Evidence that the mature form of the flavivirus nonstructural protein NS1 is a dimer

VIROLOGY

162,

187-l

Evidence

96 (1988)

that the Mature GUNTHER

Department

Form of the Flavivirus

Nonstructural

Protein

NSl Is a Dimer

WINKLER, VALERIE B. RANDOLPH, GRAHAM R. CLEAVES, TERENCE E. RYAN,’ AND VICTOR STOLLAR’

of Molecular Genetics and Microbiology, Robert Wood Johnson and Dentistry of New Jersey, 675 Hoes Lane, Piscataway, Received

July 29,

1987; accepted

September

Medical School, University New Jersey 08854

of Medicine

30, 1987

Evidence is presented which indicates that the dengue-2 virus nonstructural protein NSl (soluble complement fixing antigen) exists in infected BHK and mosquito cell cultures as part of a stable oligomer. Identification of the dissociation products of the isolated oligomer and comparison of the number of N-linked glycans in native and denatured NSl is consistent with the idea that the high-molecular-weight form of NSl is a homodimer. By analyzing lysates of BHK cells infected with St. Louis encephalitis virus or Powassan virus and proteins from dengue-2 virus-infected mouse brain we have demonstrated that the appearance of the high-molecular-weight form of NSl is a general feature of flavivirus infection. It is formed between 20 and 40 min after NSl is synthesized and before the protein passes the Golgi apparatus. Both soluble and pelletable extracellular NSl are also found as the high-molecular-weight form. o 1988 Academic

Press,

Inc.

INTRODUCTION

followed by the genes for the nonstructural proteins in the following order NSl , ns2a, ns2b, NS3, ns4a, ns4b, and NS5. Although it has not been possible so far to associate specific functions with any of the nonstructural proteins of flaviviruses, certain properties of NSl make it especially interesting. First, in contrast to the nonstructural proteins of other cytoplasmic RNA viruses, NSl is glycosylated (Westaway, 1975; Schlesinger et al., 1983; Smith and Wright, 1985), and can be found both on the cell surface and in the culture medium of infected cells (Cardiff and Lund, 1976; Stohlman et a/., 1975). Second, it has recently been shown that the dengue soluble complement fixing antigen (SCF), once thought by some to be implicated in the pathogenesis of dengue hemorrhagic fever and shock syndrome (Cardiff eta/., 1971) is actually NSl (Smith and Wright, 1985). Third, during the course of natural infection in man and experimental infections in mice, NSl evokes a strong antibody response (Russel et a/., 1980; Cammack and Gould, 1986). Fourth, it has been recently reported that antibodies to yellow fever virus NSl or dengue-2 virus NS 1, whether transferred passively or actively induced, protect mice or rhesus monkeys against lethal virus infection (Schlesinger et a/., 1985, 1986, 1987). This finding is of special interest because it demonstrates that immune protection to a virus infection can be obtained not only with neutralizing antibodies to structural proteins as is commonly observed, but in the case of flaviviruses also with antibodies to a nonstructural protein. In this report we describe a high-molecular-weight

The Flavivirus family consists of about 70 serologically distinguishable viruses including the causative agents of many serious illnesses such as dengue fever, yellow fever, St. Louis encephalitis tick-borne encephalitis, and Japanese encephalitis. The viruses in this family contain a single-stranded positive-sense RNA genome with an approximate length of 11 kb which together with the capsid (C) protein (MW 13,000-l 6,000) makes up the nucleocapsid. The nucleocapsid is surrounded by a lipid membrane which contains a large (MW 49,000-60,000) amphiphilic protein (E), which in most flaviviruses is glycosylated, as well as a smaller (MW 7000-9000) hydrophobic, nonglycosylated, integral membrane protein (M) (see Westaway et al., 1985, for review). The genomes of several flaviviruses, yellow fever virus (Rice et a/., 1985) West Nile virus (Castle et al., 1985, 1986; Wengler et al., 1985), and dengue (type 4) virus (Mackow et a/., 1987; Zhao et al., 1986) have been sequenced entirely and partial sequences of other flaviviruses have now been published. The genes for the structural proteins as well as those which encode the major nonstructural (NS) proteins have been mapped. The former are located at the 5’ end of the genome in the order (5’ + 3’) C, prM (which is later cleaved into M and a glycosylated fragment of unknown function), and E. The E protein sequence is ’ Present address: Laboratory of Biophysics, University consin, 1525 Linden Drive, Madison, WI 53706. 2 To whom requests for reprints should be addressed.

of Wis-

187

0042-6822/88 Copyright All rights

$3.00

0 1989 by Academic Press, Inc. of reproduction in any form resewed.

188

WINKLER ET AL.

form of NSl, not previously observed in flavivirus-infected cells. We believe that this band represents a dimer form of NSl and that it may represent the functional form of this protein.

