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A mutant of sindbis virus with a host-dependent defect in maturation associated with hyperglycosylation of E2
VIROLOGY
135, 331-344 (1984)
A Mutant of Sindbis Virus with a Host-Dependent in Maturation Associated with Hyperglycosylation RUSSELL K. DURBIN University
AND
of Medicine and Dentistq Department of Microbidogy,
Defect of E2
VICTOR STOLLAR’
of New Jersey, Rutgers Medical Pticatawa~, New Jersey 08854
Schd,
Received September 19, 1983; accepted March 6, 1984 Following serial passage of Sindbis virus (SV) on A& albopidus mosquito cells a mutant (SV,rIW2J was isolated which in chick cells produced small plaques and was temperature sensitive (ls). At 34.5” this mutant replicated normally in mosquito cells, but only poorly in chick or BHK cells. In the vertebrate cells SV.rrUrr was RNA+ at both 34.5 and 40” and on the basis of complementation tests carried out at 40”, was assigned to complementation group E. The block in the replication of this mutant, like that of t&O, the prototype mutant of complementation group E, was at the level of nucleocapsid envelopment. The PE2 and E2 glycoproteins of SV,,, were found to be hyperglycosylated relative to the corresponding glycoproteins of the parent virus (SV,). Analysis of revertants between PE2 and E2 hyperglycosylation and of svw5m suggests a causal relationship the host-specific defect in virus maturation. The association of a host-specific defect in virion assembly with hyperglycosylation of a viral structural protein points to the potential importance of host-specific glycosylation patterns in the determination of viral host range. INTRODUCTION
The alphaviruses, of which Sindbis virus (SV) is representative, are capable of replication in both arthropod and vertebrate hosts. (See Chamberlain, 1980,for a review of this topic.) The natural cycle of SV transmission, involving alternate growth in mosquitoes and vertebrates, undoubtedly exerts a strong selective pressure for efficient replication in both hosts. In nature, any variant which might adapt to one host type at the expense of the other would encounter an evolutionary cul-de-sac. In the laboratory, however, alphaviruses can be maintained indefinitely by passage on the cultured cells of a single species. In this way mutations which compromise the capacity of the virus to replicate in the alternate host can be maintained. We have been exploring host-virus interactions by genetic characterization of SV mutants whose growth is specifically ’ Author addressed.
to whom requests for reprints
should be
331
restricted in one type of host cell, and by biochemical characterization of the block in the replicative cycle in such nonpermissive systems. Similar approaches in other RNA animal virus systems have pointed to the involvement of host-specific factors in the replication of rhabdovirus RNA (Szilagyi and Pringle, 1975; Simpson et &, 1979), influenza virus RNA (Almond, 1977; Israel, 1980), and in the processing of both myxo- and paramyxovirus structural proteins (Lazarowitz et u& 19’73;Nagai et al, 1976). Symington and Schlesinger (1975,1978) have described an SV isolate which had been obtained by passage in mouse plasmacytoma cells, and which replicated more efficiently in mouse cells than did the starting virus. Alterations were demonstrated in both El and E2 of this virus rendering both proteins more negatively charged and resulting in more efficient adsorption to mouse cells. More recently in this laboratory, we have isolated and characterized two host-dependent mutants of SV restricted with re0042&X2/84 Copyright All rinbts
$3.00
Q 1984 by Academic Press. Inc. of reproduction in any form reserved.
332
DURBIN
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spect to growth in mosquito cells (Kowal and Stollar, 1981). We have now isolated, after serial passage on mosquito cells, a variant of SV with the reverse phenotype, i.e., restricted growth in vertebrate cells but normal growth in mosquito cells. The studies reported here associate this host restriction with an alteration in the glycosylation of the envelope glycoprotein E2. This report represents the first characterization of an alphavirus isolate whose ability to replicate is affected by host-specific patterns of glycosylation. MATERIALS
AND
METHODS
Cells, media, and viruses. The Aecks albopictus cells used in this study were C710 cells, a subclone of the LT-C7 cells described previously (Sarver and Stollar, 19’77). They were grown in E medium (MEM containing 0.2 mM of each of the nonessential amino acids) supplemented with 5% heat-inactivated fetal calf serum. BHK-21 cells and the preparation of primary chick embryo cell cultures have been described previously (Stollar et ah, 1976). Secondary chick embryo cell cultures were prepared by trypsinizing primary cultures and reseeding at a cell density of lo5 cells/ cm2 in CE medium (E medium supplemented with 5% tryptose phosphate and 5% heat-inactivated calf serum). The parental SV (SV,,,) used in these studies was a cloned derivative (Shenk and Stollar, 1973) of the HR strain of Burge and Pfefferkorn (1966a). Temperature sensitive mutants of SV, ts2, tsl0, and ts20, isolated by Burge and Pfefferkorn (1966a, b), were kindly provided by E. R. Pfefferkorn. Virus infectivity was assayed by plaque formation on CEF cells as described previously (Shenk et a& 1974). Passage of SV on Ae. albopictus cells. Cultures of Ae. aZbopictus cells (approximately lo6 cells on a 35-mm plate) were infected at a multiplicity of 0.1 plaqueforming unit (PFU) per cell, as described previously (Sarver and Stollar, 1977). At the end of 24 hr, the virus was harvested, diluted, and 0.2 ml of the diluted virus used to inoculate a new cell culture. For the first
STOLLAR
two transfers, the virus was diluted 200fold, and 2000-fold thereafter. All virusinfected cultures were maintained at 34.5”. Virus recovered after 15 passages (SVap15) was cloned by plaque purification, and one clonal derivative, SVap15,21, was used for the experiments described in this report. The properties of SVap15j21 did not, however, differ significantly from the properties of the uncloned population (SVaplB). Plaque pur$caticm, Virus was cloned by the procedure described by Kowal and Stollar (1981), but with some modifications to enhance plaque formation on Ae. albop ictus cells. Instead of agar, the overlay medium contained 1% low-melting Sea Plaque agarose (FMC Corp.) in E medium supplemented with 5% heat-inactivated fetal calf serum. After inoculation, cells were incubated for 2 days under the agarose overlay at 34.5” before staining with 0.005% neutral red. Ccmplernentatim tests. These were performed as described by Kowal and Stollar (1981) except that the adsorption was allowed to take place at 40” rather than at 4”. Polyaqdamide gel electrophoresis (SDSPAGE). Two different methods were used for electrophoresis of proteins. For electrophoresis of alkylated nonreduced proteins, monolayers of cells were washed with cold PBS, then lysed with 0.2 ml lysis buffer: 0.5% Triton X-100 (Ruger Chemical Co.), 0.05 M iodoacetamide (Sigma Chemical Co.), 0.1 M sodium acetate, 0.0025 M magnesium acetate, 0.05 M Tris acetate, pH 8.4. Nuclei were removed by low-speed centrifugation, and protein was precipitated from the supernatant by adding 1.2 ml cold acetone and chilling the mixture for at least an hour at -20”. The precipitate was pelleted by centrifugation for 5 min at 10,000 g and dissolved in sample buffer: 1% sodium dodecyl sulfate (SDS), 10% glycerol, 0.001% bromphenol blue, 0.05 M iodoacetamide, 0.125 M Tris-HCl, pH 6.8. Electrophoresis was performed on 10% polyacrylamide slab gels using the discontinuous buffer system of Laemmli (1970). Electrophoresis of thiol-reduced protein samples was as described above, except that iodoacetamide was replaced by 0.01
SINDBIS
VIRUS
M mercaptoethanol in the lysis buffer and by 0.14 M mercaptoethanol in the sample buffer. Measurement of RNA synthesis. Approximately 2 x 10r’BHK or chick cells or 4 X 105Ae. albopictus cells were seeded into each well of a 24-well multiwell tray (Costar). Twenty-four hours after seeding, cells were infected with 100 PFU/cell in 0.2 ml E medium supplemented with 0.1% bovine serum albumin. At 5 hr after infection the cells were treated with actinomycin D (4 pg/ml) for 15 min, then incubated for 1 hr in E medium containing actinomycin D and 20 &i rH]uridine/ml. Cells were washed and fixed as described by Sarver and Stollar (1977), and dissolved in 0.2 ml 0.2% SDS. After addition of 5 ml Liquiscint (National Diagnostics) radioactivity was measured in a scintillation counter. Immuno$uorescent microscopy. Viral antigen in both permeabilized and nonpermeabilized BHK cells was visualized by the technique described by Saraste et a2. (1980). The primary antiserum was from a rabbit immunized with SVata virions (Stollar et aL, 1976), and the secondary antibody was fluorescein-conjugated IgG from a goat immunized with rabbit IgG (Cwpel). Electron microscopy. This was performed as described previously (Igarashi et al, 1976). Radioactive labeling of viral proteins. Cells were seeded in 24-well cluster trays as described above, and infected with 100 PFU/cell. At 6 hr postinfection cells were refed with E medium modified by the omission of “cold” methionine and the addition of [%]methionine (20 pCi/ml). In pulse-chase studies, after labeling, cells were refed with chase medium: E medium supplemented with 1 mM “cold” methionine, and incubated for various intervals prior to preparation of lysates for electrophoresis. Chemicals and drugs. Tunicamycin was kindly provided by Dr. J. Lampen. Actinomycin D was a gift from Merck, Sharp and Dohme. [5-3H]Uridine (20 Ci/mmol) was purchased from ICN Pharmaceuticals, Inc., and [%]methionine (>lOOO Ci/mmol) from Amersham Corporation.
