infected animals - PNAS · infected animals JOSE F. RODRIGUEZ*, DOLORESRODRIGUEZ*,...

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Proc. Natl. Acad. Sci. USA Vol. 85, pp. 1667-1671, March 1988 Microbiology Expression of the firefly luciferase gene in vaccinia virus: A highly sensitive gene marker to follow virus dissemination in tissues of infected animals JOSE F. RODRIGUEZ*, DOLORES RODRIGUEZ*, JUAN-RAMON RODRIGUEZ*, ELEANOR B. MCGOWAN*, AND MARIANO ESTEBAN*t* Departments of *Biochemistry and tMicrobiology and Immunology, State University of New York Health Science Center at Brooklyn, New York, NY 11203 Communicated by Chandler McC. Brooks, November 9, 1987 ABSTRACT We have Introduced the firefly luciferase gene of Photinus pyralis into the vaccinia virus genome. This gene is expressed in a coordinate fashion during virus infec- tion. Luminescence produced by the action of luciferase [Pho- tinus-luciferin:oxygen 4-oxidoreductase(decarboxylating, ATP-hydrolyzing), EC 1.13.12.7] was easily detectable in infected cells in culture as well as in cells of tissues of infected mice. The limits of detection were about one infected cell in a background of a million noninfected cells. The luciferase assay was about 1000-fold more sensitive than that of .8- galactosidase. Our findings show that the luciferase assay can be conveniently used to follow viral gene expression and virus dissemination both in cell cultures and in tissues of infected animals. The firefly luciferase gene isolated from a cDNA library from Photinus pyralis has been expressed in bacteria (1), plants (2), and animal cells (3). This gene encodes an enzymatically active polypeptide with an apparent molecular mass of 62 kDa, which is targeted to peroxisomes in both firefly and mammalian cells (4). In the presence of luciferin and ATP, an enzyme-bound luciferyl-adenylate complex is formed, and this is followed by oxidative decarboxylation with production of C02, oxyluciferin, AMP, and light (3). The reaction catalyzed by P. pyralis luciferase (EC 1.13.12.7) emits a yellow-green light at pH 7.5 to 8.5, with the peak emission at 560 nm. This light emission can be measured spectrophotometrically or by exposure to x-ray film (2). Because luciferase assays are more sensitive than those assays for any other reporter gene, it has been pro- posed that this gene could be a new tool for studying gene expression in both plant and animal cells (2, 3). Vaccinia virus is a 185-kilobase (kb) DNA-containing virus that replicates in the cytoplasm of infected cells. During virus infection, extensive transcription occurs, and a high yield of virus, about 10,000 particles, is produced per cell (5). This virus is now being used as a eukaryotic viral vector with potential for vaccine production. This potential exists because virtually any foreign gene of prokaryotic or eukaryotic origin can be introduced into the viral genome and faithfully expressed during virus infection, and, when recombinant viruses are inoculated in animals, there is activation of both cell-mediated and humoral immune re- sponses to the foreign antigen (6). In this investigation, we have introduced the firefly luci- ferase gene into the genome of vaccinia virus and have provided direct evidence that recombinants of vaccinia virus expressing luciferase can be conveniently used to follow viral gene expression in a few infected cells. Moreover, luciferase activity can easily be measured in target tissues of experimental animals. This reporter gene could be effec- tively used to follow virus replication and levels of gene expression in animals infected with vaccinia virus recombi- nants. MATERIALS AND METHODS Virus and Cells. The WR strain of vaccinia virus was propagated in African green monkey kidney BSC-40 cells and purified as described previously (7). BSC-40 cells were grown in Dulbecco's modified Eagle's medium containing 10% newborn calf serum. Construction of Vaccinia Virus Recombinants. Recombi- nant viruses were prepared by infecting BSC-40 cells with vaccinia virus [0.01 plaque-forming unit (pfu) per cell] and transfecting them with calcium phosphate-precipitated plas- mid DNA (10 pug per 60-mm dish). Cell cultures were harvested 48 hr postinfection, and recombinant viruses were isolated by plaque assay after addition of 5-bromo-4-chloro- 3-indolyl j3-D-galactoside to the agar overlay (8). Blue plaques were picked up and were plaque purified, and stocks of recombinant viruses were prepared in BSC-40 cells. To confirm the predicted structures of the recombinant virus, DNA from infected cells was examined by restriction endo- nuclease analysis and DNA hybridization. Measurement of Luciferase and 8-Galactosidase Activities. An instrument described by Charo et al. (9) for measuring the luciferin/luciferase luminescent reaction with ATP was used to measure the level of luciferase in culture cells or in animal tissue extracts. The reactions were carried out in glass cuvettes with mechanical stirring of the contents. The reaction mixture consisted of 450 A.l of buffer containing 25 mM Hepes (pH 7.4), 136 mM NaCI, 1 mM EDTA, 6 mM MgSO4, 0.6% polyethylene glycol 6000, and 20-100 ,.l of cell extract (4.6 x 103 cells is 0.3 Ag of protein) or 75 1.l of tissue extract. The shutter was opened to establish the baseline in the absence of exogenous ATP and was closed. Then, a mixture of 25 ul 0.1 M ATP (pH 6.8) and 10 p.l of 10 mM luciferin (Analytical Luminescence Laboratory, San Diego, CA; dissolved in 10 mM Hepes, pH 7.5/150 mM NaCI/1 mM EDTA) was added to start the reaction, and the shutter was quickly opened (<2 sec) to obtain the maximum luminescence response of the sample. To quantitate the levels of luciferase present in cell cultures or tissue extracts, a standard curve was constructed from reaction mixtures containing cell extracts or tissue extracts from uninfected culture cells and spleen tissue together with purified lucife- rase (Sigma). Measurements of 83-galactosidase in cell ex- tracts were carried out as described previously (8). Abbreviations: pfu, plaque-forming unit(s); tk, thymidine kinase; AraC, cytosine arabinonucleoside. tTo whom reprint requests should be addressed. 1667 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Downloaded by guest on October 4, 2020

