VIROLOGY 184, 253-264 (1991)

A Mutation in the Ectodomain of Herpes Simplex Virus 1 Glycoproteift 8 CausesDefective Processing and Retention in the Endoplasmic Reticulum

DAVID NAVARRO, ISHTIAQ QADRI, AND LENORE PEREIRA'

Division of Oral Biology, School of Dentistry, University of California at San Francisco, San Francisco, California 94743-0512

Received March 1, 1991; accepted May 28, 1991

Herpes simplex virus 1 (HSV-1) glycoprotein B (gal is one of several envelope glycoproteins rehired for virioninfectivity and is the only one known to oligomerize into homodimers . To study the conformational constraints fortranslocation of HSV-1 gal to the surface of eldtaryotic cells, we analyzed the transport through the exocytic pathway ofthe wad-type glycoprotein and of mutant forms with insertions in the ectodomain and intracellular carbbxy terminus .Transient expression of the glycoproteins in COS-1 cells showed that an insertion at position 479 in theamino-temrinatectodomain of gB, shown previously by reactions with monoclonal antibodies to have altered the conformation ofthemolecule, also had a drastic effect on transport, precluding exit of the mutant from the endaplasmic retilailurn (ER) andtransport to the Golgi and the plasma membrane . The fact that the mutant, gB-(Lk479), formed dimefl suffillasids thatlocal changes in assembled regions caused the transport defect . Mutants containing insertions at Jof theectodomain and 810 in the intracellular domain were slightly retarded in their rate of transport from the ER toheQeigi .The glucose-regulated proteins GRP78 and GRP94, which are resident proteins of the ER, - wish psTalyglycosylated, faster-migrating forms of gB but not with the fully processed, more slowly migrating product . GRPt8 andGRP94 formed complexes with the mutant gB-(Lk479), which was degraded in the ER . Our results ladles" that GRP78,and perhaps also GRP94, acts as a chaperone in the assembly of native gB oligomers and also binds to aberrant formsof the molecule, arresting their transport from the ER and possibly serving as markers for protein degradation in thiscompartment of the exocytic pathway . 0 1991 Academic Press, Inc .

INTRODUCTION

Herpes simplex virus (HSV) glycoprotein B (gB) is amultifunctional protein in the virion envelope that playsan essential role in the penetration of host cells (Littleat al., 1981 ; Sarmiento et al., 1979). Nucleotide se-quence analysis of the HSV-1(F) gB gene predicts amembrane-anchored glycoprotein with a cleavablesignal sequence, an amino-terminal hydrophilic ecto-domain containing six potential N-linked glycosylationsites, a hydrophobic transmembrane region, and acharged intracellular carboxy terminus (Pellett at al.,1985). The predicted structures of gB encoded byHSV-1(KOS) and HSV-2(G) are similar in overall struc-ture but have some residue changes that cluster in thevery amino terminus of the molecule (Bzik et al., 1984 ;Kousoulas et al., 1988 ; Stuve at al ., 1987). HSV-1 gBforms homodimers, and studies on tsB5, a penetra-tion-defective mutant in gB which fails to form dimersat the nonpermissive temperature, indicate that oligo-merization is required for infectivity (Claesson-Welshand Spear, 1986 ; Haffey and Spear, 1980; Sarmientoand Spear, 1979) . Comparison of the conformation-de-pendent epitopes on gB specified by tsB5 showed that

1 To whom correspondence and requests for reprints should beaddressed .

253

mutant dimers made at the permissive temperaturefailed to express a subset of discontinuous epitopesrecognized by a panel of monoclonal antibodies to gB(Chapsal and Pereira, 1988) .

Our laboratory and others have used insertion anddeletion mutagenesis to perturb the structure of HSV-1gB (Car et at, 1987 ; Cai at al., 1988 ; Pereira at al.,1989; Qadri et al., 1991). We have analyzed the assem-bly of discontinuous epitopes and conformation-de-pendent neutralizing domains by constructing dele-tion, insertion, and point mutations in the gB gene andreacting the mutant gene products with monoclonalantibodies (Pereira at al., 1989; Qadri at al., 1991). Inthe course of these analyses, we found that certainmutants with insertions in the intact molecule had lostdiscontinuous epitopes and were not translocated tothe cell surface but accumulated in the cytoplasmwhere they exhibited a reticular pattern of immunofluo-rescence staining . In the present study, the processingof these mutants was further characterized to definethe conformational requirements for the transport ofg8 through the exocytic pathway to the plasma mem-brane. We found that insertion of a linker at amino acid479 precluded the transport of the mutant from theendoplasmic reticulum (ER), even though the glycopro-tein subunits were competent to form dimers . This mu-tant glycoprotein, and precursors of wild-type gB and

0042-6822/91 $3 .00Copyrght 6. 1991 by Academic Press, IncAll rghts nt reproduction In any corm reserved-

254

of the other insertion mutants, formed complexes withthe glucose-regulated proteins GRP78 and GRP94 .These results suggest that GRP78, and perhaps alsoGRP94, plays a role in the assembly of gB oligomersand also functions to prevent aberrant forms of gB fromreaching the plasma membrane .

MATERIALS AND METHODS

Cells and mediumCOS-1 cells, obtained from the American Type Cul-

ture Collection, were grown at 37° in Dulbecco's modi-fied Eagle's minimum essential medium (DME) supple-mented with 10% fetal bovine serum .

