mMTris, pH 7.0, 1 mM EDTA, pH 7.0, 0.5 M LiCI, 0.1% SDS, and 20 pg human globulin as a carrier molecule. Proteins were eluted using the same buffer without globin containing 20 mM DlT. Aliquots of the resulting fractions were analyzed for radioactivity by liquid scintillation counting and subjected to TCA precipitation followed by SDS- PAGE analysis of the precipitated proteins.

RESULTS

The carboxy-terminal sequences of the structural proteins

The sequence of the polyprotein giving rise to the WN virus structural proteins is presented in a shortened version in Fig. 1. The sequence is processed into the proteins C, pre-M (which is processed later into M), and E and contains the amino-terminus of the nonstructural protein NS 1. The amino-termini of these proteins are indicated (Castle et al., 1985; Wengler et al., 1985). We have suggested that the amino-termini of proteins pre M, E, and NS 1 are generated from the growing polypeptide chain by signalase cleavage (Wengler et al., 1985). If no other proteases are involved in the generation of the proteins C, pre-M, and E the data presented in Fig. 1 predict that these molecules have the carboxy-terminal sequences CAGA (123) PAYS (290) and NVHA (787) respectively. We have determined the carboxy-termini of these proteins, isolated from extracellular WN virus, by analyses of the amino acids released from purified protein by carboxypeptidase. Since the pre-M protein is present in extracellular WN virus only in small amounts we have instead used the M protein of extracellular virus in these experiments. The data presented in Fig. 2 show that the predicted carboxy-terminal sequences can be identified in the proteins E (Fig. 2A) and M (Fig. 28). The sequence FLSVNVHA (787) gives rise to the amino acids released from E protein: The single terminal alanine residue is released rapidly together with valine,

368 NOWAK ET AL.

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FIG. 1. Localization of the cleavages involved in the generation of the structural proteins of WN virus. The amino acid sequence of that part of theviral polyprotein which gives rise to the structural proteins is presented in a shortened version using the one letter code; the internal sequence of the E protein is not shown since it is not relevant in the context of the experiments to be reported. The amino-terminal sequences of the structural proteins identified by earlier analyses (Castle et al., 1985; Wengler et a/., 1985) are indicated by thin arrows. The carboxy-terminal sequences of the viral structural proteins C, M, and E determined in this manuscript are indicated by thick arrows. The amino-terminus of the WN virus-specific NS 1 protein is derived from alignment of the WN sequence with the corresponding YF virus derived sequence for which the amino-terminus of the NS 1 protein has been determined (Rice et a/., 1985). The six cleavage reactions inferred from these data are indicated by triangles and numbers. The only sequence of the structural polyprotein which is not found in the structural proteins of purified cell-associated WN virus particles is the region between the cleavages 2 and 3 which is underlined.

which is present twice in the sequence followed by a slower release of serine, leucine, and phenylalanine. The histadine release could not be analyzed because the corresponding region of the chromatogram was contaminated with impurities present in the protein isolated by preparative SDS-PAGE. Asparagine is not determined by the procedure used for amino acid analy-, sis. The sequence PAYS (290) gives rise to the amino acids released from M protein: Only serine and tyrosine are released. Proline cannot be released by carboxypeptidase A and inhibits the release of the preceeding amino acid (see Jones, 1986, for a review). Carboxy- peptidase A did not release amino acids from viral C protein (data not shown). The C protein sequence predicted above contains only a single cysteine residue which is part of the predicted carboxy-terminal sequence ACAGA (123) (Fig. 1). Amino acid analyses of

performic acid oxidized core protein of extracellular WN virus indicate that cysteine is absent from this molecule (data not shown). An enzyme presumably of viral origin which cleaves the viral polyprotein in positions containing two basic amino acid residues followed by an uncharged residue containing a small side chain plays an important role in the generation of the nonstructural proteins (Rice et a/., 1985; Speight et al., 1988). The sequence KKRG (106) is located in front of the C protein carboxy-terminus predicted above. Cleavage of the polyprotein within this sequence by this protease would give a carboxy-terminal sequence KKR of the viral core protein (Rice eta/., 1985). Analysis of C protein of extracellular WN virus using carboxypeptidase B, which can release only basic amino acid residues (Fig. 2C), shows that the carboxy-terminus of this protein is composed of two lysine and one arginine


