Synthesis and processing of the Marek's disease herpesvirus B antigen glycoprotein complex.

0022-538X/88/114270-10$02.00/0

Copyright C) 1988, American Society for Microbiology

Synthesis and Processing of the Marek's Disease Herpesvirus

B

Antigen Glycoprotein

Complext

I. SITHOLE,' L. F. LEE,2 AND L. F. VELICER1*

DepartmentofMicrobiology and Public Health, Michigan State University, East Lansing, Michigan48824-1101,' and

Regional Poultry Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, EastLansing, Michigan 48823-53382

Received 5 April1988/Accepted 1 August 1988

TheMarek's diseaseherpesvirus Bantigen(MDHV-B) complexwaspreviously immunologically identified andmolecularly characterizedas asetof three glycoproteinsdesignated gplOO,gp6O, andgp49onthebasis of

apparent molecular weight and immunoprecipitation with both polyclonal and monoclonal antibodies. Immunoprecipitation analysis, previously withpolyclonaland more recently with monoclonal antibodies, of infected cell lysates labeled with [35S]methionine in the presence of tunicamycin, an inhibitor ofN-linked glycosylation, revealedtwo putativeprecursormoleculesof88,000 daltons (pr88) and44,000 daltons (pr44). High-resolution pulse-chase studies revealedthatgplOOwas aglycosylated intermediate whichwasprocessed

toyield gp6O and gp49. This cleavage was inhibited by monensin, an inhibitor of glycoprotein processing.

Endo-o-N-acetylglucosaminidases

F and H(endo-F, endo-H) reduced gplOOtopr88,indicating that the latter isanintermediatein thebiosynthetic pathway. Thesesameenzymesreducedgp49,andtoalesserextentgp6O, topr44, suggestingthatpr44istheirpolypeptidebackbone.Significantsupportfor thisconceptis thefactthat the same monoclonal antibody recognized all three molecules, gp60, gp49, and pr44. In the presence of monensin, terminal addition ofcomplexsugars wasalso prevented, since gp6Owasreplacedbyaslightly faster migratingcomponent which wasinsensitivetoboth endo-F andendo-H. Cell-free translation of infected-cell mRNA, followed byimmunoprecipitation analysiswitheitherpolyclonalormonoclonalantibody, resultedin detection ofaputative unglycosylatedprecursorpolypeptideof44,000daltons. Sincepr88wasnotthe initial

precursorpolypeptide ofthe MDHV-Bcomplex, itsexistencemay have resultedfrom dimerization of pr44.

Again, detection of both pr88 and pr44 with the same monoclonal antibody is consistent with this interpretation. These collective data obtained from the cell-free and in vivo studies with polyclonal and monoclonal antibodies reactive with MDHV-B are consistent with the concept that pr44, the initial gene product, dimerizestoformpr88anddemonstrate thatpr88isactuallyaprocessing intermediate glycosylated togplOO, anotherprocessing intermediate, which isthenprocessedtogp6Oandgp49.

Marek's disease (MD) is a

lymphoproliferative

diseaseof chickensthatwasfirstdescribedbyMarek in 1907 (29,37).

The disease is caused by the Marek's disease herpesvirus

(MDHV) and represents the first neoplastic disease for which an effective vaccine has been developed (33, 35, 37). The mechanism of this immunity is not fully understood; however, it appears to be a two-step processinvolvingboth antiviral and anti-tumor-cell immunity (32, 33). Experimen-tal evidence suggests that antiviral immunity is superiorto

anti-tumor-cell immunity and that if the former occurs the

latter is not necessary (32).

Thevaccinevirus, herpesvirusofturkeys(HVT) (35), and MDHVareextensivelyrelatedimmunologically (35, 45,46),

sharing a large number ofpolypeptides that are apparently

common antigens (15, 41, 45, 46), including the two most prominent common antigens, Aand B (33, 34, 37).

In anefforttodetermine the mechanism of this protective

immunity at a molecular level, thesetwo common antigens have been the focus of recent immunologic and molecular

biologicstudies in thislaboratory (11,16-18).TheMDHV A

antigen (MDHV-A), a secretory glycoprotein of 57 to 65

(gp57-65) kilodaltons (kDa), has been extensively studied with regard toits synthesis, processing, and secretion (18). The gene encoding MDHV

gpS7-65

(i.e., MDHV-A) was

* Correspondingauthor.

tJournal article 12564 from the Michigan Agricultural Experi-mentStation.

identified(16) andsequenced (8), and similar work is almost complete for HVTgp57-65 (i.e., HVT A antigen [HVT-A]) (P. M. Coussens and L. F. Velicer, manuscript in

prepara-tion). Theprocessing studies with MDHV-A(18) serve asa useful background for designing similar studies with other antigens in this system.

The B antigens of MDHV(MDHV-B) and HVT (HVT-B) weremolecularly characterized as a complex of three immu-nologically relatedglycoproteins,gplOO,gp6O,andgp49, one of which may be a membrane component, on the basis of studies withpolyclonal seracontaininganti-MDHV-B

activ-ity (17). Tunicamycin inhibition of N-linked glycosylation resulted in either nonglycosylatedor 0-linked glycosylated putative precursor polypeptides of MDHV-B and HVT-B, called pr88 and pr44, being immunoprecipitated with the same polyclonalsera(17). Inthatsame studythe

identifica-tion, as MDHV-B, of similar-size polypeptides, previously detected by others usingtheir(15, 41) monoclonal antibod-ies, was alsoaccomplished (17), thus making it possibleto relate their subsequent virus neutralization (15, 42),

immu-noprotection (36), and pulse-chase (14, 42) studies to MDHV-B.

Silva and Lee (42) have carried out pulse-chase studies with 10-min pulses and even longer chase intervals, using

HVT-infected cells and their monoclonal antibody reactive with the three MDHV-B glycoproteins. They reported that thedisappearanceofgplOO coincided with the appearance of

gp6Oandgp49, suggestingthat the former molecule may be

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MDHV-B ANTIGEN GLYCOPROTEIN COMPLEX 4271

cleavedto generatethe lattertwomolecules. However, they

state that confirmation of therelationship of gpl00 to gp6O

and gp49 must await further experiments. Ikuta et al. (14), using HVT-infected cells, have also performed pulse-chase

studieswith similar time intervals, usingboth a monoclonal antibody and rabbit anti-MDHV-gB antiserum. They found essentially the same sequence of events as Silva and Lee (see above) and identified the larger glycoprotein as the precursorform of B antigen which is processed to the two smaller glycoproteins (14). However, in both pulse-chase studies no smaller unglycosylated precursor forms were detected, supporting the suggestion that cells may need to be pulsed foreven shorter periods of timeto visualize rapidly occurring early events (42). This may be especially true in view of the very rapid processing of MDHV-A, in which most ofthe glycosylationoccurswithin 1 min (18).

