Proc. Nat!. Acad. Sci. USAVol. 85, pp. 4455-4459, June 1988Immunology

Infection of rabbit T-cell and macrophage lines with humanimmunodeficiency virus

(acquired immunodeficiency syndrome/human T-cell leukemia virus I/herpesvirus ateles/simian virus 40)

HENRIETTA KULAGA*t, THOMAS M. FOLKSt, ROSAMOND RUTLEDGE§, AND THOMAS J. KINDT**Laboratory of Immunogenetics, Building 5, Room B1-04, tLaboratory of Immunoregulation, Building 10, Room 11C106, and §Laboratory of MolecularMicrobiology, Building 5, Room B1-29, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892

Communicated by Richard M. Krause, February 29, 1988

ABSTRACT We report the successful infection of tworabbit T-cell lines and one rabbit macrophage line with humanimmunodeficiency virus 1 (HIV-1). One T-cell line was aherpesvirus ateles transformant, the other T-cell line was ahuman T-cell leukemia virus I transformant, and the macro-phage line was a simian virus 40 transformant. After infectionwith a high-titered HIV-1 stock, the rabbit cultures exhibitedproperties that closely mimic those of HIV-1-infected humancells. Productive infection was evident in cultures 7-14 daysafter infection, as shown by an increase in reverse transcriptaseactivity, a concomitant increase in positive cells detected byindirect immunofluorescence using serum from a patient withacquired immunodeficiency syndrome, and a decrease in cellviability. RNA gel blot hybridization and protein immunoblotanalyses of infected cells indicated that all predicted viraltranscripts and proteins were synthesized during the course ofthe infection. Proof that cell-free culture supernatants of theinfected rabbit cell lines contained infectious virus was given bysuccessful passage onto a susceptible human T-cell line. Theability of HIV-1 to infect transformed rabbit cell lines in vitrosuggests that, with appropriate manipulation, the rabbit mayprovide a model for infection with HIV-1.

Human immunodeficiency virus 1 (HIV-1) has been shown tobe associated with acquired immunodeficiency syndrome(AIDS) in humans. This virus can be isolated from patientswith AIDS and with AIDS-related complex (1-3). Humanperipheral T cells and macrophages, as well as continuoushuman T-cell, B-cell, and macrophage lines, are susceptibleto HIV-1 infection (4-6). Therefore, the pathology of HIV-1infection in vivo and the cellular tropism in vitro support theimplication of this virus in this disease.A stumbling block to an understanding of the HIV-1

infection process and to the development of suitable vaccineshas been the lack of an animal model for this agent (7).Although models using related viruses have been proposedfor a variety of animal species, attempts to infect laboratoryanimals with HIV-1 have not been successful (8). Only thechimpanzee has been reported to be susceptible to HIV-1infection, but even in this model no consistent diseasesymptoms are observed (9-11). In addition, the accessibilityand handling of this endangered species limits its utility. Invitro experiments have shown that cells from certain primatesthat express the CD4 marker can be infected with HIV-1 (12).

This report presents evidence for the infection of tworabbit T-cell lines and a rabbit macrophage line with HIV-1.Sequential samples of the infected cultures were tested andshown to be positive for viral protein and RNA. Thesusceptible lines were shown to be of rabbit origin by studiesat the nucleic acid level and by cell surface markers. Theseresults suggest that, with appropriate manipulation, the

rabbit may provide an experimental model for infection withHIV-1.

MATERIALS AND METHODSCell Lines. The rabbit cell lines used were as follows: RL-5

is a T-cell line obtained by transformation with herpesvirusateles (13); 446 is a T-cell line transformed with human T-cellleukemia virus I (HTLV-I) and was a gift from A. Seto (KyotoUniversity); 6083 is a macrophage line transformed withsimian virus 40 (SV40) (14); 5943 and 6057 are adenocarci-noma lines derived from breast tumors at autopsy; UTfib isa uterine fibroma derived from a surgical specimen; Yc/cLIV liver fibroblasts and R-2 spleen fibroblasts were derivedfrom normal rabbit tissue. All rabbit lines were maintained inRPMI 1640 medium containing 10% fetal bovine serum andsupplemented with glutamine (2 mM), penicillin (1.0unit/ml), and streptomycin (100 .ag/ml). Human T-cell linesSupT1 (15) and A3.01 (16) were maintained in RPMI 1640with 10% fetal bovine serum. Rabbit peripheral blood lym-phocytes (PBLs) were isolated on Ficoll/Hypaque by pub-lished methods (14). PBLs were activated by incubation inmedium containing Con A (10 pug/ml), phytohemagglutinin(20 tkg/ml), or recombinant human interleukin 2 (IL-2, 500units/ml) or by incubation with allogeneic lymphocytes at a1:1 ratio. PBL cultures were infected with HIV-1 three daysafter initiation of treatment. Cell viabilities were determinedby trypan blue exclusion.