333
MUTANT RESULTS
Host restriction and temperature sensitivity of SV,p15j21.Yields of SVap18,zIin chick or BHK cells 24 hr after infection at 34.5“ were typically only 1% of the yield of SVstd (Fig. la). Moreover, the logarithmic phase of virus growth was delayed by some 4 to 5 hr in SVap15,21infected BHK cells as compared to SVstd-infected cells. In mosquito cells, however, SVap15,21 replicated just as well as SVsti (Fig. lb). BHK cells infected with SV,15,n and incubated at 40” produced no more than 0.03 PFU per cell, at least 200-fold less than infected BHK cells incubated at sv,1Ei/2134.5” and about 50,000-fold less than SV,tiinfected BHK cells incubated at 40” (Fig. la). Similar results were seen in experiments using chick cells (data not shown). Compared to plaques produced on chick cells by SVsti at 34.5”, those produced by were much smaller. In a typical SVap15/21 experiment, at the end of a 24-hr incubation under agar, SVstdproduced plaques 2 to 3 mm in diameter, while SVap15,21 plaques measured no more than 0.5 mm. failed to generate any plaques on SVaplW21 chick cells at 40” (PFU at 40”/PFU at 34.5” < 10-5). In mosquito cells, however, we were unable to demonstrate that the replication was temperature sensitive. At of SVaplW21 37” the yield of SVa,,15,21 was equivalent to that of SVsti (not shown); at 40” neither the standard virus nor SVap15,21 grew in mosquito cells. The results above indicate that even though the host restriction of SVap15,21 in vertebrate cells is readily demonstrable at 34.5”, at 40” the restriction is virtually complete whether assayed by virus yield or by plaque formation. S@hesis of viral RNA. To help localize the block in viral replication in SVap15,21infected vertebrate cells we compared viral RNA synthesis in SVa,,15,21and SV,*-infected cells. In both BHK and chick cells infected with the mutant virus, viral RNA synthesis, as measured by incorporation of tritiated uridine into viral RNA, was similar to that observed with cells infected with the parent virus (Table 1). Agarose
334
DURBIN
10'
AND
STOLLAR
(a)
-
2
4
6
6
10
12
04
10'
-
103
-
4
6
12
16
20
22
HOURS POST INFECTION
FIG. 1. Replication of SV.,15/n and SVsti in BHK and mosquito cells. Cells were infected at an input multiplicity of 10 PFWcell. BHK cells were incubated at either 34.5 or 40”, Ae. albspictus ceils at 34.5’. At the indicated times postinfection, samples were taken and titers were determined by plaque assay on chick cells at 34.5”. (a) BHK cells, (b) Ae. albopidus cells. (0) SV.ris,si at 34.5”; (0) SV.,l6/2l at 40”; (0) SV., at 34.5’; (m) SV,, at 40’.
gel electrophoresis demonstrated that the D) or with Ls20(group E) and maintained ratios of genomic (42 S) to subgenomic (26 at the nonpermissive temperature, the cleavage of PE2 to E2 fails to occur. S) RNA in SV,,- and SV,Is,zl-infected BHK cells were similar (not shown). Complementution analysis. Since SVap15,z1 TABLE 1 was temperature sensitive (cf. yields at VIRAL RNA SYNTHESIS 34.5” and at 40” in Fig. la), we carried out complementation tests to determine [3H]Uridine whether it could be assigned to one of the incorporation” previously established complementation groups. Because SVap15121 was clearly of the Cells 40” Virus 34.5” RNA+ phenotype, we restricted our experSVsti 2.15 X 106 1.79 x 106 iments to complementation groups C, D, and BHK 1.99 x 106 1.08 x 105 sv.p15/21 E, corresponding to defects in structural mock infected 0.15 x 106 0.07 x lo5 proteins C, El, and E2 (or PE2), respectively. SVap15121 was able to complement ts2 Chick 3.47 x 10’ 1.18 x ld SVnti 8.31 x lo’ 2.15 X lo* (Group C) and 1310(group D), but not k20 svapls/21 0.08 x ld 0.12 x lo4 mock infected (Table 2). Thus we assign SVap15,21 to complementation group E. @Incorporation of PH]uridine into acid-insoluble Viral glycoproteins. We next sought to dematerial (cpm) by infected or mock-infected cells in termine whether there were any anomalies the presence of actinomycin D was measured as dedetectable by SDS-PAGE among the struc- scribed under Materials and Methods. Since at 40” tural proteins of SVap15121, or in their pro- svapm1 induced at least 60% of the RNA synthetic . . . teolytic processing. For instance, in cells activity Induced by SV.,, it can be considered RNA+ infected with SV mutants LslOor &XI (group (Strauss and Strauss, 1930).
335
SINDBIS VIRUS MUTANT TABLE 2 COMPLEMENTATION OF SVapi5,e,WITH RNA+ SV ts MUTANTS
Yields from mixed infections”
ts2 tsl0 ts20 sv.,,m
ts2b CC)
tsl0” (D)
ts20b (E)
1.9 x 102
1.1 x 10’ (0.8) 1.3 x l@
6.0 X l@ (0.6) 1.1 x lo6 (49) 9.5 x 10s
SV.DISRI 1.6 X 8.8 X 7.4 X 2.4 X
10e (3.7; 6.0’) 10’ (66; 49”) 108(0.9; 0.6”) 102
a Secondary cultures of 2 X 105chick cells in 2-cm2 wells were coinfected with the indicated mutants. Yields (PFU/well) after ‘7 hr of incubation at 39.5’ were determined by plaque assay at 23”. The numbers in parentheses are complementation indices. Our isolate of fe2 complements poorly; we were unable to detect complementation between ts2 and either tsl0 or ts20. Complementation between ts2 and SV.p16,21 is probably detectable only because of the low leak-yield of the latter. bMutants of Burge and Pfefferkorn (1966a). Complementation groups are indicated in parentheses. ’ Complementation indices from a second experiment.
Infected BHK cells were pulse labeled C and D). Thus it appears that the difference with pS]methionine at 34.5” and the labeled between the molecular weight of the mutant proteins were analyzed by SDS-PAGE. This and standard PE2 and E2 glycoproteins is experiment demonstrated that the ap- a result of increased glycosylation of the parent molecular weight of the PE2 en- SVapi5,z1glycoproteins relative to those of was approximately 3000 the parent virus. coded by SVap15~21 greater than the SVsti coded PE2 (Fig. 2a). was also larger than The E2 of SVap15,21 SVap15j21 proteins C and El were electro- that of SVeti when both viruses were grown phoretically similar to the corresponding in mosquito cells (Fig. 4). In experiments SV,* products. During a subsequent chase similar to that shown in Fig. 2a, but perPE2 was processed to E2 with formed using mosquito cells, pulse-labeled the SVap~~/2~ kinetics similar to that seen in the case of PE2 was again larger in SV,lbj,/P1-infected SVati PE2. Again, the molecular weight of cells than in SV,ti-infected cells (not shown). the SVap15,21 E2 was approximately 3000 However, the molecular weights of all the greater than that of SVsti E2. In cells main- SV glycoproteins, both mutant and stantained at 40” (Fig. 2b) the PE2 of SVap15,21, dard, produced in mosquito cells were 1000 like that of SVsti but unlike that of ts20 to 2000 less than those of the corresponding (also of group E), was unstable (Jones et glycoproteins in vertebrate cells, consistent al, 1974). However, in contrast to the SVsti- with earlier findings (Luukonen et c& 1977; infected cells at 40°, E2 appeared in the Sarver and Stollar, 1978). !?rawport of wird antigen to the WU svapm- infected cells in lower amounts than expected. surface. The association of the defect in In order to determine whether the in- svap15/21 replication in chick and BHK cells crease in the apparent molecular weights with a mutation affecting one of the viral of the mutant PE2 and E2 was due to an glycoproteins suggested two possible exaltered pattern of glycosylation or to an planations for the failure to assemble maalteration in the amino acid sequence, we ture virions. The glycoproteins of SVap15,21 pulse labeled infected cells either with or might, like those of the complementawithout pretreatment with tunicamycin tion group D mutants, tsl0 and IX&~,fail to (TM). The PE2 “aglycoproteins” synthesized reach the cell surface. Alternatively, the in the TM-treated cells infected with mu- SVap15j21 glycoproteins might reach the cell tant and standard viruses were nearly in- surface, but fail to assemble into virions. distinguishable by SDS-PAGE (Fig. 3, lanes The latter pattern is characteristic of the
336
DURBIN AND STOLLAR
b a
MABCD
AWDEFGHIJKLMN
PE2 F
E2
FIG. 2. Pulse-chase labeling of viral proteins. (a) BHK cells were infected at an input multiplicity of 106 PFWcell with SV,, (lanes A-G) or SV*rum (lanes H-N) and incubated at 34.5”. At 6 hr postinfection, cells were labeled for 15 min with PSjmethionine, then either prepared directly for electrophoresis (lanes A and H) or first incubated with chase medium for 15 min (B and I), 26 min (C and J), 45 min (D and K), 66 min (E and L), 75 min (F and M), or 96 min (G and N). Proteins were alkylated and electrophoreaed without reduction of disulfide bonds in order to optimally resolve El and E2 (see Materials and Methods). The polypeptide marked “B” is the binary envelope protein (uncleaved PEB-El). (b) BHK cells were mock infected (lane M), infected with SV, (Lanes A and B) or with SV.,,ls,sI (lanes C and D) as above, but were maintained at 40”; at 12 hr after infection they were pulse labeled with mjmethionine for 15 min and immediately prepared for electrophoresis, as above (lanes M, A, C) or incubated in chase medium for 66 min (lanes B and D). Suppression of host synthesis is routinely less complete at 40” than at 34.5”. Prominent host proteins were marked “h”.
glycoproteins of the group E mutant, ts20, in cells incubated at the nonpermissive temperature. In order to determine whether mutant viral glycoproteins were being transported to the cell surface, we used the fluorescent antibody technique to localize the viral antigen in infected cells, with or without permeabilization of the cell membrane (Fig. 5). In light of the evidence that SV El and (P) E2 exist as a complex in infected cells (Jones et uL, 19’77;Bracha and Schlesinger, 1976; Smith and Brown, 19W, Rice and Strauss, 1982), we performed these experiments with antiserum directed against SV virions rather than with monospecific anti-El or anti-E2 serum. Fixed but
nonpermeabilized BHK cells infected with SVapwzIor with SVti showed bright surface fluorescence, both at 34.5 and at 40’ (Fig. 5B, C, and D). These results indicate that the SV,,,,, glycoproteins were in fact transported to the cell surface. As a control we also examined cells infected with SV MO. As expected (Sara&e et uL, 1980), cells infected with this group D mutant at 28” developed surface fluorescence almost as intense as in SVd-infected cells (Fig. 5E), but at 40” failed to develop surface fluorescence (Fig. 5F). Cells infected with SV,, or with either mutant showed intense fluorescenceif permeabilii prior to staining, whether incubated at the permissive temperature or at 40” (not shown).