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FIG. 2. Time course of amino acid release from the WN virus structural proteins E, M, and C by carboxypeptidase. Proteins were purified by preparative SDS-PAGE and electrophoretic elution. Purified proteins were solubilized in buffer in the presence of SDS and after removal of an aliquot for determination of protein content each protein was subjected to enzymatic digestion. Carboxypeptidase A was used to digest the proteins E and M whereas carboxypeptidase B was used to digest protein C. Aliquots were removed from the reactions prior to addition of enzyme (time 0) and at the times indicated in the figure and the amino acids released were determined by ion exchange chromatography. Each aliquot analyzed during digestion of E protein (A) contained 0.7 nmol of protein. The aliquots taken during digestion of M protein (B) contained 0.5 nmol protein. The aliquots taken during digestion of C protein (C) contained 0.7 nmol of protein. Further details are given under Materials and Methods.

residues. Since such a sequence is not present in any other region of the C protein sequence (Fig. 1) these data allow the identification of the amino acid se-

quence KKR (105) as carboxy-terminus of the C protein isolated from extracellular WN virus.

These results show that the only part of the polyprotein which is not found in the molecules E, pre-M and C, isolated from extracellular WN virus, is the hydrophobic amino acid sequence comprising the residues 106 to 123. Three possibilities exist for the fate of this segment: The core protein of intracellular virus may still contain it, it may be present as an independent molecule of 1606 Da molecular weight in intracellular and possibly extracellular virus, or the segment might be absent from both types of virus particles. The presence of this segment in the C protein of intracellular virus should give rise to molecular weights of 13.3 kDa and 1 1.7 kDa for the C proteins derived from intracellular and extracellularvirus, respectively. Furthermore it has to be expected that the 13.7-kDa molecule can be labeled with [35S]cysteine whereas the 1 1.7-kDa protein contains no cysteine (Fig. 1). In order to analyze these points a comparative SDS-PAGE analysis of purified cell-associated and extracellular WN virus was performed (Fig. 3) using virus labeled with [35S]methionine (which labels all proteins) (lanes 1 and 2), virus labeled with [35S]cysteine (lanes 4 and 5), and unlabeled virus (lanes 6 and 7). Photographs of the stained gel and of an autoradiogram of the gel are shown in Figs. 3A and 3B, respectively. It can be seen that the core protein of cell-associated and extracellular virus migrate into the same position during SDS-PAGE and that both proteins cannot be labeled with [35S]cysteine. It will be shown later that a C protein containing the amino acid residues 106 to 123 migrates slowerthan the C protein of extracellular WN virus in this type of SDS-PAGE. The data therefore indicate that the segment comprising amino acid residues 106 to 123 is absent from the C protein of cell-associated virus. If this segment were removed late during virus assembly it might be retained in the intracellular virus particle. Since the structural proteins are present in the virus in approximately equi- molar amounts (data not shown) this segment should be detectable byautoradiographic analysis of gels containing proteins of [35S]cysteine-labeled virus: Since each segment contains one residue of cysteine and since each E protein contains 12 cysteine residues the segment should contain almost one-tenth of the radioactivity present in the E protein. The autoradiogram presented in Fig. 3B and other SDS-PAGE analyses (data not shown) show that such a fragment is absent from intracellular and extracellular virus particles.

Analysis of the proteolytic cleavage by RER membranes of the structural polyprotein translated in vitro

From analyses of nucleotide sequences and of sequences of structural proteins we have suggested that

370 NOWAK ET AL.

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FIG. 3. Comparative analysis of intracellular and extracellular WN virus by SDS-PAGE. The following samples were analyzed: Lane 1, [%]methionine-labeled intracellularvirus; lane 2, [%]methionine-labeled extracellularvirus; lane 3, marker proteins: ovalbumin (43 kDa), a-chymotrypsinogen (25.7 kDa), ,&lactoglobulin (18.4 kDa), lyso- zyme (14.3 kDa), bovine trypsin inhibitor (6.2 kDa). and insulin (a mixture of A (2.3 kDa) and B (3.4 kDa) chains which are not resolved); lane 4, [35S]cysteine-labeled intracellular virus; lane 5. [%]cysteine- labeled extracellular virus; lane 6, unlabeled intracellular virus; lane 7, unlabeled extracellularvirus. A gel containing 15% acrylamide was used. The position of bromphenol blue dye at the end of electrophoresis is indicated by BPB. The gel was stained with Coomassie bril- liant blue R 250, dried, and autoradiographed. Photographs of the stained gel and the autoradiogram are shown in (A) and (B), respectively. The positions of the proteins E, pre-M, C, and M are indicated on both sides in (A) in order to allow an unambiguous identification of these molecules. The different positions of the pre-M protein present in intracellular and extracellular virus are indicated by the sub- scripts i and e, respectively.