AntibodiesThe pool of monoclonal antibodies to HSV-1 gB used

for immune reactions contained the following groupsof antibodies: D1a (H1817, H1830, H1839); D1b(H1392, H1396, H1397); Dc4a (H1394, H1399) ; Dc4b(H1163, H336); and D3c (H1393) . These antibodiesreact with continuous epitopes on mature gB and un-derglycosylated precursors (pgB) . Epitopes of these an-tibodies were mapped as previously reported (Chapsaland Pereira, 1988 ; Kousoulasetat, 1984 ; PellettetaL,1985; Pereira etal., 1989; Qadri etal., 1991). Immunerabbit serum to GRP78 and a rat monoclonal antibodyto GRP94 were gifts from Dr . William Welch .

PlasmidsDetails of the construction of the plasmids used in

this study were described previously (Qadri et al.,1991) .

DNA transfectionsCOS-1 cells were transfected using the DEAE-dex-

tran method, as reported previously (Pereira et al„1989). Briefly, COS-1 cells were grown to 6096 con-fluency and DNAs were introduced into the cells usingDEAE-dextran (100 µg/ml) . After 3 hr, the medium wasreplaced by fresh DME medium containing 10 mM ofn-butyric acid, and 44 hr later the cells were processedas indicated below .

Radiolabeling and immunoprecipitationTransfected COS-1 cells were labeled with 50 pCi/ml

[35S]methionine (sp act 800 Ci/mmol, Dupont-New En-gland Nuclear, Inc .) in methionine-free medium for 4 hrat 44 hr after transfection . Cells were harvested andlysed in phosphate-buffered saline (PBS), pH 7 .4, con-taining 1% Nonidet P-40, 1% sodium deoxycholate,0.1% sodium dodecyl sulfate (SDS), 1 mM phenyl-

NAVARRO, QADRI, AND PEREIRA

methylsulfonyl fluoride (PMSF), and 1 mg/ml aprotinin .Extracts were clarified by high speed centrifugation ina microfuge at 4° for 15 min .

To maintain the integrity of complexes of the GRPswith HSV-1 gB, transfected cells in these experimentswere lysed using mild conditions. Cell extracts wereprepared in lysis buffer consisting of PBS containing1% Nonidet P-40, 0.1% sodium deoxycholate, 1 mMPMSF, and 1 mg/ml aprotinin . ATP was not depletedfrom the cell lysates .

Extracts of transfected cells were immunoprecipi-tated as previously reported (Pereira et al., 1989). Sam-ples were electrophoresed in SDS denaturing gels (9%acrylamide crosslinked with diallyttartardiamide) . Insome experiments, samples were electrophoresed in7.5% native polyacrylamide gels; these samples wereprepared in buffer with 0 .1 % SDS, without heat or fl-mercaptoethanol, using published procedures (Chap-sal and Pereira, 1988 ; Cohen et al., 1986) .Pulse-chase experiments

Transfected COS-1 cells were pulse-labeled with100 uCVml [36S]methionine for 10 min at 44 hr aftertransfection as described above . This medium was re-moved and stored for immunoprecipitation studies .Cells were washed three times in sterile PBS, and unla-beled medium containing methionine (10 mM) wasadded. Chases were performed at various times, asdetailed in the figures . Cell extracts were prepared, im-munoprecipitated with monoclonal antibodies, andsubjected to electrophoresis as described above .

Endoglycosidase H treatmentEndoglycosidase H (Endo p-N-acetylglucosamini-

dase H) (Boehringer-Mannheim) digestions were per-formed on immunoprecipitated samples using condi-tions specified by the manufacturer . Briefly, proteinsimmunoprecipitated with monoclonal antibodies wereeluted in 2% SDS in 0 .1 M sodium citrate buffer, pH5 .5, and boiled for 3 min. The concentration of SDSwas adjusted to 0.2% in citrate buffer containing 1%Triton X-100. Endoglycosidase H (Endo H) (0 .01 units)was added to the samples and digestion was allowedto proceed for 16 hr at 37° . The samples were sub-jected to electrophoresis in 9% denaturing polyacryl-amide gels . To inhibit any endogenous proteolytic ac-tivity, all reaction mixtures contained 2 mM PMSF .Mock-treated samples (no enzyme) were processed inparallel .Carbonylcyanide m-chlorophenylhydrazonetreatment of cells

Transfected COS-1 cells were pulse-labeled as de-scribed above. They were then washed in cold PBS,

NH2

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DEFECTIVE PROCESSING OF HSV-1 gB MUTANTS

Rc. 1 . Schematic representation of HSV-1 gB and mutants . The structure of the wild-type gB molecule of 874 ammo acids is shown in A(Pellett et al., 1985) . The hydrophobic transmembrane region is depicted as a hatched box . Stick models of the insertion mutant constructs in g8are shown in B . The synthetic oligonucleotide linkers and their insertion sites in gB are shown by an inverted triangle . a4ep refers to the H1091epitope of the HSV-1 a4 protein (Hubenthal-Voss et al., 1988). Plasmids are indicated on the left of each construct . Names used to refer to themutant glycoproteins are indicated on the right .

and chase medium (4°) containing carbonylcyanide m-chlorophenylhydrazone (CCCP) (50 gg/ml, Sigma) wasadded. After 10 min of incubation at 4°, the cells werewarmed to 37° and incubated for 2 hr at this tempera-ture before lysis buffer was added . Mock-treated cells(no CCCP) were processed simultaneously .