Infection. Cell pellets were incubated with high-titeredHIV-1 stock (lymphadenopathy virus, LAV) in the presenceof Polybrene (1 jig/ml, GIBCO). After 2 hours at 370C, cellswere diluted to a density of5 x 106 per ml. The next day, cellswere pelleted and washed twice in complete medium andresuspended to a density of 106 cells per ml. Cultures wererested for 2 days before samples of cell-free medium orinfected cells were taken for various assays.

Reverse Transcriptase Assay. Reverse transcriptase (RNA-directed DNA polymerase, EC 2.7.7.49) activity was deter-mined for 10-1.l samples of cell-free supernatant by the assayof Goff et al. (17) as modified by Willey et al. (18). Quanti-tative data were obtained by liquid scintillation spectroscopyof filters.

Fluorescent Antibody Assays. For immunofluorescenceassays, infected or uninfected cells (=3 x 105) were "cy-tospun" [centrifuged (700 x g, 5 min) onto slides], fixed inacetone at 40C, washed in phosphate-buffered saline, incu-bated with a 1:1000 dilution of either normal human serum oran AIDS patient serum for 30 min at room temperature,washed in phosphate-buffered saline, and then incubated

Abbreviations: HIV-1, human immunodeficiency virus 1; HTLV-I,human T-cell leukemia virus I; AIDS, acquired immunodeficiencysyndrome; SV40, simian virus 40; IL-2, interleukin 2; PBL, periph-eral blood lymphocyte.tTo whom reprint requests should be addressed.

4455

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

with fluorescein-conjugated goat anti-human IgG for 30 minat room temperature. After washing, cells were mounted andviewed under a UV microscope.RNA Gel Blot Analyses. Total cellular RNA was isolated by

homogenization of cells in 4.23 M guanidinium isothiocya-nate and pelleting through a CsCl gradient (19). Samples (5,ug) of glyoxalated RNA were electrophoresed in a 1%agarose gel in 0.1 M sodium phosphate buffer (pH 7.2) andblotted onto a nitrocellulose filter (20). Filters were dried andbaked for 2 hr at 80'C and then prehybridized at 420C for 16hr. Probes were nick-translated with [a-32P]dCTP and addedto the filter in hybridization mixture and incubated for 18 hrat 420C. After several washes in 2 x SSC (0.3 M NaCl/0.03M sodium citrate, pH 7) at room temperature, the filters werewashed twice in 1 x SSC at 520C.Immune Blot Analyses. Cultures were harvested at various

times after infection. After washing with culture medium, 108cells were pelleted in a microcentrifuge tube and 100 jul oflysis buffer was added with vortex mixing. Samples wereplaced on ice for 15 min and then centrifuged, and thesupernatant was taken for electrophoresis. Lysis buffer was50 mM Tris-HCI, pH 8.0/5 mM EDTA/100 mM NaClcontaining 0.5% (wt/vol) 3-[3-cholamidopropyl)dimethylam-monio]-1-propanesulfonate (CHAPS; Calbiochem) and 2%(wt/vol) deoxycholate (Sigma). Proteins were separated byelectrophoresis in a NaDodSO4/polyacrylamide gel (3-27%acrylamide gradient; Tris buffer system), transferred tonitrocellulose, and allowed to react with an AIDS patientserum. 125I-labeled protein A was then used to detect immu-noreactive proteins. Filters were dried and exposed to XAR-2films with an intensifying screen at - 70°C. A lysate obtainedfrom a HIV-1-infected human T-cell culture (A3.01) was usedas a positive control.

RESULTS

Initial attempts to carry out HIV-1 infection in rabbit cellsused two previously established rabbit T-cell lines. One ofthese lines, RL-5, is a herpesvirus ateles transformant thathas been shown to be a T-cell line by numerous serologic andmolecular criteria (20-23). The other, 446, is an HTLV-Itransformant (24-26) that is not as well characterized but hascertain T-cell characteristics. Both cell lines produce RNAtranscripts that cross-hybridize with a probe specific forsequences encoding human CD4 (H.K., M. C. Rebiere, D.English, and T.J.K., unpublished data). The RL-5 and 446lines were infected with a high-titered HIV stock by amodification of a standard in vitro infection protocol (16).Cell-free supernatants were monitored daily for reversetranscriptase activity (18) (Fig. 1A). Reverse transcriptaseactivity began to rise in both T-cell lines 7-10 days afterinfection, peaked at =14 days after infection, and thendeclined. The time of occurrence and the magnitude of theactivity varied somewhat from infection to infection depend-ing on the viral stock used, but all attempts to infect RL-5 and446 have thus far been successful. No reverse transcriptaseactivity was detected in mock-infected cultures of RL-5 or446 cells.

