Immediate-Early Gene Expression Is Sufficient for Induction of

JOURNAL OF VIROLOGY, June 1991, p. 3151-31600022-538X/91/063151-10$02.00/0Copyright X 1991, American Society for Microbiology

Immediate-Early Gene Expression Is Sufficient for Inductionof Natural Killer Cell-Mediated Lysis of Herpes Simplex Virus

Type 1-Infected FibroblastsPATRICIA FITZGERALD BOCARSLY,l* DONNA M. HOWELL,' LISA PETTERA,1 SHAHRAM TEHRANI,1

AND CARLOS LOPEZ2

Department of Laboratory Medicine and Pathology, University of Medicine and Dentistry ofNew Jersey-New JerseyMedical School, Newark, New Jersey 07103-2757,1 and Virology Research, Lilly Corporate Center,

Indianapolis, Indiana 462852

Received 27 July 1990/Accepted 7 March 1991

Herpes simplex virus type 1 (HSV-1)-infected human fibroblast (HSV-FS) targets are susceptible to lysis bynatural killer (NK) cells, whereas uninfected FS are resistant to lysis. Studies were undertaken to determine themechanism of this preferential susceptibility. HSV-FS were not intrinsically less stable than FS, as determinedby a 5"Cr release assay under hypotonic shock in the presence of rat granule cytolysin and by sensitivity toanti-human leukocyte antigen class I antibody plus complement. Single-cell assays in agarose demonstratedthat although similar numbers of large granular lymphocytes bound to the HSV-FS and FS targets, theconjugates with HSV-FS were lysed at a much higher frequency than those with FS. These results suggestedthat both targets are bound by the NK cells but only the HSV-FS were able to trigger lysis. The requirementfor active virus expression was demonstrated by failure of emetine-treated HSV-FS targets or targets infectedwith UV-inactivated HSV to be lysed by NK effectors. To evaluate the role of viral glycoproteins in conferringsusceptibility to lysis, Fab were prepared from HSV-l-seropositive sera; these Fab were unable to block lysisof the HSV-FS. Furthermore, incubation in phosphonoacetic acid failed to reduce NK(HSV-FS) activity despitesharp reductions in viral glycoprotein synthesis. Finally, targets infected with tsLB2 at the nonpermissivetemperature were lysed as well as or better than targets infected with wild-type virus, indicating that HSVimmediate-early gene product expression is sufficient for conferring susceptibility to lysis. We conclude thatexpression of nonstructural viral proteins or virally induced cellular gene products early in the course ofinfection rather than structural glycoproteins is required for NK lysis of HSV-FS targets.

Natural killer (NK) cells are capable of lysing a variety ofvirus-infected target cells as well as certain tumor targetsand normal cells. Because they are relatively nonspecific,NK cells are available immediately as a first-line defenseagainst an invading viral pathogen and are postulated toserve to limit the extent of virus dissemination prior to themore specific immune responses. In vitro studies with hu-man effector cells have indicated that NK cells can effi-ciently reduce the extent of replication of certain cytopathicviruses, including herpes simplex virus type 1 (HSV-1), andadoptive transfer studies with a murine system have indi-cated a role for NK cells against a number of cytopathicviruses (10).The nature of the target antigens recognized by NK cells

on virus-infected targets has remained controversial. Rolesfor both the major structural glycoprotein G and nonstruc-tural matrix protein M have been implicated in the lysis oftargets infected with vesicular stomatitis virus (VSV) (32,37). Bishop et al. (3, 5, 6) reported that HSV glycoproteinsare the relevant structures recognized on infected fibroblastor WISH epithelial cell targets. However, Borysiewicz et al.(7) and Lopez-Guerrero et al. (31) have failed to demonstratea role for viral glycoproteins in the lysis of cytomegalovirus(CMV)- or HSV-infected targets, respectively, and haveinstead implicated a role for early viral genes or a cellulargene product. Similarly, in a bovine herpesvirus (BHV)system, Cook and Splitter (11) have suggested that viral

* Corresponding author.

glycoproteins are not essential for the lysis of targets in-fected with BHV.The issue of the target structure present on infected cells

is potentially complicated by several factors: the nature ofthe target cells to be infected, the infecting virus itself, therecent findings of the requirement for HLA-DR+ accessorycells for the lysis of many infected targets (2, 18, 33), and thestatus of the effector cells (i.e., interferon [IFN] preactivatedor not). For example, the CD16+ NK cells and the HLA-DR' accessory cells, which are both required for the lysis ofHSV-infected fibroblasts (HSV-FS), may potentially requiredifferent interactions with the virus-infected targets. Thestudies presented in this article were undertaken to evaluatethe requirements for development of susceptibility of NKtargets to lysis by human peripheral blood NK cells. Wehave evaluated the status of cellular stability of the infectedtargets compared with that of the uninfected targets and therelative target cell-binding efficiency of the effectors andhave examined the role of viral gene expression in conferringsusceptibility of the target cells to lysis. In contrast to theconclusions of Bishop et al. (3, 5, 6), we find no evidence fora requirement for viral glycoprotein synthesis for the lysis ofHSV-FS.

MATERIALS AND METHODS

Viruses. HSV-1 strain 2931 was originally isolated by C.Lopez and is grown and assayed on Vero monkey kidneycells. The temperature-sensitive mutant of HSV-1, tsLB2(24), was obtained from P. Schaffer and was propagated and

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3152 FITZGERALD-BOCARSLY ET AL.

titered at the permissive temperature of 33°C. VSV wasoriginally obtained from N. Ponzio of the New JerseyMedical School and was propagated and titered on Verocells. All virus preparations were stored at -70°C andthawed immediately prior to use.

