Immediate-early gene expression is sufficient for induction of natural killer cell-mediated lysis of herpes simplex virus type 1-infected fibroblasts.

JOURNALOFVIROLOGY, June 1991,p. 3151-3160 0022-538X/91/063151-10$02.00/0

CopyrightX 1991,American Society forMicrobiology

Immediate-Early

Gene

Expression

Is

Sufficient for Induction

of

Natural Killer

Cell-Mediated

Lysis of Herpes

Simplex

Virus

Type

1-Infected Fibroblasts

PATRICIAFITZGERALD BOCARSLY,l* DONNA M. HOWELL,' LISAPETTERA,1 SHAHRAM

TEHRANI,1

AND CARLOS LOPEZ2

Departmentof Laboratory Medicine andPathology, University of Medicine andDentistryofNewJersey-NewJersey MedicalSchool, Newark, NewJersey

07103-2757,1

and VirologyResearch, LillyCorporate Center,

Indianapolis, Indiana 462852 Received 27July1990/Accepted 7March 1991

Herpes simplex virustype1(HSV-1)-infectedhumanfibroblast (HSV-FS)targetsaresusceptibletolysis by

naturalkiller(NK)cells,whereasuninfected FSareresistanttolysis.Studieswereundertakentodetermine the mechanismof this preferentialsusceptibility.HSV-FSwerenotintrinsically less stable than FS,asdetermined

bya

5"Cr

releaseassayunder hypotonic shock in the presence ofratgranule cytolysin and by sensitivity to anti-human leukocyte antigen classIantibody plus complement. Single-cell assays inagarose demonstrated that although similar numbers oflarge granular lymphocytes bound to the HSV-FS and FS targets, the

conjugateswith HSV-FSwerelysed atamuchhigher frequencythanthose withFS. Theseresultssuggested

thatboth targetsareboundbytheNKcellsbut only theHSV-FS wereable totrigger lysis. The requirement foractive virusexpressionwasdemonstratedbyfailure of emetine-treatedHSV-FStargetsortargetsinfected

withUV-inactivatedHSVtobelysed by NK effectors. Toevaluatethe roleof viral glycoproteins in conferring susceptibilitytolysis, Fabwereprepared fromHSV-l-seropositivesera;these Fabwereunabletoblocklysis

of theHSV-FS. Furthermore, incubationinphosphonoacetic acidfailedtoreduceNK(HSV-FS)activitydespite sharp reductions in viral glycoprotein synthesis. Finally, targets infected with tsLB2 at the nonpermissive

temperature were lysedaswell as orbetter than targets infected with wild-type virus, indicating that HSV immediate-early geneproduct expressionissufficient for conferringsusceptibility tolysis. We conclude that

expression of nonstructural viral proteins or virally induced cellular gene products early in the course of

infectionrather thanstructuralglycoproteinsisrequired forNKlysis ofHSV-FStargets.

Naturalkiller(NK) cellsarecapable of lysingavariety of virus-infected target cells as well as certain tumor targets and normal cells. Because they are relatively nonspecific, NK cells are available immediately as a first-line defense against an invading viral pathogen and are postulated to serve tolimit theextent ofvirusdissemination priortothe

more specific immune responses. In vitro studies with hu-man effector cells have indicated that NK cells can effi-cientlyreduce theextentofreplicationofcertaincytopathic viruses, includingherpes simplex virustype 1(HSV-1), and

adoptive transfer studies with a murine system have indi-cated a role for NK cells against a number ofcytopathic viruses(10).

Thenature of thetargetantigens recognized byNKcells on virus-infectedtargets has remained controversial. Roles forboth the majorstructural glycoproteinG and nonstruc-tural matrix protein M havebeen implicated in the lysis of targets infected with vesicular stomatitis virus (VSV) (32, 37). Bishopetal. (3, 5, 6) reportedthat HSV glycoproteins aretherelevant structuresrecognizedon infectedfibroblast

orWISHepithelialcell targets.However,Borysiewiczetal.

(7)andLopez-Guerreroetal.(31)havefailedtodemonstrate

arole for viralglycoproteins in thelysisofcytomegalovirus (CMV)- or HSV-infected targets, respectively, and have instead implicated a role for early viral genes or a cellular

gene product. Similarly, in a bovine herpesvirus (BHV) system, Cook and Splitter (11) have suggested that viral

*Correspondingauthor.

glycoproteins are not essential for the lysis of targets in-fected with BHV.

Theissueof thetarget structure present oninfectedcells

is potentially complicated by several factors: the nature of

thetarget cells to be infected, the infecting virus itself,the recentfindingsof therequirementfor HLA-DR+ accessory cells for thelysisofmanyinfected targets(2, 18, 33),and the statusof the effectorcells(i.e.,interferon[IFN]preactivated or not). For example, the CD16+ NK cells and the

HLA-DR'

accessorycells,whicharebothrequiredfor thelysisof

HSV-infected fibroblasts(HSV-FS),maypotentially require

different interactions with the virus-infected targets. The studiespresentedinthisarticlewereundertakentoevaluate the requirements for development ofsusceptibility ofNK targets to lysis by human peripheral blood NK cells. We haveevaluated thestatus ofcellularstabilityof the infected targetscomparedwiththatof the uninfectedtargets andthe relative target cell-binding efficiency of the effectors and have examined the role of viralgeneexpressioninconferring susceptibility of the target cells to lysis. In contrast tothe conclusions ofBishopetal. (3, 5, 6),wefindnoevidencefor arequirementfor viralglycoprotein synthesisforthelysisof HSV-FS.

MATERIALSANDMETHODS

Viruses. HSV-1 strain 2931 was originally isolated by C.

Lopez and is grown and assayed on Vero monkey kidney

cells. The temperature-sensitive mutant ofHSV-1, tsLB2

(24),wasobtained fromP. Schaffer andwaspropagatedand

3151

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titered at the

permissive

temperature of

33°C.

VSV was

originally

obtained from N. Ponzio of the New

Jersey

Medical School and was

propagated

and titered on Vero

cells. All virus

preparations

were stored at

-70°C

and

thawed

immediately prior

to use.

Cell lines. Human foreskin fibroblast

(FS)

cells were

prepared

by enzymatic digest

of foreskins from full-term

infants. These cells were grown in Dulbecco's modified

Eagle's

medium

(DME;

Hazelton

Laboratories, Lenexa,

Kans.)

supplemented

with 10% heat-inactivated fetal calf

serum

(FCS;

Hyclone Labs, Logan, Utah),

penicillin,

strep-tomycin,

and

L-glutamine

in a 5%

Co2 atmosphere

in a

humidified incubator. These cells were

passaged

twice

weekly

and are used up to passage 20 to 22. GM-2767

fibroblasts,

which are trisomic for chromosome

21,

were obtained from the Human Mutant Genetic Cell

Repository

(Camden,

N.J.)

and

passaged

likeFS cells. K562 cellswere grown in

suspension

culture in RPMI 1640 medium

supple-mented with 10%

FCS,

penicillin, streptomycin,

L-glu-tamine,

and HEPES

(N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic

acid)

buffer. All cell lines were screened

routinely

for

mycoplasmas

with

Mycotrim

Culture Kits

(Hanna

Biologicals, Alameda, Calif.)

and were

negative

when used in assays.