Cell-free culture supernatants collected at the time of peakreverse transcriptase activity from HIV-infected rabbit cellswere tested for the ability to infect a highly susceptible humanT-cell line, SupT1 (17). A 1000-fold increase in reversetranscriptase activity was detected in SupT1 cultures afterincubation with RL-5 or 446-derived supernatants (Fig. 1B).Formation of syncytia was observed just before the time ofpeak reverse transcriptase activity. No reverse transcriptaseactivity was detected in SupTl cultures incubated withsupernatants from mock-infected rabbit T cells.Attempts were made to infect other continuous rabbit cell

lines and cultures of lymphoid and nonlymphoid origin (Table

A 200

a

I-9

E

0

B2000

V.

U

:t 1000.5U

1-

7 8 9 10 11 12 13 14 15 16 17 18

Days postinfection

Sup rom

* RL5 M KN RL-5 HIV* 448MKol 446 HIV

5 7 8 9 10 11 12 13 14 15

Days postinfection

FIG. 1. (A) Kinetics of appearance of reverse transcriptase (RT)activity in infected (HIV) and mock-infected (MK) cultures of therabbit T-cell lines RL-5 and 446. Data are expressed as 32p cpm per10 y4 of reaction mixture. (B) Infectivity of cell-free supernatantsfrom rabbit cell cultures in human indicator cells. Supernatants (Sup)were taken from infected (HIV) or mock-infected rabbit (MK)cultures and mixed with the human T-cell line SupT1. Reversetranscriptase (RT) activity in the SupT1 cultures is expressed as inA. Syncytia were first observed in SupT1 cultures infected withsupernatant from HIV-infected RL-5 and 446 cells on days 11 and 10,respectively. No syncytia were observed during a 20-day observationperiod in SupTl cultures given supernatants from mock-infectedrabbit cells.

1). Only the RL-5 and 446 T-cell lines were susceptible toHIV infection as detected by reverse transcriptase activityand by passage of infectious virus in cell-free supernatants toa human indicator line. One macrophage line, 6083 (14), wasnegative for reverse transcriptase activity but its supernatanttaken at day 11 was infectious for the human indicator line.Rabbit PBLs activated by mitogens, by growth in the pres-ence of recombinant human IL-2, or by mixed lymphocyteculture did not support infection even though these culturesare expected to be rich in T cells.

In subsequent large-scale infections, the relationshipamong reverse transcriptase activity, cell viability, andimmunoreactivity with an AIDS patient serum by indirectimmunofluorescence and immunoblot analysis was exam-ined. RNA was isolated from these infected cells for gel blotanalysis. Data from these infections show that for RL-5 and446 the number of viable cells fell dramatically just before

4456 Immunology: Kulaga et al.

Proc. Natl. Acad. Sci. USA 85 (1988) 4457

Table 1. Infection of rabbit cells with HIV-1Culture Description RT* Passaget

RL-5 Continuous T-cell line + +446 Continuous T-cell line + +6083 Continuous macrophage line - +PBL Unstimulated PBLs - -Con A PBL Con A-stimulated PBLs - -PHA PBL PHA-stimulated PBL - -IL-2 PBL IL-2-stimulated PBLs - -

PBL-MLR PBL mixed lymphocyte reaction - -

5943 Breast adenocarcinoma - -

6057 Breast adenocarcinoma - -

Yc/c LIV Liver fibroblast - -

UtFib Uterine fibroma - -

R-2 Spleen fibroblast - -

PHA, phytohemagglutinin.*Reverse transcriptase activity in cell-free supernatants of cultures7-60 days after infection.

tAbility of cell-free supernatants to induce syncytium formation andreverse transcriptase activity in cultures of SupT1, a human T-cellline.

peak reverse transcriptase activity (Fig. 2). Positive immu-nofluorescence was observed for cells in the HIV-infectedcultures before peak values of reverse transcriptase activity;the highest percentage of antibody-reactive cells (ap-proaching 50%) occurred just before the time ofpeak reversetranscriptase activity. Lowest values for cell viability coin-cided with peak values of reverse transcriptase activity.Syncytia were not observed in these rabbit T-cell cultures.