Transcript of infected animals - PNAS · infected animals JOSE F. RODRIGUEZ*, DOLORESRODRIGUEZ*,...

Page 1: infected animals - PNAS · infected animals JOSE F. RODRIGUEZ*, DOLORESRODRIGUEZ*, JUAN-RAMONRODRIGUEZ*, ELEANORB. MCGOWAN*, ANDMARIANOESTEBAN*t* Departments of*Biochemistry and tMicrobiology

Proc. Natl. Acad. Sci. USAVol. 85, pp. 1667-1671, March 1988Microbiology

Expression of the firefly luciferase gene in vaccinia virus: A highlysensitive gene marker to follow virus dissemination in tissues ofinfected animalsJOSE F. RODRIGUEZ*, DOLORES RODRIGUEZ*, JUAN-RAMON RODRIGUEZ*, ELEANOR B. MCGOWAN*,AND MARIANO ESTEBAN*t*Departments of *Biochemistry and tMicrobiology and Immunology, State University of New York Health Science Center at Brooklyn, New York, NY 11203

Communicated by Chandler McC. Brooks, November 9, 1987

ABSTRACT We have Introduced the firefly luciferasegene of Photinus pyralis into the vaccinia virus genome. Thisgene is expressed in a coordinate fashion during virus infec-tion. Luminescence produced by the action of luciferase [Pho-tinus-luciferin:oxygen 4-oxidoreductase(decarboxylating,ATP-hydrolyzing), EC 1.13.12.7] was easily detectable ininfected cells in culture as well as in cells of tissues of infectedmice. The limits of detection were about one infected cell in abackground of a million noninfected cells. The luciferase assaywas about 1000-fold more sensitive than that of .8-galactosidase. Our findings show that the luciferase assay canbe conveniently used to follow viral gene expression and virusdissemination both in cell cultures and in tissues of infectedanimals.

