- Email: [email protected]
lymphoma virus type I (HTLV-I) in HTLV-I-transformed cells
(~) INSTITUTPASTEUR/ELsEVIER Paris 1993
Res. ViroL 1993, 144, 185-191
In vitro thermal enhancement of human T-cell leukaemia/lymphoma virus type I (HTLV-I) in HTLV-l-transformed cells D. Sun, D.W. Archibald (*) and J.J. Sauk Department o f Pathology, School o f Dentistry, University o f Maryland at Baltimore, 666 IV. Baltimore Street, Baltimore, MD 21201 (USA)
SUMMARY
Temperature elevation constitutes a beneficial component of the host defence against viral pathogens. However, heat treatment may be detrimental to HTLV-I-infected cells by increasing virion and oncoprotein production. We investigated the effects of thermal elevation on the in vitro replication of HTLV-I (human T-cell leukaemia/lymphoma virus type I) in MT-2 cells, an HTLV-I-transformed lymphoid cell line. We found that HTLV-I replication in MT-2 cells was markedly increased as demonstrated by a nearly 2-fold increase in detection of viral p24 antigen and a 20-fold increase in reverse transcriptase activity during up to 5 h of heat treatment at 42°C. The results suggest that physiologic thermal elevations may induce viral production in HTLV-I-infected individuals. Key-words: HTLV-I, Replication, Reverse transcriptase, Heat, Fever; Enhancement, P24 antigen, Heat shock, Latent virus, Oncogenesis, Cellular SOS mechanisms, Stress.
INTRODUCTION H u m a n T-cell l e u k a e m i a / l y m p h o m a virus type I belongs to the family of exogenous Tlymphotropic type C retroviruses and is the aetiological agent of h u m a n adult T-cell leukaem i a / l y m p h o m a (ATL) (Poiesz et ai., 1980). HTLV infection is characterized by a prolonged period of latency, which may be followed by the development of T-cell l y m p h o m a (Poiesz et aL, 1980) or ATL (Hinuma et al., 1981). The detection of HTLV antigens in sera (Schupbach et al.,
Submitted March 3, 1993, accepted April 16, 1993. (*) To whomcorrespondenceaddressed.
1984) indicated a role for HTLV gene expression in the pathogenesis of ATL. It has been previously reported, using an in vitro model, that cellular heat treatment may be involved in the reactivation of latent cytomegalovirus (CMV) (Geelen et al., 1987) infection, and human immunodeficiency virus type 1 (HIV1) (Stanley et al., 1990; Geelen et al., 1988). As cellular stress factors may evoke a common induction mechanism in the reenhancement of latent CMV and HIV1, we analysed the effect of heat treatment on HTLV-I replication in HTLVI-transformed cells.
186
D. S U N E T A L . MATERIALS AND METHODS
Cells
MT-2 cells belonging to an HTLV-l-transformed human T-cell line, which were a gift from Dr. Max Essex, Harvard School of Public Health, were originally established from cord blood lymphocytes cocultivated with leukaemic cells from a patient with adult T-cell leukaemia (ATL) (Miyoshi et al., 1981). CEM-T4 cells from an ATCC-certified cell line were obtained through the AIDS Research and Reference Reagent Program, AIDS Program, N I A I D , N I H operated by ERC BioServices Corporation, Rockville, MD (contributor: Dr. J.P. Jacobs). CEM is a T-lymphoblastoid cell line and was developed from the peripheral blood buffy coat of a four-yearold Caucasian female with acute lymphoblastic leukaemia. The cells were free of virus-like particles as determined by electron microscopy (Uzman et al., 1966). H u m a n peripheral blood mononuclear cells (PBMC) were harvested from a healthy laboratory worker and separated through a Ficoll/Hypaque gradient " H i s t o p a q u e - 1 0 7 7 " (Sigma, St. Louis, MO). The cells were collected immediately before use and exhibited greater than 95 percent viability by the trypan blue exclusion assay.
Reverse transcriptase microassay
An RT microassay was performed according to an established protocol (Gregersen et al., 1988; Somogyi et al., 1990). Magnesium-dependent RT activity was measured using a synthetic RNA-template primer of poly(rA):oligo(dT)12.1s ( P h a r m a c i a / P - L Biochemicals, Piscataway, N J). Briefly, after preparation of the rA cocktail, including methyl-°H deoxythymidine triphosphate (3H-dTTP) (DuPont, NEN Res. Prod., North Billerica, MA) and poly(rA):oligo(dT)~2as template primer, 50 ~.1 samples were added to a tube containing poly(rA):oligo(dT)12.18 and v o r t e x e d . T h e s a m p l e s were incubated for 2 h in a 37°C water bath. Two 5-ram DE-81 paper discs (Schleicher & Schuell, Keene, NH), made with a standard hole punch, were added to each reaction well and mixed. The discs were then removed from the wells and placed in corresponding wells in a dot blot manifold apparatus (Bio Rad, Hercules, CA). Each sample well in the manifold apparatus was rinsed out five times with 5 % Na2HPO 4, twice with d d H 2 0 and twice with 70 % E t O H under vacuum. Each vial of triplicate samples was counted for 4 rain on a 3H channel. The non-specific background cellular polymerase activity from CEM or PBM cells was subtracted from the value from MT-2 cells. The resulting values reflected HTLV-l-specific RT activity.
P24-antigen detection assay Culture c o n d i t i o n s and heat treatment o f the cells
The cells were cultured at 37°C and 5 % CO 2 and maintained in log phase growth in RPMI-1640 medium (Gibco, Grand Island, NY) supplemented with 15 °7o foetal bovine serum (Sigma) and I00 U each of penicillin and streptomycin (Bioproducts, Inc., Walkersville, MD) per ml. Aliquots o f 5-6 x 105 cells per ml complete media were placed in sterile disposable plastic 25-cm 2 tissue culture flasks. Thermal elevations were carried out by submerging the cell culture flasks with gentle agitation in a 4 2 + 0 . 2 ° C water bath. The supernatants were immediately harvested for reverse transcriptase (RT) or p24 antigen assays.