Preparation

of effector cells. Mononuclear effector cells were

separated

from fresh

heparinized peripheral

blood obtained after informed consent from

healthy

donors

by

centrifugation

on

Lymphoprep

gradients (Accurate

Chemi-cal

Co., Westbury, N.Y.).

Mononuclear cellswere washed and enumerated with a

hemacytometer

or with a Coulter modelZBI electronic

particle

counter.

NK cell assays. NK cell assays were

performed

as de-scribed

previously (15). Briefly, newly

confluentflasks of FS cells wereeither infected with HSV-1 at the

multiplicity

of infection

(MOI)

statedforeach

experiment

for 3 hormock infected.

Simultaneously,

the cellswerelabeledwith150

,uCi

of 51Cr

(as

Na51CrO4;

New

England Nuclear, Boston,

Mass.).

The targetswerethen

trypsinized

andwashed

thor-oughly.

Nonadherent K562 cells were spun down and

la-beled with 51Crinavolumeof300 ,ul ofmediumfor1to3 h. AtotalofS x

103

targetsin 0.1 ml ofRPMI-10% FCSwere

dispensed

into each well of flat-bottomed microtiter plates

(Costar, Cambridge,

Mass.)

which

already

contained

ef-fector cells

plated

in

triplicate

in 0.1 ml to

yield

effector/

target cell

(E:T)

ratios of100:1 to 12:1.

Assay

mixes were

incubated at

37°C

for 14 h in a 5%

CO2

atmosphere in an

incubator,

after which 0.1 ml of the supernatant was

re-movedfrom each well and the

radioactivity

wasdetermined withaPackardGammaCounter.

51Cr

releasewascalculated

asfollows: %

51Cr

release =

[(experimental

cpm -

sponta-neous

cpm)/(total

cpm - spontaneous

cpm)]

x 100; where

spontaneouscpmis theamountof51Cr released

by

targetsin the presenceof medium alone and total cpm isthe amountof

51Cr

released

by

targets incubated in the presence of 5% TritonX-100

detergent. Spontaneous

releasedidnotexceed

30% forHSV-FS and FS targetsor25%for K562 targets. The data

generated

from each NKtest wereanalyzedwith the Von

Krogh equation

(35)andareexpressedasestimated kill atan E:T ratio of 50:1.

IFNassays. Atthetermination of NKcytolyticassays,an additional35

RI

ofsupernatantwascollectedfromeach 50:1 E:T wellfor the determination of antiviral activity.

Super-natantswerestored frozen at-20°C priortoquantitationof IFN

activity

with GM2767 indicator cells and VSV as the

challenge

virus in

cytopathic

effectreduction assays. Each IFN assay mix contained cell controls, virus controls, and

the internationalleukocyteIFN(IFN-ot)reference standard G-023-901-527, obtained from the National Institute of Al-lergy and Infectious Diseases.

Preparation of Fab fractions fromseropositive and

seroneg-ative donors. Fab fractions were prepared from

Sandoglob-ulin(Sandoz, East Hanover,N.J.) and fromserumobtained fromanHSV-1-seropositive and aseronegative donor. The serological status of the donors was determined by the ability of theirserumsamplestoinduceantibody-dependent

cellular cytotoxicity (ADCC) against HSV-FS and FS

tar-gets.Briefly, 20to35 ml ofserum wascollected from each of the donors. The sera were centrifuged and then passed

through a Millex GU filter. Saturated ammonium sulfatewas

added to 40%drop by drop withconstantstirring in thecold;

the mixture was centrifuged, the supernatant wasdecanted, and the pellet was resuspended in PBS anddialyzed

exten-sively against PBS. A DEAE column was prepared and equilibrated with 0.02 M sodium phosphate buffer, pH 8.0. The saturated ammonium sulfate precipitate was dialyzed

overnight in the sodium phosphate startingbuffer, the

mate-rial was passed through the column, and the first 9 ml of eluant was collected and pooled. The material was made isotonic with lOx saline, and protein concentration was

determined by OD280 measurement with a Beckman spec-trophotometer.

For preparation of Fab fractions, 0.5 ml of immobilized papain (Pierce Chemical Co., Rockford, Ill.) was added to 10 mgof immunoglobulin G (IgG) in 1.0 ml of freshly made 20 mMNaH2PH4 buffer containing 20 mM cysteine-HCl and 10

mMsodium EDTA, pH 6.2. The reaction mix was incubated for 5 h at 37°C with rocking, and then 3.0 ml of 1.0 mM Tris-HCl (pH 7.5) wasadded.The mixture was centrifuged, and the supernatant was applied to a 5-ml immobilized protein A (Pierce Chemical Co.) (22) column equilibrated with 10 nM Tris-HCl, pH 7.5. The column was washed with 15 mlof 10 nM Trisbuffer, and the eluate containing Fab was collected. Proteinconcentrations were determined with the BCA assay (Pierce Chemical Co.). The presence of Fab but notFcin the eluatefraction was confirmed by radial immu-nodiffusion (27) with goat anti-human IgG Fab and Fc reagents (Cappel Laboratories, Westchester, Pa.). The

in-tegrity ofthe Fab regions within the Fab preparations was confirmed by indirect immunofluorescence against

HSV-infected fibroblasts. The HSV-FS targets were incubated with the DEAE IgG preparation or the Fab fractions for 30 min at 4°C, followed by incubation for 45 min in a 1:20

dilution of fluorescein isothiocyanate-conjugated goat

anti-mouse IgG (Cappel Laboratories), and were visualized un-derafluorescencemicroscope.

Incubation of targetcells incytolysin. Ratgranulecytolysin

preparedfroma rat LGL tumor cell line was obtained from

Craig Reynolds, National Institutes of Health (25). The granules were stored frozen at -70°C until immediately before use and were thawed and diluted in

Ca2'-

and

Mg2+-free

PBS togiveaconcentrationof 20U/ml.Twofold dilutions of thegranulepreparationwere made in the

Ca2+-and

Mg2'-free

PBS, and 100 ,ul of cytolysin were added to 100

RI

of51Cr-labeled targets (at105 cells per ml,diluted in

RPMI-1%bovine serumalbumin) in round-bottomed plates inreplicates of six. The mixtures were incubated for various timesat37°C and centrifuged at 4°C, and 100 [L of

superna-tant washarvestedfordetermination of 51Cr release. Exposureoftargetstohypotonic shock. Forexperiments to

measure the sensitivity of targets to hypotonic shock, 5 x

10351Cr-labeledtargetcells (50

RI

perwell) were incubated in six replicates in the presence of an additional 150

RI

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NK CELL-MEDIATED LYSIS OF HSV-1-INFECTED TARGETS 3153

fluid ranging from 0 to 62% distilled, deionized water (ddH2O), with the remaining volume made up in medium. The cells were then incubated for 14 h at 37°C, after which thesupernatants were harvested and the percent51Cr release in each group was determined by dividing the mean cpm in each group by the mean cpm of a total release obtained by incubating the target cells in 5% Triton X-100 detergent.

