JOURNAL OF VIROLOGY, Jan. 1994, p. 453-462 Vol. 68, No. 10022-538X/94/$04.00+0Copyright © 1994, American Society for Microbiology

The Adenovirus E3 14.7-Kilodalton Protein Which InhibitsCytolysis by Tumor Necrosis Factor Increases the Virulence of

Vaccinia Virus in a Murine Pneumonia ModelJOANN TUFARIELLO,' SANGHO CHO,2 AND MARSHALL S. HORWITZlI3.4*

Departments of Microbiology and Immunology,' Pathology, 2 Cell Biology,3 and Pediatrics,4Albert Einstein College of Medicine, Bronx, New York 10461

Received 19 August 1993/Accepted 13 October 1993

The 14.7-kilodalton protein (14.7K protein) encoded by the adenovirus (Ad) E3 region inhibits tumornecrosis factor alpha (TNF-t)-mediated lysis of cells in tissue culture experiments, but the relevance of thiseffect in vivo is incompletely understood. To examine the effect of the ability of the Ad 14.7K protein to blockTNF lysis upon viral pathogenesis in a murine model, we cloned the 14.7K protein-encoding gene into vacciniavirus (VV), permitting its study in isolation from other Ad E3 immunomodulatory proteins. The gene formurine TNF-x was inserted into the sameW containing the 14.7K gene to ensure that each cell infected withtheW recombinant would express both the agonist (TNF) and its antagonist (14.7K).W was utilized as thevector because it accommodates large and multiple inserts of foreign DNA with faithful, high-level expressionof the protein products. In addition, infection of mice with W induces disease with quantifiable morbidity,mortality, and virus replication. The results of intranasal infections of BALB/c mice with these Wrecombinants indicate that the Ad 14.7K protein increases the virulence of W carrying the TNF-at gene byreversing the attenuating effect of TNF-et onW pathogenicity. This was demonstrated by increased mortality,pulmonary pathology, and viral titers in lung tissue following infection withW coexpressing the 14.7K proteinand TNF-ot, compared with the control virus expressing TNF-a alone. These results suggest that the 14.7Kprotein, which is nonessential for Ad replication in tissue culture, is an immunoregulatory protein whichfunctions in vivo to help counteract the antiviral effects of TNF-a.

The early region 3 (E3) of group C human adenoviruses(Ads) contains nine open reading frames, seven of which havebeen shown to be expressed in infected cells. From map units76 to 86 of Ad type 2 (Ad2), these include the 12.5-kilodaltonprotein (12.5K protein) (27), 6.7K (74), gpl9K (49), 11.6K(67), 10.4K (66), 14.5K (65), and 14.7K (69). The E3 region isdispensable for growth of the virus in tissue culture butconserved among most Ad serotypes (12, 29, 61) and appearsto play a role in the modulation of the host immune responseto infection (21, 26, 75). The best characterized of the E3proteins, gpl9K, is an integral membrane protein which local-izes to the endoplasmic reticulum. It associates with the heavychain of the class I major histocompatibility complex molecule,causing its retention within the endoplasmic reticulum; theresulting decrease in surface expression of major histocompat-ibility complex class I may allow Ad-infected cells to escaperecognition and lysis by CD8+ cytotoxic T cells (4, 8, 9, 42).The adenovirus EIA protein induces sensitivity to cytolysis bytumor necrosis factor alpha (TNF-cx) (10, 14), while threeproteins encoded by the Ad E3 region are involved in protec-tion of murine cell lines from lysis by TNF. The 14.7K proteinconfers protection upon cells spontaneously sensitive to TNF,as well as those sensitized by inhibition of protein synthesiswith cycloheximide, by disruption of microfilaments with cy-tochalasin E or by Ad ElA expression (23, 25, 29). Stabletransfectants of C3HA mouse embryo fibroblasts expressingthe 14.7K gene are also resistant to TNF cytolysis in thepresence of the above sensitizing agents (28). Additionally, the

* Corresponding author. Mailing address: Albert Einstein Collegeof Medicine, 1300 Morris Park Ave., Chanin Room 515, Bronx, NY10461. Telephone: (718) 430-2230. Fax: (718) 823-5877. Electronicmail address: horwitz@waecom.yu.edu.

E3-encoded 10.4K and 14.5K proteins function together as aplasma membrane-associated complex both to protect cellsfrom lysis by TNF (24) and to promote internalization of theepidermal growth factor receptor (68). The inhibition of TNFseems important in Ad infection, as yet another protein, the19K product of the Ad ElB transcription unit, can preventTNF cytolysis in human (22) and rat (73) cells but not in mousecells (22).

TNF-oL is a proinflammatory cytokine produced primarily byactivated monocytes and macrophages. This immune mediatoris distinguished by the highly pleiotropic nature of its actions:it activates numerous signal transduction pathways, the prod-ucts of which stimulate an array of transcription factors, whichgo on to transactivate an assortment of cellular genes (re-viewed in references 33, 60, and 71). Mature TNF-oL is anapproximately 17-kDa homotrimer (2), released via proteolysisfrom a 26-kDa membrane-bound precursor (41), which bindswith high affinity to two distinct and widely distributed recep-tors of 55 (45, 59) and 75 (62) kDa. In addition to theantitumor activity from which its name derives, TNF is also apotent immunostimulatory agent with a variety of antiviralactivities. TNF can be directly cytotoxic to infected cells; forinstance, cells infected with vesicular stomatitis virus (1),herpes simplex virus (HSV) (36), Newcastle disease virus (46),and Ad with E3 deletions (23) exhibit increased sensitivity toTNF lysis. The susceptibility to TNF lysis induced by viralinfection may be related to the shutoff of host protein synthesiswhich occurs following some viral infections, in a mannersimilar to the TNF-sensitizing effects of cycloheximide (77).TNF can also function to induce an antiviral state in uninfectedcells (5, 32, 48, 56, 57, 76), but this effect is cell type specific andmay also depend on factors such as cell age and cultureconditions (37). The importance of TNF in the immune

