Mitogen induction of murine C-type viruses

73, 17-22 (1976)

VIROLOGY

Mitogen

Induction

of Murine

II. Effect of B-Lymphocyte

C-Type Viruses Mitogens

CHRISTOPH MORONI’ Friedrich

Miescher-Institut,

P.O. Box 273, CH-4002 Basel, Switzerland AND

GEBHARD SCHUMANN Research

Department,

Pharmaceuticals

Division, Accepted

CIBA-GEIGY March

Limited,

CH-4002

Basel, Switzerland

4,1976

Various B-lymphocyte mitogens have been examined for their capacity to induce Ctype virus in mouse spleen cells. Lipopolysaccharide (LPS) Escherichia coli, LPS Salmonella abortus equi, lipid A and tuberculin (purified protein derivative, PPD) were all inductive. 5-Bromo-2’-deoxyuridine amplified virus induction but not mitogenicity of LPS and PPD, when tested in BALB/c and nu/nu cells. Antibody against reverse transcriptase from Rauscher leukemia virus inhibited the enzyme of LPS- and PPDinduced viruses. Preexposure of cells to 500 R blocked virus induction. Spleen cells of C3H/HeJ mice known to be resistant to the mitogenic action of LPS could not be induced by this agent but could still be induced by PPD. INTRODUCTION

deoxyuridine (BrdU) was added to the cultures in addition to LPS (Moroni et al., 1975). In infectivity assays, induced viruses showed a xenotropic host range (unpublished data with Dr. N. Teich). When phytohemagglutinin (PHA) and concanavalin A (Con A), two T (thymus derived)-lymphocyte mitogens, were tested (Moroni et al., 19751,no virus induction was detected by reverse transcriptase assay in the primary culture supernatant. However, Con A but not PHA became inductive in concert with BrdU. LPS is a complex molecule triggering a variety of biological effects. Its lipid A fraction is responsible for the mitogenic action (Andersson et al., 1973). In the present study, we have examined the relationship of virus induction to mitogenicity and have compared LPS with lipid A as well as with tuberculin (PPD), a chemically unrelated B-lymphocyte mitogen (Sultzer and Nilsson, 19721.

We have recently reported that lipopolysaccharide (LPSY from E. cd, a B (bone marrow derived&lymphocyte mitogen, when added to primary spleen cell cultures, induced endogenous C-type virus after 48-72 hr of culture (Moroni and Schumann, 1975). Induced virus met C-type criteria in budding from the cell membrane, banding at a density of 1.16 g/cm3, and containing an RNA-dependent DNA polymerase which behaved in templateprimer preference experiments like viral reverse transcriptase (Moroni and Schumann, 1975; Moroni et al., 1975). Virus induction was amplified when 5-bromo-2’’ Author to whom reprint requests should be sent. ’ Abbreviations used in this paper are: B&J, 5 bromo-2’-deoxyuridine; LPS, lipopolysaccharide; PPD, tuberculin (purified protein derivative); PHA, phytohemagglutinin; Con A concanavalin A; TMP thymidine monophosphate; A,,.dT,,,,, poly(A)-oligo (dT); IgG, immunoglobulin G. 17 Copyright All rights

0 1976 by Academic Press, Inc. of reproduction in any form reserved.

