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Non-productive chronic infection of human cells by a xenotropic murine type-C retrovirus
VOL.
135 E - -
No 1
JANVIER-MARS
1984
ANNALES VIROLOGIE
DE
NON-PRODUCTIVE OF BY
A
XENOTROPIC
CHRONIC
HUMAN MURINE
INFECTION
CELLS TYPE-C
RETROVIRUS
by F. Cavali&ri, F. Saal, L. D'Auriol, R. Emanoil-Ravicovitch and J. Pgri~s (*) U. 107 I N S E R M , LOI-CNRS, Ddparlrnent d' Oncologie Exp~rimentale, Inslitul de Recherches sur les Maladies du Sang, H6pilal Sainl-Louis, 75475 Paris C~dex 10
SUMMARY We report the establishment and characterization of a non-productive (NP) chronic infection of a h u m a n cell line (FL) by a xenotropic murine type-C retrovirus from M u s molossinus mouse (MoI-MuLV-X). The NP chronically infected cells (FL/Mol) do not show any virus particles detectable by electron microscopy, nor do they release any virus detectable either by reverse transcriptase activity assay or by inoculation of culture supernatants onto permissive cells. Most of the FL cells are shown to be infected, as demonstrated by specific imnlunofluorescence. The totality of provirus DNA genetic information is present, as demonstrated bv specific molecular hybridization, and infectious Mol-MuLV-X virions can be rescued from co-cultures ,~,ita permissive cells after fusion with polycthylene glycol. The integrated provirus DNA is transcribed, and both main species (24 S and 35 S) of viral R N A are synthesized. Antigenic determinants of both virus p30gag and gp70 e''v proteins are easily detected. M a n u s c r i t re~,'u le 24 m a i 1983, accept6 le 16 n o v e m b r e 1983. (-) To w h o m r e p r i n t r e q u e s t s s h o u l d be a d d r e s s e d .
4
F. CAVALII~RI AND COLL.
Polyacrylamide gel electrophoresis analvsis of i m n m n o p r e c i p i t a t e d cell lysates of NP FL/Mol cells (lid not show anv anomaly in the synthesis of the virus p30~"~ protein and its precursors. Anti-gp70 i m m u n e serum precipitated the 85 kd gp70 precursor and a gl)70 which migrated slightly faster t h a n that tound in productively infected h u m a n cell lines. I{EY-WORDS: 1-tetrovirus, Non-productive infection, Xenotropisln, lteplication; Type C, Mouse, H u m a n cells, DNA, I .NA, Proteins.
INTBODUCTION Molossinus xenotropic type-C retrovirus has been isolated from the Asian M u s rnolossinus mouse [21]. It is considered as being a xenotropic virus because of its ability to infect cells from several different non-murine species. Murine xenotropic type-C retrovirus (MuLV-X), although integrated in mouse chromosomal DNA, can productively infect cells only from species foreign to the host of origin [19]. The variety of sensitive hosts includes most m a m m a l s as well as man [24, 33]. This characteristic, along with the ubiquity of xenotropic ~'iruses in mice, was at the origin of major research efforts aimed at understanding t h e biological and pathological significance of such agents. While some data are currently available concerning t h e p u t a t i v e role(s) [20] played by MuLV-X in different life processes of their species of origin, little is known about t h e impact of the viruses on their sensitive hosts. The study of the mechanisms which permit and modulate MuLV-X virus replication in one given target cell should provide better u n d e r s t a n d i n g of these virus-host interactions. In this context, t h e establishment and characterization of the chronic non-productive (NP) infection of a h u m a n cell line by a Mol-MuLV-X described in this report offers an experimental model for the study of long-term M u L V - X - h u m a n cell association.
M A T E R I A L S AND M E T H O D S Cells and virus.
Several cell lines were employed: FL and AV3, both from human amniotic origin but showing HeLa-specific markers [7], and Cf2Th, from canine thymus [23]. cDNA EM FFU IF IP LTR MuLY-X
= = = = = = =
complementary DNA. electron microscopy. focus-forming unit. immunofluorescence. immunopreeipitation. long terminal repeat. murine xenotropic type-C virus.
retro-
i
NP PAGE PEG PTA RT RSV SDS
= = = = = = =
non-predu,:.tive. polyacrylamide gel e l e c t r o p h o r e s i s . polyethylene glycol. phosphotungstic acid. reser~,e t r a n s c r i p t a s e . Bous sarcoma virus. sodium dodecyl sulphate.
