Intracellular transport and processing of sindbis virus glycoproteins

VlROLOGY170,

ll7-122(1989)

Intracellular

Transport

and Processing

MARIE E. KNIPFER Cell Research Institute and Department

AND

of Microbiology,

Received November

of Sindbis Virus Glycoproteins

DENNIS T. BROWN’ University of Texas at Austin, Austin, Texas 78713

23, 1988; accepted January 20, 1989

The intracellular transport and processing of Sindbis virus envelope glycoproteins were studied in cells infected with Sindbis virus using the mannose-specific enzyme, endoglycosidase H (endo H). In pulse/chase labeling experiments of hamster cells with [35S]methionine, Sindbis glycoproteins PEPand El became endo H resistant in two steps at 12.5 and 20.0 min after a 5-min pulse, suggesting that the glycoproteins required this period of time to be transported to the Golgi compartments containing the enzymes which process the high mannose side chains acquired in the endoplasmic reticulum. Eacould be detected at the end of a 5-min pulse and the E2produced early was found to be endo H sensitive. The rate at which PE2was converted to E?, relative to the acquisition of endo H resistance, suggests the independence of this proteolytic event from cellular protein transport and raises the possibility that the proteolytic function is of viral origin. 0 1989 Academic Press, Inc.

and Maley, 1974) is used to determine the extent of maturation of a glycoprotein by indicating at what time point after synthesis the high mannose side chains added in the rough endoplasmic reticulum are converted to complex side chains. (See for example Rose and Bergmann, 1983). The a-mannosidases (I and II), the enzymes which process the mannose-rich side chains attached in the rough endoplasmic reticulum, reside in the proximal (cis and medial, respectively) regions of the Golgi membranes (Kornfeld and Kornfeld, 1985). The enzyme endo H may, therefore, be used to determine if a protein has passed through this intracellular compartment by determining if it has acquired resistance to this enzyme. Robbins eta/. (1977) used this enzyme to demonstrate that alphavirus glycoproteins are processed to an endo H-resistant form in a period from 30 to 180 min after synthesis. Schmidt and Schlesinger (1980), Hakimi and Atkinson (1982), and others have used this enzyme to mark the temporal occurrence of other events in glycoprotein processing (e.g., fatty acid acylation and proteolytic cleavage) relative to the conversion from high mannose to complex oligosaccharides. It is not clear at what point in the process of intracellular transport the precursor protein PE, is converted to E2 nor has it been possible to identify a protease responsible for this cleavage. Rice and Strauss (1981) and Hakimi and Atkinson (1982) have suggested that conversion of PE2 to E2 is mediated by a host cell protease located in the Golgi compartments. Our laboratory has presented a body of evidence which suggests that PE, can reach the surface of cells infected with either wild-type virus or a temperature-sensitive mutant defective in virus maturation, ts-20 (Brown and Smith,

INTRODUCTION The alphatogavirus, Sindbis, is composed of a nucleocapsid which contains a 30,000-Da capsid protein complexed with infectious single-stranded, plus-polarity RNA. The capsid is in turn enclosed within a hostderived membranous envelope consisting of the virusspecified and host-glycosylated proteins, E, and EP, which are present in equimolar ratios to capsid. The intracellular events involved in the processing, transport, and assembly of viral proteins have been extensively studied and are reviewed by Schlesinger and Schlesinger (1986). The three virus structural proteins are derived from a single 130,000-Da polyprotein which is processed to produce capsid (30,000 Da), E, (56,000 Da) and PE2 (62,000 Da), a precursor to the virus structural protein E2 (51,000 Da) and E3 (10,000 Da). Like many membrane glycoproteins the envelope proteins of alphaviruses are initially glycosylated in the rough endoplasmic reticulum of the host cell (Sefton, 1977). This first step in glycosylation produces a mannose-rich side chain which is processed to a complex oligosaccharide in the membranes of the Golgi apparatus as the protein is transported to the cell surface (Schlesinger and Schlesinger, 1986). The particular glycosylation sites on the envelope proteins of several alphaviruses have been identified (Rice and Strauss, 198 1). Sindbis has been shown to possess two glycosylation sites on each of its two envelope proteins, E, and E, (reviewed by Strauss and Strauss, 1986). The enzyme endoglycosidase H (endo H) (Tarentino ‘To whom requests for reprints should be addressed. 117