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response to viral infection is further supported by the findingthat TNF expression is induced in lymphoid (RPMI 1788) andmyeloid (HL-60) cell lines upon infection with encephalomyo-carditis virus, vesicular stomatitis virus, Ad2, or HSV type 2(76).The antiviral activities of TNF in vivo have been less

amenable to study because of the high toxicity of this cytokinefollowing systemic administration and its rapid clearance fromthe circulation, making a local antiviral effect difficult todocument (35, 63). However, it has been shown that a recom-binant VV expressing TNF-oL is highly attenuated for murineinfection and that this attenuation is not attributable toaugmentation of the cell-mediated (cytotoxic T cell, NK cell)or humoral immune responses (58). In fact, the attenuation isobserved in athymic nude or sublethally irradiated mice. Thus,local expression of TNF as directed by the VV vector results ina dramatic, and apparently direct, antiviral activity in vivo inthis model.The mechanism by which the 14.7K protein inhibits TNF-

mediated cytolysis is unclear. This protein of 128 amino acidsin Ad2 has no recognizable structural motifs with definedfunctions, has not been demonstrated to interact with anyknown cellular protein (25), and does not function by decreas-ing TNF receptor number or affinity (28). Recently, it has beendemonstrated that the 14.7K protein prevents the release of[3H]arachidonic acid upon stimulation of C3HA mouse cellswith TNF-ao plus cycloheximide (78), suggesting that the 14.7Kprotein inhibits cytosolic phospholipase A2, an enzyme which isthought to play a role in TNF-mediated cytolysis. Mutationalanalysis has shown that deletion of virtually any segment of the14.7K protein abolishes its ability to protect cells from TNF,while cysteine-to-serine point mutants retain wild-type activityas long as the resulting protein is expressed at wild-type levels(51). The redundant anti-TNF functions encoded by Ad leadone to speculate that antagonism of TNF's lytic effects isrelevant to viral pathogenesis in vivo, but this has not beendirectly examined. It has been shown that transcription of theTNF-encoding gene is induced upon high-dose Ad infection ofmice (a nonreplicating model) (20) and that intranasal infec-tion of cotton rats with an Ad5 deletion mutant lacking the14.7K, 10.4K, and 14.5K proteins results in a primarily neutro-philic pulmonary inflammatory response, in contrast to thelargely monocytic response to wild-type Ad5 (19). Direct studyof the pathogenic effects of the Ad 14.7K protein in convenientmurine models is made more difficult by the inability of humanAds to replicate in mice and the absence of the 14.7Kprotein-encoding gene or a homolog in the mouse Ad (52).Our experiments were designed to examine the effect of

antagonism of TNF cytolysis by the Ad 14.7K protein on viralpathogenicity in vivo. Because it had already been demon-strated that locally produced TNF-ot has a pronounced atten-uating effect on VV virulence, we cloned the Ad2 14.7Kprotein-encoding gene into a VV vector which also containedthe gene for murine TNF-ox to maximize the likelihood ofdetection of an antagonistic relationship between the twoproteins, as reflected by an alteration of VV virulence. Thus,the VV served a dual role: it was the pathogen capable ofreplication and induction of a lethal disease, as well as thevector for delivery of the agonist (TNF) and its antagonist (the14.7K protein). In these studies, mice were intranasally in-fected with the VV constructs and examined for illness, death,pulmonary inflammation, and virus replication in infectedtissues.

MATERIALS AND METHODS

Cell lines. The 143B tk human osteosarcoma cell line(obtained from Robert Ricciardi) was maintained in Dulbec-co's modified Eagle's medium (DME) supplemented with 10%heat-inactivated fetal bovine serum, 50 U of penicillin per ml,and 50 [Lg of streptomycin per ml. HeLa cells were maintainedin suspension culture in Joklik's modified minimal essentialmedium (Spinner medium) supplemented with 7% bovine calfserum, and penicillin and streptomycin as described above.

Plasmid construction. The BspI2861-DraI digestion productof the Ad2 EcoRI E fragment, containing the 14.7K protein-coding sequence, was cloned into the SmaI site of pUC19. Theplasmid thus generated, pUC/14.7, was digested with BamHIand EcoRI to liberate a 623-bp fragment containing the 14.7Kprotein-encoding gene. Following gel purification and bluntingof the ends with Klenow polymerase, the 623-bp fragment wasligated at the unique SmaI site of VV expression vector PSC1 1,which directs insertion into the VV tk gene of the HindIlI-Jregion. The ligation mixture was used to electroporate (2.5 kV,25 RF) competent Escherichia coli (Electromax DH1OB;GIBCO-BRL), and miniprep DNA prepared from ampicillin-resistant colonies was digested with SmaI and HinclI todetermine the presence and orientation of the insert. Twoplasmid constructs were selected for further use: one with the14.7K protein-encoding gene in the expressing orientation withrespect to the VV p7.5 promoter [PSC11/14.7(+)] and onewith the 14.7K protein-coding sequence in the nonexpressingorientation [PSC11/14.7(-)]. Both plasmids (expressing andnonexpressing) contain lacZ coding sequences behind the lateVV promoter pl1 for visual screening of recombinant plaques.Plasmid pFBX-mTNF, a kind gift of Ian Ramshaw, containsthe murine TNF-ox (mTNF-ax)-coding sequence under controlof the VV p7.5 promoter, the HSV tk gene under control ofVV promoter pF as a dominant selectable marker, and VVsequences to direct insertion into the HindIII-F region.Recombinant viruses. HeLa cells, at a concentration of 2 x