NON-PRODUCTIVE RETROVIRUS INFECTION
5
All three cell lines were obtained through the American Type Cellular Collection (Rockville, Maryland, USA). Sarcoma-positive leukaemia-negative (S+L-) mink cells [25] were kindly furnished by C. Todaro (National Cancer Institute, Bethesda, USA). All cell lines were maintained in McCoy's culture medium supplemented with 10% foetal calf serum (Flow Laboratories, Scotland). Xenotropic murine type-C retrovirus from M u s molossinus mouse (MolMuLV-X) was a kind gift from R. Callahan (National Cancer Institute, Bethesda, USA). It was produced by the chronically infected Cf2Th/Mol cell line and titred on S+L- mink cells (see ~cVirus assay ,,). Infection of human cell lines was carried out as follows: 10 ml of clarified, filtered (0.45 ~m Millipore) CI2Th/Mol supernatant fluids (105 FFU/ml) were inoculated onto 10e cells. After 1-h incubation at 370 C, the inocula were removed and fresh culture medium was added. Cultures were conserved in CO., incubator at 37 ~ C and split every 7 days. Cell cloning was performed in microplates as described elsewhere [9]. Virus assay.
The S+L - test on mink cells was carried out as described by Peebles [25]. Briefly, log~o virus dilutions were prepared in culture medium and inoculated onto S+L - mink cells, which had been incubated overnight in the presence of 1 ~g/ml polybrene. Focus-forming units (FFU) were estimated by counting the transformation loci under a light microscope 5-6 days post-infection. Titres were expressed in FFU/ml. Reverse transcriptase (RT) activity was tested in culture supernatants following the method described by Ross [31]. The clarified (10,000 g, 10 min) culture supernatants were ultracentrifuged (100,000 g, 90 rain) through a 20% glycerol cushion. Then the pellets were resuspended in Tris-HC1 50 mM pH 7.8 and disrupted by a final concentration of 0 1% NP40. The incorporation of tritiated thymidine monophosphate (from 4 mM of deoxytriphosphate nucleotide, 3H-TTP used as precursor, with specific activity 47 Ci/mM; Amersham) on polyriboadenosine as an exogenous template and oligodeoxythymidine as an exogenous primer (poly-A oligo dT(12-18)) was estimated by trichloroacet~c acid precipitation and nuclear scintillation counting. The presence of virus particles was studied by electron microcoscopy (EM) observation of cell monolayers and pelleted supernatant fluids. Cells were fixed with glutaraldehyde and osmium acid and double-stained with uranyl acetate and lead citrate as described by Hsiung el al. [13]. Pellets from supernatant fluids were stained with phosphotungstic acid (PTA). Infectious virus was also assayed by inoculation of the concentrated supernatant fluids onto canine permissive cells [32], and by direct co-cultivation of infected FL cells with the same highly permissive ceil line. Nucleic acid extraction.
The DNA of the infected cell cultures was extracted after 1 ~/o sodium dodecyl sulphate lysis followed by proteinase K (Merck, Darmstadt, RFA) digestion, phenol chloroform deproteinization and dialysis according to the method of GrossBellard el al. [10]. RNA was removed by alkaline hydrolysis (10 -~ M Na2C03, 37 ~ C, 24 h). DNA was then sonicated to 300-500 nucleotide', in length prior to liquid hybridization. Free provirus forms were extracted from acutely infected Cf2Th cells 16 h post-infection bv the method of Hirt [12]. Total cellular RNA was recovered by pelleting (16,000 g, 20 rain) the cell lysates obtained from the overnight incubation in 6 M urea 3 M lithium chloride of the mixer-blender disrupted cells. The pelleted RNA was phenol-chloroformextracted after proteinase K digestion and precipitated in cold ethanol. The polyadenylated RNA fraction was recovered by elutioa of the total RNA through an oligo-dT cellulose column at uifferent ionic strengths [1].
F. CAVALIERI AND COLL. }~estricLk.. e.~b,.,e ~cr was purcnase0 trom Boehringer Mannheim, France, and DNA digestion was carried out in the presence of k phage internal markers under conditions recommended by the manufacter.