Cell lines. Human foreskin fibroblast (FS) cells wereprepared by enzymatic digest of foreskins from full-terminfants. These cells were grown in Dulbecco's modifiedEagle's medium (DME; Hazelton Laboratories, Lenexa,Kans.) supplemented with 10% heat-inactivated fetal calfserum (FCS; Hyclone Labs, Logan, Utah), penicillin, strep-tomycin, and L-glutamine in a 5% Co2 atmosphere in ahumidified incubator. These cells were passaged twiceweekly and are used up to passage 20 to 22. GM-2767fibroblasts, which are trisomic for chromosome 21, wereobtained from the Human Mutant Genetic Cell Repository(Camden, N.J.) and passaged like FS cells. K562 cells weregrown 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 screenedroutinely for mycoplasmas with Mycotrim Culture Kits(Hanna Biologicals, Alameda, Calif.) and were negativewhen used in assays.

Preparation of effector cells. Mononuclear effector cellswere separated from fresh heparinized peripheral bloodobtained after informed consent from healthy donors bycentrifugation on Lymphoprep gradients (Accurate Chemi-cal Co., Westbury, N.Y.). Mononuclear cells were washedand enumerated with a hemacytometer or with a Coultermodel ZBI electronic particle counter.NK cell assays. NK cell assays were performed as de-

scribed previously (15). Briefly, newly confluent flasks of FScells were either infected with HSV-1 at the multiplicity ofinfection (MOI) stated for each experiment for 3 h or mockinfected. Simultaneously, the cells were labeled with 150 ,uCiof 51Cr (as Na51CrO4; New England Nuclear, Boston,Mass.). The targets were then trypsinized and washed thor-oughly. Nonadherent K562 cells were spun down and la-beled with 51Cr in a volume of 300 ,ul of medium for 1 to 3 h.A total of S x 103 targets in 0.1 ml of RPMI-10% FCS weredispensed 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 of 100:1 to 12:1. Assay mixes wereincubated at 37°C for 14 h in a 5% CO2 atmosphere in anincubator, after which 0.1 ml of the supernatant was re-moved from each well and the radioactivity was determinedwith a Packard Gamma Counter. 51Cr release was calculatedas follows: % 51Cr release = [(experimental cpm - sponta-neous cpm)/(total cpm - spontaneous cpm)] x 100; wherespontaneous cpm is the amount of 51Cr released by targets inthe presence of medium alone and total cpm is the amount of51Cr released by targets incubated in the presence of 5%Triton X-100 detergent. Spontaneous release did not exceed30% for HSV-FS and FS targets or 25% for K562 targets.The data generated from each NK test were analyzed with

the Von Krogh equation (35) and are expressed as estimatedkill at an E:T ratio of 50:1.IFN assays. At the termination ofNK cytolytic assays, an

additional 35 RI of supernatant was collected from each 50:1E:T well for the determination of antiviral activity. Super-natants were stored frozen at -20°C prior to quantitation ofIFN activity with GM2767 indicator cells and VSV as thechallenge virus in cytopathic effect reduction assays. EachIFN assay mix contained cell controls, virus controls, and

the international leukocyte IFN (IFN-ot) reference standardG-023-901-527, obtained from the National Institute of Al-lergy and Infectious Diseases.

Preparation of Fab fractions from seropositive and seroneg-ative donors. Fab fractions were prepared from Sandoglob-ulin (Sandoz, East Hanover, N.J.) and from serum obtainedfrom an HSV-1-seropositive and a seronegative donor. Theserological status of the donors was determined by theability of their serum samples to induce antibody-dependentcellular cytotoxicity (ADCC) against HSV-FS and FS tar-gets. Briefly, 20 to 35 ml of serum was collected from each ofthe donors. The sera were centrifuged and then passedthrough a Millex GU filter. Saturated ammonium sulfate wasadded to 40% drop by drop with constant stirring in the cold;the mixture was centrifuged, the supernatant was decanted,and the pellet was resuspended in PBS and dialyzed exten-sively against PBS. A DEAE column was prepared andequilibrated with 0.02 M sodium phosphate buffer, pH 8.0.The saturated ammonium sulfate precipitate was dialyzedovernight in the sodium phosphate starting buffer, the mate-rial was passed through the column, and the first 9 ml ofeluant was collected and pooled. The material was madeisotonic with lOx saline, and protein concentration wasdetermined 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 10mg of immunoglobulin G (IgG) in 1.0 ml of freshly made 20mM NaH2PH4 buffer containing 20 mM cysteine-HCl and 10mM sodium EDTA, pH 6.2. The reaction mix was incubatedfor 5 h at 37°C with rocking, and then 3.0 ml of 1.0 mMTris-HCl (pH 7.5) was added. The mixture was centrifuged,and the supernatant was applied to a 5-ml immobilizedprotein A (Pierce Chemical Co.) (22) column equilibratedwith 10 nM Tris-HCl, pH 7.5. The column was washed with15 ml of 10 nM Tris buffer, and the eluate containing Fab wascollected. Protein concentrations were determined with theBCA assay (Pierce Chemical Co.). The presence of Fab butnot Fc in the eluate fraction was confirmed by radial immu-nodiffusion (27) with goat anti-human IgG Fab and Fcreagents (Cappel Laboratories, Westchester, Pa.). The in-tegrity of the Fab regions within the Fab preparations wasconfirmed by indirect immunofluorescence against HSV-infected fibroblasts. The HSV-FS targets were incubatedwith the DEAE IgG preparation or the Fab fractions for 30min at 4°C, followed by incubation for 45 min in a 1:20dilution of fluorescein isothiocyanate-conjugated goat anti-mouse IgG (Cappel Laboratories), and were visualized un-der a fluorescence microscope.