Since their pulse-chase experiments did not detect an

unglycosylated precursor polypeptide, Silva and Lee (42)

used lysates from tunicamycin-treated infected cells to re-solve this point. Their monoclonal antibody detected a 44-kDa molecule they called pr44 on the basis of their

interpretation that it was a precursor polypeptide. In their studyapr88 molecule was notreported, although reexami-nation of their data (42) (see Fig. 4) suggests that there may

have beensomeprotein ofpr88 sizepresent, but the amount was clearly less than that of pr44 (R. F. Silva, personal

communication). On the other hand, Ikuta et al. (14) de-tected what appears to be their equivalentof pr88, but no pr44, intunicamycin-treatedinfectedcells, usingtheir

mono-clonal antibody. While our laboratory has more recently found bothputative precursorpolypeptides after

tunicamy-cin treatment (summarized above), full analysis of their

possiblerole in the MDHV-Bprecursor-processing relation-ships wasbeyond the scope of the previous study(17).

The purpose of this study was to examine the entire MDHV-Bprecursor-processing questionin more depthand answer the existing questions by using approaches not

previouslytried. Thus, the analyses ofMDHV-B pr44

syn-thesis and processing reported here were performed with a

combination of higher-resolution pulse-chase studies, two

glycosylation inhibitors (tunicamycin and monensin), two

endoglycosidases (F and H), protease inhibitors, and cell-free translation and serve to provide a more complete analysis of processing events than previously existed. Of particular significance was the cell-free translation analysis followed by immunoprecipitation, whichrevealed that pr44 is the initial unglycosylated precursor polypeptide of MDHV-B. Ofequal significancewas theuseof monoclonal

antibody, which establishedthatpr44 was a component of all

larger moleculeswith MDHV-B reactivity.Thesequenceof

events elucidated by this combination of approaches and reagentsinvolvesthe apparentdimerizationof pr44 to pr88,

followed by glycosylation ofthisintermediate togpl00and

subsequent processing ofgpl00 to yieldpg6Oand gp49.

MATERIALS ANDMETHODS

Cells and viruses. Thepreparation, propagation,and infec-tion of small-scale duckembryo fibroblast cell cultures with MDHV was generally performed as first reported by

Glau-bigeret al. (11) and more recently by Isfort et al. (17). One

exception was that the MDHV strain GA used was at passage level 6, afterisolation ofcell-free virus from feather

tipsobtained from infected birds (39).

Radiolabeling of proteins. Infected and noninfected cells werelabeled with

[35S]methionine

at 48 h postinfection for 4

hby standard methods,aspreviouslydescribed (17).

Pulse-chaselabeling was by methods already established for this system (18), with the following minor modifications. Prein-cubation without methionine was for 40 min (18); pulse labelingwasby incubation with 250

,uCi

of

[35S]methionine

(specific activity, 1,000

,uCi/mmol;

Amersham

Corp.)

perml for 1, 4, or 15 min; and chases were for the various times

indicated in the figure legends. For

labeling

in the presence of

tunicamycin (Sigma

Chemical

Co.)

ormonensin

(Calbio-chem-Behring), cellswerepreincubatedwith thedrugsfor 1 hasreportedelsewhere(18)or atvarious timesasindicated in the figure legends. Both

drugs

were present

throughout

the

remaining

courseof each

experiment.

Antisera.

Preparation

andcharacterizationof the

polyclo-nalantiserarabbitanti-MDHV-A(RoA), rabbit anti-MDHV-B(RaB), rabbit anti-MDHVinfected-cell

plasma

membrane (RaPM), and immune chicken serum have been described

previously

(17, 18). Themonoclonal

antibody 1AN86.17,

an

immunoglobulin Gl

isotype,

is a cloned derivative from hybridoma IAN86, and it

immunoprecipitated

gplOO,

gp6O,

andgp49(datanotshown),as

previously reported

for 1AN86

(41, 42). However, while this cloned monoclonal

antibody

reactedpoorlyinimmunofluorescencewith MDHV-infected

monolayer cell cultures, it exhibited a strong reaction with the surface antigen of infected cells in

suspension. Also,

it

neutralized cell-freeHVT,butnotMDHV

(data

not

shown).

This monoclonal antibodywas used becauseofits

immuno-precipitationproperties andespeciallybecause of its

ability

to

recognize

an

epitope

presentin

pr44 (42).

Immunoprecipitation and SDS-PAGE.

Immunoprecipita-tionwascarriedoutas

previously

described

(17),

exceptthat

lysate

preparation throughout

was with

detergent

buffer

containing

300 ,ug of

phenylmethylsulfonyl

fluoride per

ml,

and a second

antibody

was added as needed with mouse

monoclonal

antibody

(asdescribed

below).

The

immunopre-cipitates

were

washed, suspended

in

sample

buffer,

and

analyzed bysodium

dodecyl

sulfate-polyacrylamide gel

elec-trophoresis

(SDS-PAGE)

(24).

Acrylamide

concentrations

were 7.5% for all gels presented. Molecular sizes were calculated

by

interpolation

between

protein

standards

by

the method of Weber and Osborn(47). Fluorography was

per-formed

by

the methodofBonnerand

Laskey (4).

Autoradi-ography

ofthe dried

gels

was carried out at

-70°C

for the times indicated in the

figure legends.

