Acidotropic amines inhibit proteolytic processing of flavivirus prM protein

VIROLOGY

174, 450-458

(19!30)

Acidotropic

Amines Inhibit Proteolytic

VALERIE B. RANDOLPH,’

Processing

of Flavivirus prM Protein

GUNTHER WINKLER,’ AND VICTOR STOLLAR3

Department of Molecular Genetics and Microbiology, Robert Woodlohnson Medical School, University of Medicine and Dentistry of New Jersey, 675 Hoes Lane, Piscataway, New Jersey 08854 Received May 22, 1989; accepted October 4, 1989 Treatment of flavivirus-infected mammalian and mosquito cells with acidotropic amines (such as chloroquine, ammonium chloride, or methylamine) inhibited the normal proteolytic processing of the virus prM protein to M. As a result, virions from infected cells which had been treated with acidotropic amines late in the virus replication cycle contained prM protein rather than M protein. Identification of the prM protein was based on molecular weigh>-glycosylation, and reactivity with an anti-prM monoclonal antibody. Infected cells which had not been treated with acidotropic amines did release, along with virions which contained the mature M protein, variable amounts of virus containing the prM precursor. The relative amounts of these two types of virions were influenced both by the virus and the host cell type. Virions containing the prM protein had a lower specific infectivity than virions containing the M protein; however, in experiments with a macrophage cell line this low specific infectivity was significantly increased if the anti-prM monoclonal antibody was used to facilitate virus entry via Fc receptors. Our findings indicate that the proteolytic cleavage of prM requires an acidic environment and is necessary to generate fully infectious virus. We suggest that the cleavage of prM Q is90 Academic PRESS, inc. occurs in the acidic post-Golgi vesicles.

with Japanese encephalitis (JE) or dengue (DEN) viruses resulted in production of virus which lacked the 8-kDa M protein but contained a larger 20-kDa protein (NV2 in the earlier terminology). Recent sequence analysis has indicated that the NV2 protein is the precursor of M (Rice et al., 1986); hence it is now called prM. Intracellular virus harvested from cells which had not been treated with Tris also contained prM rather than M suggesting that Tris prevented the processing of prM to M. In addition, it was noted by these workers that virus which contained prM was less infectious than virus which contained the M protein. In the last several years much work has been performed on the effects of compounds variously termed lysosomotropic amines, acidotropic amines, or primary amines (for reviews, see Dean et al., 1984; Marsh, 1984; Steiner eta/., 1984) and their effects on the replication of certain enveloped viruses. These compounds, which act as weak bases, accumulate in acidic vesicles (endosomes and lysosomes) and raise the pH therein. We will follow the suggestion of de Duve (1983) and refer to these compounds as acidotropic rather than lysosomotropic amines. By preventing the low pH-induced fusion of viral and cell membranes which occurs in the endosome, these compounds are able to block the entry of certain viruses into the cytosol. The processing of some cellular secretory proteins whose cleavage depends on an acidic environment is also inhibited by acidotropic amines. The fact that Tris, which is a primary amine, interfered with the processing of the prM protein suggested

INTRODUCTION The flaviviruses are small (70 nm) enveloped viruses which contain a single strand of positive sense RNA. Three structural proteins are present in virions: a 13-to 16-kDa capsid protein (C), a 5 1- to 60-kDa major envelope glycoprotein (E), and a small 8-kDa transmembranal envelope protein (M) (for review, see Rice er al., 1986). The M protein is derived by proteolytic cleavage of a 19- to 23-kDa precursor protein, prM, at a site following two basic amino acid residues. The prM protein contains 1 to 3 glysosylation sites; however, these are located in the amino-terminal portion of the protein which is removed by cleavage (non-M). Little is known about the site of flavivirus assembly or the details of maturation. It is generally thought that virions mature and acquire their envelope in the cytoplasm, possibly by budding through intracytoplasmic membranes, but there is little firm evidence to support this idea. It is also postulated that the prM to M cleavage is a late event occurring shortly before virus is released from the cell (for review, see Brinton, 1986) but the significance of this cleavage and its role in virus maturation are unknown. Shapiro et al. (1972, 1973) reported that addition of 6 mM Tris to the medium of LLC-MK2 cells infected ’ Present address: Lederle Laboratories, North Middletown Road, Pearl River, NY 10965. * Present address: Biogen, Cambridge, Massachusetts 02 142. 3 To whom requests for reprints should be addressed. 0042.6822/90

$3.00

Copynght Q 1990 by Academic Press. Inc. All rights of reproduction I” any form reserved.