MATERIALS

AND METHODS

Cells and viruses The Aedes albopictus cells (C7-10 clone) and the medium in which they were grown have been described previously (Durbin and Stollar, 1984) as have the BHK cells (Stollar et a/., 1976). Dengue virus type 2 (DEN-2, New Guinea B strain, Stevens and Schlesinger, 1965) was cloned in Vero cells and passaged several times in KB cells as described by Stollar (1969). St. Louis encephalitis virus (SLE, strain LAV5-156) was received from James Hardy (Arbovirus Research Unit, University of California at Berkeley) as the second passage in mosquito cells and was passed four more times in BHK cells. Powassan virus (POW) was received from G. H. Tignor (Arbovirus Research Unit, Yale University School of Medicine) as a lOq/o suspension of infected mouse brain and was passaged three times in BHK cells.

Analysis of [35S]methionine-labeled proteins virus-infected Aedes albopictus cells

in

Mosquito cells were infected with DEN-2 at a multiplicity of about 10 PFU/cell. After an adsorption period of 90 min the cells were fed with the usual medium modified to contain only l/5 (0.02 mM) the normal concentration of methionine. Eighty hours postinfection (p.i.) the medium was removed and the cultures were refed with medium containing 1.6 PLIVI(2 pug/ml) actinomycin D (Merck, Sharp, & Dohme) and only 0.004 mM methionine. [35S]Methionine (Tran-S label, ICN) was added 96 hr p.i. at a concentration of 100 &i/l 0” cells. After a labeling period of 4 hr cold methionine was added to a final concentration of 1 mM and incubation was continued for another 2 hr. The medium was then removed and the cell monolayer was washed with a buffer containing 50 mM Tris(hydroxymethyl)aminomethane (Tris, Sigma), 100 mll/l sodium acetate, adjusted with acetic acid to pH 7.4. Cells were lysed by a IO-min incubation at 37” in a 20 mM Tris, 150 mM sodium acetate buffer, pH 8.1, containing 1.5 mM MgCl and 0.5% (w/v) Triton X-l 00 (Sigma). Insoluble cell fragments were removed by centrifugation (Beckman microfuge, 5 min at 5000 rpm) and proteins were precipitated from the supernatant by addition of 5 vol of acetone in the cold (-20”). After 2 hr, proteins were pelleted in a Beckman microfuge at 10,000 rpm for 10 min. The dried pellet was redis-

solved in Laemmli sample buffer (Laemmli and Favre, 1973) and analyzed by SDS-PAGE.

Analysis of [35S]methionine-labeled virus-infected BHK cells

proteins

in

BHK cells approaching confluency were infected with DEN-2, SLE, or POW virus at an approximate PFU/cell ratio of 1O/l. After allowing 90 min for adsorption the cells were fed with normal growth medium for 6 hr. The medium was then changed to minimum essential medium containing 5 pglml actinomytin D but without serum or tryptose phosphate. [35S]Methionine (100 PC/lo6 cells) was added 25 hr p.i. Cells were labeled for 160 min (unless otherwise indicated); they were then lysed and proteins were prepared as described for mosquito cells. To examine the state of the extracellular NSl the culture medium was clarified by centrifugation at 10,000 g for 20 min; virus was then sedimented in an SW41 rotor (Beckman) at 27,000 rpm for 3 hr. The pellet (i.e., 50,000 g pellet) was dissolved directly in Laemmli sample buffer. Soluble proteins remaining in the 50,000 g supernatant were precipitated with acetone, pelleted, and redissolved in a 20 mM Tris/HCI buffer, pH 8.8, containing 2% SDS. The solution was desalted over a Bio-Gel P-6 (Bio-Rad) column with a 5 mM sodium acetate solution, containing 0.05% SDS, as the mobile phase. The proteins were recovered by acetone precipitation and redissolved in Laemmli sample buffer.

Polyacrylamide

gel electrophoresis

Discontinous polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS) and 2-mercaptoethanol (Laemmli and Favre, 1973) was performed using linear gradient gels with 7-l 4% (w/v) acrylamide.

Western

blotting

and immunoassay

of NSl

Western blotting was performed as described by Towbin et al. (1979). Identification of protein bands on nitrocellulose by the use of NSl-specific antibodies was carried out as described by Winkler et al. (1987). The monoclonal antibody to NSl (3E9-4) was a generous gift from Erik Henchal and Mary Kay Gentry, Walter Reed Army Institute of Research, Washington, DC.