Enzyme digestions. All

digestions

were

performed

with

proteins

first bound to

Staphylococcus

aureus

protein

A

during

immunoprecipitation

as described above. For

diges-tion with

endo-,-N-acetylglucosaminidase

H, also called

endoglycosidase

H

(endo-H) (Miles Laboratories,

Inc.), immunoprecipitated

proteins

were eluted by incubating the bacterialpelletsfor 20 minat

37°C

in 50 ,ulof sodium citrate buffer(50 mM, pH 5.5)

containing

0.2%

SDS,

then

boiling

for 2 min and

centrifuging

for 3 min. The supernatant was

collected,

10 U ofendo-H was

added,

and the mixturewas incubated at

37°C

for 16 to 24 h (1). For

digestion

with

endo-3-N-acetylglucosaminidase

F,

also called

endoglycosi-dase F (endo-F) (Dupont, NEN Research Products), the

immunoprecipitated proteinswereeluted in 100 ,ulof sodium

phosphate (100 mM, pH 6.1)

containing

0.1%

SDS,

0.5% NonidetP-40,and 50mMEDTA. Endo-F

(1/4

U)wasadded to the

sample

and incubated as above. Control

samples

without endo-H orendo-F were treated the same way, and afterincubation all sampleswere

precipitated

with ice-cold acetone for 1 h at

4°C

and

centrifuged

in an

Eppendorf

microfuge

(model 5414)at top

speed

for 3 min. The

precip-VOL.62, 1988

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4272 SITHOLE ET AL.

itateswerewashedwith100%ethanol, vacuumdriedbriefly, and suspended in sample buffer for electrophoresis.

Isolation of total cellular RNA. Total cellular RNA was isolated from MDHV-infected duck embryo fibroblast cells at 48 h postinfection by the guanidinium isothiocyanate procedure described by Chirgwin et al. (6). Cells were harvested, pelleted, and suspended in 4 ml of guanidinium isothiocyanate buffer (4 M guanidinium isothiocyanate, 1% sodium sarcosyl, and 0.1 M2-mercaptoethanol) per 108 cells. A portion (8 ml) of the resulting lysate was layered on 3 ml of 5.7 M cesium chloride in a Tris-EDTA solution (TE) consisting of 10 mM Tris hydrochloride (27) and 1 mM EDTA (pH 7.5). The RNA in the lysate was pelleted at 25,000 rpm for 20 h at 22°C in a Beckman SW41 rotor. The pelleted RNA was solubilized in TE with 0.1% SDS, ex-tracted with an equal volume of phenol-chloroform, ethanol precipitated, and solubilized in H20 (27).

Cell-free translation. Total cellular RNA from MDHV-infected cells was translated by use ofa rabbit reticulocyte lysate system from Bethesda Research Laboratories, Inc. The RNA (10 ,ug) was translated in a

30-pI

reaction volume as recommended by the supplier. An equal volume of detergent buffer was added to stop the reaction. A 10-jl portion of this diluted translation mixture was analyzed directly by SDS-PAGE, and the rest was immunoprecipi-tated, as described above, with undiluted antiserum or an ascitesfluid stock solution (ascites fluid previously diluted 1: 50 in phosphate-buffered saline) at 1/20 volume (i.e., for 50

[L1

of translation mixture, 2.5

1l

ofantiserum or ascites fluid stock solution was used). To ensure successful immunopre-cipitation (see above), rabbit anti-mouse immunoglobulin G (Sigma) was added along with mouse ascites fluid stock solution at a ratio of 1:25, and rabbit anti-chicken immuno-globulin G was added along with immune chicken serum as previously described (17).

RESULTS

Kinetics ofMDHV-Bprocessing. Initially pulse-chase anal-yses were performed with 1-min pulses, as previously done for MDHV-A processing studies (18). The intent was to identify and analyze early rapid processing events, should they exist for MDHV-B, as in the case for MDHV-A. However, during preliminary experiments it became appar-ent that the appearance of molecules of interest was suffi-ciently slow(unpublished data) that a somewhat longer pulse interval could be used to facilitate isotope conservation and still critically analyze early MDHV-B processing events. Thus, for most subsequent pulse-chase analyses, infected cells were pulsed 4min and chased at various times (Fig. 1). The first molecule with MDHV-B immunological reactivity that could be detected, gplOO, was easily seen at the 12-min chase, although as early as 0-min chase trace amounts appeared (longer exposure of same gel, data not shown) as a heterogeneous slightly smaller putative glycosylation inter-mediate which acquired gplOO properties as glycosylation proceeded. The intensity of the gplOO band increased to a maximum at 45 min ofchase and declined with increasing chase periods. In contrast, gp6O and gp49 were first seen after a30-min chase and subsequently increased in amount, with the majority of the processing completed by 120min. It is important to note that the above events coincide in a manner consistent with a precursor-product relationship. The results of these pulse-chase studies with short pulses indicate that gplOO is processed relatively slowly to yield gp6O and gp49, confirming the earlier observations by Ikuta et al. (14) and Silva and Lee (42).

minutes

hours

92.5k

-

68k-43k

-chase ofter 4 minute

pulse

4

hr

0 3

6

12 30 45

2

3 4 Label

gploo

...

*,it..::,.e,ls'W,S,.>l'; W -

g

6

.

'

'

'5~~~~i15)49>g*gp

FIG. 1. KineticsofMDHV-Bprocessingasdeterminedby pulse-chase analysis in the absence oftunicamycin. A4-min pulse with

[5S]methionine was followed by chases of various times shown above each lane. Immunoprecipitationanalysis of1 mlof celllysate was done with RaPM. Fluorographic exposure was for 8 days. Molecularsizes areindicated inkilodaltons (k).

A careful examination of the data in Fig. 1 reveals two lines ofevidence that could beinterpreted tomeanthatsome gp49 forms directly by glycosylation ofasmaller precursor polypeptide, such as the pr44 described below. One obser-vation is theappearance of animmunoprecipitable

polypep-tideslightly smaller than gp49, but increasingin size, atthe 45-min and 1-h pulseintervals(Fig. 1and unpublished data).

The second observation is the accumulation ofmore radio-activity in the gp49 band than in the gp6Oband atthe 2-, 3-,

and 4-h chase intervals. Densitometric analysis (data not shown) confirms this visual observation. If gplOO is being

processed to yield gp6O and gp49, the latter two should be present in equimolaramounts. The greater accumulation of gp49could beexplainedifsome wasformedbyamoredirect route. While it is tempting to speculate about an alternate processing route, another explanation could be differential release ofgp6O and gp49 from membranes by the detergent

bufferused inthislaboratory (17), and/or

varying degrees

of susceptibility to protease degradation. In any event, if an alternate route does exist it would have to be a minor pathway and would not change the obvious existence of gplOO as aprocessingintermediate. Resolutionofthis ques-tion is beyond the scope of this study.