SINDBIS
VIRUS
C D
PEZ= El-
337
MUTANT
lication in vertebrate cells, a small plaque phenotype, temperature sensitivity, and hyperglycosylation of PE2 and E2. The coincidence of these phenotypes in a single mutant does not, however, prove a causal relation between the hyperglycosylation and the other phenotypic properties. If there is a eausal relation, reversion of the hyperglycosylation phenotype should correlate with reversion of the other phenotypes. Our stock of SVap15,w (grown in mosquito cells) when assayed on CEF was essentially free of ts+ or large plaque revertants (ts’ revertant frequency < 1.3 X 10p6).Table 3,
A
6
C
FIG. 3. Effect of tunicamycin on viral proteins. BHK cells were infected with SV,u (lanes A and C) or SVW.W~ (lanes B and D) as described under Materials and Methods. Four hours postinfection the medium in two of the wells (lanes C and D) was supplemented with 0.5 gg tunicamycin/ml. Between 6 and 6.5 hr postinfection, the cells were labeled with [86S]methionine; lysates were then prepared under reducing conditions for SDS-PAGE.
Electron micros~ characterization of the defect in SVwp,,,, assembly. To further
pinpoint the nature of the block in the replication of SVap15,z1 in vertebrate cells, we examined infected chick and BHK cells by electron microscopy. In both cell types infected with the mutant virus there was an abundance of intracellular nucleocapsids, most of them lining the inside of the plasma membrane (Fig. 6b). In contrast to the cells infected with the wild-type virus (Fig. 6a), -infected cells (Fig. 6b) viral nuin SVap15~~ cleocapsids were only very rarely seen in association with the evaginations in the plasma membrane characteristic of budding virus or on the cell surface. The electron microscopic appearance of vertebrate cells infected with SV,16,u was similar to that of published electron micrographs of cells infected with the group E SV mutant, ts20 (Brown and Smith, 1975). Revertunts. We have demonstrated that is characterized by restricted repSVaplW21
E2
E2 El
El
C FIG. 4. SDS-PAGE of purified virus produced by mosquito cells. Cells were infected with ca. 1 PFU/ cell. Medium was collected after 24 hr at 34.5” and clarified by low-speed centrifugation. Virus was pelleted, purified by centrifugation to equilibrium on a ZO-50% sucrose gradient, then repelleted. Six mierograms of viral protein were separated by nonreducing SDS-PAGE and were visualized by staining with Coomassie Brilliant Blue R-350. (A) SV., grown in mosquito cells; (B) SV.pla,21 grown in mosquito cells; (C) SV., grown in chick cells. (Yields of SV,r,Is,n from vertebrate cells were too low to generate a satisfactory sample.)
DURBIN AND STOLLAR
FIG. 5. Surface-specific immunofluorescent detection of Sindbis viral antigen. Infected BHK cells were fixed, without permeabilization, 5 hr postinfection and stained by the fluorescent antibody technique. (A) mock infected, incubation at 34.5’; (B) SV., infected, 34.5”; (C) SV,16,n infected, 34.5’; (D) SVspm infected, 40’; (E) SV f.slO infected, 28”; (F) SV MO infected, 40”.
however, shows that when SVap15,z1 was serially passaged on chick cells large plaque revertants came to dominate the population by the third passage and ts+ revertants accumulated in large numbers. A similar phenomenon was described previously by Shenk et cd (1974). To analyze the relationship between the PE2 hyperglycosylation and the other phenotypic properties of SV,r5jn, we generated ts+ and plaque size revertants by passing twice on chick cells. The chick cellsvap15/21 passaged virus was then plaque purified on mosquito cells (SVaplsIzland SV,, plaques were indistinguishable on Ae. a.lbo$&w cells) rather than on vertebrate cells, in or-
der to avoid any selective pressure toward reversion during the cloning. Twenty-six plaques were picked at random and a small stock of each was grown on Ae. albqwictus cells. These clones were characterized with respect to PE2 size as determined by SDSPAGE of infected BHK cell lysates, and with respect to yield, plaque size, and temperature sensitivity in chick cells (Table 4). A perfect correspondence was found between reversion to PE2 of the SVBti type and the relaxation of the host restriction in chick cells (P = 5.0 X lo-‘, x2 test). Of 16 isolates tested which were associated with the smaller PE2 characteristic of SV,,, all produced yields in chick cells greater
FIG. 6. Electron micrographs of infected chick cells. Secondary cultures of chick cells were infected with 100 PFU of SV., (a) or SV.pls,zI (b) per cell and incubated at 34.5”. At 7.5 hr postinfection, cells were fixed and prepared for electron microscopy. (Bar = 100 nm.) 339
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AND TABLE
STOLLAR 3
REVERSIONOFPLAQUESIZE AND ts PHENOTYPES WITH PASSAGEOFSVspls,I1AT 34.5’ ONCHICK CELLS Titer-assay temperature 34.5” Chick cell passage level Ob 1 2 3
Small plaques
Large plaques
1.6 X lo9
(Not detectable) 1.2 x 106 8.4 X lo6 3.0 x 108
5.0 x lo7 8.5 X lo6
(Not detectable)
40” Small plaques” 12 x 10’ 6.8 X ld
3.1 x 106 1.1 x 10s
o Only small plaques were produced at 40” by ts+ revertants. * Stock SV.,lWzI grown in mosquito cells.
than 10% of the yield of SV,,. Indeed, only generally without known significant bioone of the isolates (No. 25) associated with logical consequences. the larger PE2 characteristic of SVap15,z1 In some cases (Simizu and Maeda, 1981; produced a yield greater than 1% of the Eaton, 1982) the variant virus isolates were SVsti yield. Of the 15 large plaque rever- derived from persistently infected mostants, every one had also reverted to the quito cell cultures, but the significance of parental PE2 (P = 1.8 X 10-5, x2 test). Like- the alterations in the glycoproteins in wise, of the 10 ts+ revertants tested, all had terms of adaptation to mosquito cells or reverted to the parental PE2 (P = 0.0037, to the persistently infected state is not x2 test). These results strongly suggest a clear. On the other hand, it has been consistently observed that alphavirus isolates connection between the hyperglycosylation of PE2 and the other phenotypes. However, derived from long-term persistently intwo isolates (Nos. 14 and 18) which reverted fected mosquito cells typically produce with respect to the glycoprotein size and small plaques and are temperature senhost restriction remained both small plaque sitive when grown on vertebrate cells and temperature sensitive. In addition, (Shenk et al, 1974;Simizu and Maeda, 1981; three isolates (Nos. 10, 13, and 15) which Eaton, 1982). The molecular bases of these reverted in terms of host restriction, plaque phenotypic shifts have, however, not been size, and size of PE2 retained their tem- explored. Only in one case has an altered perature sensitivity. Thus of the three other alphavirus glycoprotein been tentatively phenotypic properties examined in the re- associated with a host-specific impairment vertant populations, that which best cor- in replication (Simizu et aL, 1983). In this related with loss of host-restriction phe- report, an isolate of western equine ennotype, was the presence of SVsti-type PE2. cephalitis virus derived from persistently infected mosquito cells was shown to lack the normal glycosylation of E3, and to be inefficiently released from vertebrate cells. DISCUSSION It is interesting that ts isolates of westThere have been several reports describ- ern equine encephalitis virus derived from ing alphavirus isolates which encode al- persistently infected mosquito cultures tered envelope glycoproteins. In some cases tend to be defective in the gene for PE2 (Bracha and Schlesinger, 1978; Cancedda (Simizu et d, 1981).Eaton (1982) also noted that SV glycoprotein E2 underwent a et &, 1981;Leone et d, 1980) the variations appear to arise as random clonal varia- characteristic shift in SDS-PAGE mobility with increasing passage level of persistions, probably reflecting the low fidelity of RNA-dependent RNA polymerases tently infected mosquito cultures, in this (Holland et al, 1982). These variations are case toward increased mobility. If a par-
SINDBIS TABLE
VIRUS
4
MUTANT
341
peatedly in independent passage series. In an effort to determine whether the PE2 hyperglycosylation seen in SVapi5,21was a general consequence of serial passage of Temperature PE2 Yield Plaque SV on mosquito cells, we performed six sensitivity” sized sire” IBOld (PFWml)” more independent l&transfer passages, 1 std 2.6 X 10’ L WY similar to that from which SVapi5/z1 was ts+ std 2 3.4 x 10’ L derived, each starting with a separate clone Std 3 L (NT) (NT) of sv,a. None of these generated the hySM 4 1.0 x 10’ L t-s+ 5 s mut 4.4 x lo’ WV perglycosylated PE2 or minute plaque Std 6 L ts+ 2.0 x 10’ phenotypes. Moreover, of four tempera7 ts mut 6.3 X 10’ s ture-sensitive, small plaque isolates de8 L ts+ Std 4.0 x 10’ rived from persistently infected cultures 9 ts+ Std 1.4 x 10’ L 10 6.0 X ld L ts Std (Igarashi et a& 1977), none had a hyper11 6.2 X 10’ s ts mut glycosylated (P)E2 (not shown). Thus we 12 1.2 x lti S ts mut have no evidence that the hyperglycosy13 L ts std 1.8 X 10’ lation of (P)EZ is positively favored in 14 6.3 X 10s s t8 SM mosquito cells. However, the fact that it 15 8.0 X 16 L ts Std 16 1.2 x 10’ L ts+ std is at least tolerated in mosquito cells and 17 2.8 x 10’ L t.s+ Std not in vertebrate cells reflects what may 18 2.2 x log s it.3 std be a significant difference between the two 19 5.1 x lti S ts mut types of host cell in shaping the evolution 20 1.2 x 10’ L k?+ std 21 4.6 X lo’ S ts mut of sv. 22 4.7 x lo’ S ts mut From the relative SDS-PAGE mobilities 23 L ts+ std 1.4 x 10’ of the PE2 glycoproteins of SVstd and 24 ts std 4.2 x 10’ S with and without tunicamycin 25 it.