Immunoblotting in native gelsImmunoblot reactions using native conditions were

performed as previously described (Chapsal and Per-eira, 1988). Briefly, transfected COS-1 cells were ex-tracted as described above and electrophoresed in agel of 7 .5% polyacrylamide crosslinked with bisacryla-mide. Separated proteins were electrophoreticallytransferred to nitrocellulose under native conditions(0 .1 % SDS) and reacted with a pool of monoclonal anti-bodies to HSV-1 gB .

RESULTSMutant constructs in HSV-1 gB

We previously reported the construction of a set ofmutants in the HSV-1 gB gene (Qadri et al., 1991) .Wild-type gB and the mutations in the intact gB areillustrated as stick figures in Fig . 1 . The mutants hadinsertions in-frame into different sites in the extracellu-lar domain and the intracellular carboxy terminus of themolecule. These included two mutants with linker in-sertions at residues 479 and 600, which were desig-nated respectively as gB-(Lk479) and gB-(Lk600) . Athird insertion mutant, gB-(a4ep-810), was con-structed in the carboxy terminus at residue 810 by in-serting a synthetic oligonucleotide encoding a heterol-

25 5

ogous HSV-1 a4 epitope, H1091, identified in a pre-vious study (Hubenthal-Voss et al., 1988) .

Biochemical analysis of the transport of wild-typegB and mutants

The first series of experiments was designed tostudy the transport of HSV-1 gB mutants through theexocytic pathway by analyzing the shift in electropho-retic mobility after endoglycosidase treatment . Forthese experiments, the glycoproteins were immuno-precipitated and treated with endoglycosidase H (Tar-entino et al., 1978) to determine whether they con-tained high-mannose sugars, a property of glycopro-teins whose transport is arrested in the ER (Komfeldand Kornfeld, 1985 ; Rose and Bergmann, 1983) . COS-1 cells were transfected with plasmid DNA encodingthe gB constructs, radiolabeled for 4 hr, and lysed asdescribed under Materials and Methods . Cell extractswere immunoprecipitated with a pool of monoclonalantibodies to gB reactive with partially and fully glyco-sylated forms of the molecule as described under Ma-terials and Methods. The immunoprecipitates were di-vided in two portions, one of which was digested withEndo H and the other mock-digested; both were thenelectrophoresed in denaturing polyacrylamide gels .The results (Fig . 2A) are summarized as follows .

(i) The most slowly migrating form of wild-type gB(lane 1, band 1) underwent a small mobility shift, indi-cating it remained partially sensitive to Endo H diges-tion (lane 2, band 1 E) . In contrast, a partially processedintermediate form, pgB (lane 1, band 2), underwent alarge shift, indicating it was highly sensitive to Endo Hdigestion (lane 2, band 2E), evidence of its high man-

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NAVARRO, QADRI, AND PEREIRA

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nose content and lack of complex sugars . These re-sults indicate that wild-type HSV-1 gB contains bothcomplex and high-mannose-core oligosaccharides(Wenske et a/., 1982). Similar results were reported instudies on HSV-1 gB made in a variety of virus-infectedcell lines (see review (Campadelli-Fiume and Serafini-Cessi, 1985)) .

(ii) The most slowly migrating form of mutants gB-(Lk600) and gB-(a4ep-810) (lanes 3 and 7, band 1) alsoremained partially sensitive to Endo H (lanes 4 and 8,bands 1 E). The shift in electrophoretic mobility of thetreated molecules was indistinguishable from that ob-served with wild-type gB . These results indicate thatboth mutant proteins contained complex sugars andwere transported from the ER to the Golgi .

(iii) Mutant gB-(Lk479) showed a different patternfrom that of wild-type gB and the other mutants . ThegB-(Lk479) protein in the untreated lane (lane 5, band1) migrated faster than the other untreated glycopro-teins, suggesting that terminal glycosylation had notoccurred. In contrast to wild-type gB and the other mu-tants, the mobility of band 1 of gB-(Lk479) was signifi-cantly increased after Endo H digestion (lane 5, band1 E), indicating that this mutant contained a higheramount of mannose-rich sugars . These findings sug-gest either that gB-(Lk479) was not transported fromthe ER to the Golgi or that if it was transported, theaddition of complex sugars was blocked, perhaps byaberrant folding of the mutant . The notion that the

transport of gB-(Lk479) was arrested in the ER agreeswith our previous finding that this mutant gives a reti-cular staining pattern in the cytoplasm of transfectedcells and fails to reach the plasma membrane in sur-face immunofluorescence assays (Qadri et al., 1991) .

In the second set of experiments, the cells weretreated with carbonylcyanide m-chlorophenylhydra-zone, an uncoupler of oxidative phosphorylation thatrapidly lowers ATP levels in cells treated with this drug .Since transport from the ER to the Golgi requires ATP,COOP prevents glycoprotein transport (Farquhar,1985; Tartakoff and Vassalli, 1977) . COS-1 cells weretransfected with plasmid DNA and pulse-labeled for 10min at 44 hr postransfection . The cells were thentreated with CCCP in medium without radiolabel for 10min at 4°, then shifted to 37° for 2 hr. The cells werelysed and the glycoproteins immunoprecipitated asdescribed above. Results of these experiments (Figs .2B and 2C) are summarized as follows .