The firefly luciferase gene isolated from a cDNA libraryfrom Photinus pyralis has been expressed in bacteria (1),plants (2), and animal cells (3). This gene encodes anenzymatically active polypeptide with an apparent molecularmass of 62 kDa, which is targeted to peroxisomes in bothfirefly and mammalian cells (4). In the presence of luciferinand ATP, an enzyme-bound luciferyl-adenylate complex isformed, and this is followed by oxidative decarboxylationwith production of C02, oxyluciferin, AMP, and light (3).The reaction catalyzed by P. pyralis luciferase (EC1.13.12.7) emits a yellow-green light at pH 7.5 to 8.5, withthe peak emission at 560 nm. This light emission can bemeasured spectrophotometrically or by exposure to x-rayfilm (2). Because luciferase assays are more sensitive thanthose assays for any other reporter gene, it has been pro-posed that this gene could be a new tool for studying geneexpression in both plant and animal cells (2, 3).

Vaccinia virus is a 185-kilobase (kb) DNA-containingvirus that replicates in the cytoplasm of infected cells.During virus infection, extensive transcription occurs, and ahigh yield of virus, about 10,000 particles, is produced percell (5). This virus is now being used as a eukaryotic viralvector with potential for vaccine production. This potentialexists because virtually any foreign gene of prokaryotic oreukaryotic origin can be introduced into the viral genomeand faithfully expressed during virus infection, and, whenrecombinant viruses are inoculated in animals, there isactivation of both cell-mediated and humoral immune re-sponses to the foreign antigen (6).

In this investigation, we have introduced the firefly luci-ferase gene into the genome of vaccinia virus and haveprovided direct evidence that recombinants of vaccinia virusexpressing luciferase can be conveniently used to followviral gene expression in a few infected cells. Moreover,luciferase activity can easily be measured in target tissues of

experimental animals. This reporter gene could be effec-tively used to follow virus replication and levels of geneexpression in animals infected with vaccinia virus recombi-nants.

MATERIALS AND METHODSVirus and Cells. The WR strain of vaccinia virus was

propagated in African green monkey kidney BSC-40 cellsand purified as described previously (7). BSC-40 cells weregrown in Dulbecco's modified Eagle's medium containing10% newborn calf serum.

Construction of Vaccinia Virus Recombinants. Recombi-nant viruses were prepared by infecting BSC-40 cells withvaccinia virus [0.01 plaque-forming unit (pfu) per cell] andtransfecting them with calcium phosphate-precipitated plas-mid DNA (10 pug per 60-mm dish). Cell cultures wereharvested 48 hr postinfection, and recombinant viruses wereisolated by plaque assay after addition of 5-bromo-4-chloro-3-indolyl j3-D-galactoside to the agar overlay (8). Blueplaques were picked up and were plaque purified, and stocksof recombinant viruses were prepared in BSC-40 cells. Toconfirm the predicted structures of the recombinant virus,DNA from infected cells was examined by restriction endo-nuclease analysis and DNA hybridization.Measurement of Luciferase and 8-Galactosidase Activities.

An instrument described by Charo et al. (9) for measuringthe luciferin/luciferase luminescent reaction with ATP wasused to measure the level of luciferase in culture cells or inanimal tissue extracts. The reactions were carried out inglass cuvettes with mechanical stirring of the contents. Thereaction mixture consisted of 450 A.l of buffer containing 25mM Hepes (pH 7.4), 136 mM NaCI, 1 mM EDTA, 6 mMMgSO4, 0.6% polyethylene glycol 6000, and 20-100 ,.l of cellextract (4.6 x 103 cells is 0.3 Ag of protein) or 75 1.l oftissue extract. The shutter was opened to establish thebaseline in the absence of exogenous ATP and was closed.Then, a mixture of 25 ul 0.1 M ATP (pH 6.8) and 10 p.l of 10mM luciferin (Analytical Luminescence Laboratory, SanDiego, CA; dissolved in 10 mM Hepes, pH 7.5/150 mMNaCI/1 mM EDTA) was added to start the reaction, and theshutter was quickly opened (<2 sec) to obtain the maximumluminescence response of the sample. To quantitate thelevels of luciferase present in cell cultures or tissue extracts,a standard curve was constructed from reaction mixturescontaining cell extracts or tissue extracts from uninfectedculture cells and spleen tissue together with purified lucife-rase (Sigma). Measurements of 83-galactosidase in cell ex-tracts were carried out as described previously (8).