% mut svap15/21, 1.2 x ld S 26 3.6 X lo7 L ts+ Std treatment, we have concluded that the mu4.6 X 10’ L Std t.9’ svstd tant PE2 is hyperglycosylated relative to 5.3 x l(r if.3 S mut SV,ls/sl the parental PE2. Although estimates of molecular weights of glycoproteins by Note. SV.,,,, was passaged twice on chick cells as described in the text. The resultant stock was plaqued at 34.5O on Ae. SDS-PAGE must be interpreted with caualbopidus cells and 26 plaques were picked at random. Each tion (see, for example, Segrest et d, 1971), plaque isolate was then grown into a small stock, also at it is interesting that the PE2 of the mutant 34.5” in Ae. u&opictus cells. The titers of these stocks ranged virus has an apparent molecular weight from 3 X lb to 1 X lb PFU/ml, as assayed on chick cells larger than the standard PE2 by approxat 34.5”. Each clonally derived stock was eharactariaed with respect to growth, plaque sise, and temperature sensitivity imately the size of a typical asparagineon chick cells, and with respact to PE2 size in BHK cells. linked oligosaccharide. The fact that the “Yield (PFWcell) from CEF 6 hr after infection with 10 size difference is evident even in short PFWeell and incubation at 34.5”. pulses (Fig. 2a) indicates that it does not b Plaque size was scored as small (S) if plaques were x0.5 mm after 24 hr of incubation at 34.5’ under agar, and large result from differences in late modifica(L) if plaques were >2 mm. tions such as the addition of sialic acid “Each isolate was scored as temperature sensitive (ts) if (Bonatti and Cancedda, 1982, Hakimi and its titer measured by plaque assay at 40’ was less than 1% Atkinson, 1982). This point is reinforced of its titer measured at 34.5’; if greater than 1% it was considered revertant (ts+). by the observation that the size difference ClFor each isolate, a well of BHK cells was infected with is also evident in viral proteins produced 100 PFU per cell, and labeled for 26 min with I%Jmethionine in mosquito cells (Fig. 4), since mosquito 6 hr postinfection. Labeled proteins were analyzed by SDScells are thought to produce N-linked oliPAGE under reducing conditions. PE2 was scored as “stangosaccharides only of the nonsialylated, dard” (std) or “mutant” (mut) based on comigration with the PE2 of SVati or of SV.,,r,. respectively. “high mannose” type (Stollar et d, 1976; ’ Not tested. Luukonen et al, 1977; Butters and Hughes, 1981). These considerations, taken totitular phenotypic shift results in more ef- gether, suggest that the increased size of ficient replication in mosquito cells, one the glycoprotein reflects the generation of might expect the same shift to occur re- a new glycosylation site rather than more PHENOTYPIC ANALYSIS OF ISOLATES OBTAINED PASSAGE OF SV,,s,/p ON CHICK CELLS
AFTER
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extensive glycosylation of the same sites protein could have a host-specific effect. found in the parental viral glycoprotein. Finally, it may be that the additional burFurthermore, it is easier to understand den of carbohydrate on E2 in vertebrate how a mutation in the viral genome might cells induces a shift in its tertiary strucresult in the addition of a glycosylation ture, distorting protein-protein recognisite (a function of the primary structure tion sites necessary for assembly of the of the protein), than to envision a viral envelope. mutation leading to an alteration in the In addition to its usefulness in comparcellular response to an existing glycosy- ative studies of viral assembly in insect lation site. However, we have not yet per- and vertebrate cells, SVap15j21 represents a formed the sequencing studies to distin- useful addition to the library of Sindbis guish between the two possibilities. Can- mutants, since only one other member, ts20, cedda et al. (1981) demonstrated that our of complementation group E has been reSV,d (referred to as SVm by these workers) ported. The molecular bases of the defects differs from the SVna strain from which in these two mutants appear to be different, it was derived in that E2 of SVsti has three although both affect PE2 or E2 and both instead of two glycosylation sites. If, as cause a block in viral assembly at the point seems most probable, E2 of SVap15,21 differs of envelopment. Whereas under nonpermissive conditions the PE2 of ts20 is not from that of our SVstd by an additional glycosylation site, it must differ from the cleaved, at 84.5” in BHK cells (essentially original SVns E2 by two additional sites. a nonpermissive situation for SVapl& the It seems likely that the basis of the dif- ~‘132 of Wpwa is processed to E2 with ferential response to the altered viral gly- kinetics indistinguishable from those of the coprotein in mosquito and in vertebrate SVsti PE2. It is clear, then, that normal cells is the fact that the carbohydrate moi- cleavage of PE2 can occur in the absence eties of glycoproteins synthesized in mos- of viral maturation. This conclusion conquito cells are different from the carbo- flicts with the proposal that PE2 cleavage hydrate moieties added to glycoproteins in is always tightly linked to the final stages vertebrate cells. Consequently, the same of viral morphogenesis (Jones et aL, 1974; viral gene expressed in two different cell Brown, 1980). The data in this report indicate that the types will result in chemically distinct defect in SVap15,21 maturation in vertebrate products. The mechanism by which the altered cells is related to cell-specific patterns of glycosylation pattern of the E2 of SVap15,21protein glycosylation. We hope to deterinterferes with assembly in vertebrate cells mine more precisely the feature(s) of veris unknown. Perhaps the increased number tebrate cell glycoprotein biosynthesis reof sialic acid residues per molecule of E2 sponsible for this defect by comparing the in lectin-remade possible by the addition of a new growth of SVstdand SVap15,21 glycosylation site could result in electro- sistant vertebrate cells which have specific static repulsion and therefore decreased defects affecting glycosylating enzymes affinity between peplomers. [However, (Stanley et uL, 1975). supplementation of the growth medium ACKNOWLEDGMENTS with neuraminidase did not result in an increase in the output of SVap15,21 from inThis investigation was supported by the United fected chick cells (data not shown)]. An- States-Japan Cooperative Medical Science Program other possibility is that the additional car- through Public Health Service Grant AI-05920 and bohydrate interferes sterically with the by Institutional National Research Service Award CAbinding of viral structure units to one an- 09099 from the National Cancer Institute. The authors other. Since a greater mass of carbohydrate thank Lizabeth Flares for her help with the electron is added to each glycosylation site in ver- microscopy. tebrate cells than in mosquito cells (K. REFERENCES Keegstra and V. Stollar, unpublished) it is conceivable that the addition of a glyco- ALMOND, J. W., MCGEOGH, D., and BARRY, R. D. (1977). sylation site to a viral structural glycoMethod for assigning temperature-sensitive mu-
SINDBIS VIRUS MUTANT tations of influenza viruses to individual segments of the genome. Vi7dogy 81,62-73. BRACHA, M., and SCHLESINGER, M. J. (1978). Altered E2 glycoprotein of Sindbis virus and its use in complementation studies. .Z. Vi4 26. 126-135. BROHRJ,D. T. (1980). Assembly of alphaviruses. In “The Togaviruses-Biology, Structure, Replication” (R. W. Schlesinger, ed.), pp. 4’73501. Academic Press, New York. BROWN, D. T., and SMITH,J. F. (1975). Morphology of BHK-21 cells infected with Sindbis virus temperature sensitive mutants in complement&ion groups D and E. .Z Viral 15.1262. BURCE. B. W., and PFEFFERKORN,E. R. (1966a). Isolation and characterization of conditional lethal mutants of Sindbis virus. Virdogy 30,204-213. BURGE,B. W., and PFEFFERKORN, E. R. (1966b). Cornplementation between temperature-sensitive mutants of Sindbis virus. Vi30.214-223. BU?TERS.T. D., and HUGHES, R. C. (1981). Isolation and characterization of mosquito cell membrane glycoproteins. B&him. Biophgs Acta 640.655-6’71. CANCEDDA,R., BONAT~I,S., and LEONE,A. (1981). One extra oligosaccharide chain of the high-mannose class in the E2 protein of a Sindbis virus isolate. J Vird 38, 8-14. CHAMBERLAIN, R. W. (1986). Epidemiology of arthropod-borne togaviruses. In “The Togaviruses-Biology, Structure, Replication” (R. W. Schlesinger, ed.), pp. 175-227. Academic Press, New York. EATON, B. T. (1982). Sindbis virus variants from a persistently infected mosquito cell culture contain an altered E2 glycoprotein. Vi7dogy 122. 486-491. HAKIMI, J., and ATKINSON,P. H. (1982). Clycosylation of intracellular Sindhis virus glycoproteins. Bb chemistry 21, 2140-2145. HOLLAND,J., SPINDLER, K., HORODYSKI, F., GRABAU, E., NICHOL,S., and VANDEPOL,S. (1982). Rapid evolution of RNA genomes. solace 216, 1577-1585. IGARASHI,A., HARRAP,K. A., CASAL~,J.. and STOLLAR, V. (1976). Morphological, biochemical and serological studies on a viral agent (CFA) which replicates in and causes fusion of A& albspictus (Singh) cells. Virdogy 74, 174-187. IGARASHI, A., Koo, R., and STOLLAR,V. (1977). Evolution and properties of A& albopictus cell cultures persistently infected with Sindbis virus. Virdogy 82. 69-83. ISRAEL, A. (1980). A temperature-dependent host range mutant of fowl plague virus (FPV). Virdogy 105.1-12. JONES, K. J., SOJPHAM, R. K., PFEU, J. A., WAN, K., SAGIK. B. P., and BOSE,H. R. (1977). Interaction of Sindbis virus glycoproteins during morphogenesis. J. ViroL 21.778-787. JONES,K. J., WAITE, M. R. F., and BOSE,H. R. (1974). Cleavage of a viral envelope precursor during the morphogenesis of Sindbis virus. .Z Vi&. 13, 809817.