450

AMINE

INHIBITION

OF prM PROTEIN PROCESSING

to us that maturation of prM to M might occur in an acidic compartment of the cell. To test this idea, we examined the effect of several acidotropic amines (chloroquine [CLQ], ammonium chloride, methylamine) on the cleavage of prM in flavivirus-infected cells. We found that each of these compounds interfered with the processing of prM to M, and reduced the yield of infectious virus. MATERIALS

AND METHODS

Viruses, cells, and antisera

451

medium lacking FBS but containing l/25 the normal concentration of methionine (0.004 mM) and actinomycin D (1 pg/mI). One hour later, [35S]methionine was added (100 &i/lo7 cells) and 3 hr later the medium was harvested and virus pelleted as described above. For gradient fractionation, the virus pellet was resuspended in TNE buffer (20 m/l/l Tris, pH 8.0, 100 mM NaCI, 2 mM EDTA) and centrifuged through a 20-5096 sucrose/TNE (w/w) density gradient for 16 hr. The visible virus band was removed, assayed for infectivity, and examined by electron microscopy; virus was then pelleted, resuspended in Laemmli sample buffer, and analyzed by SDS-PAGE.

The dengue type 2 (DEN, New Guinea B strain), St. Louis encephalitis (SLE), and Powassan (POW) viruses were described previously (Winkler et a/., 1988) as were the BHK and Aedes albopictus (C7 clone) cells (Stollar et a/., 1976; Durbin and Stollar, 1984). The KB cells were obtained from ATCC and were maintained in modified minimum essential medium (MEM) supplemented with 1O”b calf serum. Vero cells were obtained from Dr. James Hardy (Arbovirus Unit, University of California, Berkeley) and were maintained in MEM supplemented with 5% FBS. P388Dl cells, a mouse macrophage cell line, were obtained from ATCC and were maintained in RPMI 1640 supplemented with 10% FBS and 10 mM HEPES. The immune mouse ascites fluid to DEN and monoclonal antibodies (Mab) 2H2 and 4G2 used in the radioimmunoprecipitation and infectivity assays described below were the generous gift of Dr. E. Henchal, Walter Reed Army Institute for Research, Washington, D.C.

RIP was carried out essentially as described by Lamb et al. (1978). DEN was labeled and pelleted as described above except that the supernatant fraction following ultracentrifugation was saved, and the pellet was resuspended in RIP buffer (0.1 MTris, pH 8.3,O. 15 M NaCI, 1% sodium deoxycholate, 2% Tween 20, 100 K units Tyasylol Aprotinin/ml, 1 mM phenylmethylsulfonyl fluoride). Samples were reacted with normal mouse ascites fluid (NMAF) or with monoclonal antibody (Mab) 2H2 (ascites fluid) for 1 hr at room temperature, followed by protein A sepharose CL-4B (Pharmacia) for 1 hr at room temperature. Beads were then washed three times in RIP buffer after which the proteins were solubilized in Laemmli sample buffer and subjected to PAGE as described above. Gels were analyzed by fluorography.

Analysis of [35S]methionine-labeled extracellular virus

Analysis of glycoproteins FandH

proteins of

Cells infected with DEN, SLE, or POW viruses were labeled with [35S]methionine as described previously (Winkler et a/., 1988) but with several modifications. In the case of BHK, KB, and Vero cells, at 24 hr postinfection the cells were refed with MEM lacking methionine and not supplemented with serum. One hour later, [35S]methionine (100 &i/l O7 cells) and actinomycin D (5 fig/ml) were added. Proteins were labeled for 3 hr unless otherwise indicated. Virus was harvested from the culture media of infected cells, pelleted by ultracentrifugation, and subjected to polyacrylamide gel electrophoresis (PAGE) as described previously (Winkler et al., 1988). Labeled bands were visualized by autoradiography. To label virus produced in A. albopictus cells, the medium was removed at 24 hr postinfection and replaced with MEM containing 5% FBS and l/5 the normal concentration of methionine (0.02 mM). At 40 hr postinfection, this medium was replaced with fresh