Anti-HLA class I plus complement treatment of target cells. FS targets were infected for either 3 or 14 h or leftuninfected and labeled with 51Cr. Ten thousand targets were plated into replicate wells of round-bottomed microtiter plates and incubated for 30min at 37°C with various dilutions of rabbit anti-human leukocyte antigen (HLA) class I framework antibody (New England Nuclear, Cambridge, Mass.). Baby rabbit complement (1:4) was then added to each well at the concentrations described, and the plates werefurther incu-bated at 37°C for 1 h, after which the microtiter plates were centrifuged at 1,000 rpm for 10 min, and the top 100 ,ul of each well was harvested for determination of51Cr release. 51Cr release was calculated as for NK assays.

Single-cell assays in agarose.Peripheral blood mononuclear cells obtained from Lymphoprepgradients were allowed to adhere to nylon wool columns for 30 min and eluted with warm RPMI-10% FCS. The nylon wool-nonadherent frac-tion was separated on seven-step discontinuous Percoll gradients as described previously (15), and fractions 2 and 3, which wereenriched for large granular lymphocytes and NK activity, were collected. The large granular lymphocyte-enriched fraction was depleted of CD3 (anti-Leu-4)-positive T cells by panning (15). Target cells were prepared at an MOI of 5:1 or were mockinfected as described above. For targetbinding assays (23), targets and effectors were resus-pended at 2 x 106/ml in RPMI-10% FCS and 0.1 ml of effectorcells was incubated with 0.2 ml of target cells at 30 or 4°C for 5 min; the tubes were then centrifuged at 200 x g for 5 min, the residualmedium was decanted, and the cells were gentlyresuspended three or four times with a capillary pipette in a volume of 50

RIu

ofRPMI-10% FCS. To each tube was then added 0.1 ml of molten agarose (1.3% Sea Plaque Agarose; FMC Corp., Rockland, Maine)inRPMI-10% FCS at 39°C, and the solution was gently mixed and spread over microscope slides. After allowing the solution to harden for 2 to 3 min, the slides were placed in a staining dish with RPMI-10% FCS and incubated for 6 h at 37°C in a5%CO2

incubator. At the end of the incubation, the cells were immersed in a trypan blue-PBS solution for 5 min, after which the slides were washed threetimes, 10 min per wash, in a PBS-0.3% Formalin solution. Duplicate slides were prepared for each group. The slides were viewed with a phase-contrast microscope by an individual blind to the experimental conditions, and the percentageofeffector cells adhering to targets was determined bycounting 200 effector cells on each slide. The proportion of lysed targets was

determined by counting the number of targets inconjugates which stained with trypan blue minus the percentage of targets spontaneously staining with trypan blue on slides

incubated without effector cells. Target-effector conjugates were easily discriminated from target-target and effector-effector interactions because the target cells were much larger than the effectors.

Polyacrylamide gel electrophoresis (PAGE). Target cells in

75-cml

flaskswereinfected with HSV-1 orHSVmutantsfor 3 h in the presence or absence of antiviral drugs or mock infected and then incubatedovernightin the presenceof 100 to 300 ,uCi of

[35S]methionine

or

[3H]glucosamine

(New

England Nuclear, Cambridge, Mass.). The cells were then

gentlytrypsinizedandincubatedinlysing buffer(0.05 M Tris

base,

0.6 M KCI, 0.5% TritonX [pH 7.5]) for10minonice. The lysed cells were clarified by centrifugation (32,000 rpm for 90 min). In some experiments, the supernatants were

immunoprecipitated overnightat4°Cwith 100,llof the virus preparation and 0.1 mg of primary antibody, followed by incubation for 2 h on ice with protein A-Sepharose. The

precipitateswerewashed three times in RIPA buffer (0.05 M

Tris-HCl, 1 mMEDTA, 0.15 MNaCl, 0.25%bovine serum albumin [pH 8.0]) and incubated insamplebufferovernight.

The samples were then heated at 100°C for 5 min,

centri-fuged briefly to remove the Sepharose beads, and run on

PAGE withan8.5%resolving gel and3%acrylamide stack-ing gel cross-linked with N,N-diallyltartardiamide (Aldrich Chemical Co., Milwaukee, Wis.) in an amount correspond-ing to4%of the weight of acrylamide. Each liter ofrunning buffer contained 3 gof Tris base, 14.4 g of glycine, and 1 g of SDS.

Treatment of cells with metabolic inhibitors and antiviral drugs. Theacid formofphosphonoacetic acid(PAA; Sigma

Chemical Co., St. Louis, Mo.), which is an inhibitor of HSV-encoded DNA polymerase (26), was used ata concen-tration of300 ,ug/ml and was present throughout the course

of infection and NK assays.

Emetine-HCl(Sigma Chemical Co.), which is an irrevers-ible inhibitor of protein synthesis, was usedat a

concentra-tion 0.1 mM to treat thetarget cells priorto theaddition of virus. Following treatment with emetine for 1 h, cultures were rinsed extensively with PBS and then infected with HSV-1 as described above. Following trypsinization of FS and HSV-FS target cells or the pelleting of K562 cells, the targets receivedfour washes in 15 ml of medium to eliminate anycontaminating emetine.

UV-inactivated virus.UV-inactivated HSV-1 was prepared byirradiating HSV-1 with a UV sterilizing lamp (254 nm) at

a distance of 10 cm to yield 1,340 i±W/cm2 for the times shownin the experiments. The efficacy of the treatmentwas confirmed by titration of the UV-irradiated virus on Vero cell monolayers.

RESULTS

HSV-FS targets are not instrisically less stable than unin-fected targets. In an early study, Brooks et al. (9) demon-strated a correlation between the osmotic stability of a series of rat tumor targets and their susceptibility to lysis by rat splenic NK cells. Onepossible explanation forthe increased susceptibility of HSV-FS over uninfected FS targets was that the infected targets were unable to repair membrane damage due to shutoff of host macromolecular synthesis by

thevirus. Totestthishypothesis, we subjected infected and

uninfected targets tohypotonicshock, rat granule cytolysin, and treatment with anti-HLA class I antibody plus comple-ment. Figure 1A shows the results of a representative experiment in which 51Cr-labeled HSV-FS and FS targets were incubated in the presence of increasing amounts of

distilled water for 14 h (the time of our typical NK assay), after which the supernatants were harvested for determina-tion of51Cr release. The infected targets were found not to

be more sensitive to hypotonic lysis than the uninfected targets. In parallel studies with the same targets, infected targets werepreferentially lysed by effector cells in14-h NK assays (data not shown).

Next we tested thesensitivity of both FS and HSV-FS to

lysis by anti-HLA class I polyclonal antibody and comple-ment in 1-h 51Cr release studies. For these studies,

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3154 FITZGERALD-BOCARSLY ET AL.

A

-i- HSV-FS

-- FS

0 12.5 25 37.5

% Water

[image:4.612.92.270.77.494.2]

50 62.5

TABLE 1. Targetbinding and single-cellassays inagarosea

Target %Conjugates %of conjugates lysedb

cells

4°C

300C

4°C

300C

FS 10.8 ± 2.3 17.5 + 3.2 3.3 ± 0.8 2.0 ± 0.5

HSV-FS 11.8± 3.0 17.7± 0.7 10.7 ± 3.2 8.7 ± 2.8

aLight-density cellsfromPercoll gradients were depleted ofT cells by panning and used in single-cell target-binding assays in which the

target-effectorconjugateswereformedat4 or300C.Conjugatelysiswasdetermined

after 6 hofincubation,with 200cells countedperslide.Data arethemeansof

threeexperiments. Statisticalsignificancewasdetermined by Student'sttest;

P < 0.01 for percent lysed (FS versus HSV-FS); P > 0.1 for percent

conjugates(FSversusHSV-FS).

bPercentageoflysedtargetswithintarget-effector conjugates.