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KOWAL, K. J., and STOLLAR, V. (1981). Temperature sensitive host-dependent mutants of Sindbis virus. Vi114,140-148. LAEMMLI,U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227.686685. LAZAROWITZ, S. G., COMPANS,R. W., and CHOPPIN, P. W. (1973). Proteolytic cleavage of the hemagglutinin polypeptide of influenza virus. Function of the uncleaved polypeptide hemagglutinin. Virology 52.199-212. LEONE,A., COLANTUONI,V., PONTAREUI, G., and CANCEDDA,R. (1986). Defective mutant of Sindbis virus with a smaller molecular weight form of the El glycoprotein. .Z. Viral 34. 598-603. LUUKONEN,A., VONBONSDORFF, C-H., and RENKONEN, 0. (1977). Characterization of Semliki Forest virus grown in mosquito cells. Comparison with the virus from hamster cells. ViroZcgp 78.331335. NAGAI, Y., KLENK, H-D., and Roar, R. (1976). Proteolytic cleavage of the viral glycoproteins and its significance for the virulence of Newcastle disease virus. View 72, 474-508. RICE, C. M., and STRAUSS,J. H. (1982). Association of Sindbis virion glycoproteins and their precursors. J. Mot! Bid 154.325348. SARASPE,J., BONSDORFF, C-H., HASHIMOTO,K., KERANEN, S., and KAARIAINEN, L. (1980). Reversible transport defects of virus membrane glycoproteins in Sindbis virus mutant infected cells. CellBid Znt Z&p. 4,279-286. SARVER,N., and STOLLAR,V. (1977). Sindbis virusinduced cytopathic effect in Aeuks a&vi&us (Singh) cells. Virobgg 80. 390-400. SARVER,N., and STOLLAR,V. (1978). Virazole prevents production of Sindbis virus and virus-induced cytopathic effect in Aedes albopict2cs Virology 91,267282. SEGREST,J. P., JACKSON,R. L., ANDREWS,E. P., and MARCHESI,V. J. (1971). Human erythrocyte membrane glycoprotein: A re-evaluation of the molecular weight as determined by SDS polyacrylamide gel electrophoresis. B&him Biuphys Z&S. Commun 44.390-395. SHENK, T. E., KOSHELNYK,K. A., and STOLLAR,V. (1974). Temperature sensitive virus from Aedea albqpidus cells chronically infected with Sindbis virus. J. ViroL 13, 439-447. SHENK, T., and STOLLAR, V. (1973). Defective-interfering particles of Sindbis virus. I. Isolation and some chemical and biological properties. Virdosy 53, 162-173. SIMIZLJ,B., and MAEDA, S. (1981). Growth patterns of temperature-sensitive mutants of western equine encephalitis virus in cultured A& alxqictua cells. J. Gen Vird 56, 349361. SIMIZU, B., HASHIM~T~, K., and ISHIDA, I. (1983). A variant of western equine encephalitis virus with nonglycosylated E3 protein. Virdogy 125, 99-106.
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SIYPSON,R. W., OBIJESKI,J. F., and MC~~Z~NGIEJ&~, and SCHLESINGER, R. W. (1976). Sialic acid contents M. P. (1979).Conditional lethal mutants of vesicular of Sindbis virus from vertebrate and mosquito cells: stomatitis virus. III. Host range properties, interEquivalence of biological and immunological viral fering capacity and complementation patterns of properties. Fir69, 164-115. specific HR mutants. lrirolosy 93.493595. SYMINGTON,J., and SCHLESINGER,M. J. (1975). IsoSMITH, J. F., and BROWEI,D. T. (1977). Envelopment lation of a Sindbis virus variant by passage on mouse plasmacytoma cells. J. VkoL 15,1037-1641. of Sindbis virus: Synthesis and organization of proteins in cells with wild type and maturation-deSYMINGTON,J., and SCHLESINGER, M. J. (1978). Charfective mutants. J. Firol 22, 662-678. acterization of a Sindbis virus variant with altered STANLEY,P., CAILLIBOT,V., and SIMINOVITCH, L (1975). host range. Arch Vid 58.X37-136. Selection and characterization of eight phenotypSZILAGYI, J. F., and PRINGLE, C. W. (1975). Virion ically distinct lines of lectin resistant Chinese transcriptase activity differences in host range muhamster ovary cells. Cell 6.121-128. tants of vesicular stomatitis virus. J. I%?-& 16,927936. STOLLAR,V., STOLLAR,B. D., Koo, R., HARRAP,K. A.,
135, 331-344 (1984)
A Mutant of Sindbis Virus with a Host-Dependent in Maturation Associated with Hyperglycosylation RUSSELL K. DURBIN University
AND
of Medicine and Dentistq Department of Microbidogy,
Defect of E2
VICTOR STOLLAR’
of New Jersey, Rutgers Medical Pticatawa~, New Jersey 08854
Schd,
Received September 19, 1983; accepted March 6, 1984 Following serial passage of Sindbis virus (SV) on A& albopidus mosquito cells a mutant (SV,rIW2J was isolated which in chick cells produced small plaques and was temperature sensitive (ls). At 34.5” this mutant replicated normally in mosquito cells, but only poorly in chick or BHK cells. In the vertebrate cells SV.rrUrr was RNA+ at both 34.5 and 40” and on the basis of complementation tests carried out at 40”, was assigned to complementation group E. The block in the replication of this mutant, like that of t&O, the prototype mutant of complementation group E, was at the level of nucleocapsid envelopment. The PE2 and E2 glycoproteins of SV,,, were found to be hyperglycosylated relative to the corresponding glycoproteins of the parent virus (SV,). Analysis of revertants between PE2 and E2 hyperglycosylation and of svw5m suggests a causal relationship the host-specific defect in virus maturation. The association of a host-specific defect in virion assembly with hyperglycosylation of a viral structural protein points to the potential importance of host-specific glycosylation patterns in the determination of viral host range. INTRODUCTION
The alphaviruses, of which Sindbis virus (SV) is representative, are capable of replication in both arthropod and vertebrate hosts. (See Chamberlain, 1980,for a review of this topic.) The natural cycle of SV transmission, involving alternate growth in mosquitoes and vertebrates, undoubtedly exerts a strong selective pressure for efficient replication in both hosts. In nature, any variant which might adapt to one host type at the expense of the other would encounter an evolutionary cul-de-sac. In the laboratory, however, alphaviruses can be maintained indefinitely by passage on the cultured cells of a single species. In this way mutations which compromise the capacity of the virus to replicate in the alternate host can be maintained. We have been exploring host-virus interactions by genetic characterization of SV mutants whose growth is specifically ’ Author addressed.
to whom requests for reprints
should be
331
restricted in one type of host cell, and by biochemical characterization of the block in the replicative cycle in such nonpermissive systems. Similar approaches in other RNA animal virus systems have pointed to the involvement of host-specific factors in the replication of rhabdovirus RNA (Szilagyi and Pringle, 1975; Simpson et &, 1979), influenza virus RNA (Almond, 1977; Israel, 1980), and in the processing of both myxo- and paramyxovirus structural proteins (Lazarowitz et u& 19’73;Nagai et al, 1976). Symington and Schlesinger (1975,1978) have described an SV isolate which had been obtained by passage in mouse plasmacytoma cells, and which replicated more efficiently in mouse cells than did the starting virus. Alterations were demonstrated in both El and E2 of this virus rendering both proteins more negatively charged and resulting in more efficient adsorption to mouse cells. More recently in this laboratory, we have isolated and characterized two host-dependent mutants of SV restricted with re0042&X2/84 Copyright All rinbts
$3.00
Q 1984 by Academic Press. Inc. of reproduction in any form reserved.
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spect to growth in mosquito cells (Kowal and Stollar, 1981). We have now isolated, after serial passage on mosquito cells, a variant of SV with the reverse phenotype, i.e., restricted growth in vertebrate cells but normal growth in mosquito cells. The studies reported here associate this host restriction with an alteration in the glycosylation of the envelope glycoprotein E2. This report represents the first characterization of an alphavirus isolate whose ability to replicate is affected by host-specific patterns of glycosylation. MATERIALS
AND
METHODS
Cells, media, and viruses. The Aecks albopictus cells used in this study were C710 cells, a subclone of the LT-C7 cells described previously (Sarver and Stollar, 19’77). They were grown in E medium (MEM containing 0.2 mM of each of the nonessential amino acids) supplemented with 5% heat-inactivated fetal calf serum. BHK-21 cells and the preparation of primary chick embryo cell cultures have been described previously (Stollar et ah, 1976). Secondary chick embryo cell cultures were prepared by trypsinizing primary cultures and reseeding at a cell density of lo5 cells/ cm2 in CE medium (E medium supplemented with 5% tryptose phosphate and 5% heat-inactivated calf serum). The parental SV (SV,,,) used in these studies was a cloned derivative (Shenk and Stollar, 1973) of the HR strain of Burge and Pfefferkorn (1966a). Temperature sensitive mutants of SV, ts2, tsl0, and ts20, isolated by Burge and Pfefferkorn (1966a, b), were kindly provided by E. R. Pfefferkorn. Virus infectivity was assayed by plaque formation on CEF cells as described previously (Shenk et a& 1974). Passage of SV on Ae. albopictus cells. Cultures of Ae. aZbopictus cells (approximately lo6 cells on a 35-mm plate) were infected at a multiplicity of 0.1 plaqueforming unit (PFU) per cell, as described previously (Sarver and Stollar, 1977). At the end of 24 hr, the virus was harvested, diluted, and 0.2 ml of the diluted virus used to inoculate a new cell culture. For the first
STOLLAR
two transfers, the virus was diluted 200fold, and 2000-fold thereafter. All virusinfected cultures were maintained at 34.5”. Virus recovered after 15 passages (SVap15) was cloned by plaque purification, and one clonal derivative, SVap15,21, was used for the experiments described in this report. The properties of SVap15j21 did not, however, differ significantly from the properties of the uncloned population (SVaplB). Plaque pur$caticm, Virus was cloned by the procedure described by Kowal and Stollar (1981), but with some modifications to enhance plaque formation on Ae. albop ictus cells. Instead of agar, the overlay medium contained 1% low-melting Sea Plaque agarose (FMC Corp.) in E medium supplemented with 5% heat-inactivated fetal calf serum. After inoculation, cells were incubated for 2 days under the agarose overlay at 34.5” before staining with 0.005% neutral red. Ccmplernentatim tests. These were performed as described by Kowal and Stollar (1981) except that the adsorption was allowed to take place at 40” rather than at 4”. Polyaqdamide gel electrophoresis (SDSPAGE). Two different methods were used for electrophoresis of proteins. For electrophoresis of alkylated nonreduced proteins, monolayers of cells were washed with cold PBS, then lysed with 0.2 ml lysis buffer: 0.5% Triton X-100 (Ruger Chemical Co.), 0.05 M iodoacetamide (Sigma Chemical Co.), 0.1 M sodium acetate, 0.0025 M magnesium acetate, 0.05 M Tris acetate, pH 8.4. Nuclei were removed by low-speed centrifugation, and protein was precipitated from the supernatant by adding 1.2 ml cold acetone and chilling the mixture for at least an hour at -20”. The precipitate was pelleted by centrifugation for 5 min at 10,000 g and dissolved in sample buffer: 1% sodium dodecyl sulfate (SDS), 10% glycerol, 0.001% bromphenol blue, 0.05 M iodoacetamide, 0.125 M Tris-HCl, pH 6.8. Electrophoresis was performed on 10% polyacrylamide slab gels using the discontinuous buffer system of Laemmli (1970). Electrophoresis of thiol-reduced protein samples was as described above, except that iodoacetamide was replaced by 0.01
SINDBIS
VIRUS
M mercaptoethanol in the lysis buffer and by 0.14 M mercaptoethanol in the sample buffer. Measurement of RNA synthesis. Approximately 2 x 10r’BHK or chick cells or 4 X 105Ae. albopictus cells were seeded into each well of a 24-well multiwell tray (Costar). Twenty-four hours after seeding, cells were infected with 100 PFU/cell in 0.2 ml E medium supplemented with 0.1% bovine serum albumin. At 5 hr after infection the cells were treated with actinomycin D (4 pg/ml) for 15 min, then incubated for 1 hr in E medium containing actinomycin D and 20 &i rH]uridine/ml. Cells were washed and fixed as described by Sarver and Stollar (1977), and dissolved in 0.2 ml 0.2% SDS. After addition of 5 ml Liquiscint (National Diagnostics) radioactivity was measured in a scintillation counter. Immuno$uorescent microscopy. Viral antigen in both permeabilized and nonpermeabilized BHK cells was visualized by the technique described by Saraste et a2. (1980). The primary antiserum was from a rabbit immunized with SVata virions (Stollar et aL, 1976), and the secondary antibody was fluorescein-conjugated IgG from a goat immunized with rabbit IgG (Cwpel). Electron microscopy. This was performed as described previously (Igarashi et al, 1976). Radioactive labeling of viral proteins. Cells were seeded in 24-well cluster trays as described above, and infected with 100 PFU/cell. At 6 hr postinfection cells were refed with E medium modified by the omission of “cold” methionine and the addition of [%]methionine (20 pCi/ml). In pulse-chase studies, after labeling, cells were refed with chase medium: E medium supplemented with 1 mM “cold” methionine, and incubated for various intervals prior to preparation of lysates for electrophoresis. Chemicals and drugs. Tunicamycin was kindly provided by Dr. J. Lampen. Actinomycin D was a gift from Merck, Sharp and Dohme. [5-3H]Uridine (20 Ci/mmol) was purchased from ICN Pharmaceuticals, Inc., and [%]methionine (>lOOO Ci/mmol) from Amersham Corporation.