107/5 ml of Spinner medium (without serum), were infectedwith 0.05 PFU of VV-WR.21 per cell. After 1.75 h of incuba-tion at 37°C, the cells were pelleted and resuspended in 3 ml ofHEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic ac-id)-buffered saline containing 5 mM KCl and 6 mM glucose.Following this wash, the cells were pelleted again and resus-pended in 1.0 ml of HEPES-buffered saline and 0.5 ml wastransferred to each of two electroporation cuvettes (0.4 cm;Bio-Rad) containing 50 jg of either PSC11/14.7(+) or PSC1I/14.7( -). The cells were electroporated (Bio-Rad Gene Pulser)at settings of 0.23 kV and 250 ,F, transferred to 20 ml ofcomplete Spinner medium, and incubated on a rotator wheelfor 48 h at 37°C. The cells were then pelleted by centrifugation,and the supernatant was used to infect 143B (tk- ) monolayersin six-well plates. After 2 h of adsorption at 37°C, the inoculumwas removed and the cells were overlaid with DME containing5% fetal bovine serum, 1% low-gelling-temperature agarose(UltraPure low-melting-point agarose; GIBCO-BRL), and 25,ug of 5-bromodeoxyuridine (Sigma). After 48 h, a secondoverlay was applied containing a chromogenic ,-galactosidasesubstrate, X-Gal (5-bromo-4-chloro-3-indolyl-4-D-galactopyr-anoside; Sigma), at a final concentration of 250 p.g/ml. Isolatedblue plaques were picked and plaque purified three timesunder selective conditions. Virus stocks were prepared on143B cell monolayers; infected cells were frozen and thawedthree times to release virus and then sonicated for 4 x 15 s (5A of direct current; Branson SonicPower Sonifier), cell debriswas removed by centrifugation, and the supernatant wasdivided into aliquots and stored at - 70°C. Prior to plaque

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EFFECT OF ADENOVIRUS 14.7K PROTEIN ON VACCINIA VIRUS VIRULENCE 455

E G 1. J H 1) A B

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Ad2-14.7K

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FIG. 1. HindIII restriction map of the VV-WR genome, showingsites of insertion of foreign genes. The virus designated VV 14.7(+)/TNF expresses the mTNF-ot coding sequence from the combinedearly-late promoter p7.5 (P7.5) and the HSV tk gene from the nativepromoter pF (PF) in the HindIII-F region, as well as the Ad2 14.7Kprotein from p7.5 and ,B-galactosidase from late promoter pl 1 (P11) inthe HindIII-J region. VV 14.7(-)/TNF is identical, except that theAd2 14.7K protein-encoding gene is cloned in the reverse, nonexpress-

ing orientation relative to p7.5. Viruses VV 14.7(+) and VV 14.7(-)contain the 14.7K protein-encoding gene in either the expressing (+)or the nonexpressing (-) orientation as described above and the HSVtk gene in the HindIII-F region but lack the p7.5 promoter-TNF-Qxgene cassette. The directions of gene transcription driven by thevarious promoters are indicated with arrows.

titration, the portion of the virus stock to be utilized was

treated with an equal volume of 0.25-mg/ml trypsin at 37°C for30 min. The above-described procedure generated parentalsingle recombinant VV containing the 14.7K protein-encodinggene, in either the expressing or the nonexpressing orientation,into which the second insertion, plasmid pFBX-mTNF, was

introduced in a similar manner. This insert, which containedthe HSV tk gene as a product of early promoter pF, was

selected with medium containing 1.5 ,uM methotrexate, 15 p.Mthymidine, 50 p.M adenosine, 50 p.M guanosine, and 10 p.Mglycine (MTAGG medium) (6, 72), applied 5 to 6 h postinfec-tion. The resulting viruses, also plaque purified three times andgrown as stocks on 143B cells, are designated VV 14.7(+)/TNFand VV 14.7(-)[I)NF. In a similar fashion, we also generatedrecombinant viruses containing the 14.7K protein-encodinggene, either expressing or nonexpressing, in the HindIII-Jregion but with only the HSV tk insert in the F region (i.e., no