Radioimmunoprecipitation

(RIP)

with endoglycosidases

Treatment of viral glycoproteins pelleted from the culture media of infected cells with endoglycosidases F and H (endo F and endo H) was carried out as described previously (Winkler et a/., 1988). Virus infectivity

assays

Virus infectivity was titrated by plaque formation on Vero cells (Cahoon et a/., 1979) and by immunoperoxidase focus assay on P388Dl cells (Randolph and Hardy, 1988). RESULTS Acidotropic amines affect the relative amounts of M and prM proteins found in extracellular viral particles To determine if acidotropic amines other than Tris could affect the processing of the flavivirus PrM protein, we treated BHK cells infected with DEN, SLE, or

RANDOLPH,

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WINKLER, AND STOLLAR

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-31 -22 -14 FIG. 1. Effect of the acidotropic amines CLQ and ammonium chloride on the protein composition of extracellular virus from SLE-, POW-, and DEN-infected BHK cell cultures. Virus pellets (100K g) were obtained from the culture media of mock-infected (A), or SLE (B)-, POW (C)-, or DEN (D)-infected BHK cells 28 hr postinfection. Cultures were untreated (a) or treated with 0.1 mM CLQ (b) or 50 mM ammonium chloride (c)for 4 hr prior to harvest; all cultures were labeled with [9]Met for 3 hr prior to harvest. Samples were analyzed by SDS-PAGE.

POW with 0.1 mll/l CLQ or 50 mM ammonium chloride and then labeled them with [35S]methionine as described under Materials and Methods. Figure 1 shows the proteins found in the extracellular virus after examination by SDS-PAGE. Because treatment with CLQ and ammonium chloride reduced the total amount of labeled viral proteins to a variable extent, we tried to adjust the amounts Iof sample applied to the gel shown in Fig. 1 so that for each virus, each lane would contain roughly equivalent amounts of C protein and of E protein. With all three viruses, treatment with either CLQ or ammonium chloride led to a marked decrease in the amount of labeled M protein. Associated with this decrease in the amount of M protein, increased labeling of several proteins was noted. In the case of SLE these more heavily labeled proteins had estimated molecular weights of 23K and 24K (Fig. 1B), in the case of POW, 26K, 27K, and 28K (Fig. lC), and in the case of DEN, 22K, 23K, and 24K (Fig. 1 D). These molecular weights are compatible with what has been estimated for the molecular weights of the flavivirus prM proteins (19K23K) (Rice eta/., 1986). It should be noted that the E protein and the 22K28K proteins from ammonium chloride-treated cells migrated faster than those from either CLQ-treated cells

or untreated cells (Fig. 1, compare lanes c with lanes a and b). Additionally, in the case of the DEN-infected cells, instead of three bands with estimated molecular weights of 22K, 23K, and 24K (lanes a and b), only 1 band (MW - 22K) was seen in the ammonium chloride-treated cells. These results likely reflect an inhibition by ammonium chloride of glycosylation (see for example, Kousoulas et al., 1982). Methylamine (50 mM) gave results nearly identical to those seen with ammonium chloride, but was much more toxic to the cells (results not shown). In their experiments, Shapiro et a/. (1972, 1973) repot-ted that processing of DEN and JE prM (NV2) was inhibited by Tris buffer (a primary amine) only in LLCMK2 cells and not in CEF cells. We therefore wished to know whether CLQ would inhibit the production of M protein in other types of cells beside BHK cells. Figure 2 shows that addition of CLQ resulted in decreased quantities of DEN and SLE M protein and increased quantities of the 21K-28K proteins not only in BHK (hamster kidney) cells, but also in KB (human carcinoma), Vero (monkey kidney), and A. albopictus-clone C7 (mosquito) cell lines. Figure 2 also shows that the precise pattern of the 21 K-28K proteins is strongly influenced by the host cell. For example, in CLQ-treated DEN-infected KB cells only one protein band was seen (22K) (Fig. 2B, lane b), whereas in CLQ-treated DENinfected BHK cells, three bands (22K, 23K, and 24K) were seen (Fig. 2A, lane b, also Fig. 1). Also of interest is the variation in the amount of the 22K-28K proteins seen in untreated cells, a variation influenced both by the virus and the host cell. For example, compare DEN-infected mosquito cells (Fig. 2D, lane b) in which case a relatively large amount of 22K24K protein was seen with DEN-infected BHK and KB cells (Fig. 2A and 2B, lanes b) where these proteins were virtually absent. To assess the processing of the 22K-28K proteins as a function of the concentration of acidotropic amine used, the relative amount of SLE M protein produced was determined in BHK cells treated with various concentrations of ammonium chloride. As seen in Fig. 3A, the amount of M protein formed was inversely proportional to the concentration of ammonium chloride. In Fig. 3B this is plotted as the ratio of M protein to C protein [M/C]. Maximum inhibition of prM processing was seen beginning with 20 mM ammonium chloride. Evidence that the 22-28K proteins are forms of prM with differing degrees of glycosylation The following experiments were undertaken to clarify the nature of the multiple 22K-28K bands and their relationship to the M protein.

