In vitro replication and cytopathogenicity of the feline immunodeficiency virus for feline t4 thymic lymphoma 3201 cells

VIROLOGY

li’9,4%-497

TADAFUMI

(1990)

In Vitro Replication

and Cytopathogenicity of the Feline lmmunodeficiency for Feline T4 Thymic Lymphoma 3201 Cells

S.

KATHLEEN

TOCHIKURA,*‘t’+ JENNIFER

*Department Parasitology,

of Veterinary Yamaguchi

L. RoJKo,*W*’

A.

HAYES,*

LAWRENCE

CAROLYN

E.

MATHES,*+$

M.

CHENEY,* AND

AKIKO

RICHARD

Virus

TANABE-TocHlKuRA,*+*$ G. OLSEN,*++

Pathobiology, The Ohio State University, 1925 Coffey Road, Columbus, Ohio 43210; SDepartment of Virology and University School of Medicine, 1144 Kogushi, Ube, Yamaguchi 755, Japan; and tCenter for Retrovirus Research and +Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 432 10 Received

April 5, 1990; accepted

July 12, 1990

Cytotoxic feline immunodeficiency virus (FIV) infection was established in feline T4 thymic lymphoma 3201 cells with the Petaluma isolate of the feline immunodeficiencyvirus (FIV-Petaluma). Mg2+-dependent, reverse transcriptase (Mg*’ RT) activity and FIV p24/2&positive cells were evident beginning at 18 days postinoculation (dpi). Cell death was observed beginning at 22 dpi, with a maximum of 40% dead (trypan blue dye exclusion at 26 dpi). This cytocidal change was not observed in cultured Crandell feline kidney fibroblasts similarly infected with FIV-Petaluma. The surviving cells grew out and a chronic FIV-producer cell line was established. The 3201 cell-derived FIV (FIV-3201) was far more virulent for FIV-naive feline 3201 cells, with FIV p24/28-positive cells and Mg*+ RT activity first detectable by 4-8 dpi and subsequent loss of cell viability detectable by 8-l 2 dpi. Maximum kill (40% dead) was observed at 16 dpi. Comparison between viral infectivity of FIV-Petaluma and FIV-3201 for FIV-naive 3201 cells showed an increase of 1 log,, tissue culture infectious doses (TCID& by amplification/passage in 3201 cells. Cytologic and electron microscopic examination of 3201 cells in FIV-infected cultures showed frequent budding lentiviral particles. This lytic infection system opens the way to the routine detection, isolation, and quantitation of FIV from FIV-infected cats, to the largescale propagation of the virus, and to a system for evaluation of the mechanisms of FIV lymphocytotoxicity and the o isso Academic Press. Inc. development of therapies to counteract lentiviral cytopathicity.

with FeLV was susceptible to FIV infection (2). However, there has been little information on these virusproducing cell lines and the utility of the CrFK and FL74 cell lines for studying FIV infection and cytopathogenicity in vitro is limited. For example, neither CrFK nor FL74 cells are T4 cells (6), virus replication takes a considerable time to develop following inoculation, and infection is generally not cytopathic (2). In this report, we find that the continuous feline T4 thymic lymphoma 3201 cell line which is negative for exogenous FeLV is highly susceptible to the cytopathic effect of FIV. Moreover, the 3201 cells that survive FIV killing grow out as permanent lines that produce large amounts of FIV and the 3201-cell-derived FIV (FIV3201) is highly cytopathic for FIV-naive 3201 cells. Replication of FIV-Petaluma in 3201 cells was first evident by 18 days postinoculation (dpi) as demonstrated by induction both of Mg*+-dependent, reverse transcriptase activity and of FIV p24/28 antigen (Fig. 1A). The frequency of FIV p24/28-positive cells increased gradually with time with more than 60% of 3201 cells positive by 60 dpi. The percentage of FIV p24/28-positive cells determined using murine monoclonal anti-FIV p24/28 was identical to the percentage of FIV-positive cells determined using FIV antibodypositive feline serum (data not shown). The immuno-