(i) After treatment of transfected cells with CCCP,the electrophoretic mobility of wild-type gB (Fig . 213,lane 9, band 1) was increased (lane 10, band 1C),which indicated that the glycoprotein was blocked inthe ER. This was confirmed by the finding that Endo Htreatment of band 1C made in CCCP-treated cells in-creased the mobility of the protein to that of the un-derglycosylated form, pgB (lane 12), indicating thatband 1 C was highly sensitive to Endo H and containedonly high-mannose sugars . When compared with the

0oCoJ gB Wt gB{Lk479) gB-(Mg) gB-(

g)mrn

+ + Endo H+

+ - + CCCP -

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small mobility shift noted for Endo-H-treated gB (lane

11, band 1 E), the shift in mobility found for Endo-H-treated band 1 C was striking (lane 12) .

(ii) Analysis of CCCP-treated cells transfected withDNAs encoding gB-(Lk600) and 9B-(a4ep8l0) (Fig . 2,lanes 15 and 17) showed that the shift in electropho-retic mobilities of the mutants was comparable to thatobtained with wild-type gB (lanes 16 and 18, band 1Q .Results of these studies confirmed that, like the wild-type glycoprotein, these mutant proteins normally un-dergo transport from the ER to the Golgi complex .

(iii) Analysis of CCCP-treated cells transfected withgB-(Lk479) (lane 13, band 1) did not show any changein electrophoretic mobility of the major form of thismutant or of the underglycosylated, partially pro-cessed forms (lane 14, band 1C). These results sup-port the notion that gB-(Lk-479) acquires N-linked man-nose sugars in the ER but fails to undergo further pro-cessing coincident with transport through the exocyticpathway .

Oligomerization of the mutant proteins

A characteristic of dimers formed by HSV-1 gB isthat they are stable in the presence of a high concen-tration of sodium dodecyl sulfate and reducing agentsbut can be dissociated at high temperatures (Haffeyand Spear, 1980 ; Sarmiento and Spear, 1979) . Studieson rsBS, a mutant in gB that fails to form dimers at thenonpermissive temperature (39°), showed that oligo-merization of the glycoprotein is required for infectivity(Sarmiento and Spear, 1979) and that at the permissivetemperature (37°) tsB5 dimers have lost a subset ofconformation-dependent epitopes and readily dissoci-ate under native conditions (Chapsal and Pereira,1988). The latter finding suggests that the stability oftsB5 dimers depends on the conformation of the indi-vidual subunits .

We reported previously that linker-insertion mutantsin intact gB form dimers but that truncated mutants600 amino acids in length or smaller do not (Pereira etal., 1989 ; Qadri et al., 1991). In those studies, native(no 0-mercaptoethanol or heat) and denatured sam-ples were analyzed either as immunoprecipitates or asextracts of transfected cells in denaturing gels to de-tect dimeric forms of gB . All of the insertion mutants infull-length gB, including gB-(Lk479), formed dimers .However, immunoblots in those experiments also con-tained the monomeric form of gB, which was gener-ated from partial dissociation of dimers under the dena-turing conditions of electrophoresis used in that study(Qadri et al., 1991) .

Experiments in the present study were designed toanalyze the stability of wild-type and mutant dimers

DEFECTIVE PROCESSING OF HSV-1 gB MUTANTS

257

synthesized at different temperatures or treated with

different concentrations of ionic detergent in native

gels. For these experiments, C08-1 cells were trans-fected with plasmid DNAs, incubated at 37 or 39°, andlysed after 44 hr as described under Materials andMethods . The cells were extracted in buffer containingSDS at different concentrations, low (0 .1%) or high(2 .0%), subjected to electrophoresis under native con-ditions, transferred to nitrocellulose, and analyzed byimmunoblot . Results of these experiments are shownin Fig. 3 .

(i) Like the wild-type gB molecule, the mutants gB-(Lk600) and gB-(a4-ep8l 0) formed dimers which weredetected at both temperatures . The two high-molecu-lar-weight, electrophoreticaly distinct, dimeric formsof gB found in these immunoblots most likely corre-spond to the partially glycosylated precursors and fullyprocessed forms identified as bands 1 and 2 in Fig . 2 .This finding supports the notion that dimerization ofnascent gB molecules proceeds rapidly in the ER priorto further transit through the exocytic pathway and thatthese dimers are stable . Neither of these bands repre-sents aggregates or higher order multimers, as deter-mined by sedimentation analysis in sucrose gradients(D. Navarro, I . Qadri, and L. Pereira, manuscript in prep-aration) .

(ii) Analysis of immunoblots containing dimers ofmutant gB-(Lk479) showed that a small fraction wasunstable in buffer containing 0 .1 % SIDS at both temper-atures and that a larger proportion readily dissociatedin 2 .0% SDS. In contrast to the finding for wild-type gBand the other mutants, a single band, migrating in theposition of a partially glycosylated dirner, was observedin gB-(Lk479) immunoblots . This result indicates thatmutant gB-(Lk479) fails to reach the fully glycosylatedstate and further supports the experiments with EndoH and CCCP described above . Results of the presentstudy confirm our previous finding that gB{Lk479) mol-ecules assemble into dimers and show either that thisprocess is not as efficient as that of wild-type gB or thatthe mutant dimers are unstable in the presence ofSDS. The finding that certain monoclonal antibodiesreacted weakly or not at all with gB-(Lk479) (Qadri etal., 1991) reflects certain conformational changeswhich may result in the instability of some of thesemutant dimers .