Incubation of target cells in cytolysin. Rat granule cytolysinprepared from a rat LGL tumor cell line was obtained fromCraig Reynolds, National Institutes of Health (25). Thegranules were stored frozen at -70°C until immediatelybefore use and were thawed and diluted in Ca2'- andMg2+-free PBS to give a concentration of 20 U/ml. Twofolddilutions of the granule preparation were made in the Ca2+-and Mg2'-free PBS, and 100 ,ul of cytolysin were added to100 RI of 51Cr-labeled targets (at 105 cells per ml, diluted inRPMI-1% bovine serum albumin) in round-bottomed platesin replicates of six. The mixtures were incubated for varioustimes at 37°C and centrifuged at 4°C, and 100 [L of superna-tant was harvested for determination of 51Cr release.Exposure of targets to hypotonic shock. For experiments to

measure the sensitivity of targets to hypotonic shock, 5 x103 51Cr-labeled target cells (50 RI per well) were incubatedin six replicates in the presence of an additional 150 RI of

J. VIROL.

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 whichthe supernatants were harvested and the percent 51Cr releasein each group was determined by dividing the mean cpm ineach group by the mean cpm of a total release obtained byincubating 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 left uninfectedand labeled with 51Cr. Ten thousand targets were plated intoreplicate wells of round-bottomed microtiter plates andincubated for 30 min at 37°C with various dilutions of rabbitanti-human leukocyte antigen (HLA) class I frameworkantibody (New England Nuclear, Cambridge, Mass.). Babyrabbit complement (1:4) was then added to each well at theconcentrations described, and the plates were further incu-bated at 37°C for 1 h, after which the microtiter plates werecentrifuged at 1,000 rpm for 10 min, and the top 100 ,ul ofeach well was harvested for determination of 51Cr release.51Cr release was calculated as for NK assays.

Single-cell assays in agarose. Peripheral blood mononuclearcells obtained from Lymphoprep gradients were allowed toadhere to nylon wool columns for 30 min and eluted withwarm RPMI-10% FCS. The nylon wool-nonadherent frac-tion was separated on seven-step discontinuous Percollgradients as described previously (15), and fractions 2 and 3,which were enriched for large granular lymphocytes and NKactivity, were collected. The large granular lymphocyte-enriched fraction was depleted of CD3 (anti-Leu-4)-positiveT cells by panning (15). Target cells were prepared at anMOI of 5:1 or were mock infected as described above. Fortarget binding assays (23), targets and effectors were resus-pended at 2 x 106/ml in RPMI-10% FCS and 0.1 ml ofeffector cells was incubated with 0.2 ml of target cells at 30or 4°C for 5 min; the tubes were then centrifuged at 200 x gfor 5 min, the residual medium was decanted, and the cellswere gently resuspended three or four times with a capillarypipette in a volume of 50 RIu of RPMI-10% FCS. To each tubewas then added 0.1 ml of molten agarose (1.3% Sea PlaqueAgarose; FMC Corp., Rockland, Maine) in RPMI-10% FCSat 39°C, and the solution was gently mixed and spread overmicroscope slides. After allowing the solution to harden for2 to 3 min, the slides were placed in a staining dish withRPMI-10% FCS and incubated for 6 h at 37°C in a 5% CO2incubator. At the end of the incubation, the cells wereimmersed in a trypan blue-PBS solution for 5 min, afterwhich the slides were washed three times, 10 min per wash,in a PBS-0.3% Formalin solution. Duplicate slides wereprepared for each group. The slides were viewed with aphase-contrast microscope by an individual blind to theexperimental conditions, and the percentage of effector cellsadhering to targets was determined by counting 200 effectorcells on each slide. The proportion of lysed targets wasdetermined by counting the number of targets in conjugateswhich stained with trypan blue minus the percentage oftargets spontaneously staining with trypan blue on slidesincubated without effector cells. Target-effector conjugateswere easily discriminated from target-target and effector-effector interactions because the target cells were muchlarger than the effectors.

Polyacrylamide gel electrophoresis (PAGE). Target cells in75-cml flasks were infected with HSV-1 or HSV mutants for3 h in the presence or absence of antiviral drugs or mockinfected and then incubated overnight in the presence of 100to 300 ,uCi of [35S]methionine or [3H]glucosamine (NewEngland Nuclear, Cambridge, Mass.). The cells were then

gently trypsinized and incubated in lysing buffer (0.05 M Trisbase, 0.6 M KCI, 0.5% Triton X [pH 7.5]) for 10 min on ice.The lysed cells were clarified by centrifugation (32,000 rpmfor 90 min). In some experiments, the supernatants wereimmunoprecipitated overnight at 4°C with 100 ,ll of the viruspreparation and 0.1 mg of primary antibody, followed byincubation for 2 h on ice with protein A-Sepharose. Theprecipitates were washed three times in RIPA buffer (0.05 MTris-HCl, 1 mM EDTA, 0.15 M NaCl, 0.25% bovine serumalbumin [pH 8.0]) and incubated in sample buffer overnight.The samples were then heated at 100°C for 5 min, centri-fuged briefly to remove the Sepharose beads, and run onPAGE with an 8.5% resolving gel and 3% acrylamide stack-ing gel cross-linked with N,N-diallyltartardiamide (AldrichChemical Co., Milwaukee, Wis.) in an amount correspond-ing to 4% of the weight of acrylamide. Each liter of runningbuffer contained 3 g of Tris base, 14.4 g of glycine, and 1 g ofSDS.Treatment of cells with metabolic inhibitors and antiviral

drugs. The acid form of phosphonoacetic acid (PAA; SigmaChemical Co., St. Louis, Mo.), which is an inhibitor ofHSV-encoded DNA polymerase (26), was used at a concen-tration of 300 ,ug/ml and was present throughout the courseof infection and NK assays.Emetine-HCl (Sigma Chemical Co.), which is an irrevers-

ible inhibitor of protein synthesis, was used at a concentra-tion 0.1 mM to treat the target cells prior to the addition ofvirus. Following treatment with emetine for 1 h, cultureswere rinsed extensively with PBS and then infected withHSV-1 as described above. Following trypsinization of FSand HSV-FS target cells or the pelleting of K562 cells, thetargets received four washes in 15 ml of medium to eliminateany contaminating emetine.