Abbreviations: pfu, plaque-forming unit(s); tk, thymidine kinase;AraC, cytosine arabinonucleoside.tTo whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Page 2: infected animals - PNAS · infected animals JOSE F. RODRIGUEZ*, DOLORESRODRIGUEZ*, JUAN-RAMONRODRIGUEZ*, ELEANORB. MCGOWAN*, ANDMARIANOESTEBAN*t* Departments of*Biochemistry and tMicrobiology

1668 Microbiology: Rodriguez et al.

RESULTSIntroduction of the Firefly Luciferase Gene on Vaccinia

Virus DNA. A BamHI fragment of 1892 base pairs (bp)containing the coding region of luciferase and its putativepoly(A) signal was isolated from pD0432 (2). The presumedstart site of translation is 81 bases downstream from theBamHI site (2, 3). The 1892-bp fragment was blunt ended bytreatment with Klenow enzyme, purified by agarose gelelectrophoresis, and cloned into the Sma I site of thevaccinia virus insertion vector pSC11 (8) containing theEscherichia coli f-galactosidase gene (lacZ), a vacciniapromoter (7.5-kDa promoter) with early and late regulatorysignals (10), and flanking regions from the vaccinia thymi-dine kinase (tk) gene. As a result of this cloning strategy, weisolated two different plasmids containing the luciferasecoding sequences in both possible orientations relative to the7.5-kDa vaccinia virus promoter (Fig. 1A).

Next, we introduced the two plasmids, pSCiiJ andpSCLtC, into the tk region of vaccinia virus DNA byhomologous recombination. Selection of plaques producedby recombinant viruses was carried out by visual detectionafter incubation of infected cell cultures with 5-bromo4-chloro-3-indolyl 83-D-galactoside (8). 8-Galactosidase-pro-ducing virus plaques were picked, cloned three times, andamplified in BSC-40 cells. The genomic structure of vacciniavirus recombinants (designated WRifiii and WRLt) wasestablished by Southern blots after hybridization with either

SinaI

X psc 11

BamHI

the 32P-labeled 5-kb vaccinia virus DNA HindIII J fragmentor the firefly luciferase 1892-bp 32P-labeled BamHI frag-ment. HindIII digestion of DNA from wild-type vacciniavirus-infected cells probed with the viral HindIII J fragmentrevealed a single band of 5 kb (Fig. 1B, lane 1). The sameprobe hybridized to a larger fragment of DNA from cellsinfected with the recombinant WRiiC (Fig. 1B, lane 2) orWRLV (Fig. 1B, lane 3) due to the insertion of the plasmidDNA. To assess the orientation of the luciferase gene withrespect to the vaccinia 7.5-kDa promoter, DNAs from virus-infected cells were digested with BamHI and Sma I, andboth were hybridized with the 1892-bp labeled BamHIfragment containing the luciferase gene. As expected, DNAfrom cells infected with WRiIUl produced a fragment of1889 bp (Fig. 1B, lane 4) corresponding to the BamHI-SmaI fragment (3), whereas digestion ofDNA from cells infectedwith WRLt produced a larger fragment (Fig. 1B, lane 5).These results establish that the luciferase gene has beeninserted in the tk region of the vaccinia virus genome in bothorientations relative to the viral 7.5-kDa promoter.

Regulated Expression of the Luciferase Gene on VacciniaVirus Recombinants. Vaccinia virus gene expression is con-trolled according to a very strict temporal pattern (5). Theviral 7.5-kDa promoter has been shown to drive gene expres-sion at both early and late times after virus infection (10). Toassess temporal regulation of luciferase gene expression, weinfected BSC-40 cells with the vaccinia virus recombinant