Processing of the gB mutant proteins

To determine whether mutations in different sites ofHSV-1 gB affect the processing of the molecule fromunderglycosylated precursors to the mature product,we carried out pulse-chase experiments on cellstransfected with DNA encoding the gB mutants . For

258

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39°CFla . 3. Analysis of the stability of HSV-1 gB dimers by immunoblot . Cells transfected with wild-type HSV-1 gB and insertion mutants in gB were

incubated at 37 or 39° followed by extraction in buffer without p-mercaptoethanol but with 0 .1 or 2 .0% SIDS . They were then electrophoresed in7.5% polyacrylamide gels under native conditions, transferred to nitrocellulose, and reacted with a pool of monoclonal antibodies to continuous-epitope domains . Mutants and wild-type ga (WTgB) constructs are shown above each lane . gB and pgB refer to fully processed and partiallyprocessed dimeric forms. Numbers above each lane refer to SDS concentration in buffer .

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these experiments, COS-1 cells were labeled for 10min, rinsed, and either immediately harvested or incu-bated in medium without radiolabel (chased) forvarious intervals . Cells were then extracted, immuno-precipitated, and analyzed in denaturing polyacryl-amide gels . The results of these experiments (Fig . 4)are summarized as follows .

(i) Trace amounts of the slowest-migrating form ofwild-type gB were first detected as early as immedi-ately after the 10-min pulse and most of the faster-mi-grating, partially glycosylated forms were processedby 60 min . It should be noted that the protein precursordesignated as pgB appeared immediately after thepulse but that a fraction appeared not to undergo fur-ther processing to a more slowly migrating product,even after an 8-hr chase interval (data not shown) . Asimilar finding was reported by others using both HSV-1-infected cells and transient expression of gB (Som-mer and Courtney, 1991) . At present, we cannotascertain whether this fraction of pgB consists of asubset of malfolded forms that will not be processedfurther through the exocytic pathway .

(ii) Mutant gB-(Lk479) failed to undergo a shift in mo-lecularweight from pgB to gB and was not further modi-fied to the more slowly migrating product . Chase inter-vals up to 4 hr did not detect processing of this mutant,and after 8 hr the polypeptide band was lost and anumber of smaller bands had accumulated (data notshown), evidence that the protein was degraded .These results confirmed our previous suspicion, basedon the patterns of internal and surface immunofluores-cence, that gB-(Lk479) was retained in the ER and nottransported to the plasma membrane (Qadri et al .,1991) .

(iii) In contrast to gB-(Lk-479), mutants gB-(Lk-600)and gB-(a4ep-81 0) were efficiently processed from un-derglycosylated precursors to more slowly migratingproducts . Insofar as the processed forms were firstobserved after 20 min of chase, we concluded thatthese mutants were transported from the ER to theGolgi somewhat more slowly than wild-type gB .

Combining these results with those of experimentswith Endo H and CCCP treatment, we found that theappearance of more slowly migrating bands of gB coin-

rectly folded and glycosylated were retained in the ERand formed complexes with GRP78 (Gething et al.,1986; Hurtley et al., 1989; Kozutsumi et at, 1988). Al-though little is known about the function of GRP94, itwas also found in complexes of GRP78 and influenzaHA mutants (Kozutsumi et at, 1988). GRP94 has beenshown to be coordinately expressed with GRP78 undera variety of stress conditions (Chang et al., 1989; Lee,1987) and it has been suggested that malfolding ratherthan incorrect glycosylation is the proximal inducer ofGRP synthesis (Kozutsumi et al., 1988) .

Our experiments thus far indicated that the transportof gB-(Lk479) was arrested in the ER . The next series ofexperiments was designed to determine whether cer-tain forms of wild-type gB or the insertion mutants areretained in the ER by formation of complexes withGRP78 and GRP94. For these experiments, trans-fected C0S-1 cells expressing wild-type gB and themutants were radiolabeled at 40 hr after transfectionfor 18 hr, since the turnover time for GRP78 was re-ported to be longer than 48 hr (Hendershot and Kear-ney, 1988) . To maintain intact protein complexes, the

gB-(a4epeto) cells were lysed using mild conditions as describedunder Materials and Methods, immunoprecipitated,and subjected to electrophoresis in denaturing poly-acrylamide gels. The radiolabeled lysates were reactedwith immune reagents to gB, GRP78, and GRP94 . Theresults of these experiments (Figs . 5A-5D) are summa-rized as follows .(i) Immunoprecipitates formed by gB-specific anti-

bodies from cells expressing wild-type gB contained abroad gB band (Fig . 5A, lane 1) . Analysis of the polypep-tides precipitated by GRP78-specific antisera showedthat a protein with the electrophoretic mobility ofGRP78 coprecipitated a partially processed, undergly-cosylated band, pgB (lane 2) . This band was not pres-ent in precipitates formed by antisera to GRP78 in reac-tions with control cells transfected with the vectorplasmid alone (not shown) . Precipitates formed withGRP94-specific antisera contained the GRP94 band,which appeared as a closely migrating doublet of gly-cosylated and underglycosylated forms (lane 3) . A pro-tein band corresponding to the underglycosylated pre-cursor form, pgB, was coprecipitated with GRP94 .Only trace amounts of the more slowly migrating formof gB were contained in precipitates formed by antiserato GRP78 and GRP94 . Several background bands werealso detected in precipitates of GRP78 and GRP94, asa result of the mild washing of the precipitates to retainthe association of gB with the GRPs . This backgroundmay also have resulted from the association of GRPswith ER resident proteins, as reported by groups study-

gB4Lk479)