Analysis of asparagine-linked endoglycosidases F and H

carbohydrates

with

Lysates of [35S]methionine-labeled DEN-2-infected BHK cells were prepared as described, clarified by centrifugation, and then desalted over a Bio-Gel P-6 column into a 50 mM phosphate buffer, pH 7.0, con-

FLAVIVIRUS NSl-EVIDENCE

taining 0.1% (w/v) SDS. Proteins from the culture medium were prepared essentially as described above, but with the following changes: The 50,000 g pellet was redissolved in 50 mM phosphate buffer, pH 7.0, containing 0.1% (w/v) SDS. Proteins from the 50,000 g supernatant were precipitated with acetone and then desalted into the same buffer. Before digestion Triton X-l 00 and tetrasodium (ethylenedinitrilo) tetraacetic acid (EDTA) were added to final concentrations of 1% (v/v) and 50 mM, respectively. Endo-@-N-Acetylglucosaminidase F (endoglycosidase F, NEN) was used at a concentration of 2 units per milliliter and endo-P-/Vacetylglucosaminidase H (endoglycosidase H, NEN) was used at a concentration of 15 units per milliliter. Incubation was at 37”. Aliquots were taken at the indicated times and the reaction was stopped by precipitation with acetone. If complete digestion was desired, a similar amount of enzyme was added again after 8 hr and incubation was continued for a total of 24 hr. Isolation and 1251-labelingof the high-molecularweight complex of NSl BHK cells were infected with DEN-2, SLE, or POW in the presence of [35S]methionine, as described above. The 50,000 g pellet (containing the viral structural proteins and NSl) was prepared from the cell-free medium; the proteins were redissolved in Laemmli sample buffer and separated by SDS-PAGE. After Western blotting and exposure of the nitrocellulose to X-ray film the high-molecular-weight complex of NSl was cut from the blot using the autoradiogram as a guide. The excised nitrocellulose band was dissolved in acetone at room temperature and then cooled to -20” for 1 hr after which the proteins were pelleted in a Beckman microfuge at 10,000 rpm for 10 min and then dissolved in a 50 mM phosphate buffer, pH 7.0, containing 1% SDS. Insoluble material was removed by centrifugation. The dissolved proteins were labeled using the lodo-bead (TM, Pierce) method (Praise and Phillips, 1985) and desalted over a Bio-Gel P-6 column into a 5 mM Na-acetate solution. The iodinated proteins were recovered by acetone precipitation, dissolved in Laemmli sample buffer, boiled and analyzed by SDSPAGE. RESULTS A previously undescribed virus-specific 86,000 mol wt glycoprotein in DEN-2-infected mosquito cells, and its relationship to NSl Figure la shows a typical example of a lysate of [35S]methionine-labeled DEN-2-infected Aedes albopictus ceils and the effect of boiling the lysates prior to

FOR A DIMER

93-

6645.

-NSJ -NS3 qE

9P55

3F

-NSl

,“:

1:. 7,, ^,

1NSI

2214, 31.

~~29-31

FIG. 1. Identification of a previously undescribed 86,000 mol wt protein in DEN-2-infected mosquito cells. (a) Comparison of unboiled (open circle) and boiled (closed circle) lysates from Aedes albopicrus cells either infected with DEN-2 virus(B) or mock-infected (A). Proteins were labeled with [%]methionine in the presence of actinomycin D. Three structural proteins (E forms a double band, prM, and C) as well as seven nonstructural proteins (NS5, NS3, NSl , ~31, p29, ~20, and ~14) were identified. (b) Conversion of purified gp86 to NSl by boiling. A [36S]methionine-labeled lysate of DEN-2infected Aedes albopicfus cells was fractionated by SDS-PAGE without prior boiling. The gp86 band was excised from a Western blot and redissolved in Laemmli sample buffer. The sample was then divided into two portions one of which was heated for 5 min in a boiling-water bath (closed circle). Both samples were analyzed by SDS-PAGE. (c) The gp86 protein reacts with antibody to NSl. Aedes albopicfus cells were infected with DEN-2. labeled with [36S]methionine, and then lysed and analyzed by SDS-PAGE. Samples were boiled (closed circles) or not boiled (open circles) prior to electrophoresis. A lanes, autoradiogram; B lanes, immunoblot of the same gel with monoclonal antibody to NSl.

gel electrophoresis. In the first two lanes (A) unboiled (open circle) and boiled (closed circle) lysates of mock-infected cells were compared. No differences were seen. In contrast, boiling the lysate from DEN-2infected cells (B) had a striking effect on two virus-specific proteins. In the boiled sample the pattern of structural and nonstructural viral proteins (including NSl) was similar to that described by others for DEN-2-infected cells (Smith and Wright, 1985; Ozden and Poirier, 1985). However, in the unboiled sample, NSl (estimated mol wt of 46,000) was present only in small amounts; instead a new band (see arrow) with a mol wt of approximately 86,000 was observed. This protein was not seen in the mock-infected lysates. Since in a later section we show that the 86,000 mol wt protein is sensitive to endoglycosidase F, and thus like NSl is a glycoprotein (see Fig. 5) we shall refer to it as gp86. The finding that upon boiling of an infected cell lysate, gp86 completely disappeared and NSl appeared, along with the knowledge that both gp86 and