AMINE

(A)

INHIBITION

OF prM PROTEIN PROCESSING

(B)

453

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FIG. 2. Effect of CLQ on the protein composition of extracellular DEN or SLE obtained from four different cell lines. BHK (A), KB (B), Vero (C). and A. albopicfus (D) cell lines were mock-Infected (a) or infected with DEN (b) or SLE (c) for 28 hr. Cultures were untreated (-) or treated (+) with 0.1 mA4 CLQ and were labeled and analyzed as in the legend to Fig. 1.

First, the protein composition of gradient-purified DEN virions from untreated and CLQ-treated BHK cells was determined. (Thevirus peak was identified both on the basis of infectivity and by electron microscopy of the gradient fractions). As shown in Fig. 4, gradientpurified virus from untreated BHK cells contained E and M proteins and small amounts of the 22, 23, and 24K proteins, In contrast, virus samples from the CLQtreated cells contained E protein, no visible M protein, and significantly greater amounts of the 22K, 23K, and 24K proteins. (Under our labeling conditions, the DEN C protein is generally not identified). These results are consistent with the idea that the 22K, 23K, and 24K proteins are indeed structural components of the virion and likely represent precursors of the M protein. To see whether the 22K, 23K, and 24K proteins might represent variously glycosylated forms of the same protein, pelleted DEN virus from BHK cells treated with CLQ was treated with endoglycosidases F and H. Incubation with endo F, which cleaves both complex and high-mannose carbohydrates, resulted in the disappearance of the 22K, 23K, and 24K protein bands and the appearance of a new protein band (estimated MW -2OK) (Fig. 5). Treatment with endo H, which cleaves high-mannose but not complex carbohydrates had a similar but less marked effect. These results indicate that the multiple protein bands (22K, 23K, 24K) which are seen following CLQ treatment are indeed glycosylated, and are consistent with the idea that they represent a single protein with varying degrees of glycosylation.

In another experiment, a monoclonal antibody (Mab 2H2) thought to be specific for the DEN prM protein (Henchal eta/., 1984) was incubated with viral proteins in the medium (both the virus pellet fraction and the supernatant fraction) of DEN-infected BHK cells. Figure 6B, lane b shows that Mab 2H2 precipitated all three bands (22K, 23K, and 24K) present in the pellet fraction obtained from CLQ-treated cells. Trace amounts of these proteins were also seen in pellets from untreated cells. No proteins were precipitated by incubation with normal mouse ascitic fluid. When the supernatant fraction which remained after virus was pelleted from the medium of CLQ-treated cultures was reacted with Mab 2H2, the three bands were again precipitated (Fig. 6A, lane b, CLQ(+) lane), suggesting either that there were residual amounts of virus or slowly sedimenting hemagglutinating (SSH) particles (for review of SSH, see Russell et al., 1980) which were not pelleted under the conditions used or that some free prM protein was present in the medium. Of special interest, Mab 2H2 precipitated a large amount of labeled protein (- 15K-20K) from the supernatant of DEN-infected cultures which had not been treated with CLQ (Fig. 6A, lane b, CLQ(-) lane). We suggest that this represents the portion of the prM molecule (non-M) which is cleaved off leaving the mature M protein. This result would indicate that the non-M segment of prM is released into the medium. Kinetics of the effect of CLQ on cleavage of prM Figure 7 shows that to produce labeled SLE virus with the maximum amount of prM and no M protein

RANDOLPH,

454

WINKLER. AND STOLLAR

mM NH4 Cl 0

10

20

30

40

50

Es-

FNM

M/C

C

4

M -

-b

0

10

20

30

40

50

mM NH&l FIG. 3. Effect of different concentrations of ammonium chloride on the M protein content of SLE obtained from infected BHK cells. Ammonium chloride at concentrations of 0, 10, 20, 30, 40, and 50 mM was added to BHK cells 24 h following infection with SLE. Cultures were labeled and virus pellets harvested (28 hr postinfection) as for Fig. 1. Proteins were analyzed by SDS-PAGE (A); the relative amounts of M protein (expressed as the ratio M/C) was determined by densitometry tracing and is expressed graphically (6).