H T L V - I p24 antigen was assayed in culture supernatants as follows. A slit manifold apparatus (BioRad) was used to prepare an antigen-coated blot. Briefly, nitrocellulose membranes (Schleicher & Schuell) were loaded with 300 ~.1 of MT-2 culture supernatants under vacuum. As controls, 300 tsl of CEM supernatants were loaded into different slots on the same blot. Relative levels of p24 antigen on the blots were assayed using a mouse anti-HTLV-I p24 m A b (DuPont) and a blotting detection kit (Amersham, UK) with streptavidin alkaline phosphatase conjugate. The coiour intensities of bands on membrane were read on a "Bio-Rad Model 620 CCD Video Densitometer" in the reflectance mode.
ANOVA = ATL = CAT = CMV = HIV! = HTLV-I =
IF LTR mAb PBMC RT
analysis of variance. adult T-cell leukaemia. chloramphenicol acetyltransferase.
cytomegalovirus. human immunodeficiency virus type I. human T-cell leukaemia/lymphoma virus type I.
= = = =
immunofluorescence. long terminal repeat. monoclonal antibody. peripheral blood mononuclear cell.
= reverse transcriptase.
THERMAL ENHANCEMENT
OF HTLV-I IN HTL I/-1-TRANSFORMED CELLS
lmmunofluorescence (IF) assay for p24 antigen An 1F assay for the detection of HTLV-I p24 antigen was performed as previously described (RobertGuroff et al., 1981). Briefly, ceils (approximately l x 105 cells) were fixed in 90 °70 methanol for 20 min and then permeabilized with 100 % acetone for 10 min. Chamber slides (Nunc, Inc., Naperville, IL) were used for incubation with anti-HTLV p24 mAb and secondary rabbit anti-mouse lgG fluorescein isothiocyanate (Dakopatt, Carpinteria, CA). Slides were mounted and visualized for IF staining using an epifluorescence microscope equipped with a 40 x water immersion lens appropriate for fluorescence. CEM cells served as an HTLV-negative control. Analysis of variance (ANOVA), Duncan's multiple range test, correlation coefficients and inference test on a correlation coefficient (tr-test) were employed in data analyses. RESULTS
Effect of heat treatment on viability and kinetics of cell growth Initial experiments were carried out to determine viable cell counts and cellular growth patterns during periods o f heat treatment. MT-2 cells demonstrated a slight decrease in viable counts during up to 5 h of continuous heat treatment at 42°C without recovery compared to viable counts at 37°C. However, when heat-stressed at 48°C, MT-2 cells showed a 50 % decrease in viability after 0.5-h heat treatment and an almost 85 °7o decrease in viability after 5 h of heat treatment. Significant differences in viable counts were found between viable ceils at 48°C, and at 37°C (p < 0.01) or at 42°C (p < 0.01), as tested by A N O V A and Duncan's multiple range test. P B M C demonstrated no significant losses in viability during heat treatment at 42°C (p > 0.05) except for heat treatment at 5 h (p < 0.01). Therefore, 42°C was selected in our research as a temperature o f heat treatment.
Effects of heat treatment on RT activity and p24 antigen expression A linear increase o f approximately 20-fold in RT activity in MT-2 cells was observed during
187
5 h o f heat treatment at 42°C, as summarized in figure 1. C E M and P B M cells demonstrated a less than 2.5-fold increase in cellular polymerase activity. The difference between polymerase activity in MT-2 cells and the uninfected P B M C or C E M cells during heat treatment at 42°C was highly significant (p < 0.01), as tested by A N O V A and Duncan's multiple range test. However, no significant differences in polymerase activity were observed between MT-2 and P B M C or C E M cells at 37°C, or between P B M C and C E M cells at 42°C (p > 0.05). A nearly 2-fold increase in p24 H T L V - I protein concentrations in MT-2 cell culture supernatants as compared to non-thermostressed supernatants during up to 5 h o f heat treatment at 42°C was detected (fig. 2). This finding parallels the time course o f the appearance o f increased RT activity in thermostressed versus non-thermostressed MT-2 cells as indicated earlier ( r = 0 . 9 4 1 3 , p < 0.01, t r = 6 . 1 6 5 3 ) , as .analysed by a correlation coefficient calculation and tr-test (fig. 3).
P24 detection by IF HTLV-I p24 antigen was detected, using antiH T L V - I p24 m A b , in both cytoplasms and nuclei o f MT-2 cells during heat treatment. P24 was not found in C E M cells.
DISCUSSION We have demonstrated that H T L V - I replication can be upregulated by heat treatment in a chronically infected human cord lymphocyte cell line, as assessed by an increase in RT activity and p24 antigen expression in cell culture supernatants. Since HTLV-I is the only known viral vector (provirus) (Miyoshi et al., 1981 ; Poiesz et al., 1981) in MT-2 cells, the R T activity measured in MT-2 cell culture supernatants in excess o f those in P B M C or C E M cells is therefore regarded as HTLV-I-specific RT activity. As presynthesized H T L V - I virions are not present intracellularly, particles released from infected cells into culture supernatants following thermal