LBamHI

Bam Hi / Klenow_

~ 1 2 3 4 5

U

pSCLUC pSCLUC

FIG. 1. (A) Construction of vaccinia virus recombinants containing the firefly luciferase gene. A 1892-bp fragment containing the luciferasegene with translation and termination codons was obtained by cleavage of pDO432 (provided by S. H. Howell, University of California at SanDiego) with BamHI, and the protruding ends were filled in by using the Klenow fragment of DNA polymerase I and deoxyribonucleotidetriphosphates. The fragment was blunt-end-ligated to pSC11 (provided by B. Moss, National Institutes of Health) that had been digested withSma I. Two recombinant plasmids were obtained with the luciferase gene in both orientations relative to the 7.5-kDa viral promoter. Theresulting recombinant vectors, containing the left (tkL) and right (tkR) parts of the vaccinia tk gene, were transfected into BSC-40 cells that wereinfected with wild-type virus. After homologous recombination was allowed to occur, recombinants were selected by f3-galactosidaseexpression (8). (B) The genomic structure of vaccinia virus recombinants was determined by Southern blots. DNA from either wild-typevaccinia virus-infected cells (lane 1), WRLU -infected cells (lane 2), orWRLt(4-infectedcells (lane 3) was digested with HindlII and probedwith the 32P-labeled 5-kb vaccinia virus HindIII J fragment. DNA from cells infected with WRLU (lane 4) or WRLt (lane 5) was digestedwith BamHI and Sma I and was hybridized with the 1892-bp 32P-labeled BamHI fragment containing the luciferase gene.

Proc. Natl. Acad. Sci. USA 85 (1988)

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Page 3: infected animals - PNAS · infected animals JOSE F. RODRIGUEZ*, DOLORESRODRIGUEZ*, JUAN-RAMONRODRIGUEZ*, ELEANORB. MCGOWAN*, ANDMARIANOESTEBAN*t* Departments of*Biochemistry and tMicrobiology

Microbiology: Rodriguez et al.

@ ~~~~~10102 1103

13 mm /

eX

_o

CU

102

Cf AraC3

'D2

0 CtsC

AraC

4 8 12 16 20 24Time, hr

FIG. 2. Regulated expression of the luciferase gene duringvaccinia virus infection. BSC-40 cells (6 x 105 cells) were infected(2.5 pfu per cell) in the absence or presence ofAraC (40 iZg/ml) withrecombinant vaccinia virus WRL . At 1 hr. virus inoculum was

replaced with 4 ml of Dulbecco's modified Eagle's medium contain-ing 2.5% newborn calf serum. At various times postinfection, cellswere collected and cell extracts were tested for luciferase (Upper)and ,-galactosidase (Lower) activities. (Inset) Standard curve (pg ofluciferase vs. mm of pen deflection) was obtained with purifiedluciferase using several gain settings on the instrument. The resultsfor a gain setting of 700 are shown.

Proc. Natl. Acad. Sci. USA 85 (1988) 1669

WRL1JlC, in the absence or presence of the inhibitor of viralDNA synthesis cytosine arabinonucleoside (AraC). At var-ious times postinfection, cell extracts were prepared andtested for luciferase activity as described in Materials andMethods. The results are shown in Fig. 2 (Upper). Signifi-cant levels of luciferase activity were detected as early as 1hr postinfection. These levels increased with time postinfec-tion, reaching a maximum by 17 hr postinfection (1200 pg per2.4 x 104 cells). When late viral gene expression was

blocked with AraC, the luciferase levels did not increaseafter 6 hr postinfection and remained constant for about 24hr postinfection. Luciferase activity was not detected in cellsinfected with wild-type vaccinia virus, and only low levels(0.15 pg per 2.4 x 104 cells) of luciferase activity weredetected at late times postinfection in cells infected with thevirus recombinant WRLtC (data not shown). To show thatviral gene expression of vaccinia virus recombinants wasaccurately controlled, we measured in the same cell extractsf-galactosidase activity, which is driven by the late 11-kDaviral promoter (8). As shown in Fig. 2B (Lower), levels off3-galactosidase activity were detected only at late timespostinfection and reached a plateau by 17 hr postinfection.The appearance of j3-galactosidase was completely blockedin cells infected in the presence of AraC.We conclude that vaccinia virus recombinant WRLOCIU

expresses both luciferase and B3-galactosidase during virusinfection in a coordinate fashion.