UV-inactivated virus. UV-inactivated HSV-1 was preparedby irradiating HSV-1 with a UV sterilizing lamp (254 nm) ata distance of 10 cm to yield 1,340 i±W/cm2 for the timesshown in the experiments. The efficacy of the treatment wasconfirmed by titration of the UV-irradiated virus on Verocell 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 seriesof rat tumor targets and their susceptibility to lysis by ratsplenic NK cells. One possible explanation for the increasedsusceptibility of HSV-FS over uninfected FS targets wasthat the infected targets were unable to repair membranedamage due to shutoff of host macromolecular synthesis bythe virus. To test this hypothesis, we subjected infected anduninfected targets to hypotonic shock, rat granule cytolysin,and treatment with anti-HLA class I antibody plus comple-ment. Figure 1A shows the results of a representativeexperiment in which 51Cr-labeled HSV-FS and FS targetswere incubated in the presence of increasing amounts ofdistilled water for 14 h (the time of our typical NK assay),after which the supernatants were harvested for determina-tion of 51Cr release. The infected targets were found not tobe more sensitive to hypotonic lysis than the uninfectedtargets. In parallel studies with the same targets, infectedtargets were preferentially lysed by effector cells in 14-h NKassays (data not shown).Next we tested the sensitivity 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, 51Cr-

VOL. 65, 1991


A

-i- HSV-FS

-- FS

0 12.5 25 37.5% Water

50 62.5

TABLE 1. Target binding and single-cell assays in agarosea

Target % Conjugates % of conjugates lysedbcells 4°C 300C 4°C 300C

FS 10.8 ± 2.3 17.5 + 3.2 3.3 ± 0.8 2.0 ± 0.5HSV-FS 11.8 ± 3.0 17.7 ± 0.7 10.7 ± 3.2 8.7 ± 2.8

a Light-density cells from Percoll gradients were depleted of T cells bypanning and used in single-cell target-binding assays in which the target-effector conjugates were formed at 4 or 300C. Conjugate lysis was determinedafter 6 h of incubation, with 200 cells counted per slide. Data are the means ofthree experiments. Statistical significance was determined by Student's t test;P < 0.01 for percent lysed (FS versus HSV-FS); P > 0.1 for percentconjugates (FS versus HSV-FS).

b Percentage of lysed targets within target-effector conjugates.

B

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1009080706050403020100

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FIG. 1. Comparison of intrinsic stability of FS and HSV-FStargets. (A) HSV-FS and FS targets were incubated in variousconcentrations of ddH2O for 14 h, after which release of 51Cr intothe supematants was determined. (B) Susceptibility of HSV-FS(infected overnight) and FS targets to lysis with anti-HLA class Iand complement. Targets were incubated for 30 min at 37°C withvarious concentrations of anti-HLA class I framework followed byincubation in baby rabbit complement for 1 h. (C) Sensitivity ofHSV-FS and FS to treatment with rat granule cytolysin was

determined by incubating targets for 1 h in Ca2+- and Mg2+-free PBSin the presence of dilutions of cytolysin. Data for each experimentshown are representative of at least three similar experiments.

labeled targets were incubated with anti-HLA antiserum for30 min prior to the addition of baby rabbit complement.Figure 1B shows the results of a representative experimentdemonstrating that HSV-FS are not more susceptible tocomplement-mediated lysis than are the FS targets.HSV-FS and FS targets were also tested for their sensi-

tivity to lysis by cytolysin, the lytic material purified fromthe 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 in Fig. 1C. FS demonstrated a slight but consistentlygreater susceptibility to lysis by cytolysin than did HSV-FS.Compared with K562, both the infected and uninfected FSshowed relatively low susceptibility to cytolysin (Fig. 1C)and required longer incubation times in the presence ofcytolysin to be lysed (data not shown).

Together, the data presented in Fig. 1 indicate that al-though the HSV-FS targets are productively infected with a

lytic virus and ultimately go on to produce infectious viralprogeny (16, 30), their preferential lysis by human NK cellswithin our assay conditions cannot be explained by a greaterintrinsic instability than exists in their uninfected counter-parts.We also questioned whether trypsinization of targets had

any effect on the differential susceptibility of the targets tolysis since we observed that in wells to which trypsinizeduninfected targets were added, these targets readhered to themicrotiter plates, whereas trypsinized HSV-FS readheredless well to the microtiter wells during the course of theassay. We therefore compared the lysis of targets whichwere infected in 75-cm2 flasks followed by trypsinization andplating into microtiter plates with lysis of targets whenuninfected cells were plated in microtiter wells, allowed toreattach overnight, and then infected and 51Cr-labeled in thewells. Each of these infected populations were equallysusceptible to lysis, whereas both the recently trypsinizedand overnight-readhered, uninfected targets were insensitiveto NK lysis (data not shown.)

Similar numbers of effector cells bind to infected anduninfected FS targets. We have previously demonstrated thatuninfected targets are efficient cold competitors for the lysisof HSV-FS (15). It was therefore of interest to determinewhether there was a similar level of target binding of effectorcells to FS and HSV-FS. In these experiments, partiallyenriched large granular lymphocyte preparations were used,and the binding of the effectors to the targets and lysis oftargets were monitored in single-cell assays. There was no

significant difference in the binding of the effectors to theHSV-FS and the FS targets (Table 1). In contrast, a higherproportion of the HSV-FS than of the FS targets in conju-gates were found to be lysed at 6 h.

Active viral expression is required for making target cellssusceptible to lysis. To determine whether passive viralexpression was sufficient to make the target cells susceptibleto lysis or whether active viral expression was required,fibroblast targets were infected with either HSV-1 or HSV-1which had been UV inactivated for various amounts of time(UV-HSV) and then tested for the ability of the targets to belysed by NK cells (Table 2). Additionally, titers of virus afterUV irradiation were determined; by 5 and 10 min of irradi-

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J. VIROL.