D. S U N E T A L .
188
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Fig. 1. RT activity in thermal stressed lymphoid cells. HTLV-I RT activities in culture supernatant fluids of MT-2 cells were assayed during up to 5 h of heat treatment at 42°C. Supernatants of PBM and CEM cells were simultaneously assayed as controls. Supernatants were harvested immediately after heat treatment and analysed in an RT assay using 3H-dTTP labelling, as described in "Materials and Methods". Error bars denote the means of 3 assays plus or minus 1 standard deviation (mean _+ l SD). ANOVA and Duncan's multiple range test were carried out to determine the significance of the differences between the MT-2 and control cells. Zero hour at 42°C was equal to 37°C. Non-thermal-elevated samples maintained at 37°C were harvested at 3 h. 10
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Duration of Thermal Stress at 42° C (Hours) Fig. 2. HTLV-[ p24 antigen expression in thermal stressed MT-2 cells. Expression of HTLV-I p24 antigen in culture supernatant fluids of MT-2 cells was assayed for up to 5 h of continuous heat treatment at 42°C. The supernatants were harvested immediately after heat treatment and assayed by slot immunoblotting using an anti-HTLV-I p24 mouse mAb. The concentrations of HTLV-I p24 antigen were assayed by reflectance densitometry. Error bars denote the means of 3 assays plus or minus 1 standard deviation. Simple regression analysis was performed Of = 4.6309 + 0.92799, R = 0.932). Zero hour at 42°C was equal to 37°C. Samples maintained at 37°C were harvested at 3 h. A.U. = arbitrary unit.
T H E R M A L E N H A N C E M E N T OF H T L V-I I N H T L V-I- T R A N S F O R M E D CELLS HTLV-I p24 RT 12000
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Fig. 3. Reverse transcriptase activity and p24 expression in thermal stressed Mr-2 cells. The correlation between HTLV-I p24 expression and HTLV-I RT activity was assessed in culture supernatant fluids of MT-2 cells during 5-h heat treatment at 42°C. Both protein lysates and the supernatants were prepared immediately after heat treatment at 42°C and analysed by immunoblotting or RT assays using 3H-dTTP labelling, respectively, as described in "Materials and Methods"• Correlation coefficient calculation and t-test of coefficient were performed (r = 0.9413, p < 0.01, tr = 6.1653). Zero hour at 42°C was equal to 37°C. A.U. = arbitrary unit.
elevations would be expected to be mostly newly synthesized and assembled in response to cellular stress. However, thermal elevations may have stimulated the assembly, budding and release of both the presynthesized and newly synthesized viral components. Also, in the present study, we found that the time-dependent increase in RT activity and viral p24 antigen expression was consistent with the length of the heat treatment. The slightly reduced viability and significantly enhanced RT activity in MT-2 cells during heat treatment at 42°C as compared with control CEM and PBM cells implies that the increase in viral replication does not simply reflect a burst of virus production or release from irreversibly damaged and dying cells.
189
It has been previously reported that heat treatment can markedly increase bacteriophage production at 42-44°C (Wihberg et ai., 1988) and human CMV production at 44°C (Waghorne and Fuerst, 1985; Zerbini et al., 1986)• Our results are in agreement with those of Stanley et al. (1990), who demonstrated a marked induction of latent HIV1 replication at 41.8 and 42.8°C in HIVl-infected cells. Additionally, Geelen et al. (1988) showed a dramatic increase in the expression of an HIV long terminal repeat (LTR)-chloramphenicol acetyltransferase (CAT) construct in HIVCAT-51 cells following heat treatment at 45°C. The authors suggested that a sequence in the NF-KB binding sequence of the HIV-1 LTR is similar to the core cellular heat shock sequence, which may initiate the heat shock response in infected cells. The similarity between these two response elements may explain the induction of heat-mediated viral replication during heat stress. It is also possible that heat stress directly stimulated the viral LTR. The induction of a latent virus by heat treatment is potentially a physiologically relevant phenomenon. A characteristic of infection with HTLV-I is that the virus does not cause disease in all infected individuals. Less than one percent of individuals infected with HTLV-I eventually develop leukaemia (Yoshida et al., 1987). It is not known why only some infected individuals develop ATL or other HTLV-I-associated diseases. Furthermore, the latency between initial infection and onset of disease may be many years (Takatsuki et al., 1990). A question can be raised as to whether increased numbers of febrile periods due to an increased number of infectious diseases in HTLV-I-infected patients (Yoshioka et al., 1985) may contribute to an increase in transformation of HTLV-I-infected human lymphocytes. A possible mechanism of action of heat-mediated viral enhancement in MT-2 cells is cellular stress, or induction of the p r o p o s e d cellular " S O S " mechanism (Herrlich et al., 1984, 1986). The SOS p h e n o m e n o n consists of derepression of various cell functions initiated by a regulatory signal triggered by DNA lesions and cellular stress. The cell response includes inhibition of cellular division, enhance-