In 1986, a new feline retrovirus, feline immunodeficiency virus (FIV), was first isolated from the peripheral blood lymphocytes of cats that had a chronic acquired immunodeficiency syndrome and were negative for the feline leukemia virus (FeLV) and housed in a multiple cat household in northern California (I). FIV is highly Tlymphotropic and is morphologically and biochemically similar to, but antigenically distinct from, primate lentiviruses such as simian immunodeficiency virus (SIV) and human immunodeficiency virus (HIV) (2). Upon experimental introduction into specific-pathogen-free cats, FIV induces fever, neutropenia, and generalized lymphadenopathy (3, 4). FIV also causes specific cytopathogenic changes in feline peripheral blood lymphocytes (2) and experimentally infected cats may show inversions of T4IT8 ratios (3, 5). FIV is unrelated to other feline retroviruses including exogenous and endogenous FeLVs, endogenous type C oncornavirus (RD-1 14) and feline syncytium-forming virus. Replication of FIV in an established cell line was first achieved by Pedersen and co-workers (I, 2) using Crandell feline kidney(CrFK) cells. Subsequent work by the same investigators showed that a feline lymphoblastoid cell line FL74 which was chronically infected ’ To whom 0042-6822190

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CopyrIght Q 1990 by Academic Press, Inc. All rights of reproduction in any form resewed.

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FIG. 1. Induction of Mg’+-dependent reverse transcriptase (RT) activity (triangles) and FIV ~24728 antigen (circles) following FIV-Petaluma (A) and FIV-3201 (B) inoculation of 3201 cells. FIV-Petaluma was obtained from the NIH AIDS Research and Reference Reagent Program (Catalog No. 236) and had been grown in Con A-stimulated feline peripheral blood mononuclear cells maintained on interleukin-2, harvested, and filtered (0.45 gm). Target cells were suspended in FIV-containing medium (0.5 ml/3 X 1 O5 cells). After adsorption at 37” for 1 hr, FIV-exposed cells and sham-inoculated cells were plated at a concentration of 3 X 1 O5 cells/ml in duplicate cultures of 10 ml each in 25.cm’ culture flasks. At the designated time intervals, FIV antigen expression and activity were determined and the medium was replaced with fresh growth medium. The FIV used as inocula were FIV-Petaluma (A) as described above and the medium from FIV-infected 3201 cells (FIV-3201) (B) collected at 38 days postinoculation (dpi). The protocols for infection, maintenance, and examination of cells infected with FIV-3201 were identical to those for infection, maintenance, and examination of cells infected with FIV-Petaluma. All of the experiments using 3201 or CrFK cells were performed in culture medium consisting of equal parts of Leibovitz L-l 5 and RPMI 1640. 10% heat-inactivated fetal bovine serum 100 IU of penicillin/ml, and 100 pg of streptomycin/ml as described (6). All cultures were maintained at 37” in a humidified COP atmosphere. For indirect immunofluorescence assay, FIV-inoculated, and sham-inoculated 3201 cells were washed in phosphate buffered saline (PBS), smeared on microscope slides, air-dried, fixed with cold methanol for 10 min, and dried thoroughly. The slides were incubated with a 1: 100 dilution of anti-FIV p24/28 murine monoclonal antibodies for 30 min at 37” in a humidified chapber, washed twice with PBS, and incubated with fluorescein isothiocyanateconjugated anti-mouse IgG for 30 min. The slides were then washed twice with PBS and counterstained with 0.1% Evans blue. A minimum of 300 cells was counted and the percentage of fluorescing cells was calculated. For the RT assay, a modification of Poiesz et al. (26) was used. Briefly, 7.5 ml of culture fluid from the cells was filtered (0.22 Nm), precipitated with 30% (w/v) polyethylene glycol (PEG) on ice for 2 hr, and centrifuged at 10,000 rpm for 1 hr at 4’. The viral pellets were then disrupted by the addition of 100 ~1 of a suspension buffer containing 10 miZ/1 K,HPO, (pH 7.2) 2 mM dithtothreitol (DTT), 0.2% Tnton X-100, and 10% glycerol. The RT assay was performed at 37“ for 1 hr with a 10.~1 aliquot of the disrupted virus solution in a final volume of 50 ~1 containing 50 mM Tris-HCI (pH 8.0) 2 mlLl DTT, 20 rnn/l KCI, 60 mM MgC12, 1 &i of [3H]thymidine tnphosphate, and 50 pglml of poly(rA)-oligo(dT). The reaction was stopped with 0.2 ml of 5% trichloroacetic acid and the precipitates were collected on filters, washed with PBS, dried, and counted in a liquid scintillation counter. All RT assays were performed in triplicate.