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MUTANT RESULTS
Host restriction and temperature sensitivity of SV,p15j21.Yields of SVap18,zIin chick or BHK cells 24 hr after infection at 34.5“ were typically only 1% of the yield of SVstd (Fig. la). Moreover, the logarithmic phase of virus growth was delayed by some 4 to 5 hr in SVap15,21infected BHK cells as compared to SVstd-infected cells. In mosquito cells, however, SVap15,21 replicated just as well as SVsti (Fig. lb). BHK cells infected with SV,15,n and incubated at 40” produced no more than 0.03 PFU per cell, at least 200-fold less than infected BHK cells incubated at sv,1Ei/2134.5” and about 50,000-fold less than SV,tiinfected BHK cells incubated at 40” (Fig. la). Similar results were seen in experiments using chick cells (data not shown). Compared to plaques produced on chick cells by SVsti at 34.5”, those produced by were much smaller. In a typical SVap15/21 experiment, at the end of a 24-hr incubation under agar, SVstdproduced plaques 2 to 3 mm in diameter, while SVap15,21 plaques measured no more than 0.5 mm. failed to generate any plaques on SVaplW21 chick cells at 40” (PFU at 40”/PFU at 34.5” < 10-5). In mosquito cells, however, we were unable to demonstrate that the replication was temperature sensitive. At of SVaplW21 37” the yield of SVa,,15,21 was equivalent to that of SVsti (not shown); at 40” neither the standard virus nor SVap15,21 grew in mosquito cells. The results above indicate that even though the host restriction of SVap15,21 in vertebrate cells is readily demonstrable at 34.5”, at 40” the restriction is virtually complete whether assayed by virus yield or by plaque formation. S@hesis of viral RNA. To help localize the block in viral replication in SVap15,21infected vertebrate cells we compared viral RNA synthesis in SVa,,15,21and SV,*-infected cells. In both BHK and chick cells infected with the mutant virus, viral RNA synthesis, as measured by incorporation of tritiated uridine into viral RNA, was similar to that observed with cells infected with the parent virus (Table 1). Agarose
334
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10'
AND
STOLLAR
(a)
-
2
4
6
6
10
12
04
10'
-
103
-
4
6
12
16
20
22
HOURS POST INFECTION
FIG. 1. Replication of SV.,15/n and SVsti in BHK and mosquito cells. Cells were infected at an input multiplicity of 10 PFWcell. BHK cells were incubated at either 34.5 or 40”, Ae. albspictus ceils at 34.5’. At the indicated times postinfection, samples were taken and titers were determined by plaque assay on chick cells at 34.5”. (a) BHK cells, (b) Ae. albopidus cells. (0) SV.ris,si at 34.5”; (0) SV.,l6/2l at 40”; (0) SV., at 34.5’; (m) SV,, at 40’.
gel electrophoresis demonstrated that the D) or with Ls20(group E) and maintained ratios of genomic (42 S) to subgenomic (26 at the nonpermissive temperature, the cleavage of PE2 to E2 fails to occur. S) RNA in SV,,- and SV,Is,zl-infected BHK cells were similar (not shown). Complementution analysis. Since SVap15,z1 TABLE 1 was temperature sensitive (cf. yields at VIRAL RNA SYNTHESIS 34.5” and at 40” in Fig. la), we carried out complementation tests to determine [3H]Uridine whether it could be assigned to one of the incorporation” previously established complementation groups. Because SVap15121 was clearly of the Cells 40” Virus 34.5” RNA+ phenotype, we restricted our experSVsti 2.15 X 106 1.79 x 106 iments to complementation groups C, D, and BHK 1.99 x 106 1.08 x 105 sv.p15/21 E, corresponding to defects in structural mock infected 0.15 x 106 0.07 x lo5 proteins C, El, and E2 (or PE2), respectively. SVap15121 was able to complement ts2 Chick 3.47 x 10’ 1.18 x ld SVnti 8.31 x lo’ 2.15 X lo* (Group C) and 1310(group D), but not k20 svapls/21 0.08 x ld 0.12 x lo4 mock infected (Table 2). Thus we assign SVap15,21 to complementation group E. @Incorporation of PH]uridine into acid-insoluble Viral glycoproteins. We next sought to dematerial (cpm) by infected or mock-infected cells in termine whether there were any anomalies the presence of actinomycin D was measured as dedetectable by SDS-PAGE among the struc- scribed under Materials and Methods. Since at 40” tural proteins of SVap15121, or in their pro- svapm1 induced at least 60% of the RNA synthetic . . . teolytic processing. For instance, in cells activity Induced by SV.,, it can be considered RNA+ infected with SV mutants LslOor &XI (group (Strauss and Strauss, 1930).
335
SINDBIS VIRUS MUTANT TABLE 2 COMPLEMENTATION OF SVapi5,e,WITH RNA+ SV ts MUTANTS
Yields from mixed infections”
ts2 tsl0 ts20 sv.,,m
ts2b CC)
tsl0” (D)
ts20b (E)
1.9 x 102
1.1 x 10’ (0.8) 1.3 x l@
6.0 X l@ (0.6) 1.1 x lo6 (49) 9.5 x 10s
SV.DISRI 1.6 X 8.8 X 7.4 X 2.4 X
10e (3.7; 6.0’) 10’ (66; 49”) 108(0.9; 0.6”) 102
a Secondary cultures of 2 X 105chick cells in 2-cm2 wells were coinfected with the indicated mutants. Yields (PFU/well) after ‘7 hr of incubation at 39.5’ were determined by plaque assay at 23”. The numbers in parentheses are complementation indices. Our isolate of fe2 complements poorly; we were unable to detect complementation between ts2 and either tsl0 or ts20. Complementation between ts2 and SV.p16,21 is probably detectable only because of the low leak-yield of the latter. bMutants of Burge and Pfefferkorn (1966a). Complementation groups are indicated in parentheses. ’ Complementation indices from a second experiment.
Infected BHK cells were pulse labeled C and D). Thus it appears that the difference with pS]methionine at 34.5” and the labeled between the molecular weight of the mutant proteins were analyzed by SDS-PAGE. This and standard PE2 and E2 glycoproteins is experiment demonstrated that the ap- a result of increased glycosylation of the parent molecular weight of the PE2 en- SVapi5,z1glycoproteins relative to those of was approximately 3000 the parent virus. coded by SVap15~21 greater than the SVsti coded PE2 (Fig. 2a). was also larger than The E2 of SVap15,21 SVap15j21 proteins C and El were electro- that of SVeti when both viruses were grown phoretically similar to the corresponding in mosquito cells (Fig. 4). In experiments SV,* products. During a subsequent chase similar to that shown in Fig. 2a, but perPE2 was processed to E2 with formed using mosquito cells, pulse-labeled the SVap~~/2~ kinetics similar to that seen in the case of PE2 was again larger in SV,lbj,/P1-infected SVati PE2. Again, the molecular weight of cells than in SV,ti-infected cells (not shown). the SVap15,21 E2 was approximately 3000 However, the molecular weights of all the greater than that of SVsti E2. In cells main- SV glycoproteins, both mutant and stantained at 40” (Fig. 2b) the PE2 of SVap15,21, dard, produced in mosquito cells were 1000 like that of SVsti but unlike that of ts20 to 2000 less than those of the corresponding (also of group E), was unstable (Jones et glycoproteins in vertebrate cells, consistent al, 1974). However, in contrast to the SVsti- with earlier findings (Luukonen et c& 1977; infected cells at 40°, E2 appeared in the Sarver and Stollar, 1978). !?rawport of wird antigen to the WU svapm- infected cells in lower amounts than expected. surface. The association of the defect in In order to determine whether the in- svap15/21 replication in chick and BHK cells crease in the apparent molecular weights with a mutation affecting one of the viral of the mutant PE2 and E2 was due to an glycoproteins suggested two possible exaltered pattern of glycosylation or to an planations for the failure to assemble maalteration in the amino acid sequence, we ture virions. The glycoproteins of SVap15,21 pulse labeled infected cells either with or might, like those of the complementawithout pretreatment with tunicamycin tion group D mutants, tsl0 and IX&~,fail to (TM). The PE2 “aglycoproteins” synthesized reach the cell surface. Alternatively, the in the TM-treated cells infected with mu- SVap15j21 glycoproteins might reach the cell tant and standard viruses were nearly in- surface, but fail to assemble into virions. distinguishable by SDS-PAGE (Fig. 3, lanes The latter pattern is characteristic of the
336
DURBIN AND STOLLAR
b a
MABCD
AWDEFGHIJKLMN
PE2 F
E2
FIG. 2. Pulse-chase labeling of viral proteins. (a) BHK cells were infected at an input multiplicity of 106 PFWcell with SV,, (lanes A-G) or SV*rum (lanes H-N) and incubated at 34.5”. At 6 hr postinfection, cells were labeled for 15 min with PSjmethionine, then either prepared directly for electrophoresis (lanes A and H) or first incubated with chase medium for 15 min (B and I), 26 min (C and J), 45 min (D and K), 66 min (E and L), 75 min (F and M), or 96 min (G and N). Proteins were alkylated and electrophoreaed without reduction of disulfide bonds in order to optimally resolve El and E2 (see Materials and Methods). The polypeptide marked “B” is the binary envelope protein (uncleaved PEB-El). (b) BHK cells were mock infected (lane M), infected with SV, (Lanes A and B) or with SV.,,ls,sI (lanes C and D) as above, but were maintained at 40”; at 12 hr after infection they were pulse labeled with mjmethionine for 15 min and immediately prepared for electrophoresis, as above (lanes M, A, C) or incubated in chase medium for 66 min (lanes B and D). Suppression of host synthesis is routinely less complete at 40” than at 34.5”. Prominent host proteins were marked “h”.