TNF-ox). Plasmid pFBX-mTNF was digested with EcoRI,resulting in removal of the fragment containing the VV p7.5promoter and the mTNF-cx-encoding gene. The resulting vec-

tor was religated and used for insertion of HSV tk into theHindIII-F region of VV. Since numerous insertions into non-

essential regions of the VV genome have been shown to havean attenuating effect in vivo (7, 11, 34, 40, 43), this second setof double recombinants allowed us to compare directly thepathogenic effects of the 14.7K protein alone with those of the14.7K protein plus virally produced TNF-ox. The genomicstructures of the four VV recombirants used in these studiesare illustrated schematically in Fig. 1.Western blot (immunoblot) for detection of 14.7K protein

expression. Approximately 6 x 106 143B cells were infected

with 1 PFU of VV 14.7(+)/TNF or VV 14.7(-)/TNF per celland harvested at 48 h postinfection. The cells were scraped,centrifuged to a pellet, resuspended in 100 ,ul of cold cell lysisbuffer (100 mM Tris-HCl [pH 8], 100 mM NaCl, 0.5% TritonX-100, 1 mM phenylmethylsulfonyl fluoride), and incubated onice for 20 min. The lysate was spun for 10 min at 14,000 rpm ina microcentrifuge to pellet nuclei and clarify the supernatant,which was boiled in Laemmli sample buffer and run on asodium dodecyl sulfate-12% polyacrylamide gel. An extract ofAd2-infected HeLa cells, treated with cytosine arabinoside andcycloheximide to enhance production of early proteins afterremoval of the translational block (17), was run as a positivecontrol for the presence of the 14.7K protein. The proteinswere transferred (Bio-Rad Trans-Blot Cell apparatus) over-night at 14 V and 4°C to a nitrocellulose membrane (Amer-sham Hybond C Extra) in buffer composed of 20 mM Tris (pH8), 150 mM glycine, and 20% methanol. The blot was blockedwith phosphate-buffered saline (PBS) containing 5% nonfatdried milk (Carnation) and 0.1% Tween 20 (blocking buffer)for 1 h at room temperature. After washing with PBS contain-ing 0.1% Tween 20, the membrane was probed (1 h, roomtemperature) with a rabbit polyclonal antibody to a TrpE-14.7K fusion protein (a generous gift of William Wold) diluted1:2,500 in blocking buffer. Following washes with PBS-0.1%Tween 20, the membrane was incubated for l h with ahorseradish peroxidase-conjugated donkey anti-rabbit F(ab')2fragment (Amersham) diluted 1:2,000 in blocking buffer. Afterthe final washes with PBS-0.1% Tween 20, immunoreactivitywas detected by treating the blot with a 1:1 mixture ofdetection reagents (Amersham ECL Western Blotting Detec-tion System) and exposing it to Amersham Hyperfilm-ECL.Time course: expression of TNF (enzyme-linked immu-

nosorbent assay [ELISA]) and 14.7K protein (Western blot).Monolayers of 143B cells in 24-well tissue culture plates wereinfected with 2 PFU of VV 14.7(+)/TNF or VV 14.7(-)/TNFdiluted in 200 ,lI of serum-free DME, per cell. Following avirus adsorption period (1 h, 37°C), 600 [lI of DME supple-mented with 3% fetal bovine serum was added per well andincubation at 37°C and 5% CO2 was continued. Wells wereharvested at 4, 8, 12, 24, and 48 h postinfection and processedas described below. The supernatant was collected and passedthrough a 0.2-p.m filter, and the amount of immunoreactivemTNF-ot was quantified with an indirect ELISA (GenzymeFactorTest mTNF-ot used in accordance with the manufactur-er's instructions). The cell monolayers were scraped into 0.5 mlof cold PBS per well, transferred to Eppendorf tubes, andcentrifuged to a pellet. The cell pellet was resuspended in 50 plof cell lysis buffer, and the 14.7K protein was detected byWestern blotting as described above. Band absorbances werequantitated by densitometry on an LKB laser densitometerwith the GELSCAN XL (2.1) program.Mouse infections. BALB/c female mice, 3 to 4 weeks of age,

were purchased from either the National Cancer Institute orthe Charles River Breeding Facility and utilized for VVinfections within 1 week of arrival. Immediately prior toinfection, the recombinant VV stock was treated with trypsin(0.1 volume of a 2.5-mg/ml concentration) for 30 min at 37°Cand vortexed well. The trypsin was neutralized with 2% fetalbovine serum, and serial dilutions were prepared in PBS orDME. Mice were anesthetized via intraperitoneal injection of0.65 to 1.3 mg of pentobarbital (in PBS) and infected intrana-sally with 25 ,lI of diluted virus. For most infections, there were10 animals per group (i.e., per virus per dose) and 5 of theanimals were monitored through the course of the infection todetermine mortality and the other 5 were sacrificed at various

L

VV 14.7(+)/TNF: [ mTNF-

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VV 14.7(-)/TNF: mimTNF-PP7.5

VV 14.7(+):

VV 14.7(-):

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.1-

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times postinfection to harvest organs for determination ofpathology and virus titration.Lung titration. Mice were sacrificed by ether anesthesia

followed by cervical dislocation. Lungs from infected micewere removed aseptically, rinsed with PBS, and frozen at- 20°C until use. For titrations, lungs were thawed, maceratedwith a sterile razor blade, transferred to 1 ml of PBS (on ice),and sonicated for 4 x 15 s (5 A of direct current; BransonSonicPower Sonifier). A portion of the lung sonicate wasmixed with an equal volume of 0.25-mg/ml trypsin and incu-bated at 37°C for 30 min. The trypsinized lung was then seriallydiluted in DME containing 1% FBS, and the virus titer wasdetermined by plaque assay on 143B cell monolayers. Anagarose overlay containing 250 ,ug of X-Gal per ml was appliedat 48 h postinfection, and blue plaques were counted on thefollowing day.Lung pathology. Lungs from mice sacrificed at various times

postinfection were placed immediately into buffered formalin;following fixation, the tissue was processed and stained withhematoxylin and eosin by standard techniques. The scoring wasdone in a coded fashion by one of us (S.C.) and was based onthe percentage of bronchioles and vessels involved in theinflammatory process in a cross section of the left lung. Lungsections estimated to contain the following percentages ofinflammatory involvement were scored as follows: from 76 to100%, +4; from 51 to 75%, +3; from 26 to 50%, +2; <25%,+ 1. Sections devoid of inflammatory foci received a score of 0.The inflammation was primarily perivascular and peribronchialwith little intraalveolar inflammation. However, there wasintraalveolar edema in some samples.