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the WN virus structural polyprotein is cleaved into the proteins C, pre-M, and E bysignalase(s) (Wengleretal., 1985). In the light of the data obtained above this model must be modified. We now expect that this cleavage generates the proteins pre-M and E and a core protein containing an additional carboxy-terminal sequence comprising the amino acid residues 106 to 123. Fur- thermore we expect that this C protein contains cysteine in contrast to the viral C protein (see above) and that it is associated with the RER membrane via the hydrophobic carboxy-terminal sequence. Therefore this protein will be called the anchored C protein. In order to test these predictions we have translated a mRNA coding for the structural polyprotein in vitro in the presence of RER membranes. The mRNA used was a molecule of 2732 nucleotides in length which was transcribed in vitro by SP 6 polymerase from a cloned cDNA. This mRNA contains the nucleotide residues 1 to 271 1 of the WN viral genome which code for all viral structural proteins and forthe amino-terminal 82 amino acid residues of the NS 1 protein (see under Materials and Methods for details). An analysis of the [35S]methionine-labeled translation products obtained in the absence or presence of membranes is shown in Fig. 4A. A major translation product of about 80 kDa which re- mains in the supernatant after centrifugation is synthesized in the absence of membranes (lane 3) whereas

FIG. 4. SDS-PAGE analyses of proteins translated in V&O. (A) In the experiment shown, mRNA coding for all viral structural proteins and for the ensuing 82 amino acids of the NS 1 protein was translated in vitro in the presence of [%S]methionine in a reticulocyte lysate in the absence or presence of membranes. Following translation the material was subjected to a 15-min centrifugation at 15,000 g and the resulting fractions were analyzed by SDS-PAGE (see under Materials and Methods for details). Samples were analyzed as follows: Lanes 1 and 2, supernatant and pellet, respectively, from a reaction without mRNA and without membranes. Lanes 3 and 4. supernatant and pellet, respectively, a reaction containing mRNA but no membranes. Lanes 5 and 6, supernatant and pellet, respectively, from a reaction containing mRNA and membranes. Lanes 7 and 8, supernatant and pellet, respectively, from a reaction containing membranes but no mRNA. A fluorogram of a gel containing 12.5% acrylamide is shown. (8) A comparison of the [35S]methionine-labeled proteins present in extracellular WN virus (lane 1) with the sediment- able proteins which were translated from the polyprotein mRNA in vitro in the presence of membranes (lane 2). An autoradiogram of a gel containing 15% acrylamide is shown. The apparent molecular weights of the proteins which are synthesized in vitro were estimated from the migration of marker proteins (data not shown). (C) Electrophoretic analysis of the carbohydrate content of in vitro synthesized proteins. Proteins translated in vitro in the presence of membranes were purified by preparative SDS-PAGE and analyzed by SDS-PAGE without or after digestion with endoglycosidase H (see under Materials and Methods for details). An autoradiogram of a gel containing 12.5% acrylamide is shown. The samples were analyzed as follows: Lanes 1 and 2. p 58 protein incubated in the absence or presence of enzyme, respectively. Lanes 3 and 4, the same analyses using p 54 protein. Lanes 5 and 6, the same analyses using p 22.23 protein. Lanes 7 and 8, the same analyses using p 14 protein.


the proteins p 58, p 54, p 36, p 25, p 22,23, and p 14 accumulate in the presence of membranes. The major- ity of these proteins sediment into the pellet together with the membranes during centrifugation (lane 6). A comparison by SDS-PAGE of [35S]methionine-labeled proteins synthesized in vitro in the presence of membranes and of [35S]methionine-labeled structural proteins of extracellular WN virus is shown in Fig. 5. The p 54 protein migrates into the same position as authentic E protein. The WN virus pre M protein is represented by a closely spaced doublet after SDS-PAGE (see also

FIG. 5. Identification of proteins obtained during in vitro translation by peptide mapping. [35S]methionine-labeled proteins were obtained by preparative SDS-PAGE followed by autoradiography of the wet gel and electrophoretic elution. The proteins analyzed are indicated in the figure. Proteins p 54, p 22,23, and p 14 are isolated from in t&o translation reactions and are identified in Fig. 4. Proteins E. pre- M, and C were isolated from [?S]methionine-labeled purified extracellularvirus. Proteins were digested with trypsin (p 54, E, p 14, C) or with chymotrypsin (p 22,23, pre-M) and the resulting peptides were subjected to electrophoresis followed by chromatography as described under Materials and Methods. Autoradiograms of the resulting chromatograms are shown. The proteins analyzed are indicated In the figure.