190

WINKLER

NSl are glycoproteins, suggested to us that gp86 may be a complex which contains NSl. We attempted to evaluate this hypothesis in two ways. First we isolated radioactively labeled gp86 from a Western blot and identified the products generated by boiling. As shown in Fig. lb when gp86 was subjected to boiling, NSl was produced; no other labeled proteins were found. In a second approach we used an NSl-specific monoclonal antibody to detect the DEN-2 NSl antigen in a Western blot. As shown in Fig. lc, B lanes, the antibody bound clearly to NSl in the heat-treated sample, but even better to gp86 in the unboiled sample. The specificity of the reaction was demonstrated by exposing the nitrocellulose to X-ray film after the immunoassay was done. As shown by comparing panel A and panel B in Fig. lc, only gp86 and NSl , respectively, were stained in the immunoassay. This result shows that NSl is present in the gp86 band. From the results in Fig. 1 we conclude (1) that DEN gp86 contains NSl , and (2) since NSl is the only viral protein which appeared after heat treatment of gp86, that gp86 is not a polyprotein precursor of NSl. The two remaining possibilities were that gp86 could be a dimer of NSl or a complex formed by NSl and a cellular protein. As is apparent from Fig. la, SDS and 2-mercaptoethanol at room temperature are not sufficient to convert gp86 to NSl. We also know from analyzing cell lysates without 2-mercaptoethanol on nonreducing gels, that boiling alone is sufficient to cause this dissociation (not shown). These results strongly suggest that neither disulfide bridges nor weak hydrophobic interactions are involved in the formation of the gp86 complex. To obtain further information about the stability of gp86 we treated lysates of DEN-2-infected mosquito cells with 6 M urea, 1 M NaCI, or 5 n/r guanidine hydrochloride; each of these compounds was dissolved in 1 IV Tris/HCl, pH 8.8, containing 39/o 2mercaptoethanol. Samples were kept at 37” for 1 hr, desalted, and analyzed by SDS-PAGE. As shown in Fig. 2a, none of these commonly used denaturing reagents was able to convert gp86 irreversibly to NSl We also tested the effect of low pH on gp86. Samples were treated with a citrate/phosphate buffer (Mcllvaine, 1921) at different pH values between 6 and 2.2, at 37” for 1 hr. Again the proteins were desalted before analysis by SDS-PAGE. As shown in Fig. 2b, gp86 was stable at pH 6 but began to dissociate with decreasing pH. At pH 3 a considerable amount of gp86 was converted to NSl and at pH 2.2 no gp86 remained. The results shown in Fig. 2 suggest that the extraordinary stability of DEN-2 gp86 at neutral and basic pH may be caused by either very weak covalent or unusually strong noncovalent bonds.

ET AL.

@ A B nrornr

C

.OPSO

-NSl

FIG. 2. Stability of gp86 (a) in the presence of 6 M urea (A), 1 M NaCl (B), and 5 M guanidine/HCI (C) and (b) under different pH conditions. Lysates of DEN-2-infected [36S]methionine-labeled Aedes albopicfus cells were prepared as described under Materials and Methods. Samples of lysates were treated with urea, NaCI, or guanidine/HCI for 60 min at 37”, and then analyzed by SDS-PAGE with boiling (closed circles) and without boiling (open circles) prior to electrophoresis. In (b) samples of cell lysates were incubated at the indicated pH for 60 min at 37”, and then analyzed by SDS-PAGE with and without boiling.

NSi in DEN-2-, SLE-, and POW-infected vertebrate cells To determine whether the relationship between NSl and gp86 was unique to DEN-2-infected mosquito cells, we examined DEN-2 and two other flaviviruses, SLE and POW, in mammalian cells. BHK cells were infected with one of the three viruses and labeled with [35S]methionine at 25 hr p.i. for 120 min. Proteins were prepared from both the cell lysate and the medium; in the latter case polypeptides were separated into a pellet fraction (50,000 g, 3 hr) and a soluble fraction as described under Materials and Methods. Figure 3a shows that gp86 appears not only in DEN-2-infected mosquito cells but also in DEN-2-infected BHK cells (A lanes). As before, when the lysate was not boiled, gp86 was the predominant form of NSl; and as with the mosquito cell lysates, boiling for 5 min converted it to the 46,000 mol wt form. A similar phenomenon was seen with SLE and POW, although with these viruses the molecular weights of NSI and the corresponding high-molcular-weight forms were somewhat different (B and C lanes). Thus the presence of NSl in a highermolecular-weight complex appears to be a general feature of flavivirus-infected cells. Figure 3b shows the labeled proteins which were found in the 50,000 g pellet prepared from the medium of infected cultures. With all three viruses, we found, as expected, the viral glycoprotein E. The capsid pro-

FLAVIVIRUS

NSl-EVIDENCE

9366E E 45

31-

FOR

A DIMER

191

sedimentable fraction of the medium. Indeed it is the only viral protein with such a distribution. We next wished to know how NSl would appear in an infected animal. Suckling mice were, therefore, inoculated intracerebrally with DEN-2 and brains were prepared when the mice were moribund. After separating the mouse brain proteins by PAGE, a Western blot was performed and NSl was detected by the use of a specific monoclonal antibody. As with the infected cell cultures gp86 was detected in the unboiled sample and NSl was detected in the boiled sample (results not shown).