0042-6822/89

$3.00

CopyrIght 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

118

KNIPFER AND BROWN

1975; Smith and Brown, 1977; Erwin and Brown, 1980; Scheefers et a/., 1980; Mann et a/., 1983). Furthermore, Bracha and Schlesinger (1976) Ziemiecki et a/. (1980) and Jones eta/. (1977) have shown that antiserum against E, can block the conversion of PE2 to E2 when added to intact infected cells. The contention that PE2 can be delivered intact to the cell surface has been supported by data showing that cells transfected with an expression vector containing a cDNA sequence of only p62 (the Semliki Forest virus equivalent of PE2) transported that protein to the plasma membrane (Roman and Garoff, 1986). In the following study, we have used endo H to further elucidate the intracellular processing of Sindbis virus glycoproteins. MATERIALS

AND METHODS

Virus Sindbis virus heat-resistant (SVHR) was received from the laboratory of Elmer Pfefferkorn (Dartmouth Medical College) and serves as wild-type. The temperature-sensitive mutant ts-23 was also provided by Pfefferkorn. Virus stocks were prepared as described in Renz and Brown (1976). Tissue culture Baby hamster kidney cells (BHK-21) obtained from Peter Faulkner (Queens University, Canada) were grown in Eagle’s minimum essential medium with Earle’s salts (MEM-E) (Eagle, 1959) and supplemented with 10% fetal calf serum (FCS), 10% tryptose phosphate broth (TPB), and 2 mM glutamine (gin). Cells were grown in a 3% CO, atmosphere at 37”. Infection with SVHR and ts-23 Subconfluent monolayers in 25-cm2 flasks were treated with 4 pg/ml actinomycin D (act D) in phosphate-buffered saline (PBS) without magnesium and calcium for 90 min to shut off host cell RNA synthesis. The cells were infected with 100 plaque forming units/ cell of either SVHR or the temperature-sensitive mutant at 39.5” (nonpermissive temperature), for 90 min. MEM-E, 1% FCS, and 2 mM gln were added and the cells were incubated at 39.5” for 2 hr. The infected cells were starved for 1 hr at 39.5” in methionine-free MEM-E containing 2x amino acid concentrations, 1% FCS, and 2 mlLl gln. Radioactive

labeling

At 3 hr postinfection, the cells were pulsed for 5 min with 125 &i/ml of L-[35S]methionine (Amersham) in fresh methionine-free media at 39.5”. In samples used for initial (0) chase times, incorporation was stopped by

the addition of cold PBS, 0.2 m/l/l phenylmethylsulfonyl fluoride (PMSF) (Calbiochem), and 75 pg/ml cycloheximide (Sigma) to the flasks, and monolayers were harvested as described below. Viral proteins were chased with complete media containing 75 pg/ml cycloheximide to shut off further protein synthesis and incubated for45 min at 39.5” or shifted to 28” (permissive temperature). The chase periods were stopped by the addition of cold PBS, PMSF, and cycloheximide to the monolayers and harvesting proceeded as described below. Harvesting

and immunoprecipitation

Monolayers were washed with 0.2 mM PMSF in cold PBS, lysed in 1 ml of buffer consisting of 0.5% NonidetP-40 (Sigma), 0.02 MTris, 0.05 MNaCI, 0.2 mMPMSF, 0.2 mM N-tosyl-L-phenylalanine chloromethyl ketone (Sigma), and 0.2 mM N-cr-p-tosyl-L-lysine chloromethyl ketone (Sigma) (Kaluza et al., 1976). The nuclei were pelleted and discarded, and the supernatant was treated with 5 ~1 of rabbit anti-SVHR serum or anti-E, serum (a gift from Dr. James Strauss) and 200 pl of 10% protein A/Sepharose beads (Sigma) suspended in lysis buffer and agitated overnight at 4”. The bead/antibody complexes were washed three times with lysis buffer and resuspended in 50 ~1 buffer. Endoglycosidase