TABLE 2. NK activity against targets infected withUV-inactivated HSV-la

UV exposure HSV titer HSV-FS(min) (PFU/ml) % Lysis (SD) IFN-a (IU/ml)

0 5.0 x 106 20.0 (3.6) 3001 1.0 X 103 23.0 (3.0) 3005 <10 11.0 (4.0) 100

10 <10 6.4 (3.0)b 300

a HSV-1 was irradiated with a UV lamp at 1,380 p.W/cm2 for the times listedand used to infect FS. Values are percent lysis at an E:T ratio of 50:1 followingVon Krogh analysis for six separate blood donors. IFN generation in responseto HSV-FS and virus titers following irradiation are shown for a repre-sentative experiment.

b p = 0.05 versus lysis of uninfected FS (Student's t test); lysis ofuninfected FS was 3.2% + 1.5%, and they induced <3 IU of IFN-a per ml.

ation, infectious virus was no longer observed. Targetsinfected with UV-HSV were not lysed by the effector cells;this effect was directly related to the dose of UV, and NKsusceptibility was reduced almost to the level of uninfectedcell killing after irradiation for 10 min. In contrast, infectionof targets with UV-inactivated virus failed to decrease theproduction of IFN-a by the effector cell population whichwas induced by those targets (Table 2). The inability ofUV-inactivated virus to confer NK susceptibility to FStargets was observed at a wide range of MOIs. Even at anMOI of 20:1, UV-inactivated virus failed to make the targetssusceptible to lysis (data not shown).

Further evidence for the requirement of active viralexpression for making infected targets susceptible to lysiswas obtained by treating FS targets with the irreversibleprotein inhibitor emetine prior to infection with HSV-1. FStarget cells were incubated with 0.1 mM emetine for 1 h, andthe cultures were washed extensively and infected. As acontrol, K562 tumor targets were incubated in emetine forthe same period of time. At the end of infection and labelingwith 51Cr, the targets were thoroughly washed and used in14-h NK assays. The efficacy of emetine treatment wasconfirmed by PAGE; protein synthesis was completelyturned off in the infected targets in the presence of emetine(Fig. 2). Inhibition of protein synthesis by emetine in theK562 and HSV-FS targets was further confirmed by mea-surement of [3H]leucine incorporation into these targets. A1-h treatment of the targets with emetine resulted in a 94.6%reduction in the incorporation of [3H]leucine in the emetine-treated versus untreated K562 targets and a 100% reductionin the emetine-treated versus untreated HSV-FS targets.Treatment of the FS targets with emetine prior to infectionresulted in a dramatic reduction in NK activity against theinfected FS (Table 3). In contrast, lysis of K562 cells wasunaffected by this treatment.

Role of viral glycoproteins in making HSV-infected targetssusceptible to lysis. Bishop et al. (3, 5, 6) have suggested arole for viral glycoproteins as target structures for NK lysisof HSV-infected endothelial cells. They described lowerlysis of targets infected with viral mutants which underpro-duced glycoprotein C (gC) or gB and were partially able toblock NK lysis by the addition of Fab fragments directedagainst viral glycoproteins. To evaluate the role of viralglycoproteins in making viral targets susceptible to lysis, weused Fab fragments of serum IgG from HSV-seropositiveand seronegative donors and PAA treatment of infectedcultures to prevent viral glycoprotein expression. Fab frac-tions were prepared by papain digestion of a commercial

EXP I EXP 2

E I E

<- gB, gC

<- gD

46 -

FIG. 2. SDS-PAGE analysis of emetine-treated, HSV-infectedtargets. SDS-PAGE analysis of two experiments with 0.1 mMemetine treatment of HSV-FS targets is presented. Targets weretreated with emetine for 1 h, washed, and then infected with HSV-1at an MOI of 1. Cultures were labeled with [35S]methionine, lysed,and immunoprecipitated with a polyclonal anti-HSV antiserum.Lanes: MW, 14C-labeled molecular mass standards (shown in kilo-daltons); I, cultures infected in the absence of emetine; E, culturesinfected in the presence of emetine.

pooled human serum gammaglobulin (Sandoglobulin) orfrom an HSV-1-seropositive and a seronegative donor. Thepresence of Fab but not Fc fractions was confirmed by radialimmunodiffusion. Intact recognition of viral proteins by theFab prepared from the seropositive but not the seronegativedonor was confirmed by indirect immunofluorescence withHSV-FS; intact Sandoglobulin and the immunoglobulin pre-pared from the seropositive donor and Fab fragments pre-pared from these immunoglobulins gave strong surface im-munofluorescence staining of HSV-FS but not FS targets,whereas neither the immunoglobulin nor the Fab from theseronegative donor gave positive fluorescence. The intactIgG fraction from both the Sandoglobulin and the seroposi-tive donor but not the seronegative donor was capable ofinducing ADCC against HSV-infected targets. However,

TABLE 3. Effect of emetine on NK activitya% Lysis

Expt Donor HSV-FS K562 +HSV-FS

+ emetine K562 emetine

1 1 33.8 3.7 62.8 66.22 33.2 3.9 72.0 73.9

2 1 20.7 7.7 58.5 68.72 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)ca Target cells were infected in the presence of emetine (0.1 mM) for 1 h and

then washed extensively before being infected or used in the assay. Lysisvalues are for an E:T ratio of 50:1 for four separate mononuclear cell donors(two in each experiment). Protein synthesis was monitored by [3Hlleucineuptake. Emetine treatment of K562 cells and HSV-FS decreased proteinsynthesis by 96.4 and 100%, respectively. Failure of emetine-treated FS to

replicate HSV was also confirmed by PAGE (Fig. 2).b Significantly different from lysis of HSV-FS in the absence of emetine

(P = 0.0003).c Not significantly different from lysis of K562 in the absence of emetine.

VOL. 65, 1991

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-/- Fab Seroneg.