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DEFECTIVE PROCESSING OF HSV-1 gB MUTANTS

259

40 60 min

Chase

FIG. 4. Immunoprecipitation pattern showing the rate of process-ing of HSV-1 gB and insertion mutants in gB . C0S-1 cells were trans-fected with plasmid DNA and pulse-labeled for 10 min with lass)_methionine at 44 hr. After the chase intervals, lysates were preparedand immunoprecipitated with a pool of monoclonal antibodies to gB .Labels gB and pgB to the right designate the processed and precur-sor forms, respectively .

cides with the acquisition of complex sugars in theGolgi (Raviprakash et al., 1990) .

The glucose-regulated proteins associate withcertain forms of the wild-type and mutant gBs

Recent reports indicate that the glucose-regulatedprotein GRP78 (heavy-chain binding protein (BiP))binds to newly synthesized glycoproteins in the ER thatare incompletely assembled (Bole et al ., 1986). GRP78and GRP94 are ER resident proteins related to theheat-shock protein family whose synthesis is en-hanced in response to glucose starvation and expo-sure to inhibitors of glycosylation (Lee et al., 1984 ;Lewis et al., 1985 ; Lewis et at, 1986; Mazzarella andGreen, 1987). It was recently reported that mutantforms of influenza hemagglutinin (HA) that were incor-

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ing other viral glycoprotein mutants (Ng et al., 1989 ;Parks and Lamb, 1990) .

(ii) Analysis of precipitates formed with the mutantgB-(Lk600) showed that antibodies to gB precipitatedthe mutant glycoprotein (Fig . 5B, lane 4). Antisera toGRP78 precipitated GRP78 and a partially processedform, pgB (lane 5) . Trace amounts of more slowly mi-grating forms of gB were also detected in precipitatesformed with GRP78-specific antisera, suggesting thata small fraction of this mutant glycoprotein remainedassociated with GRP78 . Similarly, antisera to GRP94coprecipitated pgB and trace amounts of gB (lane 6) .Precipitates formed by a heterologous antibody in reac-tions with cells transfected with the plasmid did notcontain either gB or GRPs (control, Fig . 5B, lane 7) .Electrophoretic patterns comparable to those of gB-(Lk600) were also found with mutant gB-(a4ep810)(data not shown). When compared with the results ob-tained with wild-type gB, it appeared that both mutantsinteracted similarly with the GRPs .

(iii) Immunoprecipitates formed by gB-specific anti-bodies from cells expressing the mutant gB-(Lk479)contained the mutant glycoprotein band (Fig . 5C, lane8) . Analysis of proteins precipitated by GRP78-specificantibodies showed that the GRP78 band and the mu-tant gB-(Lk479) band were present (Fig . 5C, lane 9) .The control reaction of antisera to GRP78 did not showevidence of any proteins with the electrophoretic prop-

FIG . 5 . Immunoprecipitates formed by monoclonal antibodies to HSV-1 gB, GRP78, and GRP94 in reactions with wild-type gB and insertionmutants expressed transiently in COS-1 cells and labeled with [ 36S] methionine . (A) Wild-type gB, (B) gB-(Lk600), (C, D) gB-(Lk479) . Immunereagents used for the precipitation reactions are shown above each lane . GRP78 and GRP94 bands are indicated . Glycoprotein B (gB), partiallyprocessed precursor (pgB) . Molecular weights are indicated to the left . Controls indicate precipitates formed by heterologous antibody inreactions with cells . Vector (Vect), plasmid without insert .

erties of gB in precipitates obtained from cells trans-fected with the vector plasmid (Fig . 5C, lane 10) . In aseparate experiment, the gB precipitate obtained withg13-specific antibodies is shown (Fig . 5D, lane 11) . Theprecipitate formed by GRP94-specific antibody con-tained GRP94 and gB-(Lk479) (Fig . 5D, lane 12) . A het-erologous antisera failed to precipitate eitherthe GRPsor gB (not shown) .

DISCUSSIONIn earlier reports, we described the construction of a

set of deletion and insertion mutants in HSV-1 gB andreported on their antigenic properties, cell surfacetransport, and assembly into oligomers (Pereira et aL,1990; Qadri et al., 1991). Analysis of the insertion mu-tants showed that perturbations in specific regions inthe amino-terminal ectodomain of gB profoundly af-fected the conformation and intracellular localization ofthese mutants . In the present study, we further char-acterized the effects of mutations in the ectodomainand intracellular carboxy terminus of gB on its post-translational processing, transport through the exocy-tic pathway, and association with GRP78 and GRP94,which are cellular proteins resident in the ER .