H treatment

Ten microliters of the bead/antibody complex was treated with 10 ~1 of 0.003 U/10 ~1 (87 pg/ml) endo H (Sigma) suspended in 0.15 M sodium citrate, 0.1 M NaCI, pH 5.3, modified from the method of Rose and Bergmann (1983). This mixture was incubated 16 hr at 37” and 20 ~1 of 2X sample buffer (4% SDS, 0.12 M Tris, pH 6.8, 20% glycerol, 3Ob dithiothreitol, 0.0032% bromphenol blue) was added. The samples were boiled 5 min at 100” and the beads were pelleted. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis Protein analysis was performed on 14 by 12 by 0.15cm slab gels in a discontinuous system modified from Laemmli (1970). The supernatants (equalized for radioactive counts) were loaded onto a 3% acrylamide stacking gel and were resolved in 10.8% acrylamide. Electrophoresis was carried out for 4.75 hr at a constant power of 4 W. The gels were impregnated with 2,5-diphenyloxazole as described by Bonner and Laskey (1974) and autoradiographed with Kodak XOMAT AR diagnostic film. RESULTS The rate at which Sindbis virus proteins are transported from their sites of synthesis in the rough endo-

PROCESSING

PE2 El E2

C 0

7.5

60

MA

119

OF SINDBIS GLYCOPROTEINS

MO

FIG. 1. Early conversion of PE, to EZ in Sindbis virus-infected BHK21 cells. Act D-treated BHK-21 cells were pulse-labeled for 5 min after infection with Sindbis virus for 3 hr. The labeled cells were chased for 0 (pulse), 7.5, and 60 min in the presence of cycloheximide, as described under Materials and Methods. The cells were immediately lysed and were immunoprecipitated with antisera to intact SVHR, as described under Materials and Methods. Equal volumes of the precipitate were analyzed by SDS-PAGE. MA, immunoprecipitate of a 60-min label of Sindbis-infected BHK cells; MO, immunoprecipitate of mock-infected cells.

plasmic reticulum to the plasma membrane and processed into mature virions varies according to cell type and temperature during infection. This process requires 30-40 min in BHK-21 cells infected with SVHR at 37” (Erwin and Brown, 1980). Under these conditions, the conversion of PE2to E2is readily detected at 20 min after synthesis and seems to be complete at 40 min after synthesis. Figure 1 shows, however, that significant amounts of E2can be detected by immunoprecipitation with antisera prepared against intact Sindbis virus at the end of a 5-min pulse at 37”. Densitometer tracings of autoradiograms produced by these experiments revealed the amount of E2 to be from 2 to 7% of the total glycoprotein label precipitated. The amount of label in E2 increased while the amount of label in PE2decreased, relative to E, , at 7.5 min postpulse with the major conversion to E2occurring at later times. The E2made early comigrates with E2produced later. Figure 2 shows the conversion of newly synthesized Sindbis virus glycoproteins from the high mannose (endo H sensitive) form generated in the rough endoplasmic reticulum to the complex (endo H resistant) form released from the Golgi membranes with time (See also Robbins et al., 1977). At 3 hr postinfection, act D-treated BHK-21 cells were labeled with [35S]methionine for a 5-min period after which further protein synthesis was arrested by the addition of cycloheximide as described under Materials and Methods. Identical cultures were then chased for various periods

of time before being lysed. The glycoproteins in aliquots of each lysate were tested for sensitivity to endo H and analyzed by gel electrophoresis as described under Materials and Methods. After several preliminary experiments, the protocol for this experiment was set to focus on the time period between 10 and 20 min after the pulse. Figure 2 shows the result of a typical experiment. The distribution of the label in the gels produced by this experiment was determined by densitometer tracings of the original autoradiograms to establish the pattern of electrophoretic mobility of the envelope proteins in the presence and absence of endo H by measuring their migration, relative to capsid protein (i.e., the distance between the glycoprotein peak and capsid peak). The result is presented in Fig. 3 and illustrates changes in the endo H sensitivity of the various glycoprotein species with time. PE2and E, are produced in a form which is highly sensitive to endo H. The conversion of the glycoproteins from endo H sensitivity to endo H resistance was found to occur in two steps. A shift to partial resistance is consistently seen at 12.5 min with the most dramatic change occurring between 17.5 and 20 min after the completion of the 5-min pulse. No additional change in endo H sensitivity is detected after 20 min. These data suggest that the proteins have reached the cellular compartments (the proximal/medial Golgi membranes) responsible for these processing events at 20 to 25 min after their synthesis. As indicated above, the conversion of PE2to E2 has been reported to occur 20 min after synthesis. We were, therefore, surprised to find that significant amounts of E2 in an endo H-sensitive form could be

EndoH Min.