190
D. S U N E T AL.
ment o f inducible proviruses (prophages), new protein synthesis, induction o f D N A repair, m u t a g e n e s i s and inhibition o f o x i d a t i v e metabolism (Radman, 1980; Herrlich et al., 1986). Treatments o f eukaryotic cells in order to induce such stress have included the exposure o f cells to inhibitors o f replication, hypertonic solutions, mutagens, membrane active compounds, tumour-causing or t u m o u r - p r o m o t i n g agents and thermal elevation (Herrlich et ai., 1984; Friedberg, 1985). It is u n k n o w n whether heat treatment can cause cell membrane alterations, surface antigen modifications, interference with such immune functions as antigen presentation, or induction o f cytokine release, as previously reported in other biologic mechanism o f stress (Deeg et aL, 1988; Kupper et ai., 1987; Stanley et al., 1989, 1990). A direct membrane effect cannot be ruled out at this time. Surface antigen modifications and altered immune function would not be expected to play a role in the enhancement o f latent virus by a cell line in culture. Several features o f the MT-2 cell response to heat treatment would support a cellular stress response mediating viral induction. First, the kinetics o f cell growth after heat treatment clearly demonstrated an early growth arrest, a pattern repeatedly seen in response to stress (Herrlich et ai., 1984) as shown in b o t h MT-2 and P B M cells in our research. Second, the association o f slightly decreased cell viability with viral production suggests that a certain level o f cellular stress has been reached at 42°C to cause sufficient stress. Finally, we detected a marked increas,~ in Hsp72 synthesis by Western blotting during up to 5 h o f heat treatment at 42°C. In s u m m a r y , we have demonstrated the enhancement o f oncogenic H T L V - I expression in a transformed cell line by heat treatment. A possible mechanism is heat-mediated t e m p o r a r y D N A arrest and induction o f a cellular SOS response, although a direct effect o f heat treatment on the HTLV-I L T R or on cell membranes with involvement o f intracellular second messengers cannot be excluded at the present time. If physiologic thermal stress, as expected to be f o u n d during febrile episodes a m o n g certain HTLV-I-infected patients, is capable o f induc-
ing expression o f latent virus, the chances o f oncogenic transformation of HTLV-I-infected human lymphocytes and eventually A T L may be increased in those patients. Further investigation o f relationships between heat treatment and cellular transformation is needed to clarify the pathogenesis o f H T L V - I . Acknowledgements
We thank Dr. Elaine Romberg for her assistance with statistical analyses. This research was supported in part by University of Maryland Designated Research Initiative Funds, and National Institute of Health Grants NS26665, DE08553, DE08648 and AR41572.
Thermo-activation in vitro du virus HTLV-! dans des cellules transform~es par HTLV-I
Alors qu'une 616vation thermique peut favoriser les d6fenses antivirales de l'h6te, elle peut nuire aux cellules in fect6es par le virus HTLV-I en augmentant la production de virions et d'oncoprot6ines :dans les cellules MT-2, la r6plication virale est accrue (la quantit6 d'antig+ne p24 est doubl6e, i'activit6 r6verse transcriptase est multipli6e par 20) apr~s 5 h b. 42°C. Ainsi les 6tats f6briles pourraient induire une production virale chez les sujets porteurs de HTLV-I. Mots-clds: HTLV-I, R6plication, Transcriptase inverse, Chaleur, Fi~vre; Augmentation, Antig6ne p24, Choc thermique, Stress, Virus latents, Oncogen6se, Ph6nom~ne SOS.
References
Deeg, H.J. (1988), Ultraviolet irradiation in transplantation biology. Transplantation, 45, 845-851. Friedberg, E.C. (1985), DNA damage tolerance in prokaryotic cells, in "DNA repair. Chapter 7" (pp. 408-417). W.H. Freeman and Co., San Francisco. Geelen, J.L.M.C., Boom, R., Klaver, G.P.M., Minnaar, R.P., Feltkamp, M.C.W., Milligen, F.J., Sol, C.J.A. & Van Der Noordaa, J. (1987), Transcriptional enhancement of the major immediate early transcription unit of human cytomegalovirus by heat-shock, arsenite and protein synthesis inhibitors. J. Gen. Virol., 68, 2925-2931. Geelen, J.L.M.C., Minnaar, R.P., Boom, R., Van Der Noordaa, J. & Goudsmit, J. (1988), Heat-shock induction of the human immunod¢ficiency virus long terminal repeat. J'. Gen. ViroL, 69, 2913-2917.
THERMAL
ENHANCEMENT
OF HTLV-I IN HTL V-I-TRANSFORMED
Gregersen, J.P., Wege, H., Preiss, L. & Jentsch, K.D. (1988), Detection of human immunodeficiency virus and other retroviruses in cell culture supernatants by a reverse transcriptase microassay. J. Virol. Meth., 19, 161-168. Herrlich, P., Mallick, U., Ponta, H. & Rahmsdorf, H.J. (1984), Genetic changes in mammalian cells reminiscent of an SOS response. Huron. Genet., 67,360-368. Herrlich, P., Angel, P., Rahmsdorf, H.J., Mallick, U., Potting, A., Hieber, L., Luche-Huhle, C. & Schorpp, M. (1986), The mammalian genetic stress response. Adv. Enzyme Regul., 25, 485-504. Hinuma, Y., Nagata, K., Hanaoka, M., Nakai, M., Matsumoto, T., Kimoshita, K., Shirakawa, S. & Miyoshi, 1. (1981), Adult T cell leukemia: antigen in an ATL cell line and detection of antibodies to the antigen in human sera. Proc. nat. Acad. Sci. (Wash.), 78, 6476-6480. Kupper, T.S., Chua, A.O., Flood, P., McGuire, J. & Gubler, U. (1987), interleukin 1 gene expression in cultured human keratinocytes is augmented by ultraviolet irradiation. J. Clin. Invest., 80, 430-436. Miyoshi, 1., Kubonishi, I., Yoshimoto, S., Akagi, T., Ohtsuki, Y., Shiraishi, Y., Nagata, K. & Hinuma, Y. (1981). Type C virus particles in a cord T-cell line derived by co-cultivating normal human cord leukocytes and human leukemic T cells. Nature (Lond.), 294, 770-77 I. Poiesz, B.J., Ruscetti, F.W., Gazadar, A.F., Bunn, P.A., Minna, J.D. & Gallo, R.C. (1980), Detection and isolation of type C retrovirus particles from fresh and cultured lymphocytes of a patient with cutaneous T cell lymphoma. Proc. nat. Acad. Sci. (Wash.), 77, 7415-7419. Poiesz, B..I., Ruscetti, F.W., Reitz, M.S., Kalyanaraman, V.S. & Gallo, R.C. (1981), Isolation of a new type C retrovirus (HTLV) in primary uncultured cells of a patient with Sezary T-cell leukemia. Nature (Lond.), 294, 268-271. Radman, M. (1980), Is there SOS induction in mammalian cells? Photochem. Photobiol., 32, 823-830. Robert-Guroff, M., Ruscetti, F.W. & Posner, i.E. (1981), Detection of the human T cell lymphoma virus p19 in cells of some patients with cutaneous T cell lymphoma and leukemia using a monoclonal antibody. J. Exp. Med., 154, 1957-1964.