22-

Kinetics

l4-

3. Comparison of intracellular and extracellular NSl from DEN-2 (A), SLE (B), POW (C), and mock (D) infected BHK cells. BHK cells were infected with DEN-2, SLE, or POW or mock-infected and labeled with [35S]methionine as described under Materials and Methods. Cell lysates (a), 50,000 g pellets (b), and 50,000 g supernatants (c) prepared from the medium were analyzed by SDS-PAGE. All samples were examined with boiling (closed circles) and without boiling (open circles) prior to electrophoresis. The arrows indicate NSl and the high-molecular-weight form of NSl, FIG.

tein C was especially prominent in the medium from POW-infected ceils. Of greater interest, with all three viruses the high-molecular-weight form of NSl was observed in the unboiled samples, and little NSl was present. As with the cell lysates, upon boiling, gp86 or its equivalent disappeared and NSl appeared. In the case of SLE, the structural glycoprotein E and NSl bands were not well resolved (Fig. 3b, lane B) making the interpretation more difficult; but we suggest that NS1 is in the lower part of this broad band (SLE NSl is 87 amino acids shorter than E; Trent et a/., 1987). Note also that in Fig. 3a the SLE NSl moves slightly more rapidly than the E protein. Since the lower part of the band was seen in the unboiled as well as in the boiled sample it appears that in the case of SLE, before boiling, the high-molecular-weight form and NSl are present in almost equal amounts in this fraction. Figure 3c shows the labeled viral proteins in the supernatant fraction of the medium where the interfering E proteins are missing. Again, NSl was predominant only when the samples were boiled and the highmolecular-weight complex was seen only in the unboiled samples. It should be noted that with SLE, the unboiled sample did contain a considerable amount of NSl relative to the amount of the higher-molecularweight complex. The results of Fig. 3 indicate that NSl and its higher-molecular-weight form are present not only in the cell lysate, but also in the supernatant and

of gp86 formation

We next wished to learn when gp86 was formed relative to the synthesis of NSl . DEN-2-infected BHK cells were grown in the presence of actinomycin D and at 25 hr p.i. were pulsed for different times with [35S]methionine. After 10 min of labeling neither NSl nor gp86 was seen in the cell lysate (Fig. 4a). After 20 min a faint band appeared at the position of NSl and became more prominent in the 40-min sample. gp86 was not seen in the lo- or 20-min samples but was discernible after 40 min of labeling. The earlier appearance of NSl is more clearly shown in Fig. 4b where equal amounts of radioactivity were applied to each lane, rather than equal amounts of protein as in Fig. 4a. These results are consistent with the idea that NSl is the primary product and is then converted to the high-molecular-weight form. It should be noted that by 40 min the concentration of gp86 in the unboiled sample was already higher than that of NSl. These results suggest that it takes between 20 and 40 min to form the high-molecular-weight form of NSl. Returning to Fig. 4a and considering the 40-through 160-min samples it is evident that the concentration of gp86 increased with the increased time of labeling whereas the concentration of NSl in the unboiled lysates remained more or less unchanged. This suggests that gp86 is formed from NSl very quickly, keeping NSl at a low-level steady-state concentration. The time course of release of DEN-2 gp86 into the medium was followed in the same experiment (not shown). gp86 was first detected in the 50,000 g pellet and in the supernatant in the 80-min sample, that is about 40 min after it appeared in the cell lysate. The temporal relationship between the appearance of NSl and the appearance of gp86 was also examined in a pulse-chase experiment. For this purpose we wished to have a starting preparation (unboiled sample) which contained NSl and gp86 in approximately equal amounts. From the results of Fig. 4a it appeared that a labeling period between 20 and 40 min should

192

WINKLER

@ min:lO 20 40 0~0~0~0~0~0~0~0~

80

160

mock 100

@ 20

ET AL.