Processing in the presence of monensin. Monensin, a mo-novalentionophore,inhibits transport of

glycoproteins

spec-ified by vesicular stomatitis virus, Sindbis

virus,

and

herpes

simplex virus (HSV) (9, 19-21). In

addition,

this

ionophore

blocks both theprocessing of N-linked

oligosaccharides

and the addition of 0-linked oligosaccharides to HSV type 1 glycoproteins (20, 21) and varicella-zoster virus gpll8 and gp98-67 (30, 31). To further examine the

processing

of MDHV-B, lysatesfrominfected cells labeled in the presence and absence of monensin were

immunoprecipitated

with RotPM and analyzed by SDS-PAGE. At a

previously

deter-mined optimal monensin concentration of 0.5 ,uM, and at various times of pretreatment, theamountsofgp6Oandgp49

were reduced relative tothe amountofgplOO, and

gp6O

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MDHV-B ANTIGEN GLYCOPROTEIN COMPLEX 4273

minutes

chase after

15

minute

pulse 4 hr

53045615

304660

Label

_-

_ _ _

+ + +

+

-_

Monensin

92.5k-

68k-

43k-

25.7k-FIG. 2. The effect of monensin on MDHV-B processing as determined by pulse-chase analysis. A 15-min pulse with [35S]methionine was followed by chases of various times shown above each lane. Immunoprecipitationanalysis of the celllysate(1 ml) with (+) and without (-) monensin (0.5 ,uM) was done with

RoxPM.Fluorographicexposure was for 8 days. Molecular sizes are indicatedinkilodaltons(k).

replacedby aslightly faster migrating componentof about57 to58 kDa(unpublisheddata), suggesting inhibition of either

0-linked glycosylation or addition of terminal sialic acid

residues. Thisprelimninary finding suggested thatmonensin

inhibited processing of gplOO to gp6O and gp49. This was further confirmed by a modified pulse-chase experiment

performed in the presence of monensin. Since only later

processing events needed to be analyzed over a critical

period,thepulseinterval wasincreased from 4 min (Fig. 1) to 15 min to facilitate radioisotope incorporation, and only

four chase intervals were used (Fig. 2) on the basis of

information shown in Fig. 1. The appropriateness of the

conditions is shown in Fig. 2, lanes 1 through 4, where

normal processing (Fig. 1) can be seen in the absence of

monensin, with gplOO first appearing and becoming proc-essed to gp6Oand gp49.Inthe presence of monensin (Fig. 2, lanes 5through 8), however,gplOO accumulatesand no gp6O orgp49can befound.

Digestion with endo-F and endo-H. Endo-F cleaves both high-mannose and complex oligosaccharides but does not

affect 0-linked sugars (1, 23). Endo-H predominantly

cleaves between the twoproximalN-acetylglucosamine res-idues of high-mannose oligosaccharides (1, 23). Complex

oligosaccharides are resistant to endo-H, whereas

interme-diate oligosaccharides exhibit intermediate sensitivity. To

further evaluateglycoprotein processing, endo-Fand endo-HwereusedtodeglycosylateimmunoprecipitatedMDHV-B

glycoproteins (Fig. 3A and B, respectively). Both endo-F

and -H cleaved gplOO to pr88. Also, gp49, and to a lesser extentgp6O, were cleavedto pr44 by the same enzymes.

Analysis of the relationship between pr44 and pr88. The

consistent appearance of the two putative precursor

poly-peptides, pr44 and pr88, after metabolic labeling for 4 h in the presence of tunicamycin (17) (Fig. 4) suggested that

pulse-chase studies in the presence of the inhibitor might

provide information concerning the relationship between these two molecules. However, when such an experiment

was performed in parallel with, and identical to, the one

presented in Fig. 1, except in a second set of cell cultures

A

-

+

-

+

ENDO-F

- -

+ +

TM

92.5k-h

---gplO0

0..

*-pr88

68k

-_b

--gp6O

43k-

VW

25.7k-B

-

+

-

+

-

+

Endo-

H

--

---+

+

Monensin

- -

+

+

- -

TM

92.5k

-

-

-

i

gpIOO

ow

NEW40-prBB

68k--a

-gp49

-pr44

-

gp6O

.-.'-w W

_5rgp49

43k

-

W

-pr44

25.7k

-FIG. 3. Deglycosylation of MDHV-B with endo-F and endo-H. (A) SDS-PAGE analysis of MDHV-B polypeptides labeled with

[35S]methionine

for4h in thepresence (+) andabsence(-) oftunicamycin(2jj.g/ml)immunoprecipitatedwithRaPM,eluted, and incubated in thepresence (+) and absence (-) of endo F. (B)SDS-PAGEanalysis ofMDH4V-Bpolypeptideslabeled with[35S]methioninefor 4 h in the presence(+)andabsence(-) oftunicamycin(2,ug/ml) or monensin (0.05R,M),immunoprecipitatedwithRaPM, eluted, and incubated in the presence (+) and absence (-) of endo-H. For both panels, fluorographic exposure was for 1 day and molecular sizes are indicated in kilodaltons(k).

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[image:4.612.62.280.74.301.2] [image:4.612.117.487.451.670.2]

mrnuutes I m,,urs

cnhse

after 4' rfninute put

e-i'hr

o 3 6 123045 2

I,w

i-

-4

La ce

92.5k

-68k

-43k

-FIG. 4. Kinetics of MDHV-B processingasdetermined by

pulse-chase analysis in thepresenceoftunicamycin.Thisexperimentwas

performed in parallel with, and in an identical manner to, the

experiment presented in Fig. 1, exceptthat tunicamycin (2 pLg/ml)

was added to these cell cultures. Also, the fluorographic exposure

wasfor64days.

treated with tunicamycin, the hoped-fordirect evidence ofa

precursor-product relationship between pr44 and pr88 (in either direction) was not seen. The first molecule with

MDHV-B immunological reactivity that could be detected, pr88, was easily seen atthe 12-minchase and was apparent in trace amounts after the3-min chase, whena long

fluoro-graphic exposure was used (Fig. 4). The lower amount of pr88 inthe6-minchaselaneis duetolossof about50% ofthe sample,but thisdoesnot interferewith interpretation of the data. The amount of pr88 increased throughout the first3 h of chase (Fig. 4) and remained constant for hour 4 (more

apparent on a shorter exposure of the same gel, data not shown). pr44wasfirst detectedafter30min of chase (Fig. 4),

increased in amountthroughhour 3, and remained constant for hour 4 (more apparenton ashorterexposure of thesame gel, datanotshown). While pr88 increased inamountbefore pr44 did, the absence of any reduction in pr88 as pr44 increased is inconsistent with a precursor-product

relation-ship.