Probe preparalion and molecular hybridizalion. Radiolabelled viral complementary DNA (eDNA) was prepared on a 70 S genomic template obtained from purified MoI-MuLV-X. The reaction was performed in the presence of avian myeloblastosis virus RT (obtained through 3. Beard, Resources and Logistics, National Cancer Institute, Bethesda, USA) as described by Taylor el al. [35]. For Cot and Crt kinetics, the cDNA was synthesized wiLh aI:J-TT]J (50 Ci/mM; Amersham). 3~P-dCTP (25,000 Ci/mM NEN) was used to obtain radiotabelled cDNA for Southern blots. A liquid hybridization reaction were performed in a final mixture volume of 20 tzl containing 10 mM Tris-HC1 (pH 7.4) 750 mM NaCI 2 mM EDTA 0.02% SDS. Hybridization was initiated by heating the reaction mixtures at 98 ~ C for 10 nain, cooling on ice and incubating at 68 ~ C. Hybrids were tested by S: nuclease resistance [17].
Blo[ h qbridizalion. Nucleic acid electrophoresis was carried out on 0.7% agarose bed gels for DNA and 1% agarose bed gels for 1RNA. Nitrocellulose paper transfer and blot hybridization were performed according to a modification of the technique of Southern published by Thomas [37] in the presence of 5xSCC, 50% formamide at 42 ~ C.
31elabolic labelling and immunoprecipilalion (IP). IP was carried out according to Murphy el at. [22]. Briefly, radiolabelled cell cultures were pelleted, washed in a balanced salt solution and lysed in a buffer containing 1% Triton X100, 0.5% sodium deoxychlolate, 0.1% sodium dodecyl sulphate (SDS), 0.2 M sodium chloride, 0.001 M EDTA, 0.05 Tris HC1 pH 7.5. The cell lysates were cleared by overnight incubation at 4 ~ C in the presence of goat normal serum followed by protein A precipitation. The precleared supernatants were adjusted to 1M NaC1 and immunoprecipitated by specific g o a t antisera which are directed against virus gag protein precursor Pr 65 (code n ~ 76S-454) and against the viral envelope glycoprotein gpT0 (code n ~ 6 S-0431) obtained from the National Cancer Institute, Bethesda, USA. Immune complexes were recovered by centrifugation, dissociated in adequate buffer according to Laemmli [15] and resolved by electrophoresis on polyacrylamide gel gradients.
Immunofluorescence. Indirect immunofluorescence (IF) tests for detecting virus p30 protein antigens were performed according to Hilgers et al. [11], using specific anti-murine p30 goat antisera (catalogue n ~ 5 S-123) obtained from the National Cancer Institute, Bethesda, USA). RESULTS
Establishment of ihe N P
chronically infected F L / M o l cell line.
In t h e c o n t e x t of a s y s t e m a t i c s t u d y on t h e susceptibility of h u m a n cell lines to several different M u L V - X viruses, F L cells and AV3 cells were s i m u l t a n e o u s l y infected w i t h Mol-MuLV-X inocula a t a m u l t i p l i c i t y
NON-PRODUCTIVE RETROVIRUS INFECTION
7
of infection of about 1 F F U per cell. From t h e time of infection, the cell cultures were split once a week. Virus production was evaluated by the techniques mentioned in r Materials and Methods ,. Virus antigen expression was monitored by IF with anti-murine p30 virus core protein antiserum. During the first six m o n t h s post-infection, these tests were performed once every two weeks; later t h e y were carried out once every two m o n t h s o v e r one year. The results of these tests were stable and are summarized in table I. No R T activity was detected in FL/Mol culture s u p e r n a t a n t fluids over two years. No infectious viral particles detectable by the S + L - assay of culture s u p e r n a t a n t fluids were released by FL/Mol cells. Furthermore, no virus infectivity was detected in concentrated supern a t a n t fluids inoculated onto permissive cells. EM observation of cellular monolayers and of s u p e r n a t a n t pellets always gave negativc results concerning t h e presence of virus particles. Moreover, the induction of FL/Mol cells by t r e a t m e n t with halogenated pyrimidines (IdUr 40 txg/ml for 24 h) or with halogenated pyrimidines and dexamethasone (IdUr 40 ~g/ml; dexamethasone 10 -" M, 24 h) gave negative results by R T assay and EM observation (data not shown). On the contratry, AV3/Mol culture s u p e r n a t a n t fluids contained both RT activity and 10' to 106 F F U / m l . EM showed numerous particles in cells and s u p e r n a t a n t fluids. Nevertheless, viral p30 antigenic determinants were easily detected by IF in both cell lines, therefore suggesting t h a t the viral genome w a s at least partially present and active in FL/Mol cells despite the absence of virus TABLE I.