Fig. 3). The protein double-band p 22,23 migrates slightly faster than the pre-M protein of extracellular virus, The p 14 molecule which migrates slightly slower than the authentic C protein might represent the anchored C protein. In order to establish the relationships between the authentic structural proteins and the in vitro translation products the molecules were characterized further: The carbohydrate content of the proteins translated in vitro in the presence of membranes was analyzed by comparative SDS-PAGE analyses of purified proteins without or after treatment with endoglycosidase H (Fig. 4C). Endoglycosidase H does release N-linked high mannose-type glycanes which are transferred to the growing polypeptide during in vitro protein synthesis in the presence of RER membranes. The sequence content of the proteins was analyzed by two-dimensional peptide mapping (Fig. 5). The amino- terminal sequences of proteins were characterized by amino-terminal radiosequencing of [35S]methionine-labeled proteins (Fig. 6). These experiments lead to the following characterization of the in vitro synthesized proteins: The p 58 protein is an unglycosylated molecule (Fig. 4C). It contains the peptides present in authentic viral E protein (data not shown) and has the amino-terminus of authentic E protein according to the radiosequencing data (Fig. 6). The fact that this molecule has an apparent molecular weight which is some- what higher than that of authentic E protein therefore indicates that it contains an extension of corresponding size at the carboxy-terminus of the protein. A likely interpretation of these data is to assume that the (signalase) cleavage which generates the carboxy-terminus of the E protein has not been made on this protein and that this molecule therefore contains at its carboxy-terminus the short amino-terminal NS 1 protein segment coded for by the artifical mRNA used for in vitro protein synthesis. The protein p 54 is by all criteria identical to authentic E protein: The peptide maps of both proteins are similar or identical (Fig. 5); both molecules contain a methionine amino acid residue at position 6 (Fig. 6) the proteins do have an identical apparent molecular weight during SDS-PAGE (Fig. 4B) and the p 54 protein apparently does not contain carbohydrate (Fig. 4C) in accordance with a similar finding for authentic E protein (Wengler eta/., 1985). The p 36 protein contains the amino-terminus of the viral C protein (Fig. 6). An in vitro translation product in which the cleavage between the C protein and the pre-M protein sequence has not been made would have an apparent molecularweight of about 35 kDa. The pre-M protein is the only structural protein of WN virus which contains carbohydrate (Wengler et al., 1985). Since during in vitro translation carbohydrate is transferred to the appro- priate acceptor sequence of the growing polypeptide this interpretation leads to the prediction that the p 36

372 NOWAK ET AL.

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protein should contain carbohydrate attached to the pre-M sequences of the molecule. Whereas the elec- trophoretically purified p 36 protein is not efficiently converted into a faster moving molecule by endoglycosidase H (possibly because of aggregation of the denatured molecules) treatment of the complete in vitro translation reaction mixture with endoglycosidase H prior to SDS-PAGE does not alter the apparent molecular weight of the proteins p 58 and p 54 but leads to the appearance of a p 32 protein instead of the p 36 molecule (data not shown). These data indicate that the p 36 protein represents a glycosylated protein containing the sequences of the proteins C and pre-M. The p 25 protein is present in the in vitro translation in vari- able amounts (compare Figs. 4A and 48) and contains the amino-terminal sequence of the viral core protein (data not shown). We have not analyzed this molecule in more detail. The p 22,23 protein contains the peptides present in the viral pre-M protein (Fig. 5) it is represented by a closely spaced double-band during SDS-PAGE similar to the authentic pre-M protein (Fig. 4B), and it contains the amino-terminal sequence of pre-M protein (Fig. 6). A close inspection of the radiosequencing data indicates that the p 22,23 molecules analyzed were probably contaminated with small amounts of p 25 protein which gives rise to radioactive signals in the sequencing steps one and six which are characteristic for the viral C protein. Similar to authentic pre-M protein the p 22,23 protein is glycosylated (Fig. 4C). The pre-M protein of intracellular virus migrates faster than the corresponding molecule of extracellular particles (Fig. 3) and the p 22,23 doublet synthesized in vitro migrates into an intermediate position (data not shown). The slight differences in mobility during SDS-PAGE of the pre-M protein double-bands of intracellular virus, extracellular virus, and the p 22,23 protein might correspond to different stages in carbohydrate processing (see Kornfeld and Kornfeld, 1985, for a review). The finding that treatment of the pre-M protein of intracellular WN virus with endoglycosidase H converts this protein into a molecule which has the same mobility during SDS-PAGE as the endo H- treated p 22,23 protein (data not shown) strongly sup- ports this interpretation. The p 14 protein contains the amino-terminal sequence of the viral core protein (Fig. 6) and the peptides present in the authentic C protein (Fig. 5). Both proteins are unglycosylated molecules (Fig. 4). The finding that the p 14 protein has a higher apparent molecular weight than the authentic viral C protein (Fig. 4B) and does sediment with the mem-