18

MORON1

AND

SCHUMANN

from experimental values. Usually, 10 or 20 ~1 of concentrated virus-containing Mice. Specific pathogen-free 6- to losamples corresponding to 0.1 or 0.2 ml of week-old BALB/c and congenitally athymic “nude” mice (nulnu, ninth back- culture supernatant were assayed, and all cross generation onto the BALB/c back- values including the controls were normalized for 1 ml of culture supernatant. ground) were purchased from BomholdAntibody inhibition of reverse transcripgard, Denmark). C3H and C3H/HeJ mice of the same age were obtained from Insti- tase. A purified rabbit IgG fraction of antiserum to reverse transcriptase of Rauscher tut fur biologisch-medizinische Forschung leukemia virus and control IgG fraction AG, Fullinsdorf, Switzerland. were a gift of Dr. R. C. Gallo. Ten microliCell cultures. One-milliliter cultures samples were incontaining 2 * lo6 nucleated spleen cells in ters of virus-containing cubated for 15 min at 4” with 10 ~1 of a medium RPM1 1640 (Microbiological Assobuffer, pH 7.7, containing 1% Nonidet Pciates supplemented with 8% fetal bovine 600 n-&f KC1 and 40 serum (Rehatuin, Reheis), penicillin (50 40, 160 n-&f Tris-HCl, IU/ml, Hoechst) and streptomycin (50 Fg/ mg/ml of bovine serum albumin. Then, 5 ~1 of the IgG fractions containing approximl, Nova) were prepared under aseptic mately 30 Fg of protein were added toconditions as described (Schumann et al., 1973) and incubated for 3 days at 37” in 8% gether with 25 ~1 of 30 n-& Tris, and incubation was continued overnight. Reverse CO, in air without changes of medium. transcriptase was determined using the Mitogens were added at the onset of culstandard reaction mixture corrected for turing at the following optimal mitogenic already present during concentrations: LPS W Escherichia coli the constituents the overnight incubation period. Olll:B4 (Difco) at 16 pg/ml; LPS SalmoL3Hlthymidine incorporation. DNA synnella abortus equi (Dr. Ch. Galanos, MaxPlanck-Institut fur Immunbiologie, Frei- thesis was determined in parallel dupliburg i.Br., Germany) at 10 pg/ml; lipid A cate cultures by measuring incorporation of [“Hlthymidine into cellular DNA followcomplexed with bovine serum albumin ing published procedures (Vischer and Ja(Dr. Ch. Galanos) at 30 pglml; and puriquet, 1972; Schumann et al., 1973). Labelfied tuberculin (PPD, Statens Seruminstitut, Denmark) at 50 pglml. After 24 hr, 5 ing time was the last 18 hr of the 3-day Fglml of BrdU was added to some cul- culture period. tures. Control cultures contained no mitoRESULTS gens or only BrdU. At the end of culturing, the cells were removed by centrifugation In order to see whether the reported viat 1000 g for 10 min; in some experiments rus induction capacity of LPS (Moroni and the medium from corresponding cultures Schumann, 1975) resides in its mitogenic were pooled, and virus was concentrated lipid A fraction, we tested lipid A, LPS E. by centrifugation and stored in a glycerolcoli, and LPS Salmonella abortus equi for containing buffer as described (Moroni et virus induction. As shown in Table 1, lipid al., 1975). A was inductive to a degree comparable C-type virus assay. Virus was assayed as with that of the two LPS preparations, described (Moroni et al., 1975) by deter- suggesting that virus induction and mitomining reverse transcriptase activity genicity may be linked properties. Theremeasuring polymerization of [“HITMP fore, it was of interest to ask whether an(Radiochemical Centre, Amersham) on other B-lymphocyte mitogen of different A,*dT,,-,, (Collaborative Research; Miles) chemical structure would also induce vias template-primer. Specific activity of rus. We compared LPS for virus induction the loo-~1 reaction mixture was 7500 dpm/ and mitogenicity with PPD (Table 2), anpmol. Acid-insoluble radioactivity followother B-lymphocyte mitogen (Sultzer and ing a 60-min incubation was determined Nilsson, 1972). As can be seen in Table 2, using standard procedures. Values from PPD, like LPS, induced virus in spleen samples lacking virus were subtracted cell cultures of BALB/c as well as of nulnu MATERIALS

AND

METHODS

B-LYMPHOCYTE TABLE

1

BY LPS E. coli, LPS Salmonella AND LIPID A IN SPLEEN CELW OF nul

VIRUS INDUCTION

abortus equi

nu MICE Induction

method”

Reverse transcriptase activitp (pm01 of L3H]TMP)

Control LPS E. colic LPS S. abortus equid Lipid A’

0.15 4.45 7.15 6.60

a Mitogens were added at the beginning of the 3day spleen cell culture. b After 3 days, virus was concentrated from culture supernatants by centrifugation and assayed for L3HlTMP polymerization activity using A.. dT,,-,, as template-primer. Specific activity of the reaction mixture was 7500 dpm/pmol. The mean values of triplicate determinations shown correspond to activity present in l-ml culture supernatant. c 16 pg/ml. d 10 pg/ml. c Complexed to bovine serum albumin, 30 pglml. TABLE

2

OF LPS AND VIRUS INDUCTION AND MIT~GENICITY PPD IN SPLEEN CELIZ OF BALBlc AND nulnu MICE

Induction method”