-
Comparison of the viral state in PL/Mol
-
and A V 3 / M o l cells. AV3/Mol
FL/Mol Time after infection
15 days
45 clays
52 103 46
196 105 204
379 105 297
EM(,)
(+)
(+--)
(++)
I F (5)
30
60
Viral detection
R T a s s a y (1) S+L - test (2) I n o c u l a t i o n of supern a t a n t s on permissive cells (s)
3 months
60
1 year
15 days
45 days
3 months
201 104 ND
2 0 1.4
3 0 2.2
1.8 0 2.1
(+)
(-)
(-)
(--)
ND
50
50
80
1 year
2.6 0 ND
(-) ND
(t) Values r e p r e s e n t the average of results from triplicate c u l t u r e s u p e r n a t a n t s . T h e y are expressed as o p m • 103 of aH-TMP i n c o r p o r a t e d into p o l y - d T p r o d u c t in 60 min. (2) Values represent the average titre from triplicate culture s u p e r n a t a u t s expressed as F F U / m l . (a) Permissive dog-cell m o n o l a y e r s were inoculated w i t h 10 ml of filtered (0.45 ~m Miilipore) F L / M o l or A V 3 / M o l s u p e r n a t a n t s . Cultures were considered as being positive when R T activity of more t h a n 2 • 104 c p m was detected three m o n t h s after inoculation. (4) (;ells anti s u p e r n a t a n t s were studied as described in ,, Materials anti Methods ,,. ( + ) = some t y p e - C particles; ( + + ) = n u m e r o u s type-C particles ; ( - - ) = absence of particles. (s) Percentage of specifically fluorescent cells was established b y c o u n t i n g positive ceils from at least 300 ceils. N D = N o t done.
8
F. CAVALII~RI AND COLL.
production. Moreover, tile high percentage (80~ see table I) of IF-positive FL/Mol cells indicaLes that a large majority of the cells were infected. Characterizalion of lhe viral nucleic acids present in F L / M o l cells. Liquid-phase molecular hybridization experiments were performed between MoI-MuLV-X virus-specific radiolabelled eDNA and cellular DNA from both FL/Mol and AV3/Mol cells. Figure 1 shows the typical aspect of the Cot kinetics obtained in these experiments. These results show t h a t the totality of viral genetic information is present as proviral DNA in FL/Mol cells. The Cot 1/2 ratio indicates t h a t AV3/Mol contains roughly twice as many proviral genome copies as FL/Mol. In order to ascertain that proviral DNA is eovalently linked to cellular DNA in FL/Mol cells, the total cellular high inoleeular weight DNA was digested with EcoBI restriction enzyme, migrated in a 0.7% agarose gel and analysed by Southern blot hybridization with specific Mol-MuLV-X eDNA. EcoRI was
~AV/
/
J
.
MOI_
. .o {'LIMOL
/ tO
,i
O N
/,'"
,I
~5 -T
4 (.2 12.
........ 1 I . ..... .....
.......... 11)~
10 :~
cot (molex secllitcr) Fro.
Liquid phase molecular hgbridizalion between virus specific triliated 3H-eDNA and high molecular weighl celhtlar D N A , lesled by S 1 nuclease resislance.
1. - -
9 = A V J M o l D N A ; 9 = F L / M o l D N A ; 9 = u n i n f e c t e d AV~ D N A ; o = u n i n f e c t e d C o t 1 / 2 v a l u e s a r e 4.5 x 10 2 f o r F I / M o l a n d 1.9 • 10 2 f o r A ' ~ / M o l .
FL
DNA.
NON-PRODUCTIVE RETROVIRUS INFECTION
9 <
9
9
9
,_
ti
.
Ill .