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(IgG or Fab) (jg/ml)

FIG. 3. Fab prepared from HSV-seropositive sera fail to blockNK(HSV-FS) activity. IgG and Fab were prepared from Sandoglo-bulin (A) and from HSV seropositive and seronegative donors (B).The IgG and Fab were added at the concentrations shown toHSV-FS targets and effectors at an E:T ratio of 50:1. Percent 51Crrelease was determined in 14-h assays. (A) Data are representativeof four similar experiments. (B) Data are representative of twoexperiments. Lysis of uninfected FS was -5%.

when the Fab fractions were added to the NK assays, theyneither suppressed nor augmented the response at anydilution (Fig. 3). Thus, we have failed to reproduce theresults of Bishop et al. (6) in our system.PAA is a specific inhibitor of the virally encoded DNA

polymerase (26). In the absence of viral DNA replication, ithas been demonstrated that little or no HSV-1 gamma geneproducts are expressed by infected cells. We thereforeinfected FS cells and performed NK assays in the presenceof PAA (300 ,ug/ml). SDS-PAGE results confirmed that viralglycoprotein bands were sharply reduced in the PAA-treatedHSV-infected fibroblasts (Fig. 4). NK activity against theHSV-FS targets was not reduced and in some cases was

augmented by the treatment with PAA (Table 4). The lysis ofK562 cells and uninfected FS was also not affected bytreatment with PAA (data not shown). Furthermore, treat-ment with PAA had no significant effect on spontaneous 51Crrelease values for any of the targets.

Role of HSV-1 immediate-early gene expression in confer-ring target susceptibility to lysis. Convincing evidence for a

role for early viral gene expression in conferring susceptibil-ity of virus-infected targets to lysis by both NK and cyto-toxic T cells has been developed recently (8, 12, 31). Toevaluate the role of immediate-early gene expression inHSV-1-infected targets, we used tsLB2, a temperature-sensitive HSV mutant which at the nonpermissive tempera-

<- gB, gC

. c-<-gD

46 -

30 -

FIG. 4. SDS-PAGE analysis of PAA-treated, HSV-infected tar-gets. Targets were infected with HSV-1 at an MOI of 1 in thecontinuous presence of PAA (300 ,ug/ml) in low-glucose DMEmedium. The targets were labeled overnight with [3H]glucosamine,lysed, immunoprecipitated with a polyclonal anti-HSV antiserum,run on SDS-PAGE gels, and developed by autoradiography. Lanes:MW, 14C-labeled molecular mass standards (shown in kilodaltons);U, uninfected FS: I, FS; infected in the absence of PAA; +PAA, FSinfected in the presence of PAA.

ture (39°C) fails to make beta and gamma gene products andoverproduces the alpha gene product ICP4 (13, 14). At 33°C,however, viral replication proceeds normally and infectiousprogeny are produced. Targets were infected at 33 and 39°Cwith either tsLB2 or wild-type 2931 virus, and the NK assayswere carried out at these temperatures as well. At thepermissive temperature, both sets of targets were killed to anequivalent extent by the NK effectors. At the nonpermissivetemperature, however, targets infected with tsLB2 werekilled as well as or even more efficiently than the FS cellsinfected with the wild-type virus (Table 5). The failure oftsLB2 to produce normal levels of structural glycoproteinsand overproduction of ICP4 at 39°C were confirmed bySDS-PAGE (Fig. 5).

TABLE 4. Effect of PAA on lysis of HSV-FSa

% Lysis

Donor MOI 0.2:1 MOI 1.0:1

-PAA + PAA - PAA + PAA

1 35.6 34.7 56.7 51.62 60.7 50.8 74.9 67.93 35.4 50.8 34.8 37.84 33.9 37.0 28.1 33.85 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-FS targets were prepared at an MOI of 0.2:1 or 1:1. Data representestimated lysis at an E:T ratio of 50:1 following von Krogh analysis. Therewas no statistical difference (P > 0.05, Student's t test) with or without PAAat each MOI. Lysis of uninfected FS was always .5%.

J. VIROL.


TABLE 5. NK activity against FS targets infected with tsLB2a

Target cells TTemp % Lysis Virus titer(') Donor 1 Donor 2 (F/l

2931 33 18.3 21.6 1.5 x 10539 15.6 19.8 1.2 x 105

tsLB2 33 21.1 24.7 1.2 x 10539 31.1 33.7 0.2 x 102

Uninfected 33 7.8 6.6 NAb39 4.6 4.8 NA

a FS targets were infected with tsLB2 or HSV-1 strain 2931 (MOI, 1:1) at 33or 39°C, and the entire experiment was carried out at these temperatures. NKactivity and virus replication in spontaneous release wells were measuredafter 14 h of incubation. NK data are expressed for an E:T ratio of 50:1following von Krogh analysis. Data are from one experiment representative ofsix similar experiments.

b NA, not applicable.

Effect of 20-h preinfection of targets on susceptibility ofHSV-FS to NK lysis. The majority of the NK studies ofBishop et al. (3-6) were performed with targets which hadbeen infected overnight with HSV-1 before being used in NKassays. In contrast, our studies have been performed with3-h-infected targets followed by 14-h NK assays. We havenow directly compared 3- and 20-h-infected targets forsusceptibility to lysis in 14-h NK assays (Table 6). Theviability of the targets postinfection was found to be identicalfor the 3- and 20-h-infected targets (>95%), as determined bytrypan blue exclusion. Moreover, spontaneous release val-ues from the 3- and 20-h-infected targets were indistinguish-able at the end of 14 h NK assays (data not shown). When wecompared NK lysis of the 3-h and 20-h targets, however,there was a significant decrease in NK susceptibility of the20-h-infected targets; for some donors, the 20-h-infectedtargets were lysed to extremely low levels, whereas somedonors showed lesser degrees of suppression. The decreasedsensitivity of the 20-h-infected targets occurred despiteequivalent induction of IFN production and an even greatersurface immunofluorescence for HSV glycoproteins than

Unif 2931 hLB2MW 330 390 330 390 330 390

200 -<- ICP4

4 w " <-gB,gC

95.-

HV2931at<- gD

55 - " ''R'

FIG. 5. SDS-PAGE analysis of targets infected with tsLB2 orHSV 2931 at permissive and nonpermissive temperatures. Targetswere uninfected (Uninf) or infected with HSV-1 2931 or tsLB2 at anMOI of 1 at 33 or 39°C, labeled with [35 ]methionine in methionine-

free medium, lysed, and run without immunoprecipitation on an

SDS-PAGE gel. Sizes are shown in kilodaltons.