Levels of Detection of Luciferase Activity in Cultures ofVirus-Infected Cells. One of our goals was to compare thedegree of sensitivity of luciferase detection with respect tothe more commonly used f3-galactosidase reporter geneduring vaccinia virus infection (8, 11). Thus, BSC-40 cellswere infected with vaccinia recombinants WRLU andWRLlIY, ranging from 1 pfu per cell to 10-6 pfu per cell.The results are summarized in Table 1. In cells infected withWRiZU, significant levels of luciferase were detected withas little as 10-5-106 pfu per cell. In cultures infected withWRL4t, the limit of detection of luciferase activity was

0.01 pfu per cell. ,B-Galactosidase activity could only bedetected in cells infected with 1000-fold higher multiplicitiesof infection than for luciferase (Table 1). The high sensitivityof luciferase activity was further documented by lumines-cence emitted from virus plaques. Monolayers of infectedBSC-40 cells were soaked in phosphate-buffered saline con-taining 7.5 mM ATP, cells were permeabilized with 1.5%dimethyl sulfoxide, and after addition of 1 mM luciferin (in adark room), plates were exposed to Kodak Ektapan film for3 hr. Under these conditions and in cultures infected with 1

pfu per cell, luminescence could be detected by the nakedeye 18 hr postinfection. After developing the film, single

Table 1. Limits of detection of luciferase and P-galactosidase activity in cells infected withvaccinia virus recombinants

Recombinant virus

WRLUInfection, Luciferase, ng per P-Galactosidase, ,ug WRLtW luciferase,pfu per cell 3 x 10' cells per 1.5 x 106 cells ng per 3 x 105 cells

1 2.04 ± 0.3 3.4 ± 0.4 0.02 ± 0.00510-1 1.8 ± 0.3 3.02 ± 0.4 0.01 ± 0.00410-2 1.6 ± 0.25 1.3 ± 0.3 0.005 ± 0.00041o-3 0.17 ± 0.04 0.1 ± 0.05 010-4 0.01 ± 0.003 0 010-5 0.006 ± 0.0015 0 010-6 0.001 ± 0.0003 0 0

BSC-40 cells (3 x 106 cells) in 60-mm dishes were infected with serial 1:10 dilutions of vaccinia virusrecombinants. Cells were harvested 18 hr postinfection, resuspended in 500 .ul of luciferase buffer, andfreeze-thawed. Extracts were assayed for luciferase and P-galactosidase activities.

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Page 4: infected animals - PNAS · infected animals JOSE F. RODRIGUEZ*, DOLORESRODRIGUEZ*, JUAN-RAMONRODRIGUEZ*, ELEANORB. MCGOWAN*, ANDMARIANOESTEBAN*t* Departments of*Biochemistry and tMicrobiology

1670 Microbiology: Rodriguez et al.

STAIN LUMI NESCENCE

FIG. 3. Luminescence of vaccinia virus plaques. Confluentmonolayers of BSC-40 cells, grown in flexible 60-mm dishes, wereinfected with various dilutions of the vaccinia virus recombinantWRLt. The virus plaques appearing 48 hr postinfection in liquidmedium were revealed by staining with crystal violet and by theluminescence recorded on sensitive film (Kodak Ektapan, devel-oped in HC-110 dilution B for 10 min). Two different virus multi-plicities are shown.

virus plaques were easily detected as defined spots (Fig. 3).We have also determined that by infecting cell monolayers inthe presence of rifampicin, an inhibitor of virus assembly butnot of virus protein synthesis, fluorescence emitted fromsingle cells could be detected on the film (not shown). Thefindings of Table 1 establish that luciferase activity is de-tected in as little as about one infected cell in a millionnoninfected cells. Moreover, the luciferase assay is about1000-fold more sensitive than the f-galactosidase assay.Expression of Luciferase Activity in Tissues of Mice Infected