M -

+ 0

-

+ IO

- + - + - + - + 17.5 20 12.5 15

-

+

M

60

FIG. 2. Acquisition of endo H resistance of Sindbis glycoproteins and early presence of EZ in infections with BHK-21 cells. Act Dtreated, SVHR-infected cells were pulse-labeled for 5 min with t%lmethionine and chased for 0 (pulse) 10, 12.5, 15, 20, and 60 min in the presence of cycloheximide as described under Materials and Methods. The cells were lysed, immunoprecipitated, and treated with endo H (+) or not treated (-) after which SDS-polyacrylamide electrophoresis was performed. Glycoproteins from mature virus(M).

120

KNIPFER AND BROWN

of transport. The fact that cleavage of PE?to E2had not been reported previously may have resulted from the particular protocol employed and the sensitivity of the proteins to immune precipitation by a particular antisera. To accentuate the detection of Ee, we immuneprecipitated virus proteins produced in a 5-min pulse, followed by chase (in cycloheximide) using a polyclonal antiserum prepared against purified E2(Fig. 4, see Materials and Methods). When the pulse/chase experiment (described in Figs. 1 and 2) was repeated using this antisera, E2 could easily be detected at 2.5 min after the 5-min pulse. The E2produced during short labeling/chase periods does not appear to be an artifact. First, the E2protein band comigrates with the E2recovered at later time points and with the virus marker (Figs. 1 and 2). Second, it is specifically precipitated by antiSindbis serum and anti-E, serum (Fig. 4) and it is not found in mock-infected cells (Fig. 1). In addition, we were unable to detect any E2 using the above procedures in lysates of cells infected with Sindbis mutant ts-23 at nonpermissive temperatures (Fig. 5). To ensure that the E2which was detected early was not produced by fortuitous cleavage of PE2 during cell lysis (despite the presence of protease inhibitors) we conducted experiments similar to those described above with Sindbis mutant ts-23 (Fig. 5). ts-23 produces El protein containing amino acid substitutions and E2 which has a wild-type sequence (Rice and Strauss, 198 1). Cells infected with ts-23 at nonpermissive tem-

PE2 /---.

0

IO

12.5

Time

I5

17.5

20

60

(min)

FIG. 3. The rate of acquisition of endo H resistance by glycoproteins of Sindbis in infected BHK-21 cells. The distance migrated by each viral glycoprotein was determined from densitometer tracings of the gel shown in Fig. 2 and plotted against their respective time points. The relative electrophoretic mobility of each glycoprotein was determined by measuring the distance in millimeters between the center of its respective densitometer peak and the density center of the capsid protein (C), which is of constant molecularweight. Control (-) and endo H treatment (---).

detected at very short labeling times. Endo H-sensitive EZ was detected by these procedures at 0, 10, 12, 12.5, and 15 min after the pulse. At 17.5 min of chase, sensitivity to the enzyme was greatly reduced (Fig. 2) suggesting that, like E,, it is converted to an endo Hresistant form. The observation that processing of PEzto E2can occur before the acquisition of endo lj resistance can be detected in any virus glycoprotein suggests that this proteolytic processing event may occur independently

--

s E2 S 52 S 52 S E2 S E2 S E2 S E2 MA 0 2.5 5 10 15 20

FIG. 4, Temporal conversion of PE2 to E, in Sindbis virus-infected BHK-2 1 ceils. Cells were infected, as described in Fig. 1, labeled for 5 min with [%]methionine, and chased for 0, 2.5. 5, 10, 15, and 20 min in the presence of cycloheximide, as described under Materials and Methods. The cells were lysed at the end of each time point (Materials and Methods) and proteins were immunoprecipitated (indicated by CY)with monospecific antisera for PE2/E2 (E,) or antisera prepared against intact virus (S). MA, precipitation of a monolayer labeled for 45 min.

PROCESSING

PEE: E2 -

c

-

EndoH Min.