B

._ 100

90

80 70 60

50

40 30 20 10

0

1/i

-in- HSV-FS -e&- FS

.-I

1/80 1/160 1/320 1/640

AntibodyDilution

j

.~~

-m HSV-FS

. O~~-& FS

|

~~~~K562

I 80

70

.r 60A

50

40

s 40

9

30i

20

10k 110

0

1/1 1/2 1/4

Granule Dilution 1/8

FIG. 1. Comparison of intrinsic stability of FS and HSV-FS

targets. (A) HSV-FS and FS targets were incubated in various concentrations ofddH2O for 14 h, after which releaseof51Crinto

the supematants was determined. (B) Susceptibility of HSV-FS (infected overnight) and FS targets tolysis with anti-HLA class I

andcomplement. Targets were incubated for30 minat 37°C with variousconcentrations of anti-HLA class I framework followed by incubation in baby rabbit complement for 1 h. (C) Sensitivity of HSV-FS and FS to treatment with rat granule cytolysin was determinedby incubatingtargetsfor 1 hinCa2+- and Mg2+-free PBS

inthepresenceof dilutions ofcytolysin. Dataforeach experiment

shownarerepresentative ofatleast three similar experiments.

labeledtargets wereincubated with anti-HLA antiserum for 30 min prior to the addition of baby rabbit complement. Figure 1B shows the results ofarepresentative experiment

demonstrating that HSV-FS are not more susceptible to

complement-mediated lysis than arethe FStargets. HSV-FS and FS targets were also tested for their sensi-tivity to lysis by cytolysin, the lytic material purified from

the granules of a rat large granular lymphocytic leukemia (25). A representative experiment in which FS, HSV-FS, and K562 targets were incubated in cytolysin for 1 h is

shown inFig. 1C. FSdemonstrated aslightbutconsistently greatersusceptibilitytolysis bycytolysinthandid HSV-FS.

Compared with K562, both the infected and uninfected FS showed relatively low susceptibility to cytolysin (Fig. 1C)

and required longer incubation times in the presence of

cytolysin tobe lysed (datanot shown).

Together, the data presented in Fig. 1 indicate that

al-thoughthe HSV-FStargetsareproductivelyinfected witha lytic virus andultimately go on to produce infectious viral progeny(16, 30), theirpreferential lysis byhuman NK cells

withinourassayconditionscannotbe explained byagreater

intrinsic instability than exists in their uninfected counter-parts.

We alsoquestionedwhethertrypsinization of targets had anyeffect on thedifferential susceptibility of the targets to lysis since we observed that in wells to which trypsinized uninfectedtargetswereadded,these targets readheredtothe microtiter plates, whereas trypsinized HSV-FS readhered less well to the microtiter wells during the course of the

assay. We therefore compared the lysis of targets which

wereinfectedin75-cm2 flasksfollowedbytrypsinizationand

plating into microtiter plates with lysis of targets when uninfected cells were platedin microtiter wells, allowed to reattachovernight, and theninfected and 51Cr-labeled in the

wells. Each of these infected populations were equally susceptible to lysis, whereas both the recently trypsinized

andovernight-readhered, uninfected targetswereinsensitive toNKlysis (datanotshown.)

Similar numbers of effector cells bind to infected and uninfected FS targets. We havepreviouslydemonstrated that uninfected targetsare efficient coldcompetitorsfor thelysis

of HSV-FS (15). It was therefore of interest to determine whether therewasasimilar leveloftargetbindingof effector cells to FS and HSV-FS. In these experiments, partially

enrichedlargegranular lymphocytepreparationswereused, and the binding of the effectors to the targets and lysis of targets were monitored in single-cell assays. Therewas no significant difference in the binding of the effectors to the HSV-FS and the FS targets(Table 1). Incontrast, ahigher proportion ofthe HSV-FS than ofthe FS targets in conju-gateswerefoundto belysedat6 h.

Active viralexpression is required for making target cells

susceptible to lysis. To determine whether passive viral

expressionwassufficienttomake thetargetcellssusceptible to lysis or whether active viral expression was required, fibroblasttargetswereinfected with eitherHSV-1orHSV-1 whichhad beenUVinactivated for variousamountsof time

(UV-HSV)and thentested for theabilityof the targetstobe

lysed byNKcells(Table 2). Additionally,titersof virus after UV irradiation were determined; by 5 and 10 minof

irradi-08

X

Qx

_L

-50

40

30

I

20

10

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NK CELL-MEDIATED LYSIS OF HSV-1-INFECTED TARGETS 3155

TABLE 2. NK activity against targets infected with UV-inactivatedHSV-la

UVexposure HSV titer HSV-FS

(min) (PFU/ml) % Lysis (SD) IFN-a(IU/ml)

0 5.0 x 106 20.0 (3.6) 300

1 1.0 X 103 23.0 (3.0) 300

5 <10 11.0 (4.0) 100

10 <10 6.4(3.0)b 300

a HSV-1wasirradiated witha UV lamp at 1,380

p.W/cm2

forthe times listed andusedtoinfect FS.Values are percentlysisat an E:T ratio of 50:1 following

VonKroghanalysis for sixseparate blood donors. IFNgenerationin response

to HSV-FS and virus titers following irradiation are shown for a

repre-sentativeexperiment.

bp = 0.05 versus lysis of uninfected FS (Student's t test); lysis of

uninfected FSwas3.2%+ 1.5%, andtheyinduced<3 IU of IFN-a perml.

ation, infectious virus was no longer observed. Targets infected with UV-HSV were not lysed by the effector cells; thiseffect was directly related to the dose of UV, and NK susceptibility was reduced almost to the level of uninfected cellkilling after irradiation for10 min. In contrast, infection

oftargets with UV-inactivated virus failed to decrease the production of IFN-a by the effector cell population which was induced by those targets (Table 2). The inability of UV-inactivated virus to confer NK susceptibility to FS targets was observed at awide range of MOIs. Even at an

MOI of20:1,UV-inactivatedvirus failed to make the targets

susceptibletolysis (data not shown).

Further evidence for the requirement of active viral

expression for making infected targets susceptible to lysis

was obtained by treating FS targets with the irreversible proteininhibitoremetine priortoinfection with HSV-1. FS targetcellswereincubatedwith 0.1 mM emetine for1h, and the cultures were washed extensively and infected. As a

control, K562 tumor targets were incubated in emetine for thesameperiod of time. At the end of infection and labeling with 51Cr, the targets were thoroughly washed and usedin 14-h NK assays. The efficacy of emetine treatment was confirmed by PAGE; protein synthesis was completely

turned off in the infected targetsin the presenceof emetine (Fig. 2). Inhibition ofprotein synthesis by emetine in the K562 and HSV-FS targets was further confirmed by mea-surement of[3H]leucine incorporation into these targets. A 1-htreatmentofthe targets withemetine resulted ina94.6%