RESULTS

Expression of mouse TNF-a and the Ad 14.7K protein byWvirus recombinants in vitro. As described in Materials andMethods and shown in Fig. 1, the VV double recombinantswere constructed by the method of Coupar et al. (13), withselection first for disruption of the VV tk gene in the presenceof 5-bromodeoxyuridine and then for insertion of the HSV tkgene in the presence of methotrexate. Both mouse mTNF-oL-and Ad2 14.7K protein-coding sequences were cloned behindthe VV p7.5 combined early-late promoter. Expression of theinserted genes in the two recombinants, VV 14.7(+)/TNF andVV 14.7(-),TNF, was confirmed in tissue culture by infectionof 143B cells. At various times postinfection, supernatantswere collected and assayed for the presence of TNF with a

FIG. 2. Kinetics of TNF-co and Ad 14.7K protein production by VVrecombinants. (A) Human 143B cell monolayers in 24-well plates wereinfected with 2 PFU of VV 14.7(+)/TNF or VV 14.7( - )/TNF per cell,and supernatants of infected cells were collected at various timespostinfection and assayed for TNF-ot immunoreactivity with a double-sandwich ELISA system (Genzyme FactorTest mTNF-oa). (B) Afterremoval of the supernatant for the mTNF-a assay, detergent lysatesprepared from 143B cell monolayers were subjected to Western blotanalysis to detect and quantify the relative amounts of the Ad2 14.7Kprotein as described in Materials and Methods. Optical density (O.D.)units were obtained by scanning the film, which had been exposed tochemiluminescence detection reagents, by utilizing the GELSCAN XL(2.1) program on an LKB laser densitometer. The 14.7K protein was

detected only in lysates of cells infected with VV 14.7(+)/TNF, not inlysates of cells infected with VV 14.7(-)/TNF. (C) Western blotshowing lysates of VV 14.7(-)/TNF (lane 1)- or VV 14.7(+)/TNF(lane 2)-infected 143B cells or Ad2-infected HeLa cells (lane 3)probed with a polyclonal antibody to a TrpE-14.7K fusion protein. Mr(K), relative molecular weight in thousands.

A

E

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0 1 0 20 30 40 50

HOURS POST-INFECTION

B

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HOURS POST-INFECTION

C*1 2 3 Mr (K)

-106

-80

- 49.5

-32.5

'000 - 27.5

14.7K 18.5

Ad2 +vv14.7K - +TNF + +

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EFFECT OF ADENOVIRUS 14.7K PROTEIN ON VACCINIA VIRUS VIRULENCE 457

100 -

-0--- 14.7(+)/TNF* 14.7(-)/TNF Percent

Survival

80 -

60 -

204,E

2.9 x 104 2.9 x

i052.5 x 10'

Virus Dose (PFU)

IFIG. 4. Effectofthe Ad 14.7K protein on survival of mice infected0 2 4 6 8 1 0 1 2 with VV expressing TNF-ot. Mice were infected with the indicated

dilutions of VV 14.7(+)/TNF (1) or VV 14.7(-)ITNF (E), and

Day Post-infection cumulative mortality at the conclusion of the experiment (day 10) wasrecorded. There were no further deaths after this time (n = 8 to 10 for

FIG. 3. Kinetics of mortality of BALB/c mice after intranasalinfection with VV 14.7(+)/TNF or VV 14.7(-)/TNF. BALB/c femalemice 3 to 4 weeks of age were inoculated intranasally with 2.5 x 106PFU of VV 14.7(+)ITNF or VV 14.7( - ),TNF and observed daily fordeath (n = 8 to 10 mice per group).

double-sandwich ELISA technique (Fig. 2A) while detergentlysates of cell monolayers were assayed for the presence of the14.7K protein by Western blot (Fig. 2B and C). Expression ofboth proteins was detectable by 8 to 12 h postinfection, and theamounts continued to increase to 48 h, consistent with theearly-late promoter activity of p7.5. The two recombinantsproduced similar amounts of TNF (Fig. 2A), while cellsinfected with a control VV lacking the TNF insert secreted nodetectable TNF (data not shown). The 14.7K protein wasdetected only in lysates of cells infected with VV 14.7(+)/TNF,not in lysates of those infected with VV 14.7(-)/TNF; it hasthe same mobility on sodium dodecyl sulfate-polyacrylamidegel electrophoresis as the 14.7K protein produced by Ad2-infected HeLa cells (Fig. 2C). No differences were notedregarding the in vitro replicative efficiencies of VV 14.7(+)/TNF and VV 14.7(-)/TNF in 143B cells, in terms of eithervirus yield following high-multiplicity infection or plaque mor-phology. This was not unexpected, since it had been shown thatVV expressing mTNF-a (58) or gamma interferon (IFN-^y)(18, 38) replicates to wild-type levels in tissue culture systemsalthough its replication is attenuated in vivo.Ad 14.7K protein increases mortality and morbidity of mice