Total cellular RNA was prepared from HIV-infected RL-5cultures at days 7, 11, and 18 postinfection (Fig. 2A). RNApreparations from mock-infected and HIV-infected cellswere then examined by gel blot hybridization with a 6.5-kilobase (kb) cloned HIV DNA fragment, pBENN-5, thatincludes the entire HIV proviral genome except for the 3'open reading frame (B gene) and long terminal repeats (16,27). Viral transcripts were detected as early as 7 days afterinfection (Fig. 3), before reverse transcriptase activity wasdetectable above background. Five reactive RNA specieswere seen on day 11, at which time reverse transcriptaseactivity begins to approach peak levels. These included thefull-length 9.1-kb viral genomic RNA and four subgenomicRNA species of 5.5, 5.0, 4.3, and 1.8 kb (28). Viral messageswere barely detectable by day 18 postinfection, at whichpoint reverse transcriptase activity was well below peakvalues.To determine whether HIV proteins were translated and

processed in infected rabbit cultures, cell lysates weresubjected to immunoblot analyses. Lysates were preparedfrom infected RL-5 and 6083 cultures at various times afterinfection; reverse transcriptase activity in RL-5 culturesreached peak values in this experiment on about day 9 (6083cultures are reverse transcriptase-negative). Lysates derivedfrom RL-5 or 6083 infected with HIV were positive for viralproteins when immunoblotted with AIDS patient serum (Fig.4), and similar proteins were not present in lysates frommock-infected cells (data not shown). A positive-controllysate (A3.01) contained all immunoreactive proteins identi-fied by the serum used (Fig. 4A, left lane). The data shownrepresent two different autoradiographic exposure times ofthe same immunoblot. A culture ofA3.01 infected at the sametime as the rabbit cells was sampled on the same days as 6083and RL-5. There was perfect correspondence between thesizes of immunoreactive polypeptides detected in the humanand rabbit cell lysates, although the human cell line appearedto produce higher levels of viral proteins. Bands correspond-ing to p18, p24, gp41, p55, p64, and gpl20 were readilydetectable in HIV-infected rabbit cell lysates, indicating that

A

-

J, 1000-

B

30 E0

x

X20 AA1

O1. ...,.,,I 106 8 10 12 14 16 18 20

Days postinfection

2000 . , 40

L

61000

Ek

8 10 12 14 16 18

-30 'ab

SI

.20 i

20

Days postinfection

FIG. 2. Reverse transcriptase (RT) activity, immunofluores-cence, and cell viability in HIV-infected RL-5 (A) and 446 (B)cultures. Reverse transcriptase activity (o) was determined asdescribed for Fig. 1 and the number ( x 10-S) of viable cells per ml(n) was determined by trypan blue exclusion. The percentage of cellspositive by immunofluorescence was estimated: -, <10o reactivitywith AIDS patient serum; +, 10%o; + +, 25%; and + + +, 50%.

synthesis and processing of viral proteins occur in the rabbitcell lines (29).

DISCUSSIONThe results indicate that, unlike cells from other nonprimateexperimental animal systems, at least two rabbit T-cell linesand one rabbit macrophage cell line are susceptible to HIVinfection in vitro. With the exception of the macrophage cellline, 6083, attempts to infect continuous rabbit cell lines ofnonlymphoid origin as well as PBL cultures were not suc-cessful. Verification that the cell lines RL-5, 446, and 6083 areof rabbit origin was given by reactivity with rabbit-specificantibodies. RL-5 has been characterized extensively withrespect to the presence and expression of immunoglobulin,major histocompatibility, and T-cell antigen-receptor genes(20-23). The macrophage line 6083 has likewise been inves-tigated extensively in this laboratory with regard to deriva-tion and lineage (14). The possibility of contamination withhuman cells was eliminated by the failure of DNA fromnormal and infected RL-5, 446, and 6083 cells to hybridize

4 +4 4+4+4.

Immunology: Kulaga et al.

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

AJ9 rl - _u >, > >0

Xm m

E *o O

6083 A301 RL-5

11 17 9 1 1 1 4 7 9 11 1 4 9 11 14 days p inf.

9p120 - f....

- 5 5- 50- 4 .3

--

__

28S . s_._._.

_._.