fluorescence pattern resembled that of HIV and consisted of ringlike or crescent-shaped staining of the cytoplasm with an eccentric round spot of fluorescence (data not shown). Western blot assay of FIV purified from the supernatant of infected 3201 cells typically showed a strong reaction to the FIV gag protein p24/28 and variable reactivity to other FIV proteins including those of M, 10,000, 17,000, 32,000, 52,000, 55,000, and 62,000

when monoclonal antibody to FIV p24/28 (4F2) and FIV antibody-positive feline serum were used as the primary reagents, respectively (Fig. 2). In contrast, FIV antigens did not react when FIV antibody-negative feline serum was used as the primary reagent. The induced reverse transcriptase activity preferred Mg2+ to Mn2+; the optimum conditions for reactivity resembled those of HIV and FIV reverse transcriptases (1) and differed from those of FeLV (Table 1). The ability

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FIG. 2. Western blot of purified FIV using monoclonal antibody to FIV core protein p24/28 (lane l), and serum from an FIV-infected cat (lane 2) and an FIV-negative cat (lane 3). FIV (FIV-3201) was precipitated with PEG from 300 ml of supernatant fluid from an infected 3201 cell culture by centrifugation at 10,000 rpm for 1 hr. The virus pellet was layered on a discontinuous gradient of 20 and 50% sucrose and centrifuged at 30,000 rpm for 1 hr. The interface was harvested, pelleted by centrifugation, resuspended in 200 ~1 of PBS, and used as the virus preparation. Ten microliters of virus suspension was disrupted with sodium dodecyl sulfate (SDS) and 2-mercaptoethanol and separated on a 12% SDS-polyacrylamide gel. After electrophoresis, the proteins were transferred to nitrocellulose membrane filters by electroblotting. As the primary antibody, mouse anti-FIV p24/28 monoclonal antibody (4F2; IgG 2b) or FIV antibodypositive feline serum was used and, as secondary, alkaline phosphatase-conjugated goat anti-mouse IgGAM or goat anti-feline IgG was used. The 5-bromo+chloro-3-indoyl-phosphate/nitroblue tetrazolium phosphatase substrate system was used to visualize the reactive bands.

of the induced reverse transcriptase to also utilize Mn2+ may reflect coinduction of endogenous RD-114 feline retrovirus (7) which is unrelated to FeLV but related to baboon endogenous virus (8). FIV was cytopathic for 3201 cells 22 dpi with FIVPetaluma (determined by trypan blue dye exclusion, data not shown). The maximum decrease in cell viability (60% live in FIV-infected cultures versus 96% live in sham-inoculated cultures) was seen at 26 dpi. Cytopathicity followed the evidence of viral replication; changes observed by inverted phase microscopy included ballooning degeneration and appearance of small refractile cells and occasional giant cells (data not shown). Aggregates of cells were not prominent. FIV p24/28 antigens were present in 320 1 cells showing cytopathic effects and were especially noted in giant cells (data not shown). Electron microscopic examination of infected cultures revealed surface-associated and budding lentiviral particles with characteristic 1 OOto 140-nm diameters, and cylindrical to bullet-shaped or condensed, circular, eccentric nucleoids (Fig. 3).