with Recombinant Vaccinia Virus. Knowing the high sensi-tivity of luciferase detection in cultures of virus infectedcells, it was important to establish if the luciferase assaycould also be used to follow vaccinia virus replication intarget tissues of infected animals. Thus, female BALB/cmice were inoculated i.p. with various doses of WRiIUC,and the extent of virus replication in different organs wasmeasured by standard plaque assays and by the luciferase

assay. Typical results obtained two days after virus infectionare shown in Table 2. This is the time postinfection at whichmaximum levels of luciferase were consistently detected intissues. In mice infected at sublethal virus doses (1 x 105 pfuper mouse), luciferase activity was easily detectable in thespleen but not in other tissues. In mice inoculated with highvirus doses (108 pfu per mouse), high levels of luciferasewere detected in spleen, kidney, and liver and low levels inthe lung. As expected, there was a good correlation betweenluciferase activity and virus titers in the different infectedorgans (Table 2). Similar dissemination of infectious virus indifferent organs was found in mice inoculated with wild-typevaccinia virus at 108 pfu per mouse (not shown).These findings establish that luciferase activity can be

used as a reporter gene to follow vaccinia virus replication ininfected animals. Moreover, expression of two recombinantgenes (luciferase and p-galactosidase) does not alter tissuetropism of the recombinant virus., The ratio between lucifer-ase level and virus titer provides a good indicator for theability of vaccinia virus recombinants to replicate in differenttissues.

DISCUSSIONIn this study we provide direct evidence that the fireflyluciferase gene can be expressed as an active enzyme invaccinia virus-infected cells in culture and in target organs ofinfected mice. The luciferase gene was introduced in the tkregion of vaccinia DNA and fused to the viral regulatorysequences of the 7.5-kDa promoter. Luciferase was detectedspectrophotometrically and by exposure of infected cellcultures to sensitive film. Luciferase was detected in cells inculture as early as 1 hr postinfection and accumulatedthroughout the virus replication cycle. We have been able todetect luciferase in as little as one virus-infected cell in abackground of a million noninfected cells (Table 1). Simul-taneous comparison of the levels of detection of luciferaseand 13-galactosidase revealed that the luciferase assay isabout 1000-fold more sensitive than the ,B-galactosidaseassay. In mice infected with sublethal virus doses, presenceof luciferase was detected only in the spleen. In time-coursestudies, maximum levels of luciferase were detected in thespleen on the second day after virus inoculation. Since thespleen is a target tissue for poxvirus multiplication in mice(12), our findings provided evidence that the tissue tropismof recombinants of vaccinia virus expressing luciferase andalso,-galactosidase is not altered. As expected, in miceinfected with high doses of recombinant virus that causedgeneralized dissemination of vaccinia, luciferase was de-tected in several tissues (Table 2).

Table 2. Detection of luciferase activity in tissues of mice inoculated with vaccinia virus recombinant WRLTWRLUC infection

105 pfu per mouse 108 pfu per mouse

Luciferase, Virus yield, Luciferase, Virus yield,Organ ng/mg of tissue pfu/mg of tissue ng/mg of tissue pfu/mg of tissue

Spleen 0.02 + 0.005 5 ± 2 0.08 ± 0.005 5.7 ± 1.8 x 103Liver 0 0 0.1 ± 0.03 1.6 ± 0.5 x 103Kidney 0 0 0.1 ± 0.04 2.5 ± 0.8 x 104Lung 0 0 0.013 ± 0.004 1.5 ± 0.5 x 102BALB/c female mice (4 to 6 weeks old) (Charles River Breeding Laboratories) were inoculated i.p. with two different

doses of vaccinia virus recombinant WRL, and 2 days later tissues were removed from four mice per group, washedin phosphate-buffered saline (PBS), and stored at - 700C. Tissues were divided into two aliquots and were weighed. Onealiquot was homogenized in luciferase extraction buffer (5 Iul per mg of tissue) containing 1 mM phenylmethylsulfonylfluoride and leupeptin (10 ,ug/ml) and was sonicated. Cell debris was removed by centrifugation, and the supernatant wasassayed for luciferase activity. Another aliquot of tissue was homogenized in PBS (5 gl per mg of tissue), and virus yieldsin the supernatant were determined by plaque assays. Values are the mean ± SD.