infected with W expressing mTNF-a. Mice were infected todetermine the effect of antagonism of TNF cytolysis by the14.7K protein on VV virulence in vivo. It had been shown thatvirus-directed TNF expression has a pronounced attenuatingeffect on VV pathogenesis in normal, euthymic, and immuno-deficient (nude or sublethally irradiated) mice (58). Therefore,we hypothesized that if TNF directly causes the lysis ofVV-infected cells and if the Ad 14.7K protein is able toabrogate this cytotoxicity, then the VV recombinant coexpress-ing TNF and the 14.7K protein should be able to replicate tohigher titers and cause greater morbidity and mortality thanthe virus expressing TNF alone. That is, if the 14.7K protein isable to rescue VV-infected cells from premature lysis by TNF,a lysis occurring prior to the production of a full complementof viral progeny, then virus replication and pathogenicityshould be correspondingly enhanced. Figure 3 shows that,

the two largest doses of VV administered and 3 to 5 for the smallestdose).

indeed, VV 14.7(+)/TNF was lethal to 3- to 4-week-oldBALB/c mice infected intranasally while VV 14.7(-)/TNF atan equivalent input dose (2.5 x 106 PFU) was not lethal. Miceinfected with 2.5 x 106 PFU of VV 14.7(+)/TNF, the largestdose administered, began to manifest illness on day 3 to 4postinfection, as evidenced by decreased activity, ruffling offur, hunched posture, and rapid, labored respiration. Theillness was progressive, with deaths occurring on days 5, 6, and7. Animals infected with a 10-fold-lower input dose showedsimilar symptoms in milder form, with a peak of illness on day5 or 6 postinfection and recovery thereafter. Mice infected withVV 14.7(-)/TNF manifested a consistently milder illness, andthere were no deaths. Mice inoculated with 2.5 x 106 PFU ofVV 14.7(-)rTNF appeared similar, in terms of clinical illness(hunching, ruffling, etc.), to those receiving a 10-fold lowerdilution of VV 14.7(+)/TNF. The cumulative mortality afterinfection with three different doses of virus (Fig. 4) corre-sponded to a 50% lethal dose (LD50) of 8 x 105 PFU for VV14.7(+)/TNF, while the LD50 for VV 14.7(-)/TNF wasgreater than or equal to 8 x 106 PFU. The exact end pointcould not be determined, since no animals died when given thehighest virus concentration that could be administered.Ad 14.7K protein increases intrapulmonary replication ofW containing the mTNF-a-encoding gene. VV replicationwithin the lungs of infected mice was quantified, and the resultswere found to concur with the preceding data concerningmorbidity and mortality in that the VV expressing both the14.7K protein and TNF replicated to higher titers than did theVV expressing TNF alone. Mice infected with 10-fold serialdilutions of VV were sacrificed at various times postinfection,lung sonicates were prepared, and virus titers were determinedas described in Materials and Methods. For infections with thelargest virus dose shown in Fig. SA, lung titers were lowest on

day 1 (most of the virus presumably represented the input) androse by day 4, reaching a plateau and remaining elevatedthrough day 6 or 7. By day 7 postinfection, 100% of the VV14.7(+)/TNF-infected animals had succumbed to the infectionwhile all of the VV 14.7(-)/TNF-infected mice remainedalive. From day 4 onward, lung titers were 2 or more orders ofmagnitude greater following infection with VV 14.7(+)/TNFthan after infection with VV 14.7(-)/TNF. Infections with

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10-fold less virus (Fig. 5B), which all of the animals survived,

108 showed that in the presence of virally produced TNF, 14.7KA protein expression caused a marked delay in virus clearance

107 4 from the lungs. Virus was undetectable in pulmonary sonicatesof VV 14.7(-)/TNF-infected mice by day 8 postinfection,

106 while titers remained greater than 105 PFU per lung for VV/1-1 14.7(+)/TNF-infected mice. Infections with a 100-fold dilutionqZ 105 of stock virus confirmed this trend (Fig. SC); although there

was no mortality and minimal morbidity at this dose, lung virus.@ 104 Utiterswere still greater for mice infected with the VV recom-

3 binant expressing the 14.7K protein than for those infected= 10 with the nonexpressing control.

102 Ad 14.7K protein increases the pulmonary inflammatoryresponse after intranasal infection with W expressing

101 mTNF-ot. Consistent with the increased morbidity and mortal-ity, the pulmonary inflammatory response was more severe for

10°- ., ., , ,., mice infected with the two largest doses of VV 14.7(+)/TNF0 2 4 6 8 1 0 (2.5 x 10" and 2.9 x 10: PFU) than for those infected with

equivalent doses of VV 14.7( - )/TNF. At the peak of infectionDay Post-infection (days 4 to 6), there was prominent peribronchial and perivas-

108 cular inflammation affecting the larger vessels and bronchi.The alveolar septae were widened, with some intra-alveolar

107 edema, indicating vascular congestion, but there was no intra-alveolar inflammatory response. The extent of inflammation

i6o was typically scored as 1 or 2 grades more severe (on a scale of0 to 4) for mice infected with VV expressing the 14.7K protein

I 105lo than for those infected with the control virus. These differencesS& 4 /4 in the severity of pulmonary inflammation were greatest

10 between days 4 and 6 postinfection and with the largest inputdoses of VV. Photographs illustrating the comparative pulmo-

= i03 \nary histopathology in VV 14.7(+)/TNF- and VV 14.7(-)-102 infected animals are shown in Fig. 6A and B, respectively.m0 Spleens of infected mice were also submitted for pathologic

101 examination and were noted to be markedly hypocellular, withdepletion of lymphoid follicles, following infection with the

100 largest dose (2.5 x 10" PFU) of recombinant VV. This0 2 4 6 8 1 0 pathology also occurred more frequently after infection with

VV 14.7(+)/TNF than after infection with VV 14.7(-)/TNFDay Post-infection (data not shown).