Cell cultures showing FIV p24/28 antigens, Mg2+-dependent reverse transcriptase activity, and cytopathic effects were negative for FeLV p27 by ELISA, negative for infectious FeLV by S+/L- assay, and negative for feline syncytium-forming virus and feline infectious peritonitis virus by indirect immunofluorescence (data not shown). All evidence of cytopathic effect disappeared by 50 dpi and more than 60% of the remaining viable cells became positive for FIV antigens. The chronically infected cell line that grew out was designated FIV-3201 and has been in continuous culture for 3 months with stable FIV infection as assayed by reverse transcriptase activity, FIV antigen assay, and electron microscopy and has not shown further evidence of FIV-induced cytopathicity. Next, a similar experiment was carried out using the culture supernate from the FIV-infected 3201 cells at 38 dpi. As compared to FIV-Petaluma, FIV-3201 was far more virulent and induced FIV replication (Mg2+-dependent reverse transcriptase activity and FIV p24/28 by 4-8 dpi and cytopathicity by 12 dpi (Fig. 16). The percentages of infected and killed cells obtained following FIV-3201 infection were similar to those obtained following FIV-Petaluma infection; however, infection with FIV-3201 progressed much more rapidly. As the 3201 cells are a cloned feline thymic lymphoma cell line which expresses two epitopes of feline CD4 (9, Rojko, Ackley, O’Brien, and Cooper, unpublished), this may reflect selection for T4-tropic FIV strains. We have confirmed that our laboratory passage of 3201 cells is

TABLE COMPARISON

OF Mg”+TRANSCRIPTASE

1

AND Mn’+-DEPENDENCE OF REVERSE ASSAYS OF FIV AND FELV

Cell of origin Concentration hM) ~~CIz 300 60 6 0 MnCI, 6 0.06 0

3201

32Ol/FIV’

FL-74’

1.1= NDd 2.6 1.8

1.8 134.5 375.3 3.0

1.7 1.4 5.0 1.7

2:7 3.0 2.8

2.8 136.7 3.1

8.1 100.5 3.3

a For this experiment, FIV-naive 3201 cells with FIV-3201. * FeLV-producer cell line. c Reverse transcriptase activity (X 1 O3 cpm). d Not done.

were

newly

infected

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FIG. 3. TI ransmission electron micrograph nucleoids. Osmium tetroxide stain

showing

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mature

65 to 99% positive for feline CD4, depending on culture (data not shown). Alternatively, it may merely indicate FIV preference for the higher frequency of CD4-positive cells in the clonal 3201 cell population as opposed to the frequency in uncloned feline peripheral blood lymphocytes (about 20-40% CD4-positive) ((3, 9); Rojko, Ackley, O’Brien, and Cooper, unpublished) and, hence, reflect an improved target cell. The yields of FIV from the FIV-infected 3201 cells and the FIV-infected CrFK cell lines were assessed by reverse transcriptase assay. As shown in Table 2, the reverse transcriptase activity of 320 1-cell-derived FIV was about 2- to 3.5-fold higher than that of CrFK-derived FIV. This suggests that the 3201 T4 lymphoid cells were more permissive to FIV than were the CrFK kidney cells. The increased permissiveness of the 3201 cell line to FIV also was indicated using a tissue culture infectious dose assay to quantify biologic activity of the released FIV. As shown in Table 2, the TCID5,, of the 3201 -cell-derived FIV was 1 loglo higher than the

to recently

495

budded

lentiviral

particles

with

cylindrical

to bullet-shac

red

TCIDSo of the peripheral blood mononuclear cell-derived FIV-Petaluma. There was a parallel relation between TCIDSo titer and reverse transcriptase activity, suggesting that TCIDSO assays were useful for quantitation of FIV biologic activity. As described above, our results indicate that 3201 cell line is readily infected and killed by FIV. Others have previously reported that the 3201 cells do not support FIV replication (2); however, these investigators now find that the 3201 cells are permissive to FIV (Pedersen, personal communication). It has been demonstrated that susceptibility to HIV infection and cytopathicity correlates with the expression of the human CD4 antigen on the cell surface and that HIV replication and RT activity are observed in fractionated T4 but not in similarly fractionated T8 cells (70). Like HIV infection of human T4 cells such as MOLT-4 and H9 ( 1 I- 74) and FIV infection of feline peripheral blood lymphocytes (1, Z), FIV infection of feline T4 thymic lymphoma 3201 cells leads to syncytial cell