it was necessary to add CLQ at least 60 min before harvesting the virus. When CLQ was added 15 min before harvest, the pelleted virus contained only M protein and little of the prM proteins. When added 30 and 45 min before harvest the virus contained progressively less M protein and more of the prM proteins. Finally when CLQ was added 60 or 75 min prior to harvest, the viral particles contained only the prM protein and no M protein. Effect of acidotropic

amines on flavivirus

infectivity

Shapiro eta/. (1972) reported that JE containing prM had a lower specific infectivity than normal JE. To test whether the absence of mature M protein had a similar effect on DEN we compared the infectivity of virus from CLQ-treated cells and normal virus (designated prM and M virus) on Vero cells. In light of evidence that im-

mune enhancement may play an important role in flavivirus infection of cells with Fc receptors, we also tested the effect of adding a low concentration of monoclonal antibody reactive with the prM protein (Mab 2H2) or the E protein (Mab 4G2) on the infectivity of prM and M virus for P388Dl cells (a mouse macrophage cell line). Henchal er al. (1984) had previously reported that these Mabs had enhanced the infectivity of DEN type 2 virus for the U-937 human monocyte cell line. As shown in Table 1, when tested on Vero cells the specific infectivity of the prM virus (PFU/cpm of [35S]methionine) was only about l/8 that of the M virus. Although the specific infectivities of the M and prM DEN when assayed on P388Dl cells (after incubation with NMAF) were about 1OO-fold lower than on Vero cells, the prM virus again showed a significantly (6-to 7-fold)

AMINE

INHIBITION

OF prM PROTEIN PROCESSING

455

DISCUSSION The effects of acidotropic amines, such as chloroquine, on the early stages of virus replication have been well studied. By raising the pH of acidic endosomes they prevent the acid-dependent fusion of virus and cell membranes and thereby inhibit the entry of the viral nucleocapsid into the cytoplasm. We show in this report that with flaviviruses, acidotropic amines also exert effects at a late stage in virus replication. We suggest that the prM to M cleavage, which follows two basic amino acid residues, requires an acidic environment. This may be due to either a low pH-dependent protease activity or to low pH-induced conformational changes in the PrM protein which must occur to ex-

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FIG. 4. Effect of CL0 on protein composition of gradient-purified DEN. BHK cells were infected with DEN and were either untreated (-) or treated (+) with 0.1 rnA,J CLQ and labeled with [35S]Met as described in the legend to Fig. 1. Virus was harvested 28 hr after infection, pelleted, and subjected to equilibrium centrifugation through 20-5096 sucrose gradients; the fraction containing the peak of infectious virus was analyzed by SDS-PAGE.

lower specific infectivity than the M virus. Addition of the Mab 2H2 (monoclonal antibody against prM) increased the specific infectivity of the prM virus for P388Dl cells about 1O-fold but had little effect on the M virus. This result raised the ratio of specific infectivities (prM/M) from 0.14 following incubation with NMAF to 0.82 following incubation with Mab 2H2. Thus in the presence of anti-prM antibody the infectivity of the prM virus was only slightly less than that of the M virus. In contrast, although the Mab 4G2 (monoclonal antibody against the E protein) did enhance virus infectivity for P388Dl cells, the effect was nonspecific, raising the specific infectivities of both prM and M virus about 5-to 7-fold, but not significantly altering the ratio of specific infectivities.

FIG. 5. Treatment of DEN from CLQ-treated BHK cell cultures with endoglycosidases F and G. [?3]Met-labeled virus from the medium of CLQ-treated DEN-infected BHK cell cultures was either untreated (U), treated with endo F (F), or treated with endo H (H). Products were analyzed by SDS-PAGE. Procedures were otherwise as described in the legend to Fig. 1.