TABLE 6. Extended infection of targets decreases sensitivity ofHSV-FS to lysisa

3-h-infected targets 20-h-infected targets

Donor IFN IFN% Lysis generated % Lysis generated

(IU/ml) (IU/ml)

1 30.2 300 13.1 2002 42.0 300 23.2 2003 18.4 1,000 3.9 1,0004 16.2 3,000 5.0 3,0005 26.6 700 17.6 7006 15.1 1,000 6.8 1,0007 26.6 1,000 21.0 1,0008 26.0 1,000 16.8 1,0009 41.4 10,000 21.1 10,00010 28.2 300 17.9 30011 22.9 300 6.6 30012 19.3 300 20.4 300x (SD) 26.1 (8.8) 780 14.5 (7.1)b 729

a FS targets were infected with HSV-1 for 3 or 20 h at an MOI of 1 and thenused in 14-h NK assays. Data represent estimated lysis at an E:T ratio of 50:1following von Krogh analysis and IFN generated at an E:T ratio of 50:1 for 12different donors. Arithmetic mean NK activity and geometric mean IFNproduction are shown in the last line. Uninfected FS lysis was '10% for alldonors.

b Significantly different from lysis of 3-h-infected targets as determined byStudent's t test (P < 0.05).

observed for the 3-h-infected targets when stained withanti-HSV antiserum.

HSV-seropositive serum induces ADCC but blocks induc-tion of IFN. Serum from HSV-1-seropositive or seronegativeindividuals or reconstituted Sandoglobulin immune globulinwere added to 14-h NK cells against HSV-FS targets.Sandoglobulin and serum from seropositive but not seroneg-ative donors augmented lysis of the HSV-FS targets viaADCC. In contrast, IFN production in the presence of theHSV-immune sera was depressed, whereas HSV-seronega-tive serum had no effect on IFN production (Table 7). Thus,antibodies against viral proteins expressed on the cell sur-face blocked the ability of the infected cells to provide aninductive signal to the IFN-producing cells.

DISCUSSION

In this study, we have examined the mechanism of thepreferential lysis of HSV-infected fibroblasts over theiruninfected counterparts. Since HSV-1 efficiently turns offhost cell macromolecular synthesis (28), it was possible thatthe increased lysis of the infected targets could be due to aninherent fragility of these targets and an inability to repairmembrane damage. Using three different measurements ofsensitivity to lysis, we have failed to demonstrate increasedsensitivity of the HSV-FS: they showed similar sensitivity tohypotonic shock and to treatment with anti-HLA class I pluscomplement and were not more susceptible to rat granulecytolysin, the lytic material from a rat large granular lym-phocyte tumor line (25) (Fig. 1). The sensitivity to lysis bycytolysin of both HSV-FS and FS targets was far lower,however, than that of K562, a finding which is in agreementwith the report of the relative insensitivity of fibroblasts tothe action of cytolysin. We also observed no significantdifference in the percentage of effector cells which bound toHSV-FS and FS targets, whereas the HSV-FS targets were

preferentially lysed (Table 1). These results support our

previous observations that uninfected FS targets are efficient

VOL. 65, 1991


TABLE 7. Suppression of IFN production by HSV-immune butnot nonimmune serum7

Expt Serum or Amt % Lysis' IFN-a produceddantibody addedb (>tg/ml) s (IU/ml)

1 None 26.9 3,000Seropositive 46.2 100

2 None 37.4 10,000Seropositive 49.3 1,000

3 None 27.5 3,000Seropositive 67.2 700

53.0 700Seronegative 31.8 3,000

23.0 7,000

4 None 23.8 10,000Sandoglobulin 750 52.6 300

75 58.4 100

S None 37.7 1,000Sandoglobulin 750 80.7 <3

75 79.9 <3

6 None 35.7 10,000Sandoglobulin 750 71.4 <3

75 71.4 30a Serum (5 ,ul) from HSV-immune (seropositive) or nonimmune (seronega-

tive) donors was added to NK(HSV-FS) assays at the beginning of culture.Sandoglobulin was added to the assays at a final concentration of 750 or 75jig/ml. Lysis of uninfected FS was -5%.

b In experiment 3, sera from two each seropositive and seronegative donorswere tested.

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

d IFN production at an E:T ratio of 50:1.

cold competitors for the lysis of HSV-FS targets (15) and theresults of Borysiewicz et al., who demonstrated competitionbetween CMV-infected and uninfected fibroblasts (7, 8). Ourcompetition and target binding results support the conceptthat the initial recognition structure is present on the unin-fected targets, whereas the infected targets are selectivelytriggered to preferentially lyse the infected targets. In con-trast to our target-binding results, Borysiewicz et al. (7, 8)have reported a significantly greater (approximately 50%)binding of effector cells to human CMV-infected fibroblaststhan to uninfected fibroblasts. These latter results, however,were obtained with IFN-pretreated effectors, which mayhave influenced the target-binding profile.The role of active expression of virus in making the targets

susceptible to lysis was evaluated by using UV-irradiatedvirus to infect targets. Our results indicate that adsorption ofthe UV-damaged virus to the FS targets was insufficient tomake the target cells susceptible to lysis. In contrast, theUV-inactivated virus induced levels of IFN-a similar tothose induced by nonirradiated virus. It has previously beensuggested that lysis of virus-infected targets occurs nonspe-cifically and is due to promiscuous killing by NK cells whichhave been activated by the IFN induced by the viral infec-tion (38, 39). However, the observation that high levels ofIFN were induced by UV-treated virus-infected targets butthat these targets were not lysed indicates that IFN induc-tion alone is insufficient to lead to lysis of the fibroblasttargets (Table 2). Rather, active expression of the viralgenome, perhaps leading to the induction of a trigger struc-ture on the infected targets, is also required. That preferen-