CELLS
191
Schupbach, J., Sarngadharan, M.G. & Gallo, R.C. (1984), Antigens on HTLV-infected cells recognized by leukemia and AIDS sera are related to HTLV viral glycoprotein. Science, 224, 607-610. Somogyi, P.A., Gyuris, A. & Foldes, A.l. (1990), A solid phase reverse transcriptase micro-assay for the detection of human immunodeficiency virus and other retroviruses in cell culture supernatants. J. Virol. Meth., 27, 269-276. Stanley, S.K., Folks, T.M. & Fauci, A.S. (1989), Induction of expression of human immunodeficiency virus in a chronically infected promonocytic cell line by ultraviolet irradiation. A I D S Res. Hum. Retroviruses, 5, 375-384. Stanley, S.K., Folks, T.M. & Fauci, A.S. (1990), Stressmediated induction of HIV expression from chronically infected promonocytic T cell lines. A I D S Res. Hum. Retroviruses, 6, 52-53. Takatsuki, K., Yamaguchi, K. & Hattori, T. (1990), Adult T-cell leukemia/lymphoma, in "Retrovirus Biology and Human Disease" (Gallo, R.C. & Wong-Staal, F.) (pp. 147-159). Marcel Dekker Inc., New York. Uzman, B.G., Foley, G.E., Farber, S. & Lazarus, H. (1966), Morphologic variations in human leukemic lymphoblasts (CCRF-CEM cells) after long-term culture and exposure to chemotherapeutic agents. Cancer, 19, 1725-1747. Waghorne, C. & Fuerst, C.R. (1985), Involvement of the htpR gene product of Escherichia coil in phage lambda development. Virology, 141, 51-64. Wihberg, J.S., Mowrey-McKee, M.F. & Stevens, E.J. (1988), Induction of the heat shock regulon of Escherichia coil markedly increases production of bacterial viruses at high temperatures..L ViroL, 62, 234-245. Yoshida, M. & Seiki, M. (1987), Recent advances in the molecular biology of HTLV-I: trans-activation of viral and cellular genes. Ann. Rev. Immunol., 5, 451-459. Yoshioka, R., Yamaguchi, K., Yoshinaga, T. & Takatsuki, K. (1985), Pulmonary complications in patients with adult T-cell leukemia. Cancer, 55, 2491-2494. Zerbini, M., Musiani, M. & La Placa, M. (1986), Stimulating effect of heat shock on the early stage of human cytomegalovirus replication cycle. Virus Res., 6, 21 !-216.
Res. ViroL 1993, 144, 185-191
In vitro thermal enhancement of human T-cell leukaemia/lymphoma virus type I (HTLV-I) in HTLV-l-transformed cells D. Sun, D.W. Archibald (*) and J.J. Sauk Department o f Pathology, School o f Dentistry, University o f Maryland at Baltimore, 666 IV. Baltimore Street, Baltimore, MD 21201 (USA)
SUMMARY
Temperature elevation constitutes a beneficial component of the host defence against viral pathogens. However, heat treatment may be detrimental to HTLV-I-infected cells by increasing virion and oncoprotein production. We investigated the effects of thermal elevation on the in vitro replication of HTLV-I (human T-cell leukaemia/lymphoma virus type I) in MT-2 cells, an HTLV-I-transformed lymphoid cell line. We found that HTLV-I replication in MT-2 cells was markedly increased as demonstrated by a nearly 2-fold increase in detection of viral p24 antigen and a 20-fold increase in reverse transcriptase activity during up to 5 h of heat treatment at 42°C. The results suggest that physiologic thermal elevations may induce viral production in HTLV-I-infected individuals. Key-words: HTLV-I, Replication, Reverse transcriptase, Heat, Fever; Enhancement, P24 antigen, Heat shock, Latent virus, Oncogenesis, Cellular SOS mechanisms, Stress.
INTRODUCTION H u m a n T-cell l e u k a e m i a / l y m p h o m a virus type I belongs to the family of exogenous Tlymphotropic type C retroviruses and is the aetiological agent of h u m a n adult T-cell leukaem i a / l y m p h o m a (ATL) (Poiesz et ai., 1980). HTLV infection is characterized by a prolonged period of latency, which may be followed by the development of T-cell l y m p h o m a (Poiesz et aL, 1980) or ATL (Hinuma et al., 1981). The detection of HTLV antigens in sera (Schupbach et al.,
Submitted March 3, 1993, accepted April 16, 1993. (*) To whomcorrespondenceaddressed.
1984) indicated a role for HTLV gene expression in the pathogenesis of ATL. It has been previously reported, using an in vitro model, that cellular heat treatment may be involved in the reactivation of latent cytomegalovirus (CMV) (Geelen et al., 1987) infection, and human immunodeficiency virus type 1 (HIV1) (Stanley et al., 1990; Geelen et al., 1988). As cellular stress factors may evoke a common induction mechanism in the reenhancement of latent CMV and HIV1, we analysed the effect of heat treatment on HTLV-I replication in HTLVI-transformed cells.