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PRODUCTION

2

OF FIV BY VARIOUS CELL LINES Quantitation

Cell None 3201

Virus Medium Sham FIV-Petaluma FIV-3201

CrFK

FIV-Petaluma

Days postinoculation

% FIV p24/28-positive cells immunofluorescence 0 0 0 46.4 1.2 60.0 0 2.2 10.8

14 22 4 36 4 14 46

a TCID,, were assayed in 3201 cells. b ND, not done. c RT activity was measured with 60 mn/r MgClp

Reverse

of FIV transcriptase (X 1 O3 cpm)C

activity

1.2 2.2 2.9 53.4 9.0 72.3 2.5 ND 25.4

TCID,,/ml” 0 0 ND* 2 x 1 o”.5 ND 2 x 1o’.5 ND ND ND

for Mg’+.

formation and cytopathicity, followed by disappearance of cytopathicity and viral spread throughout many, but not all, of the cells. Interestingly, although the cell number expressing viral antigens gradually increased with time in our study, 30 to 35% of the 3201 cells remained negative for FIV ~24128 and budding virus throughout the culture period. Cloned FIV probes have recently been developed (15, 16) which will permit us to determine whether these FIV antigen-negative cells are innately resistant similar to the intrinsic resistance of cloned variants of human CEM cells to HIV infection (17). Alternatively, these FIV p24/28 antigennegative cells may actually harbor latent FIV infection as has been described for certain emerging subpopulations of cultured human T4 cells (18, 19). The most important point here, though, is that the 3201 cell line may be used to amplify FIV, potentially enhance the lymphocytopathicity of the virus, and provide large quantities of virus for in viva and in vitro pathogenicity studies. First, this may allow better determination of the in viva significance of FIV infection. At present, FIV-Petaluma isolates are only mildly pathogenic after experimental introduction into specificpathogen-free cats (3-5, 20) and only become fulminant in cats coinfected with FeLV (3). Future studies will determine whether the increased virulence of 3201 -cell-derived FIV for feline T4 cells in vitro reflects an increased virulence of 3201 -cell-derived FIV for cats in vivo. Our study also suggests that FIV infection of feline T4 thymic lymphoma 3201 cells may alter endogenous retroviral expression. Endogenous proviruses related to FeLV are normal constituents of the cat genome (27-24) and are noninfectious and close to full-length

in size. Subgenomic expression occurs in feline placental trophoblasts but expression does not correlate with any disease process. Endogenous feline retroviral sequences, designated RD-1 14, are rarely expressed in adult cat tissues but are expressed in some lymphomas (2 1,26). An etiologic role for RD-1 14 in cat disease has not been demonstrated. In the present study, we find that both Mg’+- and Mn*+-dependent reverse transcriptase activities were induced in FIV-inoculated 3201 cells as compared to sham-inoculated 3201 cells (Table 1). On the contrary, Mn’+-dependent reverse transcriptase activity was not observed in FIV- or shaminoculated CrFK cells (data not shown). Although there is no report of interactions between FIV and endogenous cat retroviruses, we feel our data raise the possibility that such interactions may occur. Alternatively, the FIV reverse transcriptase may somehow acquire the ability to use Mn*+ during its replication in feline T4 thymic lymphoma 3201 cells. Future studies will ascertain whether the shift in reverse transcriptase cofactor usage reflects interaction with endogenous RD-1 14 or FeLV or whether it is associated with increasing virulence. Taken together, our study establishes a feline lentiviral Iytic infection system for evaluation of the mechanisms of FIV lymphocytotoxicity and for the development of therapies to combat lentiviral cytopathicity. ACKNOWLEDGMENTS This project was funded, in part, by Public Health Service Grant UOl Al-25722 and Contract NO1 Al-62525 from the Developmental Therapeutics Branch, AIDS Program, National Institute of Allergy and Infectious Disease. We thank Dr. P. Andersen for two anti-FIV ~241 28 murine monoclonal antibodies (2D4[lgG2] and 4F2[lgG2b]), Dr. R.