40

FIG. 4. Kinetics of DEN-2 NSl and gp86 formation in BHK cells. (a) BHK cells were infected with DEN-2 and labeled with [35S]methionine for the indicated length of time prior to harvest. Cell lysates were then prepared and analyzed by SDS-PAGE. Each sample was examined with boiling (closed circles) and without boiling (open circles) prior to electrophoresis. Equal amounts of protein were applied to each lane. (b) The analysis of the 20- and 40-min samples shown in (a) but now with the application of equal amounts of radioactivity to each lane. (c) BHK cells were infected with DEN-2, labeled for 30 min with [35S]methionine beginning at 25 hr p.i., and then chased with cold methionine for the indicated periods of time. Cell lysates were prepared and analyzed by SDS-PAGE with boiling (closed circles) and without boiling (open circles) prior to electrophoresis. Equal amounts of protein were applied to each lane.

provide us with such a sample. As seen in Fig. 4c when DEN-2-infected cells were labeled with [35S]methionine for 30 min both proteins, NSl and gp86, were indeed present in the unboiled sample in comparable amounts. As the chase period was increased, the concentration of NSl in the unboiled samples decreased progressively so that after a 40-min chase the NSl band had become quite faint and after 80 min had almost completely disappeared. At the same time the concentration of gp86 in the unboiled lysates showed a progressive increase for at least 40 min. By examining the boiled samples, it is apparent that the total amount of NSl changed very little during the chase period indicating that there was little net loss of NSl . The disappearance of NSl during the chase period along with the increased amount of gp86 gives strong support to the idea that NSl has a precursor-product relationship to gp86. gp86 is formed

before NSl

passes the

Golgi apparatus Since NSl is a secretory glycoprotein it must traverse the endoplasmic reticulum and the Golgi system on its way to the cell surface (Griffiths and Simons, 1986). The question arises, however, as to when during this migration NSl moves into the gp86 complex. It should be possible to obtain information on this question by determining the nature of the N-linked carbohydrates of NSl and gp86. Such an analysis would allow one to distinguish between events taking place

before or after the polypeptide has passed the Golgi apparatus, where high-mannose oligosaccharides are processed to complex sugars. Accordingly, we prepared 35S-labeled proteins (labeling times, 160 min) from a lysate of DEN-2-infected BHK cells as well as from the medium and treated them with endoglycosidase H, which cleaves high-mannose but not complex types of asparagine-linked oligosaccharides (TarenF which cleaves tino et al., 1974). Endoglycosidase high-mannose and complex oligosaccharides equally well (Elder and Alexander, 1982) was used for purposes of comparison. Figure 5A shows that the apparent molecular weight of gp86 from DEN-2-infected cell lysates was reduced by approximately 8000 after treatment with either endoglycosidase H or endoglycosidase F. The single band resulting from endoglycosidase F digestion also suggests that we achieved complete deglycosylation. After endoglycosidase H treatment the major new band comigrated with the endoglycosidase F-generated band indicating that all glycosylation sites in the labeled intracellular gp86 have high-mannose types of N-linked glycans. There was, however, a small portion of the protein which seems to form a diffuse intermediate band, suggesting that a change in the glycosylation pattern was underway. This change became more evident when we analyzed the pellet (Fig. 5B) and the supernatant fractions (Fig. 5C), prepared from the culture medium. Again the carbohydrates of the extracellular gp86 showed complete accessibility to endogly-

FLAWVIRUS

+LLA&B-C

NSl-EVIDENCE

b

UFHUFHUFHUFHUFHUFH

gpa -NSl

FIG. 5. Susceptibility of cell-associated and extracellular NSl to endoglycosidases F and H. BHK cells were infected with DEN-2 and labeled with [%]methionine for 160 min beginning at 25 hr p.i. (see Materials and Methods). Cell lysates (A, a) were prepared as well as the 50,000 g pellets (B, b) and 50,000 g supernatants (C, c) from the culture medium. Unboiled (A, 6, C) and boiled (a, b, c) samples were untreated (U), or treated with endoglycosidase F (F) or endoglycosidase H (H) prior to analysis by SDS-PAGE. Note that the apparent molecular weights of the extracellular forms of gp86 and NSl are slightly greater than those of the respective cell-associated proteins. This is likely due to the presence of complex oligosaccharides in the extracellular form.

cosidase F (in this case the molecular weight difference between the treated and untreated gp86 was estimated to be about 12,000) but in contrast to gp86 in the lysate, were partially resistant to endoglycosidase H. This partial resistance was shown by the band intermediate between the untreated gp86 and the completely deglycosylated (endoglycosidase F treated) form. Estimates of the molecular weights of these various forms of gp86 suggest that approximately half of the glycosylation sites were involved in the processing to complex oligosaccharides. This finding would also explain the increased molecular weight of extracellular gp86 relative to that of gp86 in the lysate (compare Figs. 5A and 5B). After removal of the carbohydrate from the extracellular gp86 by endoglycosidase F, however, the molecular weight was the same as that of deglycosylated intracellular gp86. When we heated the various samples before gel electrophoresis (so that the samples contained NSl rather than gp86), the results obtained were basically similar to those just described for gp86 (Figs. 5a-c). Most of the NSl in the cell lysate contained only highmannose glycans whereas the extracellular NSl was partially resistant to endoglycosidase H indicating that approximately half the sites had been processed to a complex form.