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WINKLER, AND STOLLAR

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Earlier experiments reported by Shapiro et al. (1972, 1973) indicated that the primary amine Tris prevented processing of PrM, however, only in LLC-MK2 cells and not in CEF cells. We found in our initial studies (data not shown) that Tris was relatively ineffective at preventing the cleavage of PrM in Vero and BHK cells. This may be because Tris is relatively hydrophilic, even in its neutral form, and does not pass through membranes easily (Ohkuma and Poole, 1981). In contrast to Tris, other acidotropic primary amines, such as chloroquine, ammonium chloride, and methylamine, inhibited the cleavage of prM equally well in all cell types used (Vero, KB, BHK, and A. albopictus). Little is known concerning how flaviviruses are assembled. This is especially true with respect to the details of how and where the viral envelope is acquired. Current thinking, however, suggests that flavivirus particles assemble in association with cytoplasmic vesicles, that they accumulate in these vesicles, and that, upon fusion of these vesicles with the plasma membrane, virions are released from the cell. Since cell-as-

14-

FIG. 6. Immunoprecipitation of viral proteins by monoclonal antibody to prM. Cultures of DEN-infected BHK cells were either untreated (-) or treated (+) with 0.1 ml\/l CLQ, then labeled and harvested 28 hr postinfection as described In the legend to Fig. 1. The supernatants (A) and pellets (6) obtained following centrifugation at 100,000 g for 3 hr were reacted with normal mouse ascites fluid (a) or Mab 2H2 (b). Proteins precipitated by reaction with Protein A sepharose were analyzed by SDS-PAGE.

pose the cleavage site. By raising the pH of the vesicles in which this cleavage occurs, acidotropic amines are able to prevent the processing of prM to M. There are numerous examples of both cellular and viral proteins which are cleaved after double basic amino acids (for reviews, see Steiner et al., 1984; Strauss et a/., 1987). Such cleavages are likely mediated by trypsin-like host cell enzymes, although viral enzymes may also be involved. Processing of at least some of these proteins probably occurs in the acidic trans or post-Golgi compartment of the cell (Anderson and Pathak, 1985; Griffith and Simons, 1986; Orci et a/., 1987). Several reports have indicated that the processing and/or the secretion of certain of these proteins can be inhibited by acidotropic amines, e.g., the cleavage of Sindbis virus pE2 to E2 (Coombs et al., 1981), the processing of the fusion protein of Newcastle disease virus (Yoshida et al., 1989), the proteolytic conversion of proalbumin to albumin (Oda and Ikehara, 1985), and the processing of complement components (Hot-tin and Strauss, 1986).

prM= CM0 1530456075 Time(min) FIG. 7. The’ effect of the addition of CLQ at different times prior to harvest on cleavage of PrM in SLE-infected BHK cells. BHK cells infected with SLE virus were pulse-labeled with [35S]Met for 10 min at 25 hr postinfection and then chased with cold methionine for 75 min. CLQ (0.1 mn/l) was added at 0, 15,30,45,60, and 75 min before the media were harvested. The virus pellets were analyzed by SDSPAGE.

AMINE

INHIBITION

OF prM PROTEIN PROCESSING

TABLE1 EFECTOF CLQ ON DEN VIRUS INFECTIVITY Specific infectivity (PFU/cpm) Cell

line

Vero P388Dl P388Dl P388Dl

Antibody

M-DEN

PrM-DEN

PrMlM

NMAF 2H2 4G2

226.3 1.3 2.8 6.2

27.6 0.2 2.3 1.2

0.12 0.15 0.82 0.19

Note. BHK cells were infected with DEN and labeled with [35S]methionine as described under Materials and Methods. 24 hr after infection one culture was treated with CLQ (0.1 rnw) and one culture received no drug. Virus harvested 4 hr later from the medium of the CLQ-treated and the untreated cultures (designated prM and M virus, respectively) was purified by equilibrium centnfugation through ZO-50% sucrose gradients. Infectivity was determined by plaque assay on Vero cells or by immunoperoxidase focus assay on P388Dl cells in the presence of normal mouse ascites fluid (NMAF), Mab 2H2, or Mab 4G2. Results are presened as PFU/cpm.