glycoproteins of the group E mutant, ts20, in cells incubated at the nonpermissive temperature. In order to determine whether mutant viral glycoproteins were being transported to the cell surface, we used the fluorescent antibody technique to localize the viral antigen in infected cells, with or without permeabilization of the cell membrane (Fig. 5). In light of the evidence that SV El and (P) E2 exist as a complex in infected cells (Jones et uL, 19’77;Bracha and Schlesinger, 1976; Smith and Brown, 19W, Rice and Strauss, 1982), we performed these experiments with antiserum directed against SV virions rather than with monospecific anti-El or anti-E2 serum. Fixed but
nonpermeabilized BHK cells infected with SVapwzIor with SVti showed bright surface fluorescence, both at 34.5 and at 40’ (Fig. 5B, C, and D). These results indicate that the SV,,,,, glycoproteins were in fact transported to the cell surface. As a control we also examined cells infected with SV MO. As expected (Sara&e et uL, 1980), cells infected with this group D mutant at 28” developed surface fluorescence almost as intense as in SVd-infected cells (Fig. 5E), but at 40” failed to develop surface fluorescence (Fig. 5F). Cells infected with SV,, or with either mutant showed intense fluorescenceif permeabilii prior to staining, whether incubated at the permissive temperature or at 40” (not shown).
SINDBIS
VIRUS
C D
PEZ= El-
337
MUTANT
lication in vertebrate cells, a small plaque phenotype, temperature sensitivity, and hyperglycosylation of PE2 and E2. The coincidence of these phenotypes in a single mutant does not, however, prove a causal relation between the hyperglycosylation and the other phenotypic properties. If there is a eausal relation, reversion of the hyperglycosylation phenotype should correlate with reversion of the other phenotypes. Our stock of SVap15,w (grown in mosquito cells) when assayed on CEF was essentially free of ts+ or large plaque revertants (ts’ revertant frequency < 1.3 X 10p6).Table 3,
A
6
C
FIG. 3. Effect of tunicamycin on viral proteins. BHK cells were infected with SV,u (lanes A and C) or SVW.W~ (lanes B and D) as described under Materials and Methods. Four hours postinfection the medium in two of the wells (lanes C and D) was supplemented with 0.5 gg tunicamycin/ml. Between 6 and 6.5 hr postinfection, the cells were labeled with [86S]methionine; lysates were then prepared under reducing conditions for SDS-PAGE.
Electron micros~ characterization of the defect in SVwp,,,, assembly. To further
pinpoint the nature of the block in the replication of SVap15,z1 in vertebrate cells, we examined infected chick and BHK cells by electron microscopy. In both cell types infected with the mutant virus there was an abundance of intracellular nucleocapsids, most of them lining the inside of the plasma membrane (Fig. 6b). In contrast to the cells infected with the wild-type virus (Fig. 6a), -infected cells (Fig. 6b) viral nuin SVap15~~ cleocapsids were only very rarely seen in association with the evaginations in the plasma membrane characteristic of budding virus or on the cell surface. The electron microscopic appearance of vertebrate cells infected with SV,16,u was similar to that of published electron micrographs of cells infected with the group E SV mutant, ts20 (Brown and Smith, 1975). Revertunts. We have demonstrated that is characterized by restricted repSVaplW21
E2
E2 El
El
C FIG. 4. SDS-PAGE of purified virus produced by mosquito cells. Cells were infected with ca. 1 PFU/ cell. Medium was collected after 24 hr at 34.5” and clarified by low-speed centrifugation. Virus was pelleted, purified by centrifugation to equilibrium on a ZO-50% sucrose gradient, then repelleted. Six mierograms of viral protein were separated by nonreducing SDS-PAGE and were visualized by staining with Coomassie Brilliant Blue R-350. (A) SV., grown in mosquito cells; (B) SV.pla,21 grown in mosquito cells; (C) SV., grown in chick cells. (Yields of SV,r,Is,n from vertebrate cells were too low to generate a satisfactory sample.)
DURBIN AND STOLLAR
FIG. 5. Surface-specific immunofluorescent detection of Sindbis viral antigen. Infected BHK cells were fixed, without permeabilization, 5 hr postinfection and stained by the fluorescent antibody technique. (A) mock infected, incubation at 34.5’; (B) SV., infected, 34.5”; (C) SV,16,n infected, 34.5’; (D) SVspm infected, 40’; (E) SV f.slO infected, 28”; (F) SV MO infected, 40”.
however, shows that when SVap15,z1 was serially passaged on chick cells large plaque revertants came to dominate the population by the third passage and ts+ revertants accumulated in large numbers. A similar phenomenon was described previously by Shenk et cd (1974). To analyze the relationship between the PE2 hyperglycosylation and the other phenotypic properties of SV,r5jn, we generated ts+ and plaque size revertants by passing twice on chick cells. The chick cellsvap15/21 passaged virus was then plaque purified on mosquito cells (SVaplsIzland SV,, plaques were indistinguishable on Ae. a.lbo$&w cells) rather than on vertebrate cells, in or-
der to avoid any selective pressure toward reversion during the cloning. Twenty-six plaques were picked at random and a small stock of each was grown on Ae. albqwictus cells. These clones were characterized with respect to PE2 size as determined by SDSPAGE of infected BHK cell lysates, and with respect to yield, plaque size, and temperature sensitivity in chick cells (Table 4). A perfect correspondence was found between reversion to PE2 of the SVBti type and the relaxation of the host restriction in chick cells (P = 5.0 X lo-‘, x2 test). Of 16 isolates tested which were associated with the smaller PE2 characteristic of SV,,, all produced yields in chick cells greater
FIG. 6. Electron micrographs of infected chick cells. Secondary cultures of chick cells were infected with 100 PFU of SV., (a) or SV.pls,zI (b) per cell and incubated at 34.5”. At 7.5 hr postinfection, cells were fixed and prepared for electron microscopy. (Bar = 100 nm.) 339
DURBIN
340
AND TABLE
STOLLAR 3
REVERSIONOFPLAQUESIZE AND ts PHENOTYPES WITH PASSAGEOFSVspls,I1AT 34.5’ ONCHICK CELLS Titer-assay temperature 34.5” Chick cell passage level Ob 1 2 3
Small plaques
Large plaques
1.6 X lo9
(Not detectable) 1.2 x 106 8.4 X lo6 3.0 x 108
5.0 x lo7 8.5 X lo6
(Not detectable)
40” Small plaques” 12 x 10’ 6.8 X ld
3.1 x 106 1.1 x 10s
o Only small plaques were produced at 40” by ts+ revertants. * Stock SV.,lWzI grown in mosquito cells.