1o8 Ad 14.7K protein has no effect on W infection in vivoC 1 without coexpression of the TNF-ot-encoding gene. We also

io7 examined the effect on VV pathogenesis of antagonism ofendogenous TNF-ox by the 14.7K protein. For these studies,

106 BALB/c mice were infected with VV 14.7(+) and VV 14.7( -),the recombinants which contain the 14.7K protein-encoding

1105 gene (either expressing or nonexpressing) but have only theX1043 \ HSV tk insert (without TNF) in the Hindlll F region. Mice

.i0 bsuccumbed at nearly equivalent rates following infection with103 2.5 x 10" PFU of VV 14.7(+) or VV 14.7(-); the final,

cumulative mortalities after infections with serial dilutions of102 D ' hvirus were also very similar for the two VV recombinants (Fig.

7). The LD5(s derived from these data were 0.9 x 105 for VV101 14.7(+) and 1.4 x 105 for VV 14.7(-). Pulmonary virus titers

were also very similar at all three input doses following100 infection with VV 14.7(+) or VV 14.7(-) (data not shown).

0 2 4 6 8 1 0 Thus, the biologic effects of the Ad 14.7K protein on VVpathogenesis required concomitant virus-directed expression

Day Post-infection of TNF-o.FIG. 5. Virus titers in lungs after infection with VV 14.7(+)/TNF

or VV 14.7( - )/TNF. Mice infected intranasally with VV 14.7(+)/TNF DISCUSSION(D) or VV 14.7(-)/TNF (-) were sacrificed at various times postin-fection, and lungs were removed for virus titration as described in Following Ad infection, there can exist a prolonged periodMaterials and Methods. Shown are the total numbers of PFU present (months to years) of viral persistence and/or latency (15, 44,in the right lungs of mice infected with 2.5 x 1(0 (A), 2.9 x 105 (B),Or 2.9 x 1()4 (C) PFU of recombinantVV./ 55). Gene products encoded by the Ad E3 region are thought

mbiantVVto play a role in the evasion of host immune surveillance thatmay be required for viral persistence, including escape from

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EFFECT OF ADENOVIRUS 14.7K PROTEIN ON VACCINIA VIRUS VIRULENCE 459

A BF-

5 , __ .

FIG. 6. Pulmonary histopathology following infection with VV recombinants. The sections shown are from mice infected with 2.9 x 10 PFUof VV 14.7(+)/TNF (A) or VV 14.7(-)/TNF (B) and sacrificed on day 6 postinfection. The letter B indicates bronchioles, and the letter Vindicates a blood vessel.

cytokine-mediated antiviral effects. One of these gene prod-ucts, the Ad 14.7K protein, functions as a general inhibitor ofTNF cytolysis in tissue culture systems. However, its influenceon viral pathogenesis in an infected host has remained unchar-acterized, apart from the finding that deletion of the Ad514.7K protein (as well as the other TNF antagonists, the 10.4Kand 14.5K proteins) alters the cellular nature of the inflamma-tory response from largely monocytic to primarily neutrophilicin cotton rat lungs.We have shown that expression of the Ad2 14.7K protein by

a recombinant VV which also expresses mTNF-cx results insignificant reversal of the TNF-mediated viral attenuationinitially described by Sambhi et al. (58). Intranasal inoculationof BALB/c mice with VV 14.7(+)/TNF resulted in greatermorbidity and mortality [LD50s, 8 x 105 PFU for VV 14.7(+)/TNF versus -8 x 106 PFU for VV 14.7(-)/TNF], enhancedinfiltration of inflammatory cells in perivascular and peribron-chial regions, and increases of approximately 2 orders of

100 -

PercentSurvival

80 -

60 -

40 -

20 -

* VV 14.7(-)

N VV 14.7(+)

O 4-2.5 x 104 2.5 x I05 2.5 x 106

Virus Dose (PFU)FIG. 7. Final percent survival after infection with VV 14.7(+) or

VV 14.7(-). Mice were infected with the indicated dilutions of VV14.7(+) or VV 14.7(-), and cumulative mortality at day 12 postinfec-tion was recorded (there were no deaths after this time). For the twolargest doses of VV administered, n = 9 or 10, and for the smallestinput dose, n = 5.

magnitude in peak viral lung titers compared with infectionwith VV 14.7(-)/TNF. The Ad 14.7K protein presumablyachieves this effect by inhibiting premature lysis of infectedcells by TNF prior to the production of a full complement ofviral progeny. Our findings regarding the enhanced pathoge-nicity of VV coexpressing the 14.7K protein and TNF relativeto that of the control virus expressing TNF alone lend supportto the hypothesis that virally produced TNF attenuates, at leastin part, through direct lysis of infected cells, because inhibitionof TNF-mediated cytolysis is the only effect shown to bereversed by the 14.7K protein in tissue culture assays. The Ad14.7K protein has not been shown to affect other actions ofTNF, such as transcriptional activation. However, the increasein viral virulence is insufficient to restore the LD50 to that ofVV lacking the TNF insertion [LD5,,, approximately 105 PFUfor VV 14.7(+) and VV 14.7(-); Fig. 7]. The fact that reversalof the TNF-mediated attenuation by 14.7K is incomplete maybe a quantitative effect, on the basis of the levels of TNF andthe 14.7K protein expressed, or may indicate a cell type-specific sensitivity to lysis. It is also possible that secreted TNFacts on uninfected neighboring cells to induce an antiviralstate, an effect which the nonsecreted 14.7K protein wouldhave no opportunity to reverse.The fact that the differences in virus growth potential