reductionin theincorporation of[3H]leucine in the emetine-treated versusuntreated K562 targets anda100%reduction in the emetine-treated versus untreated HSV-FS targets. Treatment of the FS targets with emetinepriortoinfection resulted ina dramatic reduction in NKactivity against the infected FS (Table 3). In contrast, lysis of K562 cells was

unaffected by thistreatment.

Roleof viral glycoproteinsinmaking HSV-infected targets susceptible to lysis. Bishop et al. (3, 5, 6)have suggested a rolefor viralglycoproteins astargetstructures for NKlysis

of HSV-infected endothelial cells. They described lower

lysis oftargets infected with viralmutants which underpro-ducedglycoprotein C(gC) orgBandwere

partially

ableto block NK lysis by the addition of Fab fragments directed

against viral glycoproteins. To evaluate the role of viral

glycoproteinsinmakingviral targetssusceptibletolysis,we

used Fab fragments of serum IgG from

HSV-seropositive

and seronegative donors and PAA treatment of infected cultures toprevent viral

glycoprotein expression.

Fab frac-tions were prepared by papain digestion ofa commercial

EXPI EXP2

E I E

<-gB,gC

<-gD

46

-FIG. 2. SDS-PAGE analysis of emetine-treated, HSV-infected targets. SDS-PAGE analysis of two experiments with 0.1 mM emetine treatment of HSV-FS targets is presented. Targets were treatedwith emetine for 1h, washed,and then infected with HSV-1

at anMOI of 1. Cultures werelabeled with[35S]methionine, lysed, and immunoprecipitated with a polyclonal anti-HSV antiserum.

Lanes: MW, 14C-labeled molecularmassstandards(shownin kilo-daltons);I,culturesinfected in the absence ofemetine; E,cultures infectedin the presence of emetine.

pooled

human serum

gammaglobulin

(Sandoglobulin)

or

froman

HSV-1-seropositive

and a

seronegative

donor. The presenceof Fab butnotFcfractionswasconfirmed

by

radial immunodiffusion. Intact

recognition

of viral

proteins by

the Fab

prepared

fromthe

seropositive

butnotthe

seronegative

donorwas confirmed

by

indirect immunofluorescence with

HSV-FS;

intact

Sandoglobulin

andthe

immunoglobulin

pre-pared

from the

seropositive

donor and Fab

fragments

pre-pared

from these

immunoglobulins

gave strongsurface im-munofluorescence

staining

of HSV-FS but not FS targets, whereas neither the

immunoglobulin

nor the Fab from the

seronegative

donor gave

positive

fluorescence. The intact

IgG

fractionfrom both the

Sandoglobulin

and the

seroposi-tive donor but not the

seronegative

donor was

capable

of

inducing

ADCC

against

HSV-infected targets.

However,

TABLE 3. Effect of emetineonNKactivitya

%Lysis

Expt Donor HSV-FS K562 +

HSV-FS +

emetine

K562 emetine

1 1 33.8 3.7 62.8 66.2

2 33.2 3.9 72.0 73.9

2 1 20.7 7.7 58.5 68.7

2 31.1 7.0 52.7 55.9

Mean(SEM) 29.7(3.1) 5.6(1.0)b 61.5(4.1) 66.2(3.8)c

aTargetcellswereinfectedin the presenceofemetine(0.1mM)for1 hand

then washedextensivelybefore beinginfectedorusedinthe assay. Lysis

valuesareforanE:Tratioof 50:1 for fourseparatemononuclear cell donors

(two in eachexperiment). Protein synthesiswasmonitoredby [3Hlleucine

uptake. Emetine treatment of K562 cells and HSV-FS decreased protein synthesis by96.4and100%,respectively. Failure ofemetine-treatedFSto replicateHSVwasalsoconfirmedbyPAGE(Fig.2).

bSignificantly different from lysis ofHSV-FSin theabsence of emetine (P=0.0003).

cNotsignificantlydifferentfrom

lysis

of K562 in the absenceof emetine.

VOL.65, 1991

mw I

IRP-111,

A.

".,

". joibk,b

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70 60

,50

-a40

20

10

0

80 70 60

~.5

dE40

a

20

10 n

MW U I +PAA

A

- ii1'

L + Fab

200

-97

-69

-MIl'l 500 166 55.5

(IgGorFab) (jg/mi)

18.5 6.2

B -in- IgG Seroneg.

-/- Fab Seroneg.

-/- IgG Seropos.

-e/- Fab Seropos.

iI

X

it

I 500 100 20 4

(IgGorFab) (jg/ml)

FIG. 3. Fab prepared from HSV-seropositive serafail toblock NK(HSV-FS) activity. IgG and Fabwereprepared from Sandoglo-bulin(A) and from HSV seropositiveandseronegativedonors(B). The IgG and Fab were added at the concentrations shown to HSV-FStargetsand effectorsatanE:Tratio of 50:1. Percent 51Cr

releasewasdetermined in 14-hassays.(A) Dataarerepresentative of four similar experiments. (B) Data are representative of two experiments. Lysis of uninfected FSwas -5%.

when the Fab fractions wereaddedtotheNKassays, they neither suppressed nor augmented the response at any dilution (Fig. 3). Thus, we have failed to reproduce the

resultsof Bishopetal. (6)inoursystem.

PAA is a specific inhibitor of the virally encoded DNA

polymerase (26). Inthe absence of viral DNAreplication, it has been demonstrated thatlittleorno HSV-1gamma gene

products are expressed by infected cells. We therefore infected FS cellsandperformedNKassaysin thepresence of PAA(300,ug/ml). SDS-PAGE results confirmed that viral

glycoprotein bandsweresharplyreduced in the PAA-treated HSV-infected fibroblasts (Fig. 4). NK activity against the HSV-FS targets was not reduced and in some cases was augmented bythetreatmentwith PAA(Table 4).Thelysisof K562 cells and uninfected FS was also not affected by treatmentwith PAA (datanot shown). Furthermore, treat-mentwith PAAhadnosignificant effectonspontaneous51Cr

release valuesfor anyof thetargets.

Role ofHSV-1 immediate-early geneexpression in confer-ring targetsusceptibility tolysis. Convincing evidence fora roleforearlyviralgeneexpressioninconferring susceptibil-ity ofvirus-infected targets tolysis by both NK and cyto-toxic T cells has been developed recently (8, 12, 31). To evaluate the role of immediate-early gene expression in HSV-1-infected targets, we used tsLB2, a temperature-sensitive HSVmutantwhichatthenonpermissive

tempera-<-gB,gC

. c-<-gD

46

-30

-FIG. 4. SDS-PAGEanalysis ofPAA-treated, HSV-infected

tar-gets. Targets were infected with HSV-1 at an MOI of 1 in the continuous presence of PAA (300 ,ug/ml) in low-glucose DME medium.The targetswerelabeledovernight with[3H]glucosamine,

lysed, immunoprecipitated withapolyclonalanti-HSV antiserum,

run onSDS-PAGEgels, anddevelopedbyautoradiography.Lanes: MW, 14C-labeledmolecularmassstandards(showninkilodaltons); U, uninfectedFS:I,FS;infectedintheabsence ofPAA;+PAA,FS infected in thepresenceofPAA.

ture(39°C)failstomake betaand gamma geneproductsand overproducesthealphagene

product

ICP4(13,14).At33°C,

however, viralreplication proceeds normallyand infectious progenyareproduced. Targetswereinfectedat33 and39°C

with either tsLB2orwild-type2931virus,and the NK assays were carried out at these temperatures as well. At the

permissivetemperature,bothsetsof targetswerekilledtoan equivalentextentbythe NKeffectors.Atthe

nonpermissive

temperature, however, targets infected with tsLB2 were killedas well as or even more efficiently than the FS cells infected with the wild-type virus (Table 5). The failure of tsLB2 to produce normal levels of structuralglycoproteins