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FIG. 6. Characterization of amino-terminal sequences of in vitro translated proteins and authentic viral structural proteins by radiosequencing. [%]Methionine-labeled proteins were isolated by preparative SDS-PAGE of purified extracellularvirus (proteins E. pre-M, and C) and of the reaction products translated in vitro (proteins p 58, p 54, p 36, p 22,23, and p 14) as shown in Fig. 4. In the representa- tion of the data obtained during analyses of the authentic structural proteins the amino-terminal sequence of the corresponding protein is indicated on the abscissa and the positions of sequencing cycles in which a signal derived from [%]methionine is expected are marked by asterisks. All three amino-terminal sequences present in authentic viral C protein (Fig. 1) are given for this molecule. In order to simplify the comparison of the authentic proteins with those prod-

ucts of in vitro translation which correspond most closely to these molecules the data obtained for these molecules are presented on top of each other (p. 54 and E; p 22,23 and pre-M; p 14 and C).


branes after in vitro synthesis (Fig. 4A) indicates that the p 14 molecule represents the anchored C protein molecule. In view of the importance of the identification of the anchored C protein we have made two types of experiments in order to show more directly that the p 14 protein contains a cysteine residue (the residue 120) in contrast to the viral C protein which is free of cysteine (Fig. 1). A comparison of the SOS-PAGE pat- terns of the proteins synthesized in vitro in the presence of radioactively labeled methionine or cysteine is shown in Fig. 7A. It can be seen that all proteins which were identified using the pattern of [35S]methionine-labeled proteins are also labeled by [35S]cysteine includ- ing the p 14 molecule. Evidence for the presence of cysteine in a protein can be obtained by chromatography of the corresponding molecule on organomercurial agarose which covalently and selectively binds the cysteine SH groups. The bound protein can be specifically eluted under reducing conditions. Such a chromatography using [35S]methionine-labeled authentic C protein and [35]methionine-labeled p 14 protein is shown in Fig. 7B. It can be seen that these proteins show a very different behavior in this analysis: Whereas the authentic viral core protein does not bind, the [35S]methionine-labeled p 14 protein does bind efficiently albeit not quantitatively to the column. The fact that the p 14 protein does not bind in a quantitative manner probably

A

we .23

does not reflect a heterogenity of the cysteine content of the protein since approximately only half of the small amount of human globin molecules which were used as carrier during chromatography and which contain one and two free cysteine residues per CY and p chain, respectively, bind to the column (data not shown). The proteins used in these experiments were purified by preparative SOS-PAGE and SOS was included in the chromatography in order to keep the protein in a denatured soluble state. The available experimental evidence indicates that some oxidation of cysteine does occur during the preparation of the molecules and that SOS bound to the proteins interferes to some extent with the binding of these molecules to the affinity chromatography matrix (data not shown). Taken together the experiments reported above indicate that the p 14 molecule represents an unglycosylated protein which contains the amino-terminal and the internal sequences of the authentic C protein and a carboxy-terminal extension which includes the cysteine residue 120 of the polyprotein.