observed in vivo are not apparent during propagation of theviruses in cultured cells is not unexpected, as the same findinghas been made for VV encoding mTNF-ox and IFN-y (18, 38,58). These viruses are attenuated for murine infections butreplicate to wild-type titers in vitro. It may be that the fullytransformed cell lines used to culture the virus are not lysed byTNF, even when infected with VV, but that the primary lungcells (type I and II pneumocytes, fibroblasts, etc.) are sensi-tized to TNF cytolysis by VV infection. It is also possible thatTNF-mediated lysis of VV-infected cells in vivo requiresparticipation of another host-derived factor, perhaps IFN-y,which is known to be synergistic with TNF in lysis of virus-infected cells (76). However, it seems unlikely that the cell-mediated or humoral components of the adaptive immuneresponse are directly involved in mediating the effects of the14.7K protein in this system because the TNF-expressing VVstudied by Sambhi et al. (58) was attenuated equally forinfections of nude, sublethally irradiated, and euthymic mice,and the immunocompetent mice showed no evidence of aug-

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460 TUFARIELLO ET AL.

mented cytotoxic-T-cell, NK-cell, or antibody responses to theinfection. In addition, preliminary results from intranasalinfections of SCID mice with our VV constructs indicateenhanced pathogenicity of VV 14.7(+)ITNF compared withthe controls, which is consistent with the pattern observedupon infection of immunocompetent BALB/c mice. Thus, the14.7K protein does not appear to require the presence of T orB cells (or their products) to reverse the TNF-induced atten-uation of VV, just as the attenuation itself does not appear toinvolve these cell populations.Lee et al. (43) have reported that the LD50 was 6.3 x i04 for

2- to 3-week-old BALB/c mice intranasally infected withVV-WR, whereas the LD50 for those infected with a tkdeletion mutant was greater than 108. It is interesting that thedouble recombinants VV 14.7(+) and VV 14.7(-), whichhave the HSV tk gene in the HindIII-F region, have LD50s onthe order of 105, much closer to the wild-type than to the tkmutant values. These results suggest that expression of theHSV tk gene can restore virulence to VV in which the native tkgene has been disrupted by insertional mutagenesis.The finding that a viral gene product (the Ad 14.7K protein)

which antagonizes an antiviral cytokine (TNF) significantlyenhances virus replication in the presence of that cytokine isconsistent with much recent literature supporting the impor-tance of soluble mediators in the immune response to poxvirusinfection. These data are derived from a wide variety ofexperimental approaches. Recombinant VV expressing inter-leukin 2 (16, 50), IFN-y (18, 38, 58), and TNF-ao (58) areattenuated for infection of nude mice, and the interleukin-2-induced reduction in virulence depends, in part, on NK cellsand production of IFN- y. VV replicates to higher titers and ismore lethal in mice that are homozygous for disruption of theIFN-,y receptor gene (IFN--yR0/0) (31). In addition, pretreat-ment of mice with IFN-ao/1 inhibits viral gene expression,determined by assaying the activity of a luciferase reportergene inserted into the VV genome (54).

Study of poxvirus genes and their expression has alsosupported the importance of cytokine antagonists in evasion ofthe host immune response (3, 53, 64, 70). For example,secreted fragments of the receptors for various cytokinespresumably bind to and neutralize their cognate cytokines,preventing access to the membrane-bound cellular receptors.Shope fibroma virus contains an open reading frame (SFVT-2) with homology to the extracellular portion of the TNFreceptor (TNF-R), but significantly for our studies, this openreading frame (Sal F 19R) is disrupted by stop codons in theVV-WR genome (30). Thus, VV is not known to express theTNF-R homolog or any other TNF antagonist which may havecontributed to the effects of the Ad 14.7K protein in vivo.The interplay between Ad infection and TNF action is

complex. TNF expression is induced by Ad infection in vitroand in vivo. TNF lyses cells infected with Ad E3 deletionmutants (23); is antiviral in vitro, especially in synergy withIFN--y (76); and augments the IFN-y-induced block to Adassembly (47). Three Ad E3 genes (the 14.7K, 10.4K, and14.5K protein-encoding genes) are involved in antagonism ofTNF cytolysis, and the E3 promoter from which these genesare expressed is activated by TNF (39). We have removed the14.7K protein from this network of interactions and shown thatwhen isolated and transplanted into the VV genome in com-bination with TNF-co, the Ad protein counteracts the attenu-ating effect of TNF on VV virulence.

ACKNOWLEDGMENTSM.S.H. was supported by Public Health Service grant Al 27199 and

Cancer Center grant P01 CA-13330. J.T. was supported by Medical

Scientist Training grant 5T32 GM07288 from the National Institutes ofHealth.We thank Bernard Moss of the National Institutes of Health for

plasmid pSC11 and Ian Ramshaw and William Wold for generouslyproviding plasmid pFBX-mTNF and the polyclonal antibody to the14.7K protein, respectively. We also thank Betty Diamond and Mat-thew Scharff for critical reading of the manuscript and many helpfulsuggestions.

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