tial lysis of virus-infected targets is not due solely to IFN-induced NK cytotoxicity is further supported by theobservations that IFN-pretreated effectors still preferentiallylyse virus-infected over uninfected targets (4, 17) and thatinclusion of anti-IFN antiserum in NK assays fails to reducelysis of the virus-infected targets despite neutralizing detect-able supernatant IFN (2, 17).The suppression of NK(HSV-FS) but not NK(K562) ac-

tivity by emetine (Fig. 2, Table 3) further indicates thatactive 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, wasfound to be similar for the HSV-FS and K562 targets, rulingout a differential effect of the emetine on the two targets.These results confirm and extend those of Bishop et al. (3),who demonstrated that 18-h treatment of HSV-infectedtargets with emetine also diminished NK susceptibility.These investigators, however, failed to report control exper-iments with a noninfected NK target cell such as K562. Thisis an important control, since blockade of protein synthesisin targets for such an extended period prior to the NK assaywould also be expected to affect cellular protein turnover.Although both the seropositive antiserum and Sandoglob-

ulin (but not the seronegative serum) induced ADCC of theHSV-FS targets, the Fab failed to block cytotoxicity despitetheir retaining the ability to recognize virus-infected targets.This failure to inhibit lysis is in contrast to the partialinhibition observed by Bishop et al. (6) with Fab preparedfrom a seropositive donor or a monoclonal antibody to gB orgC.

Further evidence for a lack of requirement for structuralglycoproteins comes from our data demonstrating intactNK(HSV-FS) activity in the presence of PAA (Table 4) andwith the temperature-sensitive mutant tsLB2 (Table 5) de-spite the reduced production of viral glycoproteins. Theseresults are similar to those indicating a lack of requirementfor structural glycoproteins for the lysis of CMV-infectedhuman fibroblasts (8) or BHV-infected fibroblasts (11). Thedata we have obtained with the tsLB2 mutant at the nonper-missive temperature further indicate that expression of theimmediate-early genes of HSV is sufficient to make thetargets susceptible to lysis. In fact, the tsLB2-infectedtargets were killed better at the nonpermissive temperaturethan the targets infected with wild-type virus despite thefailure of the tsLB2 virus to replicate or express structuralglycoproteins (Table 5, Fig. 5). These results are consistentwith the observation that expression of immediate-earlygene products is sufficient to induce lysis of CMV-infectedFS targets (8).Our findings of a lack of requirement for structural glyco-

proteins of HSV-infected targets for conferring susceptibilityto NK lysis contradict those of Bishop et al. (3, 5, 6). Onedifference in our assay systems is that they routinely usedtargets that were infected overnight, whereas we usedacutely infected targets. However, when we directly com-pared 3- and 20-h-infected targets in 14-h NK assays, wefound 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 surfaceviral glycoproteins (Table 6). These results again argue forNK recognition of a relatively early event in viral replication(whether it be a cellular or viral gene product) which is atleast 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.

J. VIROL.


Although we have demonstrated that expression of theHSV genome is required to make the infected targets sus-ceptible to lysis, the nature of the target signal and/ortriggering molecules remains unclear. It is possible that theimmediate-early gene products are the actual target struc-tures recognized by NK cells. Peptide sequences of imme-diate-early gene products of a number of viruses are knownto be capable of inducing cytolytic T cells. These peptidesequences are thought to associate with major histocompat-ibility complex (MHC) antigens inside the cell and to betransported to the cell surface. It is possible that somesimilar mechanism might occur for the recognition of in-fected cells by NK cells; however, NK cells are not MHCrestricted, nor would they be expected to have the samedegree of fine specificity as cytotoxic T cells. Alternatively,expression of immediate-early gene products of HSV, someof which are known to affect cellular gene expression (13,20), may be altering a cellular gene product (either upregu-lating, downregulating, or modifying it) that may influencetarget susceptibility.NK lysis was found to correlate with increased total and

recycling transferrin receptor expression by CMV-infectedtargets by Borysiewicz et al. (7); however, these authorsconcluded that the NK target structure was not the transfer-rin receptor itself, since they were unable to inhibit lysis witheither iron-saturated transferrin or affinity-purified transfer-rin receptor. Lopez-Guerrero et al. (31) have also implicatedhost cell transferrin receptor in the killing process of bothtumor and virus-infected targets, yet were unable to blocklysis of the HSV-infected targets with certain anti-transferrinreceptor monoclonal antibodies which were able to blocklysis of the tumor targets. These authors have also demon-strated that inhibition of N-linked glycosylation of HSV viralglycoproteins leads to decreased sensitivity of the targets tolysis, yet, like us, were unable to correlate sensitivity withlevels of surface expression of viral glycoproteins B, C, andD(31).The conflicting data in the literature regarding the relative

role of viral structural proteins, immediate-early genes, andhost 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+ accessorycells, both of which are required for lysis of HSV-FS andCMV-FS (2, 18, 33). Our data with UV-inactivated virusindicates that input viral glycoproteins themselves are suffi-cient to induce IFN production by the HLA-DR+ accessorycells 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 predictthat Fab against viral glycoproteins would also decrease IFNproduction, but we have not yet performed these experi-ments. In one report, blocking of gD with monoclonalantibody 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 ofinduced 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 thatstimulation of accessory cell activity may be dependent uponviral glycoprotein expression, whereas the NK cells them-selves require recognition of the infected target cell, forwhich de novo immediate-early gene expression, eitherdirectly or indirectly, is required. Studies are under way toevaluate the relative roles of specific immediate-early geneproducts 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 ofcellular proteins which are up- or downregulated by HSV(e.g., heat shock proteins, which are upregulated, and MHCantigens, which are downregulated) in HSV-infected cells isbeing evaluated.

ACKNOWLEDGMENTSWe thank Joan Mack for typing the manuscript, Michael Feldman

for preparing the figures, and Monica Mendelsohn and Shelley Curlfor technical assistance.

This study was supported USPHS grant CA 42093 from the NCI.

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J. VIROL.

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Immediate-Early Gene Expression Is Sufficient for Induction of