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Ayl for serum from a pet cat with a natural FIV infection, Dr. J. Gaskin for anti-FeSFV(+) serum, Dr. T. lshida for anti-FIPV(+) serum, and Dr. M. Tanaka for anti-FIV(-) cat sera. We are grateful to M. K. Pollman and L. J. Rezankafor help with Western blotting assays. We acknowledge the support of the Center for Retrovirus Research and Comprehensive Cancer Center of The Ohio State University.

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12. KOYANAGI, Y., HARADA, S.. and YAMAMOTO, N., Cancer Lett. 30, 299-310(1986). 13. TOCHIKURA, T. S., NAKASHIMA, H., TANABE, A., and YAMAt.,ioTo, N., Wrology 164, 542-546 (1988). 14. TOCHIKURA, T. S.. NAKASHIMA, H.. and YAMAMOTO, N., /. Ac9. lmmun. Defic. Syndrome 2,44 l-447 (1989). 15. OLMSTED, R. A., BARNES, A. K., YAMAMOTO, J. K., HIRSCH, V. M., PURCELL, R. H., and JOHNSON, P. R., Proc. Nat/. Acad. SC;. USA 86,2448-2452 (1989). 16. TALBO~, R. L., SPARGER, E. E., LOVELACE, K. M., FITCH, W. M.. PEDERSEN, N. C., Luctw, P. A., and ELDER, J. H., Proc. Nat/. Acad. Sci. USA 86, 5743-5747 (1989). 17. CASAREALE, D., STEVENSON, M., SAKAI, K., and VOLSKY, D. J.. Virology 156,40-49 (1987). 18. FOLKS, T., POWELL, D. M., LIGHTFOOTE, M. M., BENN, S., MARTIN, M. A., and FAUCI, A. S., Science 231,600-602 (1986). 19. ZAGURY, D., BERNARD, J., LEONARD, R.. CHEYNIER, R., FELDMAN, M., SARIN, P. S., and GALLO, R. C., Science 231, 850-853 (1986). 20. RIDEOUT, B. A., LOWENSTINE, L. J., MOORE, P. F., and PEDERSEN, N. C., “Proceedings, 40th Meeting American College of Veterrnary Pathology, Baltimore, MD, 1989.” 21. NIMAN, H. L., GARDNER, M. B., STEPHENSON, J. R., and ROY-BURMAN, P.. J. Virol. 23, 578-586 (1977). 22. SOE, L. H., DEVI, B. G., MULLINS, 1. I., and ROY-BURMAN, P., J. Viral. 46,829-840 (1983). P., 23. SOE, L. H., SHIMIZU, R. W., RANDOLPH, 1. R., and ROY-BURMAN, J. Viral. 56,701-710 (1985). 24. BERRY, B. T., GHOSH, A. K., KUMAR, D. V., SPODICK, D. A., and ROY-BURMAN, P., /. Viral. 62, 3631-3641 (1988). 25.

CHENEY, C. M., ROJKO, J. L., KOCIBA, G. J.. WELLMAN, M. L., DIBARTOLA, S. P., REZANKA, L. J., FORMAN, L., and MATHES, L. E., ln Vitro Gel/. Dev. Biol. 26, 455-463 (1990).

26.

POIESL, B. J., RUSCE~I, F. W., GAZDAR, A. F.. BUNN. P. A., MINNA, J. D., and GALLO. R. C., Proc. Natl. Acad. SC;. USA 77, 7415-7419(1980).