BALBlc Control LPSd PPD

Reverse transcriptase activity (pmol zfs[hyJTMP -

t3Hlthymidine incorporationC (cpm x 103)

19

INDUCTION

mice, the latter ones lacking differentiated T lymphocytes and consisting predominantly of B lymphocytes. BrdU, although inhibiting r3Hlthymidine incorporation, did not inhibit the mitogen-induced stimulation of DNA synthesis and amplified virus induction of both mitogens (Table 2). Since PPD represents a novel inducing agent, a specificity control was necessary to show that the induced virus indeed belongs to the class of murine C-type viruses. Antibody against reverse transcriptase from Rauscher leukemia virus was tested for reactivity with reverse transcriptase of PPD- and LPS-induced viruses. As shown in Table 3, enzymes from both sources crossreacted with the enzyme from Rauscher leukemia virus. The fact that LPS and PPD, two chemically unrelated mitogens, are both virus inducers suggests that induction may be linked to cell proliferation. We therefore tested whether induction could still be detected in cells whose proliferative capacity had been blocked by exposure to 500 R of irradiation prior to culturing. As can be seen in Table 4, no virus induction could be detected. To analyze further the relationship beTABLE

0.25 k 0.02 1.35 + 0.01 0.65 5 0.02

5.1 35.5 19.9

0.40 5 0.05 6.75 2 0.35 2.35 2 0.15

0.4 3.6 1.8

Control LPSd PPD

0.45 k 0.05 2.15 ” 0.25 0.80 2 0.05

12.5 64.7 19.2

Control/BrdU LPS/BrdU PPD/BrdU

0.25 f 0.15 2.85 k 0.25 1.85 ? 0.20

5.9 44.7 11.3

Control/BrdU LPS/BrdU PPD/BrdU

VIRUS

ANTIBODY

Induction

method”

Reverse transcriptase activity

Inhibition (o/o)

,~~I~%ig

nulnu

a Mitogens were added at the beginning of the 3day spleen cell culture; BrdU after 24 hr. * See Table 1, footnote b. c Duplicate parallel cultures were labeled with 0.5 &!i of r3H]thymidine for the last 18 hr of the 3day culture period and processed for acid-precipitable radioactivity determination. The standard error was less than 20% of the mean. d LPS E. coli.

3

INHIBITION OF LPS- AND PPD-INDUCED VIRAL REVERSE TRANSCRIPTASE

BALB/c LPS-BrdUd BALB/c PPD-BrdU nulnu LPS-BrdUd nulnu PPD-BrdU Rauscher leukemia viruse

Antibody”

Control”, e

0.30 0.16 0.20 0.17 0.52

2.00 0.80 0.93 0.61 4.21

85 80 78 72 88

(1See Table 2, footnote a. b Prior to reverse transcriptase assay, disrupted virus (see Materials and Methods) was incubated with 30 pg of a purified IgG fraction of rabbit antibody to reverse transcriptase prepared from Rauscher leukemia virus or of control IgG fraction. c Activity in the absence of IgG differed by 4 10%. d LPS E. coli. e Prepared from JLS-V5 culture supematant (Moroni, 1972).