DISCUSSION

Together with earlier sequence analyses (Castle et a/., 1985; Wengler et al., 1985) the experiments described above allow us to propose a complete scheme

u, 2 L 6 8 x) 12 1L 16 li 20 22

Fractrons

FIG. 7. Analyses of the content of cysteine of the p 14 protein translated in vitro. (A) Comparative SDS-PAGE analysis of the proteins translated in vitro in the presence of RER membranes which were labeled either with [35S]methionine or with [35S]cysteine. An autoradiogram of a gel containing 12.5% acrylamide is shown. Experimental details are described under Materials and Methods. The following samples were analyzed: Lane 1, protein translated in vifro in the presence of ?S cysteine in a reaction containing no added mRNA. Lane 2, [36S]methionine-labeled purified extracellular WN virus. Lanes 3 and 4, proteins translated in vitro in the presence of mRNA coding for the structural polyprotein in the presence of either [35S]cysteine (lane 3) or [35S]methionine (lane 4). The authentic structural proteins are indicated on the left according to the proteins separated in lane 2. The in vitro translated proteins are indicated on the right. (B) Behavior of [35S]methionine-labeled authentic C protein isolated from extracellular virus and of [35S]methionine-labeled p 14 protein isolated from in vitro translation during covalent chromatography on organomercurial agarose. The material is loaded onto the column in the absence of a reducing reagent and bound molecules are eluted by DTT (see under Materials and Methods for details). (0) [35S]methionine-labeled authentic C protein; (A) [35S]Methionine-labeled p 14 protein.

374 NOWAK ET AL.

of the proteolytic cleavages involved in the synthesis and assembly of the structural proteins of WN virus. The processes can be described in three steps: in the first step, which is cotranslational, cleavage of the viral polyprotein, probably by cellular signalase(s), generates the proteins anchored C, pre-M, E, and the amino-terminus of the nonstructural protein NS 1. The amino-terminal methionine residue is removed from part of the anchored C protein molecules. In the second step cleavage of the anchored C protein between amino acid residues 105 and 106 generates (mature) C protein. This cleavage occurs prior to or during virus assembly. The cell-associated virus contains the proteins C, pre-M, and E. The C protein anchor sequence (residues 106-l 23) is the only part of the viral structural polyprotein which is not present in this virus. Cleavage of the viral pre-M protein between amino acid residues 2 15 and 2 16 during or shortly after release of virus from infected cells represents the third proteolytic step. The amino-terminal part of the pre-M protein (residues 124-2 15) is lost from the virus. In the case of WN virus this cleavage does not occur on all pre-M protein molecules.

Although the experiments reported above are not concerned directly with the identification of the proteases which take pat-t in the processing of viral proteins the data indicate that four different proteolytic activities are involved: The analyses of the amino acid sequences surrounding the cleavage sites (Castle et al., 1985; Wengler et al., 1985) and the studies concerning the in vitro synthesis of the structural proteins reported above indicate that cellular signalase(s) cleave the polypeptide at three places giving rise to the proteins anchored C, pre-M, and E. Removal of the amino-terminal methionine from the primary translation product by a cellular protease which is commonly performed in un- infected cells also occurs to some extent on the C protein in viva (Castle et al., 1985) and in vitro (Fig. 6). A protease of presumably viral origin which cleaves the polyprotein after two basic amino acid residues if an amino residue containing an uncharged small side- chain follows plays an important role in the synthesis of the nonstructural proteins (Rice eta/., 1985; Speight eta/., 1988). The sequence specificity and the localization in the cellular cytoplasm make this enzyme a good candidate for the protease involved in the conversion of anchored into mature C protein. Analyses of the Yellow fever virus polyprotein for the presence of such cleavage sites already have led Rice et al. (1985) to suggest that this enzyme might be responsible for the cleavage of the C protein from the polyprotein. Inspection of the sequences of flavivirus polyproteins determined so far (see references given under the Introduction) shows that this cleavage site is conserved (data not shown). The fourth protease involved in the generation of struc-

tural proteins is the enzyme which converts pre-M into M. The results reported above do not contain any new information concerning the nature of this enzyme. Cel- lular proteases are present in the lumen of the Golgi vesicles which cleave polyproteins at the peptide bond following two basic amino acid residues (see Herbert and Uhler, 1982, and Bond and Butler, 1987, for reviews). If the virus particles pass through this compart- ment this enzyme could possibly convert pre-M into M. Further aspects of the enzymes involved in the proteolytic processing of the flavivirus polyprotein might be obtained from recent reviews on cellular proteases (Bond and Butler, 1987) on the proteolytic processing of viral polyproteins in general (Wellink and van Kam- men, 1988), and of flaviviruses in particular (Strauss et a/., 1987).