1 8S . I:

*3w:r

p64 -

P55 - _.....- * 9.>..-

p241- *1_Ale-71

p24-4- _EIMU 0

p18-

B 6083 A301

7 9 11 14 7 9 11 14

FIG. 3. RNA gel blot analysis of HIV-infected RL-5 cells. Cellswere infected and reverse transcriptase activity was monitored.RNA was obtained at the indicated time points (which correspond todays postinfection in Fig. 2A) and subjected to electrophoresisfollowed by blot hybridization with HIV probe (pBENN 5, a 6.5-kbfragment from a HindIll digest) (17, 28). Sizes (kb) of HIV RNAspecies were estimated by comparison with 28S and 18S ribosomalsubunits ofRNA subjected to electrophoresis in the same gels. RNAfrom HIV-infected human T-cell line A3.01 was included as positivecontrol (pos con) and RNA from mock-infected RL-5 was includedas negative control (mock).

with a probe for the human Alu repeat sequence (data notshown) (30).

It is of interest that the majority of cellular prerequisitesnecessary for productive infection are similar for rabbit andhuman cells. For example, HIV has been shown to bepredominantly T-cell- or macrophage/monocyte-tropic forreplication in the human and appears to be so in the rabbit.In addition, the transforming viruses for the RL-5, 446, and6083 cell lines-herpesvirus ateles, HTLV-I, and SV40,respectively-belong to viral families that have been shownto up-regulate HIV production in human systems (31, 32).Events that occur after HIV infection of rabbit T-cell cul-tures, such as appearance of reverse transcriptase activity,viral RNA production, decreased cell viability, and positiveimmunofluorescence, are similar to those seen in humaninfections (16).

Levels of reverse transcriptase activity ==3 times back-ground (as opposed to 30 times background in Figs. 1 and 2)were detected when rabbit cell-derived HIV stocks werepassed back to RL-5 or 446 cell cultures (data not shown). Bycontrast to human T-cell lines, a relatively high amount ofinput virus is necessary for productive infection of rabbitcultures, indicating that HIV is not as efficient an infectiousagent in rabbit cells as in human cells. Nevertheless, wild-type viral progeny were generated after passage through therabbit lines as shown by infection of the human indicator lineSupT1 with cell-free culture supernatants.The receptor for HIV-1 on human and primate cells is CD4

(33). The rabbit homolog of CD4 has not been characterized.

RL-5

9 11 14 days p int.

..g

FIG. 4. Immunoblot analysis of total cellular protein obtained

from HIV-infected 6083, A3.01, and RL-5 cells. Numbers at left give

molecular mass values for viral proteins (kDa) determined by

comparison of bands in human control sample (left lane) to molecular

mass standards (not shown). Filter was exposed overnight. (B) As in

A but with 2-day exposure of filter.

RL-5, 446, and 6083 express RNA transcripts that cross-

hybridize with a probe specific for human CD4, the putative

receptor for HIV on lymphocytes, although no reactivity with

monoclonal antibodies directed against human CD4 was

found (H.K., D. English, and T.J.K., unpublished data). It is

possible that differences in the CD4 equivalent in the rabbit

account for the lower efficiency of infection in this species.

Although HIV-infected 6083 cultures yielded reverse tran-

scriptase negative supernatants, immunoblot analysis of cell

lysates and passage of infectious virus indicated substantial

synthesis of viral products. Preliminary experiments have

shown that noninfected 6083 supemnatants decrease reverse

transcriptase activity in known positive supemnatants. This

could explain the failure to detect reverse transcriptase

activity in HIV infected-6083 cultures. Since the polymerase

is present in HIV-infected cell lysates as shown by immu-

c0

00~CL

MP

4458 Immunology: Kulaga et al.

Proc. Natl. Acad. Sci. USA 85 (1988) 4459

noblot analysis (p64 in Fig. 4; see ref. 29), this cell line maybe producing an inhibitor of the enzyme rather than causingproduction of defective virus.The observation of HIV infection of rabbit-T cell lines

opens the possibility that in vivo infection of this species maybe accomplished. Rabbit T cells transformed with herpesvi-rus ateles or HTLV-I and a macrophage line transformed bySV40 are permissive to infection with HIV. It has beenreported that HTLV-I-transformed human T-cell lines arehighly susceptible to HIV infection in vitro (34). Rabbits canbe routinely infected with HTLV-I, and T-cell lines such as446 can be cultured from their blood (24-26). In our hands,five of five rabbits injected with a human HTLV-I-producingcell line contained serum antibodies to HTLV-L within 2months. If in vivo infection of HTLV-I-positive rabbits withHIV-1 proves successful, such animals would provide amodel of great value for vaccine trials and for drug testing.

We thank Audrey Kinter, Delores Dobson, and Dr. Guido Poli forhelp with experimental procedures and Drs. K. Strebel and A.Rabson for help with data interpretation. We thank Drs. M. Martin,J. Gallin, and A. Fauci for helpful comments regarding the manu-script. The editorial assistance ofMs. G. Shaw is gratefully acknowl-edged.

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Immunology: Kulaga et al.