[image:6.612.53.302.72.378.2]

and

overproduction

of ICP4 at

39°C

were confirmed by SDS-PAGE

(Fig.

5).

TABLE 4. Effectof PAAonlysis of HSV-FSa

% Lysis

Donor MOI 0.2:1 MOI 1.0:1

-PAA +PAA -PAA +PAA

1 35.6 34.7 56.7 51.6

2 60.7 50.8 74.9 67.9

3 35.4 50.8 34.8 37.8

4 33.9 37.0 28.1 33.8

5 25.9 35.0 27.8 34.2

Mean(SEM) 38.3(5.9) 41.7(3.8) 44.5 (9.3) 45.1(6.6)

a PAA (300 ,ug/ml) was present throughout infection and NK assays.

HSV-FStargets wereprepared at an MOIof0.2:1or 1:1. Datarepresent

estimatedlysisatan E:Tratio of 50:1followingvonKroghanalysis.There

was nostatistical difference(P>0.05,Student's ttest)with orwithoutPAA

ateach MOI.Lysisof uninfected FSwasalways.5%.

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NK CELL-MEDIATED LYSIS OF HSV-1-INFECTED TARGETS 3157

TABLE 5. NKactivityagainstFStargetsinfectedwithtsLB2a

Target cells

TTemp

% Lysis Virus titer

(') Donor 1 Donor 2 (F/l

2931 33 18.3 21.6 1.5 x 105

39 15.6 19.8 1.2 x 105

tsLB2 33 21.1 24.7 1.2 x 105

39 31.1 33.7 0.2 x 102

Uninfected 33 7.8 6.6 NAb

39 4.6 4.8 NA

aFS targets were infected with tsLB2 or HSV-1 strain 2931 (MOI, 1:1) at 33 or39°C, and the entireexperiment was carriedout atthese temperatures.NK

activity and virus replication in spontaneous release wells were measured after 14 h of incubation. NK data are expressed for an E:T ratio of 50:1 following von Krogh analysis.Data arefromoneexperiment representative of six similar experiments.

bNA, notapplicable.

Effect of 20-h preinfection of targets on susceptibility of HSV-FS to NK lysis. The majority of the NK studies of

Bishop et al. (3-6) wereperformed with targets which had

been infectedovernight with HSV-1 before being used inNK assays. In contrast, our studies havebeen performed with 3-h-infected targets followed by 14-h NK assays. We have now directly compared 3- and 20-h-infected targets for susceptibility to lysis in 14-h NK assays (Table 6). The

viability of the targetspostinfection was found to beidentical forthe 3- and20-h-infectedtargets

(>95%),

asdeterminedby

trypan blue exclusion. Moreover, spontaneous release val-uesfromthe 3- and20-h-infectedtargets were

indistinguish-able at the endof14h NK assays(datanotshown).When we

compared NK lysis ofthe 3-h and 20-h targets, however,

there was asignificant decrease in NK susceptibility ofthe

20-h-infected targets; for some donors, the 20-h-infected

targets were lysed toextremely low levels, whereas some

donorsshowed lesser degrees ofsuppression. Thedecreased sensitivity of the 20-h-infected targets occurred despite equivalent induction ofIFN

production

andan evengreater

surface immunofluorescence for HSV

glycoproteins

than

Unif 2931 hLB2

MW 330 390 330 390 330 390

200- <-ICP4

4 w " <-gB,gC

95.

-H

V

2931

at

<- gD

55-

"

''R'

FIG. 5. SDS-PAGE analysis of targets infected with tsLB2 or HSV 2931 atpermissiveand nonpermissive temperatures. Targets

wereuninfected(Uninf)orinfected with HSV-1 2931 or tsLB2 at an

MOI of1 at33or39°C, labeled with[35 ]methioninein methionine-free medium, lysed, and run without immunoprecipitation on an

SDS-PAGEgel. Sizes are shown in kilodaltons.

TABLE 6. Extendedinfection of targets decreasessensitivityof HSV-FStolysisa

3-h-infected targets 20-h-infectedtargets

Donor IFN IFN

%Lysis generated %Lysis generated

(IU/ml) (IU/ml)

1 30.2 300 13.1 200

2 42.0 300 23.2 200

3 18.4 1,000 3.9 1,000

4 16.2 3,000 5.0 3,000

5 26.6 700 17.6 700

6 15.1 1,000 6.8 1,000

7 26.6 1,000 21.0 1,000

8 26.0 1,000 16.8 1,000

9 41.4 10,000 21.1 10,000

10 28.2 300 17.9 300

11 22.9 300 6.6 300

12 19.3 300 20.4 300

x(SD) 26.1(8.8) 780 14.5(7.1)b 729

aFS targetswereinfected with HSV-1 for3or20hat anMOI of1andthen

usedin 14-h NK assays. Data represent estimatedlysisat anE:Tratio of 50:1

followingvonKroghanalysisand IFNgeneratedatanE:Tratio of 50:1 for12

different donors. Arithmetic mean NK activity and geometric mean IFN productionareshown in the lastline. Uninfected FSlysiswas '10%for all donors.

bSignificantlydifferent fromlysisof 3-h-infectedtargetsasdeterminedby

Student'sttest(P<0.05).

observed for the 3-h-infected targets when stained with

anti-HSV antiserum.

HSV-seropositive

serum induces ADCC but blocks induc-tionofIFN.Serumfrom

HSV-1-seropositive

or

seronegative

individuals orreconstituted

Sandoglobulin

immune

globulin

were added to 14-h NK cells

against

HSV-FS targets.

Sandoglobulin

andserumfrom

seropositive

butnot

seroneg-ative donors

augmented

lysis

of the HSV-FS targets via

ADCC. Incontrast, IFN

production

in the presence ofthe

HSV-immunesera was

depressed,

whereas

HSV-seronega-tiveserumhadnoeffectonIFN

production (Table 7). Thus,

antibodies

against

viral

proteins expressed

on the cell

sur-face blocked the

ability

ofthe infected cells to

provide

an

inductive

signal

tothe

IFN-producing

cells.

DISCUSSION

In this

study,

we have examined the mechanism of the

preferential

lysis

of HSV-infected fibroblasts over their

uninfected counterparts. Since HSV-1

efficiently

turns

off

hostcellmacromolecular

synthesis (28),

itwas

possible

that the increased

lysis

ofthe infected targets could be duetoan

inherent

fragility

ofthese targets and an

inability

to

repair

membrane

damage.

Using

three different measurements of

sensitivity

to

lysis,

wehavefailedtodemonstrateincreased

sensitivity

ofthe HSV-FS:

they

showedsimilar

sensitivity

to

hypotonic

shockandto treatmentwithanti-HLAclass I

plus

complement

and were not more

susceptible

to rat

granule

cytolysin,

the

lytic

material from a rat

large

granular

lym-phocyte

tumorline

(25)

(Fig. 1).

The

sensitivity

to

lysis

by

cytolysin

of both HSV-FS and FS targets was far

lower,

however,

than thatof

K562,

a

finding

which is in

agreement

with the report of the relative

insensitivity

offibroblasts to the action of

cytolysin.

We also observed no

significant

difference in the percentageof effector cells which boundto HSV-FS andFS targets, whereas the HSV-FS

targets

were

preferentially

lysed

(Table 1).

These results

support

our

previous

observationsthatuninfected FStargetsareefficient

VOL.65, 1991

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TABLE 7. Suppression ofIFNproductionbyHSV-immune but notnonimmuneserum7

Expt Serum or Amt %Lysis' IFN-aproducedd

antibodyaddedb (>tg/ml) s (IU/ml)

1 None 26.9 3,000

Seropositive 46.2 100

2 None 37.4 10,000

Seropositive 49.3 1,000

3 None 27.5 3,000

Seropositive 67.2 700

53.0 700

Seronegative 31.8 3,000

23.0 7,000

4 None 23.8 10,000

Sandoglobulin 750 52.6 300

75 58.4 100

S None 37.7 1,000

Sandoglobulin 750 80.7 <3

75 79.9 <3

6 None 35.7 10,000