Withtwoputativeprecursorpolypeptides detected invivo

after labeling in the presence oftunicamycin, and only one

seen invitro (preliminary cell-free translation dataobtained

with polyclonal sera, not shown), there was concern that

pr44 might be an artifact of pr88 degradation by cellular proteasesin theformer situation. We examined the effect of four classes ofprotease inhibitors (2, 40)on theappearance ofpr44invivo, synthesized in thepresence oftunicamycin.

In all cases the ratio of pr44 to pr88 remained very similar (unpublished data), as would be expected if pr44 is a precursorpolypeptide andnotadegradation productof pr88.

Cell-free translation. Interpretation of data obtained from cell-free translation and immunoprecipitation analysis of the translation product is aided by careful choiceofantiseraand

ideally some prior informationconcerning the putative pre-cursorpolypeptides. While the RcxB and RoLPM sera gener-atedin thislaboratoryhavebeen useful in MDHV-B studies and detect pr44 made intunicamycin-treated cells, they are

of low titer(17). Thus, even the higher-titered RaPM must

be used in such quantities that the amountof

immunoglob-ulin G heavy chain present causes a distortion of protein

migration in that region ofthe gel, which in turn interferes with normal migration of MDHV-B pr44. Fortunately, a

panel ofmonoclonal antibodies that neutralize MDHV and

immunoprecipitate the MDHV-B polypeptides gplOO, gp6O, andgp49 has been prepared (41). The monoclonal antibody

1AN86.17 was ofparticular interest because the antibody

from the original hybridoma, 1AN86, was one of the more

extensively used and better-characterized members of the panel (41) andbecause itcouldimmunoprecipitate pr44 from

tunicamycin-treated cells (42), implying that it should be

possibleto useitsderivative (lAN86.17) to do the same from cell-free translation products. This antibody was first com-pared directly with RaPM (analysis in the same gel) with respect to their ability to immunoprecipitate the known MDHV-B polypeptides. As would be predicted from the

independent analyses (17, 41), both antibodies immunopre-cipitated gplOO, gp6O, and pg49 from MDHV-infected cells labeled in the absence of tunicamycin (data not shown). Both RoxPM and 1AN86.17 alsoimmunoprecipitated pr88 andpr44 formedin the presence of tunicamycin (data not shown). As predicted, there was no distortion of protein migration with 1AN86.17.This feature, and itsability toimmunoprecipitate

both pr88 and pr44, made 1AN86.17 the reagent ofchoice for identification of the MDHV-B precursor polypeptide made

bycell-free translation.

Once work shifted to analysis of the products resulting from cell-free translation of MDHV-infected-cell mRNA,

another point required clarification. The MDHV-A precur-sor polypeptide is very nearly the size (16, 18) of MDHV-B pr44 (17). In fact, its previous size estimate as pr47 was based on the previous estimate of the size of the ovalbumin marker as 46 kDa. Now that the supplier has changed this marker size to 43 kDa (17), on the basis of amino acid sequence data, it might be predicted that the MDHV-A precursorwill migrate close to MDHV-B pr44 in SDS-PAGE and that confusion could exist if both were present in the same gel because of either coprecipitation or nonspecific trapping. On the basis of our previous cell-free translation studies (16, 18) the MDHV-A precursor will definitely be present in any translation product resulting from translation of unselected MDHV-infected-cell mRNA. Furthermore, it will be immunoprecipitated with immune chicken serum, which has both MDHV-A and MDHV-B reactivity. There-fore, intentional coimmunoprecipitation of the two precursor polypeptides was done with immune chicken serum, with and without priorimmunoprecipitation by RotA, to differen-tiate the MDHV-B pr44 from the MDHV-A precursor. The results (data notshown) indicate that the apparent molecular weight of the MDHV-A precursor polypeptide (16, 18) is more appropriately presented as approximately 43 kDa (if ovalbumin marker is reported as 43 kDa), since it migrates veryclose toovalbumin and just below MDHV-B pr44. Now that the actualdifferences in the apparent molecularweights

of these two precursor polypeptides have been shown in the same gel lane, it is clear that MDHV-B pr44canbe identified without confusion.

Immunoprecipitation analysis of the product of cell-free translation of MDHV-infected-cell mRNA, with the mono-clonalantibody 1AN86.17, revealed a precursorpolypeptide

-

pr88

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[image:5.612.70.309.71.329.2]

MDHV-B ANTIGEN GLYCOPROTEIN COMPLEX 4275

_1., ,~,1, ,~

L

1

Q Y

N

N

S

F

pr88-*:@

-

92.5k

-

68k

pr44-

_-43k

FIG. 5. Identification of the [35S]methionine-labeled MDHV-B precursorpolypeptidepr44 bycell-free translation and immunopre-cipitation. Immunoprecipitation and SDS-PAGE analysis of cell-free translation products (*) from infected (INF*) and uninfected (CON*) cellmRNAandoflysates (LYS**) of infectedcells treated with tunicamycin (2 ,jg/ml) by using monoclonal antibody 1AN86.17. Fluorographic exposure was for 8 days. Molecular sizes are asindicated in kilodaltons(k).

of 44 kDa(Fig. 5, lane INF) which comigrated with pr44 seen after immunoprecipitation of lysates labeled with [35S]meth-ioninein the presence of tunicamycin (Fig. 5, lane LYS). In contrast, the larger putative precursor molecule, pr88, also detected by immunoprecipitation analysis of these lysates

from tunicamycin-treated cells (Fig. 5, lane LYS), was not seen after cell-free translation and immunoprecipitation

analysis(Fig. 5, lane INF). This observation establishes pr44 astheprimary product of the MDHV-B gene.

pr88

*_

Formed in

presence

of

Tucmcn

DISCUSSION

Processing of the MDHV-B glycoproteins. The already published detailed study of MDHV-A antigen synthesisand processing (18), which was done with the polyclonal antise-rumRoA,serves asprecedence for similar analyses involv-ing other MDHV-encoded antigens. In the current study, MDHV-B synthesis andprocessing events were elucidated by using similarhigh-resolution pulse-chase studies, a vari-etyofother methodsnew tothe system, andacombination of the polyclonal antibody RaPM and the monoclonal anti-body 1AN86.17. These two sera were useful because they were both known to immunoprecipitate the MDHV-B gly-coproteins gp 100, gp6O, and gp49 and theMDHV-Bputative precursorpolypeptides pr88 and pr44. The results obtained with this combination of approaches and reagents are sum-marized in Fig. 6.