186
D. S U N E T A L . MATERIALS AND METHODS
Cells
MT-2 cells belonging to an HTLV-l-transformed human T-cell line, which were a gift from Dr. Max Essex, Harvard School of Public Health, were originally established from cord blood lymphocytes cocultivated with leukaemic cells from a patient with adult T-cell leukaemia (ATL) (Miyoshi et al., 1981). CEM-T4 cells from an ATCC-certified cell line were obtained through the AIDS Research and Reference Reagent Program, AIDS Program, N I A I D , N I H operated by ERC BioServices Corporation, Rockville, MD (contributor: Dr. J.P. Jacobs). CEM is a T-lymphoblastoid cell line and was developed from the peripheral blood buffy coat of a four-yearold Caucasian female with acute lymphoblastic leukaemia. The cells were free of virus-like particles as determined by electron microscopy (Uzman et al., 1966). H u m a n peripheral blood mononuclear cells (PBMC) were harvested from a healthy laboratory worker and separated through a Ficoll/Hypaque gradient " H i s t o p a q u e - 1 0 7 7 " (Sigma, St. Louis, MO). The cells were collected immediately before use and exhibited greater than 95 percent viability by the trypan blue exclusion assay.
Reverse transcriptase microassay
An RT microassay was performed according to an established protocol (Gregersen et al., 1988; Somogyi et al., 1990). Magnesium-dependent RT activity was measured using a synthetic RNA-template primer of poly(rA):oligo(dT)12.1s ( P h a r m a c i a / P - L Biochemicals, Piscataway, N J). Briefly, after preparation of the rA cocktail, including methyl-°H deoxythymidine triphosphate (3H-dTTP) (DuPont, NEN Res. Prod., North Billerica, MA) and poly(rA):oligo(dT)~2as template primer, 50 ~.1 samples were added to a tube containing poly(rA):oligo(dT)12.18 and v o r t e x e d . T h e s a m p l e s were incubated for 2 h in a 37°C water bath. Two 5-ram DE-81 paper discs (Schleicher & Schuell, Keene, NH), made with a standard hole punch, were added to each reaction well and mixed. The discs were then removed from the wells and placed in corresponding wells in a dot blot manifold apparatus (Bio Rad, Hercules, CA). Each sample well in the manifold apparatus was rinsed out five times with 5 % Na2HPO 4, twice with d d H 2 0 and twice with 70 % E t O H under vacuum. Each vial of triplicate samples was counted for 4 rain on a 3H channel. The non-specific background cellular polymerase activity from CEM or PBM cells was subtracted from the value from MT-2 cells. The resulting values reflected HTLV-l-specific RT activity.
P24-antigen detection assay Culture c o n d i t i o n s and heat treatment o f the cells
The cells were cultured at 37°C and 5 % CO 2 and maintained in log phase growth in RPMI-1640 medium (Gibco, Grand Island, NY) supplemented with 15 °7o foetal bovine serum (Sigma) and I00 U each of penicillin and streptomycin (Bioproducts, Inc., Walkersville, MD) per ml. Aliquots o f 5-6 x 105 cells per ml complete media were placed in sterile disposable plastic 25-cm 2 tissue culture flasks. Thermal elevations were carried out by submerging the cell culture flasks with gentle agitation in a 4 2 + 0 . 2 ° C water bath. The supernatants were immediately harvested for reverse transcriptase (RT) or p24 antigen assays.
H T L V - I p24 antigen was assayed in culture supernatants as follows. A slit manifold apparatus (BioRad) was used to prepare an antigen-coated blot. Briefly, nitrocellulose membranes (Schleicher & Schuell) were loaded with 300 ~.1 of MT-2 culture supernatants under vacuum. As controls, 300 tsl of CEM supernatants were loaded into different slots on the same blot. Relative levels of p24 antigen on the blots were assayed using a mouse anti-HTLV-I p24 m A b (DuPont) and a blotting detection kit (Amersham, UK) with streptavidin alkaline phosphatase conjugate. The coiour intensities of bands on membrane were read on a "Bio-Rad Model 620 CCD Video Densitometer" in the reflectance mode.
ANOVA = ATL = CAT = CMV = HIV! = HTLV-I =
IF LTR mAb PBMC RT
analysis of variance. adult T-cell leukaemia. chloramphenicol acetyltransferase.
cytomegalovirus. human immunodeficiency virus type I. human T-cell leukaemia/lymphoma virus type I.
= = = =
immunofluorescence. long terminal repeat. monoclonal antibody. peripheral blood mononuclear cell.
= reverse transcriptase.
THERMAL ENHANCEMENT
OF HTLV-I IN HTL I/-1-TRANSFORMED CELLS
lmmunofluorescence (IF) assay for p24 antigen An 1F assay for the detection of HTLV-I p24 antigen was performed as previously described (RobertGuroff et al., 1981). Briefly, ceils (approximately l x 105 cells) were fixed in 90 °70 methanol for 20 min and then permeabilized with 100 % acetone for 10 min. Chamber slides (Nunc, Inc., Naperville, IL) were used for incubation with anti-HTLV p24 mAb and secondary rabbit anti-mouse lgG fluorescein isothiocyanate (Dakopatt, Carpinteria, CA). Slides were mounted and visualized for IF staining using an epifluorescence microscope equipped with a 40 x water immersion lens appropriate for fluorescence. CEM cells served as an HTLV-negative control. Analysis of variance (ANOVA), Duncan's multiple range test, correlation coefficients and inference test on a correlation coefficient (tr-test) were employed in data analyses. RESULTS
Effect of heat treatment on viability and kinetics of cell growth Initial experiments were carried out to determine viable cell counts and cellular growth patterns during periods o f heat treatment. MT-2 cells demonstrated a slight decrease in viable counts during up to 5 h of continuous heat treatment at 42°C without recovery compared to viable counts at 37°C. However, when heat-stressed at 48°C, MT-2 cells showed a 50 % decrease in viability after 0.5-h heat treatment and an almost 85 °7o decrease in viability after 5 h of heat treatment. Significant differences in viable counts were found between viable ceils at 48°C, and at 37°C (p < 0.01) or at 42°C (p < 0.01), as tested by A N O V A and Duncan's multiple range test. P B M C demonstrated no significant losses in viability during heat treatment at 42°C (p > 0.05) except for heat treatment at 5 h (p < 0.01). Therefore, 42°C was selected in our research as a temperature o f heat treatment.