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Page 5: infected animals - PNAS · infected animals JOSE F. RODRIGUEZ*, DOLORESRODRIGUEZ*, JUAN-RAMONRODRIGUEZ*, ELEANORB. MCGOWAN*, ANDMARIANOESTEBAN*t* Departments of*Biochemistry and tMicrobiology

Proc. Natl. Acad. Sci. USA 85 (1988) 1671

Vaccinia virus is now being tested as a eukaryotic expres-sion vector with potential usage as a vaccine against a broadspectrum of infectious diseases of animal and human origins(6). Because of this potential, the World Health Organization(WHO) has recommended the development of attenuatedstrains of vaccinia virus for vaccination purposes (13), withthe aim of avoiding complications generated during smallpoxvaccination campaigns (14). Another important issue raisedby WHO is to assess that introduction of new geneticmaterial into vaccinia virus does not alter the virus tropismfor target tissues. This is of importance in view of the factthat many of the inserted foreign genes encode antigenicproteins that interact with cell membrane components (6).Recent progress has been made in the generation and char-acterization of attenuated variants of vaccinia virus (15-18).Since, on the whole, attenuated viruses will multiply lessefficiently in infected animals than the wild-type virus, witha concomitant decrease in viral antigen production, it be-comes important when using vaccinia virus recombinants todefine both the tissue tropism and levels of viral geneexpression. Our findings described in this report show thatthe luciferase gene can be effectively used as a reporter geneto follow viral tropism and gene expression in tissues ofinfected animals. These findings provide several applicationsfor the luciferase assay in studies of vaccinia virus recombi-nants. The luciferase assay could be used to compare theextent of replication and gene expression of various attenu-ated recombinants of vaccinia virus. For these assays,expression of the luciferase gene could be placed under thesame viral promoter (i.e., the 7.5-kDa promoter), and resultscould easily be compared between variants, along with otherrelevant parameters such as LD50 and antibody production.This approach would assess, for the purpose of vaccination,a means of selecting the most effective vaccinia virus recom-binant. The luciferase assay could also be used after primaryimmunizations to assess the level of gene expression ofrecombinant virus after repeated virus inoculations in thesame animals. Another application of the luciferase assay isthat it can be used to define functional properties of thevaccinia virus promoters. Viral promoter sequences appearto be contained within 30 nucleotides upstream of the firstATG (19-21). These viral promoters could be geneticallymanipulated, and their strength could be measured by fusionwith the luciferase gene in both virus infected cells and ininfected animals. Since luciferase activity is noninvasive andnondestructive (2, 3), recombinants of vaccinia virus couldalso be used in the detailed studies of the early events ofvirus dissemination in a whole animal.

We thank Dr. Stephen H. Howell (Department of Biology,University of California at San Diego) for the generous gift ofplasmid pD0432. We thank Vincent Garofalo for excellent photo-graphic assistance and Victoria Jiminez for technical assistance.This investigation was supported by grants from the NationalInstitute of Health CA 44262, National Science Foundation DMB8609236, and by the U.S.A.-Spain Joint Committee for Scientificand Technological Cooperation CCA 8510 to M.E.

1. deWet, J. R., Wood, K. V., Helinski, D. R. & DeLuca, M.(1985) Proc. Natl. Acad. Sci. USA 82, 7870-7873.

2. Ow, D. W., Wood, K. V., DeLuca, M., deWet, J., Helinski,D. R. & Howell, S. H. (1986) Science 234, 856-859.

3. deWet, J. R., Wood, K. V., DeLuca, M., Helinski, D. R. &Subramani, S. (1987) Mol. Cell. Biol. 7, 725-737.

4. Keller, G. A., Gould, S., DeLuca, M. & Subramani, S. (1987)Proc. Natl. Acad. Sci. USA 84, 3264-3268.

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2642-2647.19. Hanggi, M., Bannwarth, W. & Stunnenberg, H. G. (1986)

EMBO J. 5, 1071-1076.20. Bertholet, C., Stocco, P., van Meir, E. & Wittek, R. (1986)

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