20

MORON1

AND

SCHUMANN

tween virus induction and mitogenicity, DISCUSSION we compared C3H to C3H/HeJ mice. The Before studying the mechanism by latter but not the former strain is genetiwhich LPS induces virus in primary culcally resistant to the mitogenic action of LPS, while responding normally to PPD tures of mouse spleen cells (Moroni and (Sultzer and Nilsson, 1972, Watson and Schumann, 1975; Moroni et al., 19751, it Riblet, 1974). When both agents were was necessary to obtain information on the of its mitogenic and virogenic tested with C3H cells (Table 51, each relationship properties. LPS is a complex molecule conproved to be both mitogenic and virus inductive. In contrast, C3H/HeJ cells proved sisting of various regions of differing antigenic and properties resistant to both virus-inductive and mito- chemical (Luderitz et al., 1971): Polysaccharide regenie effects of LPS, while responding like gions carrying the serological specificities C3H cells to PPD. Analogous to AKR cells are linked to the lipid component termed (Moroni et al., 1975), the BrdU amplificalipid A which plays a role in endotoxicity tion effect was not observed with C3H nor (Galanos et al., 1972) and is the motogenic C3H/HeJ cells. principle (Andersson et al., 1973). Our results indicate a correlation between virus TABLE 4 VIRUS INDUCTION AND MITOGENICITY OF LPS AFTER induction and mitogenicity: X RADIATION” OF BALBlc SPLEEN CELLS 1. Lipid A was inductive just as LPS E. Induction Reverse tran13H]thymidine incoli and LPS Salmonella abortus equi (Tamethod0 scriptase activitf corporationd (cpm ble 11, two LPS molecules differing in their (pmol of L3HlTMP) x 103) serological specificities but sharing the XL2 ,” C;zltrsol X-rayed Control same lipid A part. f cells cells 2. PPD, another B-lymphocyte mitogen Control/ 0.05 0.30 0.3 9.7 of different chemical composition was also BrdU inductive (Table 2). LPS/BrdU’ 0.10 2.30 1.1 24.9 3. No induction was detected in cultures ” Prior to induction, the spleen cell suspension whose proliferative capacity had been was exposed to 500 R using a Muller RT 250 X-ray blocked by 500 R of irradiation prior to machine with the following physical constants: 250 induction (Table 4). kV, 15 mA, 0.2 mm Cu added filtration, 0.8 mm half4. In C3HlHeJ mice, the absence of provalue layer, 60-cm focal distance, dose rate 98 Rpro liferative response to LPS was accompamin. nied by absence of virus induction. * See Table 2, footnote a. 5. When subpopulations of lymphoid c See Table 1, footnote b cells were analyzed (Schumann and Mod See Table 2, footnote c. ’ LPS E. coli. roni, 19761, virus induction occurred only TABLE

5

VIRUS INDUCTION AND MIT~CENICITY OF LPS AND PPD IN SPLEEN CELLT OF C3H AND C3H/HeJ Induction

method”

Reverse transcriptase (pm01 of PHITMP

activitp s SD)

MICE

13Hlthymidine incorporation (cpm x 10s)

C3H

C3H/HeJ

C3H

Control LPSd PPD

0.60 zt 0.05 7.20 k 0.95 1.40 % 0.10

0.20 i 0.01 0.25 ” 0.02 0.70 k 0.05

13.6 73.2 28.1

10.9 8.0 24.9

Control/BrdU LPS/BrdW PPD/BrdU

0.80 k 0.10 5.35 ? 0.95 5.40 r 0.15

0.20 -c 0.03 0.20 2 0.03 0.45 r 0.05

5.8 33.6 14.1

3.5 3.2 11.3

’ See Table 2, footnote a. b See Table 1, footnote b. c See Table 2, footnote c. d LPS E. coli.