Effects of heat treatment on RT activity and p24 antigen expression A linear increase o f approximately 20-fold in RT activity in MT-2 cells was observed during
187
5 h o f heat treatment at 42°C, as summarized in figure 1. C E M and P B M cells demonstrated a less than 2.5-fold increase in cellular polymerase activity. The difference between polymerase activity in MT-2 cells and the uninfected P B M C or C E M cells during heat treatment at 42°C was highly significant (p < 0.01), as tested by A N O V A and Duncan's multiple range test. However, no significant differences in polymerase activity were observed between MT-2 and P B M C or C E M cells at 37°C, or between P B M C and C E M cells at 42°C (p > 0.05). A nearly 2-fold increase in p24 H T L V - I protein concentrations in MT-2 cell culture supernatants as compared to non-thermostressed supernatants during up to 5 h o f heat treatment at 42°C was detected (fig. 2). This finding parallels the time course o f the appearance o f increased RT activity in thermostressed versus non-thermostressed MT-2 cells as indicated earlier ( r = 0 . 9 4 1 3 , p < 0.01, t r = 6 . 1 6 5 3 ) , as .analysed by a correlation coefficient calculation and tr-test (fig. 3).
P24 detection by IF HTLV-I p24 antigen was detected, using antiH T L V - I p24 m A b , in both cytoplasms and nuclei o f MT-2 cells during heat treatment. P24 was not found in C E M cells.
DISCUSSION We have demonstrated that H T L V - I replication can be upregulated by heat treatment in a chronically infected human cord lymphocyte cell line, as assessed by an increase in RT activity and p24 antigen expression in cell culture supernatants. Since HTLV-I is the only known viral vector (provirus) (Miyoshi et al., 1981 ; Poiesz et al., 1981) in MT-2 cells, the R T activity measured in MT-2 cell culture supernatants in excess o f those in P B M C or C E M cells is therefore regarded as HTLV-I-specific RT activity. As presynthesized H T L V - I virions are not present intracellularly, particles released from infected cells into culture supernatants following thermal
D. S U N E T A L .
188
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4
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Fig. 1. RT activity in thermal stressed lymphoid cells. HTLV-I RT activities in culture supernatant fluids of MT-2 cells were assayed during up to 5 h of heat treatment at 42°C. Supernatants of PBM and CEM cells were simultaneously assayed as controls. Supernatants were harvested immediately after heat treatment and analysed in an RT assay using 3H-dTTP labelling, as described in "Materials and Methods". Error bars denote the means of 3 assays plus or minus 1 standard deviation (mean _+ l SD). ANOVA and Duncan's multiple range test were carried out to determine the significance of the differences between the MT-2 and control cells. Zero hour at 42°C was equal to 37°C. Non-thermal-elevated samples maintained at 37°C were harvested at 3 h. 10
m t-
r,o A
HTLV-I p24
O a.
O
0
i
I
i
Duration of Thermal Stress at 42° C (Hours) Fig. 2. HTLV-[ p24 antigen expression in thermal stressed MT-2 cells. Expression of HTLV-I p24 antigen in culture supernatant fluids of MT-2 cells was assayed for up to 5 h of continuous heat treatment at 42°C. The supernatants were harvested immediately after heat treatment and assayed by slot immunoblotting using an anti-HTLV-I p24 mouse mAb. The concentrations of HTLV-I p24 antigen were assayed by reflectance densitometry. Error bars denote the means of 3 assays plus or minus 1 standard deviation. Simple regression analysis was performed Of = 4.6309 + 0.92799, R = 0.932). Zero hour at 42°C was equal to 37°C. Samples maintained at 37°C were harvested at 3 h. A.U. = arbitrary unit.
T H E R M A L E N H A N C E M E N T OF H T L V-I I N H T L V-I- T R A N S F O R M E D CELLS HTLV-I p24 RT 12000
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~6000
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O U •
i 1
•
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i
2
3
•
!
i
4
5
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Duration of Thermal Stress at 42°C
(Hours)
Fig. 3. Reverse transcriptase activity and p24 expression in thermal stressed Mr-2 cells. The correlation between HTLV-I p24 expression and HTLV-I RT activity was assessed in culture supernatant fluids of MT-2 cells during 5-h heat treatment at 42°C. Both protein lysates and the supernatants were prepared immediately after heat treatment at 42°C and analysed by immunoblotting or RT assays using 3H-dTTP labelling, respectively, as described in "Materials and Methods"• Correlation coefficient calculation and t-test of coefficient were performed (r = 0.9413, p < 0.01, tr = 6.1653). Zero hour at 42°C was equal to 37°C. A.U. = arbitrary unit.
elevations would be expected to be mostly newly synthesized and assembled in response to cellular stress. However, thermal elevations may have stimulated the assembly, budding and release of both the presynthesized and newly synthesized viral components. Also, in the present study, we found that the time-dependent increase in RT activity and viral p24 antigen expression was consistent with the length of the heat treatment. The slightly reduced viability and significantly enhanced RT activity in MT-2 cells during heat treatment at 42°C as compared with control CEM and PBM cells implies that the increase in viral replication does not simply reflect a burst of virus production or release from irreversibly damaged and dying cells.