M

-t-+-e 0

M 45

45

39.5”C

Shift

121

OF SINDBIS GLYCOPROTEINS

FIG. 5. Processing of proteins synthesized in BHK-21 cells infected with Sindbis mutant ts-23. Act D-treated ts23infected cells were pulsed (0) for 5 min with [35S]methionine and chased at nonpermissive temperature (39.5’) or shifted and chased at permissive temperature for 45 min in the presence of cycloheximide. The cells were lysed, immunoprecipitated with anti-Sindbis serum, and treated (-I-) or not treated (-) with endo H. Glycoproteins from mature virus (M).

perature produced PE2 and E,. These proteins were maintained in an endo H-sensitive form when chased at either permissive or nonpermissive temperature. Erwin and Brown (1980) have shown that at nonpermissive temperature, ts-23 proteins are transported to Golgi membranes but not to plasma membranes. The data presented here show that despite their presence in Golgi membranes these proteins are not processed to an endo H-resistant form and that this condition is not reversed on shift to permissive temperature. The specific detection of endo H-sensitive E2 in the pulse, lo-, 12.5, and 15min chase periods in wild-type-infected cells but not in cells infected with ts-23 argues against this protein representing a random degradation product of PE, or E, . These data suggest that the proteolytic cleavage of PE2to E2can take place at a variety of locations in the cell and is not wholly dependent upon the processing of the glycoprotein side chains. DISCUSSION We have used the enzyme endo-N-acetylglucosaminidase H (endo H) to expand upon existing obsetvations of the rate of processing and cellular site of processing (see Introduction) of glycoproteins of Sindbis virus. The data show that at 37’the processing of Sindbis virus glycoprotein oligosaccharides, from the high mannose form to the complex form, takes place during a period 12.5 to 20 min after a 5-min pulse. The processing of the oligosaccharides takes place in two steps (Fig. 3) as determined by acquisition of resistance to endo H. This stepwise conversion to endo H resistance may reflect the result of processing events occurring in separate stacks of the Golgi complex.

The data presented above also show that conversion of PEPto E2 by proteolysis can be detected within 510 min of synthesis and that this early product of proteolytic processing exists as an endo H-sensitive protein (Figs. 1, 2, 3). E2 produced in an endo H-sensitive form was not detected late in the chase, indicating that it was either degraded or converted to an endo H-resistant form. The detection of E2 at 5 to 10 min after synthesis of PEZ,combined with the existence of the product E2in an endo H-sensitive form, suggests that some conversion of PE2to E2 occurs before the processing of oligosaccharides in the Golgi membranes, which occurs at 12.5-20 min after the pulse (Fig. 2). These data suggest that the conversion of PE2to E2does not take place at a single location in the infected cell and raise the possibility that this cleavage is not carried out by a host enzyme which is restricted to a particular cellular compartment. These data suggest the possibilities that host cell enzymes capable of mediating the cleavage of PE, to E2 are present in the rough endoplasmic reticulum or that a virus-mediated (rather than a cell-mediated) proteolytic event may be responsible for the conversion of PE* to EP. In the latter case, the proteolysis may be mediated by an activity associated with E, (defective in ts-23) and the conversion of PE2to E2occurs following the pairing of E, and PE2. The assumption of a particular conformation by this pair may be essential for proteolysis. Following this logic, PE2 of ts-20 (a mutant which has amino acid substitutions in PE,) (Strauss and Strauss, 1986) would be a defective substrate for a normal E,. Alternatively, both mutants may be defective in establishing a pair or, if pairing does take place, in achieving a conformation satisfactory for the E,-mediated proteolytic event. The host range of the alphaviruses spans phyla to include vertebrate and invertebrate hosts. The ability of these viruses to replicate in diverse biochemical environments may be facilitated by an ability to conduct certain essential processing events independently of the host. ACKNOWLEDGMENTS This research was supported by Public Health Service Grants All 9545 and All 4710 from the National Institutes of Health, Grant F-l 017 from the Robert A. Welch Foundation, and through generally appropriated funds from the State of Texas to the Cell Research Institute.

REFERENCES BONNER,W. M., and LASKEY,R. A. (1974). Afilm detection method for tritium-labelled proteins and nucleic acids in polyacrylamida gels. Eur. 1. Biochem. 46, 83-88. BRACHA, M., and SCHLESINGER,M. 1. (1976). Defects in RNA+ temperature sensitive mutants of Sindbis virus and evidence for a complex of PE,-E, viral glycoproteins. Virology 74,441-449.