than 10% of the yield of SV,,. Indeed, only generally without known significant bioone of the isolates (No. 25) associated with logical consequences. the larger PE2 characteristic of SVap15,z1 In some cases (Simizu and Maeda, 1981; produced a yield greater than 1% of the Eaton, 1982) the variant virus isolates were SVsti yield. Of the 15 large plaque rever- derived from persistently infected mostants, every one had also reverted to the quito cell cultures, but the significance of parental PE2 (P = 1.8 X 10-5, x2 test). Like- the alterations in the glycoproteins in wise, of the 10 ts+ revertants tested, all had terms of adaptation to mosquito cells or reverted to the parental PE2 (P = 0.0037, to the persistently infected state is not x2 test). These results strongly suggest a clear. On the other hand, it has been consistently observed that alphavirus isolates connection between the hyperglycosylation of PE2 and the other phenotypes. However, derived from long-term persistently intwo isolates (Nos. 14 and 18) which reverted fected mosquito cells typically produce with respect to the glycoprotein size and small plaques and are temperature senhost restriction remained both small plaque sitive when grown on vertebrate cells and temperature sensitive. In addition, (Shenk et al, 1974;Simizu and Maeda, 1981; three isolates (Nos. 10, 13, and 15) which Eaton, 1982). The molecular bases of these reverted in terms of host restriction, plaque phenotypic shifts have, however, not been size, and size of PE2 retained their tem- explored. Only in one case has an altered perature sensitivity. Thus of the three other alphavirus glycoprotein been tentatively phenotypic properties examined in the re- associated with a host-specific impairment vertant populations, that which best cor- in replication (Simizu et aL, 1983). In this related with loss of host-restriction phe- report, an isolate of western equine ennotype, was the presence of SVsti-type PE2. cephalitis virus derived from persistently infected mosquito cells was shown to lack the normal glycosylation of E3, and to be inefficiently released from vertebrate cells. DISCUSSION It is interesting that ts isolates of westThere have been several reports describ- ern equine encephalitis virus derived from ing alphavirus isolates which encode al- persistently infected mosquito cultures tered envelope glycoproteins. In some cases tend to be defective in the gene for PE2 (Bracha and Schlesinger, 1978; Cancedda (Simizu et d, 1981).Eaton (1982) also noted that SV glycoprotein E2 underwent a et &, 1981;Leone et d, 1980) the variations appear to arise as random clonal varia- characteristic shift in SDS-PAGE mobility with increasing passage level of persistions, probably reflecting the low fidelity of RNA-dependent RNA polymerases tently infected mosquito cultures, in this (Holland et al, 1982). These variations are case toward increased mobility. If a par-
SINDBIS TABLE
VIRUS
4
MUTANT
341
peatedly in independent passage series. In an effort to determine whether the PE2 hyperglycosylation seen in SVapi5,21was a general consequence of serial passage of Temperature PE2 Yield Plaque SV on mosquito cells, we performed six sensitivity” sized sire” IBOld (PFWml)” more independent l&transfer passages, 1 std 2.6 X 10’ L WY similar to that from which SVapi5/z1 was ts+ std 2 3.4 x 10’ L derived, each starting with a separate clone Std 3 L (NT) (NT) of sv,a. None of these generated the hySM 4 1.0 x 10’ L t-s+ 5 s mut 4.4 x lo’ WV perglycosylated PE2 or minute plaque Std 6 L ts+ 2.0 x 10’ phenotypes. Moreover, of four tempera7 ts mut 6.3 X 10’ s ture-sensitive, small plaque isolates de8 L ts+ Std 4.0 x 10’ rived from persistently infected cultures 9 ts+ Std 1.4 x 10’ L 10 6.0 X ld L ts Std (Igarashi et a& 1977), none had a hyper11 6.2 X 10’ s ts mut glycosylated (P)E2 (not shown). Thus we 12 1.2 x lti S ts mut have no evidence that the hyperglycosy13 L ts std 1.8 X 10’ lation of (P)EZ is positively favored in 14 6.3 X 10s s t8 SM mosquito cells. However, the fact that it 15 8.0 X 16 L ts Std 16 1.2 x 10’ L ts+ std is at least tolerated in mosquito cells and 17 2.8 x 10’ L t.s+ Std not in vertebrate cells reflects what may 18 2.2 x log s it.3 std be a significant difference between the two 19 5.1 x lti S ts mut types of host cell in shaping the evolution 20 1.2 x 10’ L k?+ std 21 4.6 X lo’ S ts mut of sv. 22 4.7 x lo’ S ts mut From the relative SDS-PAGE mobilities 23 L ts+ std 1.4 x 10’ of the PE2 glycoproteins of SVstd and 24 ts std 4.2 x 10’ S with and without tunicamycin 25 it.% mut svap15/21, 1.2 x ld S 26 3.6 X lo7 L ts+ Std treatment, we have concluded that the mu4.6 X 10’ L Std t.9’ svstd tant PE2 is hyperglycosylated relative to 5.3 x l(r if.3 S mut SV,ls/sl the parental PE2. Although estimates of molecular weights of glycoproteins by Note. SV.,,,, was passaged twice on chick cells as described in the text. The resultant stock was plaqued at 34.5O on Ae. SDS-PAGE must be interpreted with caualbopidus cells and 26 plaques were picked at random. Each tion (see, for example, Segrest et d, 1971), plaque isolate was then grown into a small stock, also at it is interesting that the PE2 of the mutant 34.5” in Ae. u&opictus cells. The titers of these stocks ranged virus has an apparent molecular weight from 3 X lb to 1 X lb PFU/ml, as assayed on chick cells larger than the standard PE2 by approxat 34.5”. Each clonally derived stock was eharactariaed with respect to growth, plaque sise, and temperature sensitivity imately the size of a typical asparagineon chick cells, and with respact to PE2 size in BHK cells. linked oligosaccharide. The fact that the “Yield (PFWcell) from CEF 6 hr after infection with 10 size difference is evident even in short PFWeell and incubation at 34.5”. pulses (Fig. 2a) indicates that it does not b Plaque size was scored as small (S) if plaques were x0.5 mm after 24 hr of incubation at 34.5’ under agar, and large result from differences in late modifica(L) if plaques were >2 mm. tions such as the addition of sialic acid “Each isolate was scored as temperature sensitive (ts) if (Bonatti and Cancedda, 1982, Hakimi and its titer measured by plaque assay at 40’ was less than 1% Atkinson, 1982). This point is reinforced of its titer measured at 34.5’; if greater than 1% it was considered revertant (ts+). by the observation that the size difference ClFor each isolate, a well of BHK cells was infected with is also evident in viral proteins produced 100 PFU per cell, and labeled for 26 min with I%Jmethionine in mosquito cells (Fig. 4), since mosquito 6 hr postinfection. Labeled proteins were analyzed by SDScells are thought to produce N-linked oliPAGE under reducing conditions. PE2 was scored as “stangosaccharides only of the nonsialylated, dard” (std) or “mutant” (mut) based on comigration with the PE2 of SVati or of SV.,,r,. respectively. “high mannose” type (Stollar et d, 1976; ’ Not tested. Luukonen et al, 1977; Butters and Hughes, 1981). These considerations, taken totitular phenotypic shift results in more ef- gether, suggest that the increased size of ficient replication in mosquito cells, one the glycoprotein reflects the generation of might expect the same shift to occur re- a new glycosylation site rather than more PHENOTYPIC ANALYSIS OF ISOLATES OBTAINED PASSAGE OF SV,,s,/p ON CHICK CELLS
AFTER
242
DURBIN
AND
STOLLAR
extensive glycosylation of the same sites protein could have a host-specific effect. found in the parental viral glycoprotein. Finally, it may be that the additional burFurthermore, it is easier to understand den of carbohydrate on E2 in vertebrate how a mutation in the viral genome might cells induces a shift in its tertiary strucresult in the addition of a glycosylation ture, distorting protein-protein recognisite (a function of the primary structure tion sites necessary for assembly of the of the protein), than to envision a viral envelope. mutation leading to an alteration in the In addition to its usefulness in comparcellular response to an existing glycosy- ative studies of viral assembly in insect lation site. However, we have not yet per- and vertebrate cells, SVap15j21 represents a formed the sequencing studies to distin- useful addition to the library of Sindbis guish between the two possibilities. Can- mutants, since only one other member, ts20, cedda et al. (1981) demonstrated that our of complementation group E has been reSV,d (referred to as SVm by these workers) ported. The molecular bases of the defects differs from the SVna strain from which in these two mutants appear to be different, it was derived in that E2 of SVsti has three although both affect PE2 or E2 and both instead of two glycosylation sites. If, as cause a block in viral assembly at the point seems most probable, E2 of SVap15,21 differs of envelopment. Whereas under nonpermissive conditions the PE2 of ts20 is not from that of our SVstd by an additional glycosylation site, it must differ from the cleaved, at 84.5” in BHK cells (essentially original SVns E2 by two additional sites. a nonpermissive situation for SVapl& the It seems likely that the basis of the dif- ~‘132 of Wpwa is processed to E2 with ferential response to the altered viral gly- kinetics indistinguishable from those of the coprotein in mosquito and in vertebrate SVsti PE2. It is clear, then, that normal cells is the fact that the carbohydrate moi- cleavage of PE2 can occur in the absence eties of glycoproteins synthesized in mos- of viral maturation. This conclusion conquito cells are different from the carbo- flicts with the proposal that PE2 cleavage hydrate moieties added to glycoproteins in is always tightly linked to the final stages vertebrate cells. Consequently, the same of viral morphogenesis (Jones et aL, 1974; viral gene expressed in two different cell Brown, 1980). The data in this report indicate that the types will result in chemically distinct defect in SVap15,21 maturation in vertebrate products. The mechanism by which the altered cells is related to cell-specific patterns of glycosylation pattern of the E2 of SVap15,21protein glycosylation. We hope to deterinterferes with assembly in vertebrate cells mine more precisely the feature(s) of veris unknown. Perhaps the increased number tebrate cell glycoprotein biosynthesis reof sialic acid residues per molecule of E2 sponsible for this defect by comparing the in lectin-remade possible by the addition of a new growth of SVstdand SVap15,21 glycosylation site could result in electro- sistant vertebrate cells which have specific static repulsion and therefore decreased defects affecting glycosylating enzymes affinity between peplomers. [However, (Stanley et uL, 1975). supplementation of the growth medium ACKNOWLEDGMENTS with neuraminidase did not result in an increase in the output of SVap15,21 from inThis investigation was supported by the United fected chick cells (data not shown)]. An- States-Japan Cooperative Medical Science Program other possibility is that the additional car- through Public Health Service Grant AI-05920 and bohydrate interferes sterically with the by Institutional National Research Service Award CAbinding of viral structure units to one an- 09099 from the National Cancer Institute. The authors other. Since a greater mass of carbohydrate thank Lizabeth Flares for her help with the electron is added to each glycosylation site in ver- microscopy. tebrate cells than in mosquito cells (K. REFERENCES Keegstra and V. Stollar, unpublished) it is conceivable that the addition of a glyco- ALMOND, J. W., MCGEOGH, D., and BARRY, R. D. (1977). sylation site to a viral structural glycoMethod for assigning temperature-sensitive mu-
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SIYPSON,R. W., OBIJESKI,J. F., and MC~~Z~NGIEJ&~, and SCHLESINGER, R. W. (1976). Sialic acid contents M. P. (1979).Conditional lethal mutants of vesicular of Sindbis virus from vertebrate and mosquito cells: stomatitis virus. III. Host range properties, interEquivalence of biological and immunological viral fering capacity and complementation patterns of properties. Fir69, 164-115. specific HR mutants. lrirolosy 93.493595. SYMINGTON,J., and SCHLESINGER,M. J. (1975). IsoSMITH, J. F., and BROWEI,D. T. (1977). Envelopment lation of a Sindbis virus variant by passage on mouse plasmacytoma cells. J. VkoL 15,1037-1641. of Sindbis virus: Synthesis and organization of proteins in cells with wild type and maturation-deSYMINGTON,J., and SCHLESINGER, M. J. (1978). Charfective mutants. J. Firol 22, 662-678. acterization of a Sindbis virus variant with altered STANLEY,P., CAILLIBOT,V., and SIMINOVITCH, L (1975). host range. Arch Vid 58.X37-136. Selection and characterization of eight phenotypSZILAGYI, J. F., and PRINGLE, C. W. (1975). Virion ically distinct lines of lectin resistant Chinese transcriptase activity differences in host range muhamster ovary cells. Cell 6.121-128. tants of vesicular stomatitis virus. J. I%?-& 16,927936. STOLLAR,V., STOLLAR,B. D., Koo, R., HARRAP,K. A.,