While the pulse-chase studies were set up in a manner verysimilar to that ofstudiesused for MDHV-A (18), some results obtained during MDHV-B processing analysis were very different. First, no immunoprecipitable MDHV-B pu-tative precursorpolypeptides(either pr44orpr88)were seen in the absence oftunicamycin (Fig. 1), apparently because

glycosylationis cotranslational. In contrast, some MDHV-A precursor polypeptide was detected after a 1-min pulse, despite rapid glycosylation kinetics consistentwith cotrans-lationalglycosylation(18). The first MDHV-B moleculeseen was an apparent glycosylation intermediate slightly smaller than gplOO, and eventhen it was present in an immunopre-cipitable form in only trace amounts after a 4-min pulse (eightfold-longer fluorographic exposure of same gel used in Fig. 1). Labeled precursor molecules must have been present in normal amounts from the start, since MDHV-B intermediates and products appeared in expected amounts

later,butthey apparentlyare notaccessible to

immunopre-cipitation at theseearly times.

Presumablyduringcotranslationalglycosylation, the pre-cursor molecules were membrane bound and were not

solubilized efficiently by our detergent buffer; hence the failuretodetectexpected levels ofsignal,aswasfoundtobe thecase with HSV type 1 precursor to gB (43) and withthe latent membraneprotein, Imp63,of Epstein-Barr virus (28). The presumption that MDHV-B precursor polypeptides

N-LINKED GLYCOSYLATlON

Endoglycosidce

F and H

HYPOTHESIZED

DMERIZATION STEP

*gp)OO

Mon

Tr

hhtion

fI2~~~~~~~~~~~~~~~W

pr44

g4Endoglyc

ses F and H

g49

Alsoformed by

Cell-free

tromotin

FIG. 6. MDHV-Bprocessing sequence summary. Heavy arrows denote biological pathway. VOL.62, 1988

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[image:6.612.122.246.77.313.2] [image:6.612.155.484.526.718.2]

were present but undetectable at these early times was

supported by pulse-chasestudiesperformed in thepresence

oftunicamycin (Fig. 4), in which pr88 wasfirstdetected at the 3-min chase after a 4-min pulse, 3-min after a partly

glycosylated form of gplOO appeared in the absence of tunicamycin (Fig. 1). pr44 was first detectedafter a30-min

chase,after which it increased in amountin theabsence of

anyreduction in pr88. This is notthe resultexpectedif pr88 cleavage (degradation) resulted in pr44 formation. Appar-ently pr44 and pr88 are also membrane bound and not solubilized efficiently in our detergent buffer. While this

pulse-chase experiment in the presence oftunicamycin did

notdemonstrateaprecursor-processing relationship

consis-tent with dimerization of pr44 to pr88, as hypothesized

below, that fact cannot be interpreted as evidence against

such asequenceofevents. Insteadit is nowapparent, both in thepresence(Fig. 4) andabsence(Fig. 1)oftunicamycin,

that the earliest appearance ofeach MDHV-B molecule in

the gel is determined by when it becomes available for immunoprecipitation (by a mechanism yet to be deter-mined), notby the actual sequenceof processingevents.

The comparison of early processing events of MDHV-A and -Bthatinvolvedprimarilyglycosylationwarrantsfurther discussion. The heterogeneousMDHV-B molecules slightly smallerthan gplOO that were seen first (Fig. 1) were most probably glycosylation intermediates. While some details differed, suchdiffuse glycosylation intermediates were also

seenwith MDHV-A (18). MDHV-B wasglycosylated to its full-sizedgplOOform aftera3-min chase aftera4-min pulse. Similarly,MDHV-A reached its full-size form aftera6-min chase following a 1-min pulse. Possibly when similarities

occur,suchasglycosylationkinetics, theirexistence reflects

the involvement of the same cellular (duck embryo

fibro-blast) glycosylation mechanism. On the other hand, when differencesoccur,suchasfailuretodetect primaryprecursor

polypeptides (see above), fundamental polypeptide

differ-ences may be involved, since MDHV-A is predominantly

secreted (18), whereas MDHV-B appears to be membrane associated (17).

The pulse-chase studies reported here suggest gplOO is processed to form gp6O and gp49. The amount of gplOO molecule increases to a maximum at 45 min ofchase and then decreases. This decrease coincides very closely with the appearance of gp6O and gp49, in a manner consistent with a precursor-product relationship, thus confirming the

earlier observations ofSilva and Lee (42) and Ikutaet al. (14).Thisinterpretation isfurther substantiated in thisstudy by the observation that monensin inhibited processing of gplOO to gp6O and gp49 (Fig. 2). Use ofthis inhibitor is a majorextension of thepublished work referredtoabove and leadstoa veryimportant conclusionthatgplOOisactuallyan intermediate intheoverall biosynthesis ofMDHV-B. Since processing isslow,and gplOOisanintermediate, someof the moleculeis alwayspresentinthe cell. This conclusionraises

animportantnewquestion. Up untilnow we(17)and others (15, 41) have referred to a complex ofthe three glycopro-teins, designated gplOO, gp6O, and gp49 (17, 41), that we

identifiedasMDHV-B(18). Thesenewdata(summarized in Fig. 6) suggestthat themature forms ofMDHV-B are only gp6O and gp49, since gplOO is apparently an intermediate. Thepreviousinterpretationthatit ispartof thefinalcomplex

canbe understoodwhenoneconsiders the slowprocessing kinetics of gplOO and the long (4-h) labeling periods used previously (17). Possibly the profile of three polypeptides seenpreviously simply reflects the presence ofasignificant

amountofradiolabeled intermediate gplOOunder the

condi-tions

previously

used. On the other

hand,

when chase intervals as

long

as 20 h were used

(data

not

shown),

the amountof

gplOO

was notreducedtozero,

although

lesswas detected thanwasfound after the 4-h chase

presented

in

Fig.