122

KNIPFER AND BROWN

BROWN, D. T., and SMITH, J. F. (1975). Morphology of BHK-21 cells infected with Sindbis virus temperature-sensitive mutants in complementation groups D and E. J. Viral. 15, 1262-l 266. EAGLE,H. (1959). Amino acid metabolism in mammalian cell cultures. Science 130,432-437. ERWIN,C., and BROWN, D. T. (1980). Intracellular distribution of Sindbis virus membrane proteins in BHK-21 cells infected with wildtype virus and maturation-defective mutants. /. Viral. 36,775-786. HAKIMI, J., and ATKINSON, P. H. (1982). Glycosylation of intracellular Sindbis virus glycoproteins. Amer. Chem. Sot. 21, 2140-2145. JONES,K. J., SCUPHAM, R. K., PFEIL.J. A., WAN, K., SAGIK, P., and BOSE, H. R. (1977). Interaction of Sindbis virus glycoproteins during morphogenesis. J. Viral. 21,778-787. KALUZA, G.. KRAUS, A. A., and Roar, R. (1976). Inhibition of cellular protein synthesis by simultaneous pretreatment of host cells with fowl plague virus and actinomycin D: A method for studying early protein synthesis of several RNA viruses. J. Viral. 17, l-l 9. KORNFELD, R., and KORNFELD, S. (1985). Assembly of asparaginelinked oligosaccharides. Annu. Rev. Biochem. 54, 631-664. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685. MANN, E., EDWARDS,J., and BROWN, D. T. (1983). Polycaryocyte formation mediated by Sindbis virus glycoproteins. J. Viral. 45, 10831089. RENZ, D., and BROWN, D. T. (1976). Characteristics of Sindbis virus temperature-sensitive mutants in cultured BHK-2 1 and Aedes albopictus (mosquito) cells. J. Viral. 19, 775-781. RICE, C. M., and STRAUSS,J. H. (1981). Nucleotide sequence of the 26s mRNA of Sindbis virus and deduced sequence of the encoded virus structural proteins. Proc. Nat/. Acad. Sci USA 78, 20622066. ROBBINS,P. W., HUBBARD,S. C., TURCO, S. J.. and WIRTH, D. F. (1977). Proposal for a common oligosaccharide intermediate in the synthesis of membrane glycoproteins. Cell 12, 893-900.

ROMAN, L. M., and GAROFF, H. (1986). Alteration of the cytoplasmic domain of the membrane-spanning glycoprotein p62 of Semliki Forest virus does not affect its polar distribution in established lines of Madin-Darby canine kidney cells. 1. Ce// Biol. 103, 26072618. ROSE,J. K., and BERGMANN,J. E. (1983). Altered cytoplasmic domains affect intracellular transport of the vesicular stomatitis virus glycoprotein. Cell 34, 513-524. SCHEEFERS,H., SCHEEFERS-BORCHEL,U., EDWARDS,J., and BROWN, D. T. (1980). Distribution of virus structural proteins and proteinprotein interactions in plasma membrane of baby hamster kidney cells infected with Sindbis or vesicular stomatitis virus. Proc. Nat/. Acad. Sci. USA 77,7277-7281. SCHLESINGER,M. J., and SCHLESINGER,S. (1986). Formation and assembly of alphavirus glycoproteins. In “The Togaviridae and Flaviviridae” (S. Schlesinger and M. J. Schlesinger, Eds.), pp. 12 l-l 48. Plenum, New York. SCHMIDT, M. F. G., and SCHLESINGER,M. J. (1980). Relation of fatty acid attachment to the translation and maturation of vesicular stomatitis and Sindbis virus membrane glycoproteins. J. Biol. Chem. 255,3334-3339. SEFTON, B. M. (1977). Immediate glycosylation of Sindbis virus membrane proteins. Cell 10, 659-668. SMITH, 1. F., and BROWN, D. T. (1977). Envelopment of Sindbis virus: Synthesis and organization of proteins in cells infected with wildtype and maturation-defective mutants. J. Viral. 22,662-678. STRAUSS.E. G., and STRAUSS,J. H. (1986). Structure and replication of the alphavirus genome. ln “The Togaviridae and Flaviviridae” (S. Schlesinger and M. J. Schlesinger, Eds.), pp. 35-l 19. Plenum, New York. TARENTINO,A. L., and MALEY, F. (1974). Purification and properties of an endo-(@)-acetylglucosaminidase from Streptomyces griseus. J. Biol. Chem. 249,8 1 l-8 17. ZIEMIECKI, A., GAROFF, H.. and SIMONS, K. (1980). Formation of the Semliki Forest virus membrane glycoprotein complexes in the infected cell. 1. Gen. Viral. 50, 11 l-l 23.