The data reported above represent the first experiments in which the synthesis of the three proteins anchored C, pre-M, and E has been identified in vitro. The data extend earlier experiments on the translation of Tick-borne encephalitis virus genome RNA in the presence of membranes in vitro (Svitkin et a/., 1984; Lya- pustin et a/., 1986). At first sight the pattern of proteins synthesized in vitro in our experiment is rather different from the proteins present in extracellularvirus (Fig. 4B). The following interpretation of the pattern of proteins synthesized in vitro which follows from the data described above evaluates this difference. Our analyses indicate that the E protein has been synthesized in vitro in the form of the p 54 molecule. The p 22,23 protein synthesized in vitro represents the pre-M protein. The p 22,23 protein and the pre-M proteins of extracellular and intracellular virus particles are glycosylated. The data described above strongly indicate that the small differences in the mobility of these proteins during SDS-PAGE reflect differences in carbohydrate processing. The C protein is synthesized in vitro in the form of the p 14 molecule, the anchored C protein. Complete cleavage of the polyprotein in vitro, presumably by signalase(s), generates the proteins p 54, p 22,23, and p 14. In view of the complexities involved in the membrane-associated synthesis of these molecules it is not surprising that all three cleavages do not occur in all polyprotein molecules in vitro: if the cleavage which generates the carboxy-terminus of the p 54 protein is omitted the protein p 58 is made in vitro and omission of the cleavage which generates the amino- terminus of the p 22,23 protein probably leads to synthesis of the protein p 36. The p 36 protein probably represents a protein which contains the sequence of the core protein and the pre-M protein since it contains the amino-terminal sequence of the core protein, since its apparent molecular weight corresponds to the com- bined apparent molecular weight of the core protein and the pre-M protein of 35 kDa, and since the p 36


protein is synthesized as a glycosylated molecule in vitro. The protein M is generated from pre-M during the release of virus from the cells. Therefore it cannot be expected that the p 22,23 protein is processed further into M protein in the in vitro reaction analyzed in our experiments. Further experiments will be necessary in order to find conditions in which the efficiency of signalase cleavage in vitro might be increased. We expect that under these conditions the synthesis of the proteins p 58 and p 36 will be reduced in favor of the synthesis of the protein p 54 (E), p 22,23 (pre-M), and p 14 (anchored C).

The biological consequences of the conversion of the anchored C protein into the viral C protein remain to be determined but the chemical consequences of this reaction and their localization during virus synthesis, assembly, and release allow the suggestion of the following hypothesis: Fusion of the viral membrane with a cellular membrane followed by release and dis- assembly of the core within the cytoplasm is necessary during the early stages of infection. On the other hand, later on the viral genome RNA has to be assembled into stable viral particles during virus replication. It has been shown that not only the viral membrane proteins but also the newly synthesized viral core protein molecules are associated to cellular membranes in flavivirus-infected cells(Shapiro eta/., 1972; Stohlman eta/., 1975; Boulton and Westaway, 1976). The data reported above then suggest that at least some of the earlier steps of virus assembly make use of the membrane- associated anchored C protein. Since the C protein present in virus particles does not contain the carboxy- terminal hydrophobic sequence, the core which is released into the cellular cytoplasm in the early stages of infection then is of a different structure than the corresponding complex during virus assembly. The removal of the carboxy-terminal sequence of anchored C protein could convert a stable membrane-associated intra- cellularly assembling RNA-protein complex into a core which can be released from the viral envelope into the cytoplasm early during infection and which is able to liberate the genome RNAfor translation. Further experiments will be necessary to clarify these questions.

ACKNOWLEDGMENTS

We thank Dr. R. Rott for continuous support. We thank Mr. M. Dreisbach for the performance of amino acid analyses and Mrs. A. Krauss for excellent technical assistance. This study was supported by the Sonderforschungsbereich 47 and by the Fonds der Chem- ischen Industrie. This work is in partial fulfillment of the requirements for the degree Dr. rer. nat. of P. M. FBrber, Fachbereich Biologic, University of Giessen.

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Documents

Analyses of the terminal sequences of west nile virus structural proteins and of the in vitro translation of these proteins allow the proposal of a complete scheme of the proteolytic