1. Final resolutionof this

question

may

require

determina-tion of the arrangement of MDHV-B

polypeptides

in the infected-cell nuclear and

plasma

membranes.

Endoglycosidase digestion of the MDHV-B

glycoproteins.

The useof

endoglycosidase

digestion

alone and in combina-tion with monensin was also a new

approach

to

studying

MDHV-B

processing

and also

generated

additional informa-tion that contributes to our overall

understanding

of this

system. Thefacts that

gplOO,

andnot

pr88,

wassensitiveto

endo-H and endo-F

(Fig. 3)

and that the former

yields

the latter upon

digestion

suggest that

pr88

is the immediate precursor of

gplOO

and thus is an intermediate in the

biosynthetic

pathway.

The fact that

gp6O

exhibited

partial

sensitivity

to both endo-F and

especially

endo-H indicated

that it had

undergone

some modifications such as the addi-tion of

complex

sugarsor0-linked

glycosylation (9, 20, 21).

At lowerconcentrations of monensin

(.0.5

pLM)

(data

not

shown)andatincreasedtimesofits pretreatment,

gp6O

was

replaced by

a

faster-migrating

molecule

(data

not

shown)

which does not

comigrate

with any of the endo-H- or

endo-F-resistant

gp6O

species

(Fig.

3A and

B).

However,

gp49 was

completely

sensitiveto both endo-F and

endo-H,

indicating

that

only

N-linked sugarswereaddedtothe

gp49

portion

of the moleculebefore

cleavage.

On thebasis ofdata

described

above,

taken

together,

itappearsthat

pr88

under-goescotranslationaladdition of N-linked

high-mannose

sug-ars to

yield

gplOO,

which in turn is

processed

to

gp6O

and

gp49.

Apparently

gp6O

and

gp49

are

differentially

glycosyla-ted,

withgp49

retaining

N-linked

high-mannose

sugars

(en-tirely

endo-H and endo-F

sensitive)

and with

gp6O

presum-ably

undergoing trimming

and

possibly

addition of0-linked and

complex

sugars, as

suggested

by

both the effect of

monensin and

partial

sensitivity

to endo-H andendo-F.

Identification of

pr44

as the

primary

MDHV-B precursor

polypeptide.

The role of

pr44

as the

primary

precursor

polypeptide

becomes clear when the full range ofdatanow

available is considered.

First,

the cell-free-translation data

(Fig. 5) clearly

indicate that

only pr44,

andnot

pr88,

is made invitro. This is not dueto premature termination of

trans-lation,

because othermolecules

larger

than

pr88

aremade in our cell-free translation system.

Also,

the

pr44

comigrates

with thepr44found in cells after

tunicamycin

treatment

(Fig.

5).

Second,

studies on the effects ofprotease inhibitors on

pr44and

pr88

(data

not

shown)

indicate thatboth molecules are made in

vivo,

since the former is not a

degradation

product ofthe latter.

Third,

preliminary

partial

proteolytic

cleavage

analysis

of

pr44

and

pr88

(data

not

shown)

suggests

that the two molecules are related.

Fourth,

the results obtained

by

deglycosylation

of

gp6O

and

gp49

by

endo-Fand -H

(Fig.

3) and the

immunoprecipitation

ofall three mole-cules with the same monoclonal

antibody

(41, 42;

data not

shown)

indicate that

pr44

is the

polypeptide

backbone of each

glycoprotein.

Fifth,

the gene

encoding

MDHV-B has been

identified,

and

analysis by

hybrid

selection,

cell-free

translation,

and

immunoprecipitation

(I.

Sithole,

P. M.

Coussens,

and L. F.

Velicer,

manuscript

in

preparation)

revealed a 44-kDa

polypeptide

that

comigrates

with

pr44

seen after

tunicamycin

treatment.

Sixth,

the fact that

pr44

from both the in vitro and in vivo sources are

immunopre-cipitated

by

thesamemonoclonal

antibody

(Fig.

5)

indicates that

they

areidentical.

Finally,

Northern blot

analysis

witha MDHV-B gene

probe (Sithole

et

al.,

in

preparation)

detected

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MDHV-B ANTIGEN GLYCOPROTEIN COMPLEX 4277

atranscript of 1.8 kilobases, a size appropriate toencode a

polypeptide of 44 kDa, as is the case for the MDHV-A

precursor polypeptide (16). The conclusion based on these

data is that pr44 is the MDHV-B primary gene product.

Thus, it is tempting to speculate (Fig. 6) that the initial processingstepinvolvesformation of pr88asadimer oftwo pr44 molecules. The finding that only pr44, and not pr88, forms in a cell-free-translation system suggests that this hypothesized dimerization may occur on the rough

endo-plasmic reticulum.

Theconceptof glycoprotein oligomerizationisnotwithout precedent in biology. Malek-Hedayat et al. (26) have char-acterized an endogenous lectinfrom cultured soybean cells

whichcanbe found asbotha30-kDamonomeranda60-kDa

dimer. Surprisingly, the dimerwas stable underconditions

such as heating in buffers containing SDS,

,-mercapto-ethanol, andurea. The two forms are interconvertible, with

theextentofinterconversion dependentontheconditionsof

prolonged incubation. The interconversion establishes that the dimer forms by noncovalent association (26). The fact that thedimercanbefoundinrootextractsisconsistentwith the idea that it may be more than an artifact that occurs duringstorage.Glycoprotein B (gB) encoded byHSVcanbe

found in both virions and infected cells in the form of detergent-stable oligomers (7)that have certain similarityto

MDHV-Bwithrespect tothe processing datareported here. Specifically, for both HSV gB (7) and MDHV gplOO, dimer-izationoccurred withinminutesofpolypeptidesynthesis and did not depend on N-linked glycosylation, since their pre-cursors both formed in the presence oftunicamycin. How-ever, HSV gB differs in one important way: it can be

disassociated by heat (7), whereas MDHV-Bpr88andgplOO

canwithstand boiling in SDS sample buffer (17).