C3H/HeJ

B-LYMPHOCYTE

VIRUS

21

INDUCTION

The excellent technical assistance of Miss D. in populations showing proliferative reMartin, Mrs. C. Kristofic, and Mr. P. Hofer is gratesponse to the mitogens tested. fully acknowledged. In summary, it would appear that inducWe are indebted to Drs. P. Dukor and N. N. tion of DNA synthesis and cellular prolifIscove for stimulating discussions. eration may be a prerequisite for virus induction by a mitogen. Whether or not REFERENCES DNA synthesis and mitosis are fundamental components of the inductive process or AARONSON, S. A., TODARO, G. J., and SCOLNICK, E. whether cell proliferation simply amplifies M. (19711. Induction of murine C-type viruses a process already occurring below the from clonal lines of virus-free Balbi3T3 cells. Science 174, 157-159. threshold of detection remains to be seen. In this communication, we report a new ANDERSSON, J., MELCHERS, F., GALANOS, C., and LODERITZ, 0. (1973). The mitogenic effect of lipoC-type virus-inducing agent, namely PPD. polysaccharide on bone marrow-derived mouse It behaved similarly to LPS, in that it was lymphocytes. J. Exp. Med. 137, 943-953. inductive in cultures of BALB/c and nulnu GALANOS, C., RIETSCHEL, E. T., LODERITZ, O., WES’D cells and showed a synergistic effect when PHAL, O., KIM, Y. B., and WATSON, D. W. (19721. used with BrdU in BALB/c or nulnu culBiological activities of lipid A complexed to bovine tures (Table 2). C3H/HeJ cultures, resistserum albumin. Eur. J. Biochem. 31, 230-233. ant to LPS induction, could still be induced LIN, S. Y., and RIGGS, A. D. (19721. Lac operator by PPD. Antibody against reverse trananalogues: Bromodeoxyuridine substitution in the lac operator affects the rate of dissociation of the scriptase of Rascher leukemia virus inlac repressor. Proc. Nat. Acad. Sci. USA 69, 2574hibited the enzymes of viruses induced by 2576. both mitogens (Table 3). In preliminary LOWY, D. R., ROWE, W. P., TEICH, N., and HARTLEY, infectivity studies (in collaboration with J. W. (19711. Murine leukemia virus: High freDr. N. Teich), PPD as well as LPS induced quency activation in vitro by 5-iododeoxyuridine xenotropic viruses in BALB/c and AKR and 5-bromodeoxyuridine. Science 174, 155-156. cultures. Viruses induced by these two mi- L~~DERITZ, O., WESTPHAL, O., STAUB, A. M., and togens alone and in collaboration with NIKAIDO, H. (1971). Isolation and chemical and BrdU could not be distinguished yet. immunological characterization of bacterial lipoOther criteria will have to be used to see polysaccharides. In “Microbial Toxins” G. Weinbaum, S. Kadis, and S. J. Ajl, (eds.), Vol. 4, pp. whether the different inducers activate the 145-233. Academic Press, New York, 1971. same or different endogenous viral geMGLLER, G. (1974). Effect of B-cell mitogens on lymnomes. phocyte subpopulations possessing C’3 and Fc reThe mechanism by which a mitogen inceptors. J. Exp. Med. 139, 969-982. duces endogenous virus is still obscure. MORONI, C. (1972). Structural proteins of Rauscher The only induction system with a plausileukemia virus and Harvey sarcoma virus. Virolble explanation is the fibroblast-BrdU sysogy 47, 1-7. tem (Lowy et al., 1971; Aaronson et al., MORONI, C., SCHUMANN, G., ROBERT-GUROFF, M., 1971). Here, induction is known to depend SUTER, E., and MARTIN, D. (19751. Induction of on incorporation of BrdU into cellular endogenous C-type virus in spleen cell cultures treated with mitogens and 5-bromo-2’-deoxyuriDNA (Teich et al., 19731, which can alter dine. Proc. Nat. Acad. Sci. USA 72, 535-538. the binding of regulatory proteins to DNA (Lin and Riggs, 1972). Mitogens may have MORONI, C., and SCHUMANN, G. (1975). Lipopolysaccharide induces C-type virus in short term cula dual effect. They seem to induce in a still tures of Balb/c spleen cells. Nature (London) 254, unexplained way virus from cells entering 60-61. mitogen-induced DNA synthesis. In addi- SCHUMANN, G., SCHNEBLI, H. P., and DUKOR, P. tion, they may act by promoting incorpora(1973). Selective stimulation of mouse lymphocyte tion of BrdU into genes controlling endogpopulations by lectins. Znt. Arch. Allergy 45, 331enous viruses. 340. ACKNOWLEDGMENTS We thank Dr. R. C. Gallo for his gift of antibody to reverse transcriptase and Dr. Ch. Galanos for his gift of purified LPS Salmonella and lipid A.

SCHUMANN, G., and MORONI, C. (1976). Mitogen induction of murine C-type viruses. I. Analysis of lymphoid cell subpopulations. J. Zmmunol. 116, 1145-1150. SULTZER, B. M.,

and NILSSON,

B. S. (1972). PPD

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tuberculin-a B-cell mitogen. Nature New Biol. 240, 198-200. TEICH, N., LOWY, D. R., HARTLEY, J. W., and ROWE, W. P. (1973). Studies of the mechanism of induction of infectious murine leukemia virus from AKR mouse embryo cell lines by Fj-iododeoxyuridine and 5-bromodeoxyuridine. Virology 51, 163173. VISCHER, T. L., and JAQUET, C. (1972). Effect of

SCHUMANN antibodies against immunoglobulins and the theta antigen on the specific and non-specific stimulation of mouse spleen cells in vitro. Immunology 22, 259-266. WATSON, J., and RIBLET, R. (1974). Genetic control of responses to bacterial lipopolysaccharides in mice. I. Evidence for a single gene that influences mitogenic and immunogenic responses to lipopolysaccharides. J. Erp. Med. 140, 1147-1161.