FOR

A DIMER

193

These results together with the results of our kinetic studies suggest that NSl forms gp86 before it enters the Golgi apparatus, where the glycosylation pattern is changed. The processing to complex-type glycan structure would also appear to account for the observed heterogeneity of the extracellular gp86. As shown in both Fig. 3 and Fig. 5, whereas the intracellular gp86 forms a sharp single band the extracellular gp86 is present as a broader diffuse band. However, the demonstration that after endoglycosidase F treatment the arrowed band in Fig. 5 (B, b and C, c) which represents the completely deglycosylated form appears to be a sharp band indicates that the heterogeneity of this protein is indeed due to glycosylation. Since endoglycosidase H-digested extracellular gp86 shows the same heterogeneity as the untreated protein we believe that microheterogeneities in the structure of complex glycans are responsible for the broad nature of the band. This would explain why the intracellular material which carries only high-mannose sugars appear to be more homogenous than the extracellular sp86. Evidence

that gp86 is a dimer of NSl

Although we have shown that the high-molecularweight complex contains NSl we cannot say from the results so far whether it is simply a dimer of NSl or a complex formed by NSl and a cellular protein. The following experiments were carried out in order to distinguish between these two possibilities. First, instead of labeling predominantly viral proteins, we labeled DEN-2-, SLE-, and POW-infected cells so that cellular and viral proteins would be labeled equally well. This was achieved by omitting actinomycin D and by prelabeling the almost confluent BHK cells with [35S]methionine for 6 hr prior to infection. The radioactive label was also present for the entire 28-hr period following infection until the time of harvest. Proteins were then prepared from the cell lysate and from the medium and analyzed by PAGE as already shown in Fig. 3. Although the overall background was markedly increased due to the incorporation of label into cellular proteins, the results (not shown), as far as NSl was concerned, were similar to those shown in Fig. 3. Furthermore, heat denaturation of the samples did not generate any proteins which were not seen in Fig. 3. Thus this experiment provided no evidence that gp86 contained a cellular protein which was labeled under the conditions used in this experiment. In a second approach we exploited what we learned about the number of glycosylation sites in DEN-2 gp86 and NSl Analysis of the time course of endoglycosi-

194

WINKLER

dase F digestion of a glycoprotein allows one to determine the number of oligosaccharides based on the appearance of intermediate degradation products (Elder and Alexander, 1982). As starting material we used gp86 prepared from the soluble fraction of the medium. This preparation has a very low background of other proteins (see Fig. 3c) and is therefore ideally suited for reading a stepwise deglycosylation pattern. To examine NSl , half of the sample was boiled prior to digestion. As shown in Fig. 6a, including the control sample, gp86 yielded five different bands. Assuming that after the 24-hr digestion the most rapidly moving species represents the completely deglycosylated form of gp86, we conclude that the untreated gp86 contained four oligosaccharides. From the results shown in Fig. 6b we conclude that NSl contains only two oligosaccharides. The demonstration that the high-molecular-weight complex contained twice the number of glycans present in NSl is consistent with the hypothesis that gp86 is a dimer of NSl A third approach involved the in vitro labeling of the purified NSl high-molecular-weight complex with 1251. For this experiment, DEN-2, SLE, and POW high-molecular-weight complexes of NSl were isolated from a gel as described under Materials and Methods. This material was then iodinated, heated, and analyzed by PAGE. Lanes a-c in Fig. 7 show that with each virus, heat-induced dissociation generated only one major iodinated band, namely NSl. No distinct iodinated

ET AL

a

b

c

def

14FIG. 7. Analysis of products derived from ‘251-labeled isolated dimer forms of NSl after boiling. BHK cells were infected with DEN-2 (a, d), SLE (b, e), or POW (c, f) and labeled with [35S]methionine for 160 min. The 50,000 g pellets were prepared from the culture medium and fractionated by SDS-PAGE, after which the proteins were transferred to a nitrocellulose membrane. The dimer forms of NSl were identified by autoradiography, cut from the blot, iodinated, boiled, and then analyzed by SDS-PAGE. A second X-ray film inserted between the gel (lanes a-c) and the film shown in the figure served to filter out the radioactivity due to [%]methionine. Thus the bands shown in lanes a-c represent only ‘z51-radioactivity. The diffuse bands found in the 66-93,000 mol wt region likely represent cellular proteins which comigrated and were excised together with the high-molecular-weight form of NSl Lanes d-f represent the same samples but without iodination (% activity). For these lanes the film was directly exposed to the gel.

band other than NSl was observed. Thus it is likely that the high-molecular-weight form of NSl is a homodimer.