Sandoglobulin 750 71.4 <3

75 71.4 30

a Serum(5 ,ul) fromHSV-immune(seropositive)ornonimmune (seronega-tive) donorswasaddedtoNK(HSV-FS)assays at thebeginning ofculture.

Sandoglobulinwasaddedto theassaysat afinalconcentration of 750 or 75

jig/ml.Lysis of uninfected FSwas -5%.

bInexperiment 3,serafromtwoeachseropositiveandseronegative donors

weretested.

c NK or ADCC activity at an E:T ratio of 50:1 following von Krogh

analysis.

dIFNproductionat anE:Tratioof 50:1.

coldcompetitorsfor thelysisof HSV-FS targets(15)and the resultsof Borysiewicz etal.,who demonstratedcompetition between CMV-infected and uninfected fibroblasts(7,8).Our

competition and targetbinding results support the concept thatthe initial recognition structureis presenton the

unin-fected targets, whereas the infected targets are selectively triggered to preferentially lyse theinfected targets. In con-trast to ourtarget-binding results, Borysiewiczet al. (7, 8)

have reported a significantly greater (approximately

50%)

bindingofeffector cells to human CMV-infected fibroblasts thantouninfected fibroblasts. These latterresults, however,

were obtained with IFN-pretreated effectors, which may have influenced thetarget-binding profile.

The role ofactiveexpression of virus inmakingthe targets

susceptible to lysis was evaluated by using UV-irradiated virus toinfect targets. Our results indicate that adsorption of the UV-damaged virus to the FS targets was insufficient to make the target cells susceptible to lysis. In contrast, the

UV-inactivated virus induced levels of IFN-a similar to thoseinduced by nonirradiated virus. It has previously been suggested that lysis ofvirus-infected targets occurs

nonspe-cificallyandis due to promiscuous killing by NK cells which have been activated by the IFN induced by the viral infec-tion (38, 39). However, the observainfec-tion that high levels of IFN wereinduced by UV-treatedvirus-infected targets but thatthese targets were notlysedindicates that IFN induc-tion alone is insufficient to lead to

lysis

of the fibroblast targets (Table 2). Rather, active expression of the viral genome,perhaps leadingtotheinductionofatrigger

struc-tureontheinfectedtargets, is alsorequired. That

preferen-tiallysis of virus-infected targets is not due solelyto

IFN-induced NK cytotoxicity is further supported by the

observationsthatIFN-pretreatedeffectors stillpreferentially

lyse virus-infected over uninfected targets (4, 17) and that

inclusion ofanti-IFN antiserum in NK assays failstoreduce

lysis ofthevirus-infectedtargetsdespite neutralizing detect-able supernatant IFN(2, 17).

The

suppression

ofNK(HSV-FS) but notNK(K562)

ac-tivity by emetine (Fig. 2, Table 3) further indicates that active viral expression is required to make the virally

in-fected targets susceptible to lysis. Protein synthesis

inhibi-tion

by

emetine,

as measured

by

[3H]leucine uptake, was

found tobe similarfor the HSV-FS and K562 targets, ruling out a differential effect of the emetine on the two targets. These results confirm and extend those ofBishop etal.

(3),

who demonstrated that 18-h treatment of HSV-infected targets with emetine also diminished NK susceptibility.

Theseinvestigators, however,failedtoreportcontrol exper-iments with anoninfected NK target cell such as K562. This is an important control, sinceblockade of protein synthesis

in targetsforsuch an extendedperiod prior to the NK assay would also be expected to affect cellular proteinturnover.

Althoughboth the seropositive antiserum and

Sandoglob-ulin(but not the seronegative serum) induced ADCC of the HSV-FS targets, the Fabfailed to block cytotoxicitydespite theirretainingtheability torecognize virus-infected targets. This failure to inhibit lysis is in contrast to the partial inhibition observed by Bishop et al. (6) with Fab prepared

from a seropositive donor oramonoclonal antibody to gBor

gC.

Further evidence for alack of requirement for structural glycoproteins comes from our data demonstrating intact NK(HSV-FS) activity in the presence of PAA (Table 4) and with the temperature-sensitive mutant tsLB2 (Table 5) de-spite the reduced production of viral glycoproteins. These results are similar to those indicating a lack ofrequirement for structural glycoproteins for the lysis of CMV-infected human fibroblasts (8) or BHV-infected fibroblasts (11). The data we haveobtained with the tsLB2 mutant at the nonper-missive temperature further indicate that expression of the immediate-early genes of HSV is sufficient to make the targets susceptible to lysis. In fact, the tsLB2-infected targets were killed better at the nonpermissive temperature than the targets infected with wild-type virus despite the failure of the tsLB2 virus to replicate or express structural glycoproteins (Table 5, Fig. 5). These results are consistent with the observation that expression of immediate-early gene products is sufficient to induce lysis of CMV-infected FS targets (8).

Our findings of a lack of requirement for structural glyco-proteins of HSV-infected targets for conferringsusceptibility to NK lysis contradict those of Bishop et al. (3, 5, 6). One difference in our assay systems is that they routinely used targets that were infected overnight, whereas we used acutely infected targets. However, when we directly com-pared 3- and 20-h-infected targets in 14-h NK assays, we found that the 20-h-infected targets demonstrated signifi-cantly less susceptibility to NK lysis despite strong induc-tion of IFN by these targets and high levels of cell surface viral glycoproteins (Table 6). These results again argue for NKrecognition of a relatively early event in viral replication (whether it be a cellular or viral gene product) which is at least partially downregulated following 20 h of infection. Studies are under way to more fully characterize the bio-chemical differences between the 3- and 20-h-infected tar-gets.

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NK CELL-MEDIATED LYSIS OF HSV-1-INFECTED TARGETS 3159

Although we have demonstrated that expression of the

HSV genome is required to make the infected targets sus-ceptible to lysis, the nature of the target signal and/or

triggeringmolecules remains unclear. It is possible that the immediate-early gene products are the actual target struc-tures recognized by NK cells. Peptide sequences of

imme-diate-earlygeneproductsofanumber of viruses are known to be capable of inducing cytolytic T cells. These peptide sequences are thought to associate with major

histocompat-ibility

complex (MHC) antigens inside the cell and to be transported to the cell surface. It is possible that some

similar mechanism might occur for the recognition of