Viral envelope glycoproteins have been used as mod-els for studying the assembly, processing, and trans-port of oligomeric glycoproteins through the exocytic

260 NAVARRO, QADRI, AND PEREIRA

A B C D

WTgB gB-(Lk600) gB-(Lk479) Vect gB-(Lk479)

rv 0

n a P2a a n a 0a aAntisera : m ¢

m¢0 mm

-Cr Qa C7

0z mrn ac7 t7 IM

C

pathway of eukaryotic cells . Vesicular stomatitis virusglycoprotein (VSV G) and hemagglutinin glycoproteinof influenza virus have received the most attention .Studies on these glycoproteins have shown that com-plete processing and transport to the plasma mem-brane require the correct conformation of monomersand assembly of oligomeric structures (Gething et al.,1986 ; Kreis and Lodish, 1986) . Mutations in the amino-terminal ectodomain that block the initial folding of themolecule perturb its oligomerization and transport tothe cell surface (Copeland et al., 1986; Doms et al.,1988; Doyle et at, 1986 ; Gething at al., 1986 ; Guan etal., 1988a; Machamer and Rose, 1988) . Mutant formsof influenza HA whose transport is blocked in the ERform complexes with GRP78 and GRP94 (Hurtley etal.,1989; Kozutsumi et al ., 1988) .It has been postulated that members of the heat-

shock protein (HSP) families, HSP70 and HSP90, areinvolved in the assembly and disassembly of proteinsand protein-containing structures (Pelham, 1986) .GRP78, or the heavy-chain binding protein, is amember of the HSP70 family (Haas and Meo, 1988 ;Munro and Pelham, 1986) and plays a role in the as-sembly of immunoglobulin (1g) heavy chains in B-lym-phocytes (Bole et at, 1986; Haas and Wabl, 1983 ;Hendershot and Kearney, 1988) . Recently, it wasshown that GRP78 is displaced from the Ig heavychains by the addition of newly synthesized lightchains in the ER, thus confirming the role of GRP78 inthe normal assembly of this oligomeric glycoprotein(Hendershot, 1990) . In light of the finding that GRP78and GRP94 are expressed ubiquitously in many differ-ent cell types, it is likely that these proteins play a moregeneral role in stabilizing membrane-bound and se-creted proteins by forming soluble complexes utilizingATP (Lee, 1987) . Cumulative data from many laborato-ries indicate that GRP78 regulates transport of bothnormal and abnormal proteins . It appears to functionas a molecular chaperone (see review by Ellis andHemmingsen (1989)), which assists in the assembly ofoligomeric proteins and blocks the transit of misfoldedproteins from the ER through the exocytic pathway(see review by Pelham (1989)) .

In the present study, we observed that GRP78 andGRP94 readily formed complexes with partially glyco-sylated forms of wild-type gB but not with the fully pro-cessed molecule . At this time we have not determinedwhether GRP78 binding to precursor gB is transitory,although this seems likely insofar as GRP78 was notassociated with significant amounts of the fully glyco-sylated form of the glycoprotein . Based on studies ofother mutant viral glycoproteins, it appears that GRP78binds to partially folded assembly intermediates of gB,preventing their aggregation prior to oligomerization .

DEFECTIVE PROCESSING OF HSV-1 gB MUTANTS

261

The finding that a fraction of the underglycosylated gBforms fails to undergo additional processing suggeststhat some nascent gB molecules may be stably asso-ciated with GRP78. When compared with the amountof fully processed gB, however, the unprocessed frac-tion, pgB, coprecipitating with GRP78 is small . Itshould be noted that studies on the activation ofGRP78-promoter constructs by transient expression ofwild-type HSV-1 gB or the mutant gB-(Lk479) showedthat transient expression of this mutant, but not of wild-type gB, transactivates the GRP78 promoter (A . Lee etal., manuscript in preparation). We have begun to ana-lyze GRP-protein complexes in HSV- 1 -infected cells todetermine whether such complexes are formed in thepresence of other glycoproteins . GRP94 may also playa role in sorting transport-competent proteins from theER to the Golgi, since it is coordinately regulated withGRP78 (Chang etat, 1989; Lee, 1987 ; Lee etal., 1984)and is found in GRP78 complexes with aberrantlyfolded HA molecules (Kozutsumi et al., 1988) .

Both Class I and Class II molecules of the major his-tocompatibility complex present antigen for recogni-tion by T-cell receptors (Brodsky and Guagliardi, 1991 ;Townsend and Bodmer, 1989 ; Unanue, 1984) . Class Imolecules generally bind endogenous peptides de-rived from proteins synthesized by virus-infected cells,which present portions of newly synthesized polypep-tides on the cell surface (Morrison at al-, 1988; Town-send and Bodmer, 1989) . Several studies have demon-strated that HSV-1 glycoproteins on the plasma mem-brane are targets for binding of Class I- and ClassII-restricted cytotoxic T-lymphocytes (CTLs) (Carter etal., 1981 ; Lawman et al., 1980) (see review (Zarling,1986)). HSV-1 gB has been shown to be a major targetof CTL activity (Blacklaws et al., 1987 ; Witmer et at,1990). It was recently reported that gB peptides fromamino acids 489 to 515 associated with the Kb Class Igene product and that 1-1-2d-restricted CTLs recog-nized an immunodominant region of gB mapping be-tween residues 233 and 379 (Hanke et al., 1991). Ourcurrent results suggest that in HSV- 1 -infected cells, afraction of the nascent gB molecules may be seques-tered by the cellular GRPs in the ER and is conse-quently destined for degradation . The fragmented pro-teins associate endogenously with cellular Class I mol-ecules undergoing processing and assembly in thiscompartment of the exocytic pathway . It follows, then,that Class I molecules that have bound gB peptides aretranslocated to the plasma membrane, where thesepeptides are presented on the cell surface as targetsfor CD8' cytotoxic T-cells. It would be of interest tofurther define the T-cell-responsive epitopes on HSV-1gB and to relate their sequence to the antigenic struc-ture of the molecule delineated by monoclonal anti-

262

NAVARRO, OADRI, AND PEREIRA

body binding sites and to malfolded regions recog-nized by GRPs .