DISCUSSION

-NSl

FIG. 6. Estimation of the number of N-linked oligosaccharides of extracellular DEN-2 gp86 (a) and NSl (b). BHK cells were infected with DEN-2 and labeled with [36S]methionine as described under Materials and Methods. The 50,000 g supernatant fraction was prepared from the culture medium and divided into two portions one of which was boiled (b) and the other was not (a). Samples of each portion were treated as indicated on the figure with endoglycosidase F for the indicated times (hours), with endoglycosidase H for 24 hr, or were untreated. Samples were then analyzed by SDSPAGE.

As recounted in the Introduction, the flavivirus nonstructural protein NSl has certain features which make it of special interest among the flavivirus nonstructural proteins. Most puzzling is the question of its role in virus replication. Given its distribution both on the cell surface and in the medium, that it is glycosylated, and that other cytoplasmic RNA viruses (e.g., alphaviruses) apparently do not generate glycosylated nonstructural proteins, it seems unlikely that NSl plays a role in viral RNA synthesis or in the post-transcriptional modification of RNA. It has, on the other hand, been suggested that NSl is involved in virus assembly (Rice et al., 1986) but no specific models have been proposed. Depending on the particular virus, NSl has been found by PAGE to have a molecular weight of about

FLAVIVIRUS

NSl-EVIDENCE

40-50,000. This estimate is consistent with the size of the protein deduced from nucleotide sequencing and limited sequencing of the protein. The results which we have presented, however, indicate that NSl is present in infected cell cultures as an oligomer with an apparent molecular weight in the 75-86,000 range. It is likely that the reason this high-molecular-weight form of NSl has not been generally recognized previously is that it is common practice to boil protein samples before analysis by PAGE. Our results clearly show that boiling converts the high-molecular-weight form to the monomer form of NSl . What remained to be explained was the precise relationship between NSl and its high-molecular-weight form. The fact that when [35S]methionine-labeled gp86 was isolated from gels and then heat denatured, NSl was the only product seen argues against the possibility that gp86 could be a polyprotein precursor of NSl . The results of the pulse-chase experiment are also inconsistent with this idea. On the other hand, the results of the iodination experiment and the determination of the number of glycans on DEN-2 NSl and gp86 (Fig. 6) are consistent with the hypothesis that the high-molecular-weight form of NSl is a homodimer. The idea that oligomerization of NSl is a general feature of flavivirus infection is supported by the fact that we observed the high-molecular-weight form with three different members of the Flavivirus family belonging to three different serogroups (DeMadrid and Porter-field, 1974). Furthermore, although we first observed the dimer form of NSl in mosquito cells, subsequent experiments showed that a similar form exists in infected mammalian cells and in infected mouse brain. These findings raise the possibility that the high-molecular-weight form of NSl is the functional form of this protein. Interestingly, it has been shown with influenza and vesicular stomatitis viruses that oligomerization of viral membrane proteins is required for the efficient export from the endoplasmic reticulum and for transport through the Golgi apparatus (Gething et a/., 1986; Kreis and Lodish, 1986). The nature of the bonds involved in the formation of the high-molecular-weight form is not clear and requires further investigation. Covalent bonds involving proteins are usually not cleaved by boiling or low pH. Furthermore, the stability of gp86 in the presence of 2-mercaptoethanol and the heat-induced dissociation in the absence of reducing reagents indicate that disulfide bridges are not involved. On the other hand, noncovalent interactions would usually not be resistant to SDS, urea, guanidine/HCI, and high salt. This leaves the possibility that the bonds involved are either unusually strong noncovalent or very weak covalent bonds.

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A DIMER

195

The distribution of NSl in the infected cultures, i.e., in the cell lysate, and in the culture medium is also curious, especially for a nonstructural protein. It would be interesting to know whether there are any significant differences, i.e., post-translational modifications, between the extracellular and the cell-associated forms. Also we would like to know why some of the extracellular NSl protein is pelletable and some is not. Is the extracellular NSl found in the 50,000 g pellet anchored in cellular membranes which have budded off from the infected cells, or could it, in fact, be loosely associated with the virus particles? As to NSl in the 50,000 g supernatant, the possibility has been raised that this form has lost the sequence of amino acids which anchor it in the membrane (Rice et al., 1985). However, the fact that extracellular NSl from the 50,000 g pellet and from the 50,000 g supernatant migrated identically when analysed by PAGE (Fig. 5) makes this unlikely. The most fascinating question about NSl concerns its function in virus replication. By learning more about the properties of NSl we hope to clarify what this function is.

ACKNOWLEDGMENTS This investigation was supported by the United States-Japan operative Medical Science Program through Grant Al-05920 the National Institute of Allergy and Infectious Diseases.

Cofrom

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