Despite this difference it is tempting to speculate that dimerization is a step that is needed before glycosylation, since MDHV-B pr88 forms in the presence oftunicamycin

(Fig. 4). Experiments to resolve questions concerning the detailed nature of the dimerization process, such as the

nature ofthe bonds,the partof the peptideinvolved, andin the case of MDHV-B, the mechanism of separation of the

dimer(i.e., the processingofgplOOto yield gp6O and gp49),

are beyond the scope of the current work on MDHV-B.

Possibly dimerization of pr44 to pr88 and subsequent

proc-essing ofgplOO occur as the molecules move through

dif-ferent environments within the cell, and the existence of MDHV-Bdimersresistanttodissociation by heatrepresents

some difference between the properties of the polypeptides

of MDHV-B and HSV gB unrelatedtosomesortofcommon processing mechanism. However, it must be emphasized thatthenamesofthesetwoherpesvirus polypeptidesinclude the letter designation B for entirely unrelated reasons, not because they are homologs. The comparison ofthese two herpesvirus polypeptidesis thereforeinrelationtoapossible

common glycoprotein-processing mechanism, not because theyare genesconserved between the twoherpesviruses. In fact,werecentlylearned (T. M. O'Tal,P. M. Coussens, and

L. F.Velicer, unpublished data)thattheMDHVhomologof HSVtype 1 gB is located inadifferentregion of the MDHV genome than the geneencoding MDHV-B (Sithole et al., in preparation).

Possiblerole of MDHV-Bin MDHVmembrane biology and immunobiology. Our pulse-chase data (Fig. 1) indicate that

mature MDHV-B may not be the three polypeptides as we

have recently reported (17). Theresults summarized in Fig. 6indicatethat gp49 and gp6Oarethe mature fully processed final form of MDHV-B, whereas gplOO is an intermediate

that is processed slowly, and therefore small amounts are present atall times. Now that we know that gplOO is really an intermediate, and that a slow processing sequence is involved, we can moreaccuratelyplan future experiments to monitor the processing of MDHV-B polypeptides into the nuclear and plasma membranes. We predict thatgp6O and gp49 will both be in the membrane as mature forms of MDHV-B and that they will appear slowly accordingto the kinetics seenfor their processing. This leads to an interesting question: doesgplOO move tothe membrane, where it is then processed, or doesprocessing occurfirst, and then do gp49 and gp6O move to the membrane? On the basis of the monensin data, processing appears to occur in the Golgi apparatus, which would suggest that gplOO is processed to gp60and gp49, which then move to the membrane. Alterna-tively, disruption of the Golgi apparatus (20, 21) by

monen-sincould prevent transport of gpl00 to a site where process-ing can occur. We do not know whether processing is brought about by a cellular or virus-specific protease, or even if proteolysis is involved. Processing ofa larger mole-cule to two smaller molemole-cules has been observed for Rous sarcoma virus (22) and for at least four members of the herpesvirus family, pseudorabies virus (13, 25), bovine her-pesvirus (44), varicella-zoster virus (12), and cytomegalovi-rus (3, 5, 10). In some cases the processing involves

poly-peptide cleavage andformation ofdisulfidelinkages(3, 5, 10,

12, 13, 44). However, gp6O and gp49 are not linked by disulfide bridges (17). In vitro artifactual cleavage has been documented to be cell-line specific for HSV glycoproteins (38, 48). Althoughartifactual cleavage has not been demon-strated in human fibroblasts, gpl60ofhuman

cytomegalovi-rus is cleaved togpll6 and gp55 (5). Inthis MDHV study,all steps involving cell lysis and immunoprecipitation were carried out at4°C inbuffer containing the protease inhibitor phenylmethylsulfonylfluoride to preventnonspecific proteo-lytic cleavage.

The evidence that virus neutralization could be achieved with monoclonal antibody that also immunoprecipitated the three glycoproteins previously identified as the MDHV-B complex (17), as originally presented by Silva and Lee (41)

andIkuta et al. (15), suggests that someform of MDHV-B is the primaryantigen responsible for generating virus-neutral-izing antibody in vivo. Ono et al. (36) reported that

glyco-proteins purified by affinity chromatography with monoclo-nal antibody to gB (their terminology for the B antigen

complex) elicited partial protection against MD. While the relationship between viralneutralization and

immunoprotec-tion remains to be elucidated, it is assumed to be highly

significant (36). On the other hand, cell-mediated immunity

may be a factor as well. In either case the probable mem-brane location of these glycoproteins becomes important.

Determining the relationship of the MDHV-B polypeptides

to the cell membranes is part of the planned future

experi-ments on the kinetics of their insertion into the plasma and nuclearmembranes. Regardlessof the relativeimportance of virus neutralization and cell-mediated immunity, the ques-tion of whether all three glycoproteins are involved in protective immunity or whether just asingle one is involved must beasked. Since they all seem to contain thesamepr44 polypeptide backbone,eitherindividually(gp6Oandgp49)or in a dimer form (gplOO), they should all have the same epitopes that are determined by the polypeptide backbone. Possibly their role willbe determined more by their location inrespect to the surface of relevant membranes. The

signif-icance of the ideas presented above become more apparent when the observation (L. F. Lee, data not shown) that the VOL.62, 1988

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monoclonal antibody used in this study, 1AN86.17, reacts well with the surface of MDHV-infected cells in a membrane immunofluorescence assay is taken into consideration. Clearly the epitope recognized by this antibody is exposed on the cell surface. However, presenting a more complete answer to these questions concerning possible membrane locations and possible epitopes involved inprotective immu-nity is beyond the scope of this article.

ACKNOWLEDGMENTS

We thank R. A. Stringer for technical supportof the project. We also thank R. F. Silva of the U.S. Department of Agriculture Regional Poultry Research Laboratoryforevaluationof thiswork in relation to his own and Paul M. Coussens of the Department of Animal Science formanuscript review.

This research was supported primarilyby a grant from theElsa U. Pardee Foundation and partly by grant 86-CRSR-2-2870 under the Special Research Grants programadministered by theCooperative State Research Services, Sciences and Education, of the U.S. DepartmentofAgriculture.

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