In a recent study, we analyzed the antigenic struc-ture and assembly of mutant gB-(Lk479) with monoclo-nal antibodies to discontinuous epitopes and foundthat it had lost a subset of discontinuous epitopes thatmap in a major neutralizing domain (Qadri et al., 1991) .Transfected cells expressing gB-(Lk479) stained with areticular pattern by immunofluorescence and failed toshow surface staining, which suggested that the trans-port of this mutant was blocked in the ER . Our presentstudy revealed that the GRIPS formed complexes withthe mutant gB-(Lk479) . Sensitivity of this glycoproteinto Endo H, failure of the ATP uncoupler CCCP to affectits processing, and pulse-chase experiments showingthat gB-(Lk479) failed to undergo a shift in mobility andwas lost after longer chases support the notion that theprotein was degraded in the ER . Thus, local conforma-tional changes in the monomer that did not preventoligomerization of gB-(Lk479) drastically affected itstransport through the exocytic pathway . It is likely thatperturbations in the polypeptide chain and local mal-folding of this mutant exposed hydrophobic sites notfound on the exterior of native gB and that these re-gions bound irreversibly to the GRPs . The fact that gB-(Lk479) formed dimers but was nevertheless not trans-ported indicates that these dimers might not be stable,as our present results indicate . Thus, the proper con-formation of individual subunits, which also plays a rolein the stability of nascent dimers, may be as importantin controlling exit of a protein from the ER as the tertiaryor quaternary structure of the oligomer . It is likely thatassembly of full-length gB subunits into stable dimersis required priorto transit of this protein from the ER butis not sufficient for surface transport .

Studies on mutant forms of viral glycoproteins indi-cate that the cytoplasmic domain may carry a signalwhich influences the targeting to vesicular carriers andthe transport rate of glycoproteins through the exocyticpathway (see review by Rose and Doms (1988)) . Muta-tions in the cytoplasmic domain of VSV G glycoproteinhad no effect on the folding or assembly of oligomersbut did slow or prevent their exit from the ER (Guan atal., 1 988b; Puddington et al., 1986 ; Rose and Berg-mann, 1983). Studies on carboxy-terminal mutants inthe paramyxovirus hemagglutinin-neuraminidase pro-tein showed that their transport from ER to Golgi wassignificantly retarded (Parks and Lamb, 1990) . Insofaras the association of these mutants with GRP78 wastransitory, it was concluded that altered transport dueto mutations in the cytoplasmic domain was notcaused by binding to GRP78 . We previously found thatinsertions in the cytoplasmic domain of HSV-1 gBfailed to alter the local folding and oligomerization of

the ectodomain and that these mutants were translo-cated to the plasma membrane (Qadri at at, 1991) .Results of the present study indicated, however, thatmutant gB-(a4ep810), which contains an insertion inthe intracellular carboxy terminus, was retarded in itstransport from the ER to the cell surface . These resultssuggest that the cytoplasmic domain of gB plays a rolein transport and are supported by studies from othergroups (Cai at al., 1988 ; Raviprakash at al., 1990). Fur-thermore, the finding that the transport of mutant gB-(Lk600) was also slowed indicates that mutations incertain regions of the ectodomain that do not signifi-cantly alter the conformation of gB may neverthelesshave an impact on the translocation of the molecule .

In this and other studies (Pereira at al., 1989 ; Qadriat al., 1991), we expressed constructs of HSV-1 gBthat encode mutant glycoproteins in transient assaysand established that the proper assembly of discon-tinuous domains, particularly those designated D2aand D2b, is required for translocation to the cell sur-face . In earlier studies, nucleotide sequence analysisof HSV-1 gB mutants resistant to potent neutralizingantibodies showed that conservative amino acidchanges in discontinuous epitopes mapping in certainneutralizing domains (identified later as D2a and D2b)altered antibody binding but not transport of the glyco-proteins or infectivity of the mutants at the permissivetemperature (Kousoulas at al., 1984; Kousoulas et al.,1988 ; Pellett et at, 1985). Further analysis showed,however, that the altered residues affect the process-ing of the mutant gB glycoproteins in infected cellsmaintained at the nonpermissive temperature (D . Na-varro, P . Paz, and L . Pereira, manuscript in prepara-tion). It remains to be determined how conformationalchanges that significantly alter the antigenic propertiesof the gB subunits and preclude cell surface transportaffect viral infectivity . To answer this question, we arecarrying out cotransfection experiments with intactviral DNA and mutant gB genes encoding glycopro-teins with various transport defects to obtain recombi-nants with defined defects in the domain of the gBgene .

ACKNOWLEDGMENTS

We thank Pedro Paz for excellent technical assistance and BillWelch and Amy Lee for their gifts of antisera to GRP78 and GRP94 .These studies were supported by Public Health Service Grants DE-08275 from the National Institutes of Dental Research and Al-30873and Al-23592 from the National Institute of Allergy and InfectiousDiseases . D .N. was supported by a fellowshipfrom the Spanish Min-istry of Education and Science . I .Q . was supported by a CRCC fel-lowship from the Academic Senate of the University of California,San Francisco .

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