in-fected cells by NK cells; however, NK cells are not MHC restricted, nor would they be expected to have the same degree of fine specificity as cytotoxic T cells. Alternatively, expressionof immediate-early gene products of HSV, some

of which are known to affect cellular gene expression (13, 20), may be altering a cellular gene product (either upregu-lating, downreguupregu-lating, or modifying it) that may influence targetsusceptibility.

NK lysis was found to correlate with increased total and recycling transferrin receptor expression by CMV-infected targets by Borysiewicz et al. (7); however, these authors concluded that the NK target structure was not the transfer-rin receptor itself, since they were unable to inhibit lysis with either iron-saturated transferrin or affinity-purified transfer-rin receptor. Lopez-Guerrero et al. (31) have also implicated host cell transferrin receptor in the killing process of both tumor and virus-infected targets, yet were unable to block lysis of the HSV-infected targets with certain anti-transferrin receptor monoclonal antibodies which were able to block lysis of the tumor targets. These authors have also demon-strated that inhibition of N-linked glycosylation of HSV viral glycoproteins leads to decreased sensitivity of the targets to lysis, yet, like us, were unable to correlate sensitivity with levels of surface expression of viral glycoproteins B, C, and D

(31).

The conflicting data in the literature regarding the relative role of viral structural proteins, immediate-early genes, and host factors in making HSV- or CMV-infected targets sus-ceptible to lysis may reflect different requirements for acti-vation of the

CD16+

effector cells and HLA-DR+ accessory cells, both of which are required for lysis of HSV-FS and CMV-FS (2, 18, 33). Our data with UV-inactivated virus indicates that input viral glycoproteins themselves are suffi-cient to induce IFN production by the

HLA-DR+

accessory cells but not NK susceptibility of the targets. Similarly, blocking of viral glycoproteins with antibody led to a dra-matic suppression of IFN production (Table 7). We predict that Fab against viral glycoproteins would also decrease IFN production, but we have not yet performed these experi-ments. In one report, blocking of gD with monoclonal antibody was found to result in a decrease in IFN production (3). In the case of tsLB2-infected targets or assays per-formed in the presence of PAA, we observed levels of induced IFN similar to those obtained with wild-type virus (data not shown), again indicating that input virus is suffi-cient to stimulate IFN production. Thus, we propose that stimulation of accessory cell activity may be dependent upon viral glycoprotein expression, whereas the NK cells them-selves require recognition of the infected target cell, for which de novo immediate-early gene expression, either directly or indirectly, is required. Studies are under way to evaluate the relative roles of specific immediate-early gene products of HSV in making targets susceptible to lysis, utilizing newly available HSV deletion mutants and vectors

containing specific HSV-1 genes. In addition, the role of

cellular proteins which are up- or downregulated by HSV

(e.g., heatshockproteins, which areupregulated,andMHC

antigens, which aredownregulated) in HSV-infectedcellsis being evaluated.

ACKNOWLEDGMENTS

Wethank JoanMack fortyping themanuscript, Michael Feldman for preparingthefigures,andMonicaMendelsohnandShelleyCurl for technicalassistance.

This study wassupportedUSPHSgrantCA42093fromthe NCI.

REFERENCES

1. Abb, J., H.Abb,and F. Deinhardt. 1983. Phenotypeofhuman alpha-interferon producingleucocytesidentifiedby monoclonal antibodies. Clin. Exp. Immunol.52:179-184.

2. Bandyopadhyay,S., B.Perussia,G.Trinchieri,D.S.Miller,and S. Starr. 1986. Requirement forHLA-DR+ accessory cells in natural killing ofcytomegalovirus-infected fibroblasts. J. Exp. Med. 164:180-195.

3. Bishop,G. A., J.Glorioso,and S. A. Schwartz. 1983. Relation-shipbetween expression ofherpes simplex virusglycoproteins andsusceptibility oftargetcellstohumannatural killeractivity.

J. Exp. Med. 157:1544-1561.

4. Bishop,G. A., J. C. Glorioso,and S. A. Schwartz. 1983. Roleof interferon in human natural killer activity against target cells infected withHSV-1. J. Immunol. 131:1849-1853.

5. Bishop, G. A., G. Kumel, S. A. Schwartz, andJ. C. Glorioso. 1986.Specificityof humankiller cellsinlimitingdilution culture fordeterminantsofherpessimplex virustype 1glycoproteins. J. Virol. 57:294-300.

6. Bishop, G. A., S. D.Marlin,S. A. Schwartz,andJ. C.Glorioso. 1984. Human natural killer cell recognition ofherpes simplex

virus type 1 glycoproteins: specificity analysis with the use of monoclonal antibodies and antigenic variants. J. Immunol. 133:2206-2214.

7. Borysiewicz, L. K., S. Graham, and J. G. P. Sissons. 1986. Human natural killer cell lysis of virus-infected cells. Relation-ship toexpression of thetransferrinreceptor. Eur. J. Immunol. 16:405-411.

8. Borysiewicz, L. K., B. Rodgers, S. Morris, S. Graham, and J. G. P. Sissons. 1985.Lysisofhumancytomegalovirus-infected fibroblasts by naturalkiller cells: demonstration ofan interfer-on-independent component requiring expression ofearly viral proteins and characterization of effector cells. J. Immunol. 134:2695-2701.

9. Brooks, G. C., E. A. Wayner, P. J. Webb, J. D. Gray, S. Kenwick,and R. W. Baldwin. 1981. Thespecificityofratnatural killer cells and cytotoxic macrophages on solid tumor derived target cells and selected variants. J. Immunol. 127:2477-2483. 10. Bukowski,J. F., J. F. Warner, G. Dennert, and R. M. Welsh.

1985. Adoptive transfer studies demonstrating the antiviral effect ofnatural killer cells in vivo. J. Exp. Med. 161:40-52. 11. Cook, C. G., and G. A. Splitter. 1989. Characterization of

bovine mononuclear cell populations with natural

cytolytic

activity against bovineherpesvirus 1-infected cells. Cell. Immu-nol. 120:240-249.

12. Cook,J. L., D.May,A.Lewis,andT.Walker. 1987.Adenovirus ElA gene induction ofsusceptibility to lysis by natural killer cells and activated macrophages in infected rodent cells. J. Virol. 61:3510.

13. Estridge, J. K., L. M. Kemp, N. B. La Thangue, B. S. Mann, A. S.Tyms, and D. S.Latchman.1989. Theherpessimplexvirus type 1immediate-early protein ICP27isobligatelyrequired for the accumulation of a cellular protein during viral infection. Virology 168:67-72.

14. Feldman, M., and P. Fitzgerald-Bocarsly. 1990.

Sequential

en-richment and immunocytochemical visualization ofhuman in-terferon-a producing cells. J. InterferonRes. 10:435-446. 15. Fitzgerald,P.A.,R.Evans,D.Kirkpatrick,andC.Lopez.1983.

Heterogeneity ofhumanNKcells: comparisonof effectorsthat lyse HSV-1-infected fibroblasts and K562

erythroleukemia

tar-VOL.65, 1991

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