sociated virus contains predominantly prM rather than M, it appears that the cleavage of prM occurs after the virus particle has been assembled, and, as has been suggested by the work reported here, in acidic vesicles probably between the Golgi compartment and the plasma membrane. Following cleavage and fusion of the vesicle with the plasma membrane, the amino-terminal end of prM (non-M) is released into the medium. In an analogous manner, the E3 protein of many alphaviruses (E3 is derived from the amino-terminal portion of the pE2 protein) is also released into the medium. Semliki Forest virus is an exception to this rule in that E3 remains associated with the viral particle. Recent work indicates, however, that in the case of Sindbis virus, the presence of uncleaved pE2 does not lower the specific infectivity of the virus (Presley and Brown, 1989; Russell et al., 1989). With flaviviruses, on the other hand, our work shows that the cleavage of prM is required for maximal infectivity, possibly because uncleaved prM does not permit efficient interaction of the virus with the normal receptors on the cell. The cleavage of prM is not required, however, for infectivity per se, since prM virus was nearly as infectious as M virus if allowed to enter cells via Fc receptors. In other work we have obtained evidence that the flavivirus E protein is capable of inducing cell fusion at pH’s slightly below neutrality, presumably by bringing about fusion of viral and cell membranes (manuscript in preparation). This observation raises the question as to why, if virus particles exit the cell via acidic vesicles (flaviviruses are also generally unstable at low pH), the E protein does not cause fusion of viral and cell membranes during virus exit as appears to occur in endo-

457

somes during virus entry. It may be that uncleaved prM prevents fusion by in some way interfering with the interaction of the E protein with vesicular membranes. This idea fits with the suggestion by Wengler and Wengler (1989) that, in the case of West Nile virus, the cleavage of prM leads to the disocciation of prM and E heterodimers which are present in cell-associated virus. Alternatively such a role might be associated with the flavivirus NSl protein for which no function is known. Evidence supporting or refuting these speculations awaits a better understanding of how and where flavivirus particles are assembled.

ACKNOWLEDGMENTS This investigation was supported by the U.S.-Japan Medical Science Program through Public Health Service Grant Al-05920 from the National Institutes of Health and by Institutional National Research Service Award CA-09069 from the National Cancer Institute. We are grateful to Florence Szymanski for her help in the preparation of this manuscript.

REFERENCES ANDERSON, G. W., and PATHAK, R. K. (1985). Vesicles and cisternae in the ffans Golgi apparatus of human fibroblasts are acidic compartments. Cell40, 635-643. BRINTON, M. (1986). Replication of flaviviruses. ln “The Togaviridae and Flavivlridae” (S. Schlesinger and M. 1. Schlesinger, Eds.), pp. 327-374. Plenum, New York. CAHOON, B. E., HARDY, J. L.. and REEVES,W. C. (1979). Growth of California encephalitis and other viruses in Aedes dorsalis (Dipter: Culicotdae) cell cultures. 1. Med. fnfomol. 16, 104-l 11. COOMBS, K., MANN, E., EDWARDS.J.. and BROWN, D. T. (1981). Effects of chloroquine and cytochalasin B on the infection of cells by Sindbls virus and vesicular stomatitis virus. J. Vifol. 37, 1060-l 065. DE DUVE, C. (1983). Lysosomes revisited. Eur. 1. Biochem. 137, 391397. DEAN, R. T., JESSUP,W., and ROBERTS,C. R. (1984). Effects of exogenous amines on mammalian cells, with particular reference to membrane flow. Biochem. 1. 217, 27-40. DURBIN. R. K., and STOLLAR,V. (1984). A mutant of Sindbis virus with a host-dependent defect in maturation associated with hyperglycosylation of E2. Virology 135, 331-344. GRIFFITHS,G., and SIMONS. K. (1986). The vans Golgi network: Sorting at the exit site of the Golgi complex. Science 234, 438-443. HENCHAL, E. A.. MCCOWN. J. M., BURKE, D. S., SEGUIN. M. C., and BRANDT, W. E. (1984). Epitopic analysis of antigenic determinants on the surface of dengue-2 virions using monoclonal antibodies. Amer. J. Trop. Med. Hyg. 34, 162- 169. HORTIN, G., and STRAUSS,A. W. (1986). Effects of acidotropic compounds on the secretory pathway: Inhibition of secretion and processing of the third and fourth components of complement. Biothem. Biophy. Res. Commun. 136,603-609. Kousoul~s, K. G., PERSON, S., and HOLLAND, T. C. (1982). Herpes simplex virus type 1 cell fusion occurs in the presence of ammonium chloride-inhibited glycoproteins. L/iro/ogy 123, 257-263. LOMB. R. A., ETKIND. P. R., and CHOPPIN, P. W. (1978). Evidence for a ninth influenza viral polypeptide. Virology 91, 60-78. MARSH, M. (1984). The entry of enveloped viruses into cells by endocytosis. Biochem. /. 218. l-10.

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