189
It has been previously reported that heat treatment can markedly increase bacteriophage production at 42-44°C (Wihberg et ai., 1988) and human CMV production at 44°C (Waghorne and Fuerst, 1985; Zerbini et al., 1986)• Our results are in agreement with those of Stanley et al. (1990), who demonstrated a marked induction of latent HIV1 replication at 41.8 and 42.8°C in HIVl-infected cells. Additionally, Geelen et al. (1988) showed a dramatic increase in the expression of an HIV long terminal repeat (LTR)-chloramphenicol acetyltransferase (CAT) construct in HIVCAT-51 cells following heat treatment at 45°C. The authors suggested that a sequence in the NF-KB binding sequence of the HIV-1 LTR is similar to the core cellular heat shock sequence, which may initiate the heat shock response in infected cells. The similarity between these two response elements may explain the induction of heat-mediated viral replication during heat stress. It is also possible that heat stress directly stimulated the viral LTR. The induction of a latent virus by heat treatment is potentially a physiologically relevant phenomenon. A characteristic of infection with HTLV-I is that the virus does not cause disease in all infected individuals. Less than one percent of individuals infected with HTLV-I eventually develop leukaemia (Yoshida et al., 1987). It is not known why only some infected individuals develop ATL or other HTLV-I-associated diseases. Furthermore, the latency between initial infection and onset of disease may be many years (Takatsuki et al., 1990). A question can be raised as to whether increased numbers of febrile periods due to an increased number of infectious diseases in HTLV-I-infected patients (Yoshioka et al., 1985) may contribute to an increase in transformation of HTLV-I-infected human lymphocytes. A possible mechanism of action of heat-mediated viral enhancement in MT-2 cells is cellular stress, or induction of the p r o p o s e d cellular " S O S " mechanism (Herrlich et al., 1984, 1986). The SOS p h e n o m e n o n consists of derepression of various cell functions initiated by a regulatory signal triggered by DNA lesions and cellular stress. The cell response includes inhibition of cellular division, enhance-
190
D. S U N E T AL.
ment o f inducible proviruses (prophages), new protein synthesis, induction o f D N A repair, m u t a g e n e s i s and inhibition o f o x i d a t i v e metabolism (Radman, 1980; Herrlich et al., 1986). Treatments o f eukaryotic cells in order to induce such stress have included the exposure o f cells to inhibitors o f replication, hypertonic solutions, mutagens, membrane active compounds, tumour-causing or t u m o u r - p r o m o t i n g agents and thermal elevation (Herrlich et ai., 1984; Friedberg, 1985). It is u n k n o w n whether heat treatment can cause cell membrane alterations, surface antigen modifications, interference with such immune functions as antigen presentation, or induction o f cytokine release, as previously reported in other biologic mechanism o f stress (Deeg et aL, 1988; Kupper et ai., 1987; Stanley et al., 1989, 1990). A direct membrane effect cannot be ruled out at this time. Surface antigen modifications and altered immune function would not be expected to play a role in the enhancement o f latent virus by a cell line in culture. Several features o f the MT-2 cell response to heat treatment would support a cellular stress response mediating viral induction. First, the kinetics o f cell growth after heat treatment clearly demonstrated an early growth arrest, a pattern repeatedly seen in response to stress (Herrlich et ai., 1984) as shown in b o t h MT-2 and P B M cells in our research. Second, the association o f slightly decreased cell viability with viral production suggests that a certain level o f cellular stress has been reached at 42°C to cause sufficient stress. Finally, we detected a marked increas,~ in Hsp72 synthesis by Western blotting during up to 5 h o f heat treatment at 42°C. In s u m m a r y , we have demonstrated the enhancement o f oncogenic H T L V - I expression in a transformed cell line by heat treatment. A possible mechanism is heat-mediated t e m p o r a r y D N A arrest and induction o f a cellular SOS response, although a direct effect o f heat treatment on the HTLV-I L T R or on cell membranes with involvement o f intracellular second messengers cannot be excluded at the present time. If physiologic thermal stress, as expected to be f o u n d during febrile episodes a m o n g certain HTLV-I-infected patients, is capable o f induc-
ing expression o f latent virus, the chances o f oncogenic transformation of HTLV-I-infected human lymphocytes and eventually A T L may be increased in those patients. Further investigation o f relationships between heat treatment and cellular transformation is needed to clarify the pathogenesis o f H T L V - I . Acknowledgements
We thank Dr. Elaine Romberg for her assistance with statistical analyses. This research was supported in part by University of Maryland Designated Research Initiative Funds, and National Institute of Health Grants NS26665, DE08553, DE08648 and AR41572.
Thermo-activation in vitro du virus HTLV-! dans des cellules transform~es par HTLV-I
Alors qu'une 616vation thermique peut favoriser les d6fenses antivirales de l'h6te, elle peut nuire aux cellules in fect6es par le virus HTLV-I en augmentant la production de virions et d'oncoprot6ines :dans les cellules MT-2, la r6plication virale est accrue (la quantit6 d'antig+ne p24 est doubl6e, i'activit6 r6verse transcriptase est multipli6e par 20) apr~s 5 h b. 42°C. Ainsi les 6tats f6briles pourraient induire une production virale chez les sujets porteurs de HTLV-I. Mots-clds: HTLV-I, R6plication, Transcriptase inverse, Chaleur, Fi~vre; Augmentation, Antig6ne p24, Choc thermique, Stress, Virus latents, Oncogen6se, Ph6nom~ne SOS.
References
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THERMAL
ENHANCEMENT
OF HTLV-I IN HTL V-I-TRANSFORMED
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CELLS
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