:"';~ "
-6.0
,
I
Fro. 2. - -
I :
-2.9
Nitrocellulose paper blot hybridisation between oirus-specific a~P-cDNA and cellular D N A .
Free proviral DNA intermediate from the Hirt s u p e r n a t a n t fraction of Mol-MuLV-X acutely infected dog cells (lane 1). EcoR[ digests of high molecular weight DNA from: Cf2Th/Mol cells (lane 2), AVJ.Mol cells (lane 3) and FL/Mol cells (lane 4). No hybridization bands were detectable in uninfected negative controls.
chosen because it cuts only once into the provirus of MoI-MuLV-X [5]. Figure 2 shows t h a t the specific virus eDNA probe recognizes fragments of a molecular weight higher than that of free proviral forms, as expected in the case. of integrated provirus. The results of liquid-phase hybridization between FL/Mol cytoplasmic RNA and Mol-MuLV-X cDNA are shown in figure 3. Crt kinetics demonstrate, within the limits of tile technique, that the proviral DNA present in NP FL/Mol cells is transcribed into virus-specific FINA. The Crt 1/2 ratio shows t h a t virus RNA expression is about 10 times lower in FL/Mol than in AV3/Mol cells. The largest families of viral RNA transcripts were analysed by Northern blot hybridization (fig. 4). Both 24 S and 35 S transcripts are present in FL/Mol cells. Moreover, both transcripts are recovered only in poly-A-enriched fractions of cellular RNA, which suggests t h a t they may be able to serve as mRNA. When several clones derived from the original FL/Mol population (see below) were analysed, the results obtained concerning RNA species were
10
F. CAVALII~RI AND COLL.
..o ......... ~ AV IMOL
lOtl
.,.'
f
2 /2 o,.
~FLJ MOL
/ .,:""
/~
-t 40
~ .~176
g.
~176
..-" 2o
FL ~0.e
10 3
i04
Crt (mole x ser,liler) Fla.
3.
~
Liquid phase molecular hybridization belween virus-specific tritiated aH-cDNA and total cellular ICNA, tested by S~-nucleasc resislance.
9 == A V a / M o l F I N A ; o = F L / M o l t : I N A ! V -~ u n i n f e c t e d A V 3 o r F L Y I N A . Crt 1 / 2 v a l u e s are 2 • 10 s Io:' F L / M o l a n d 2 • 10 ~ f o r AV.~/Mol.
strictly similar to those obtained in the total population (results not shown).
Evaluation of the infectious potential of FL/MoI virus information. Our results showed that the totality of proviral D N A information is present in FL/Mol cells, and is apparently fully and normally transcribed into virus-specific 24 S and 35 S poly-A RNA. Nevertheless, no virions are detectable in FL/MoI cultures by any of the very sensitive methods employed in virus assays. However, the hybridization techniques employed do not allow the detection of mutations or small deletions in the virus nucleic acids, while such events could lead to virus defectiveness. The infectious potential of FL/Mol virus information was therefore tested by co-culture of FL/Mol cells with permissive dog cells in the presence
NON-PRODUCTIVE RETBOVIRUS INFECTION
11
of a fusing agent (PEG 1550). The co-culture s u p e r n a t a n t fluids always contained both detectable R T activity and infectious virions, as shown by the S+L - test. Moreover, ten out of ten one-cell-derived FL/Mol sub-, clones which were also totally negative in terms of virus production gave rise to productive infection when co-cultivated and fused with the permissive dog cell line. The rescued infectious v i r u s presented t h e same host range and antigenic determinants as Mol-MuLV-X (data not shown). Since FL/Mol clones were picked at random, this result suggests t h a t at least a large majority of the FL/Mol cells contain functional virus information. :
A::
9 /[
C
"
i
.
D
,
FIG, 4. - - Nitrocellulose paper blot hybridization between virus-speeifie 3~P-eDNA and eellutar R N A .
Poly-A-enriehed lqNA: FL/Mol cells (lane A) and AV#Mol cells (lane B). Poly-A-negative RNA: FL/Mol cells (lane C) and AVz/Mol ceils (lane D). Arrows indieate the positions of 28 S and 18 S ribosomal RNA.
Characterization of the virus-coded polgpeptides present in FL/Mol cells. The total absence of m a t u r e virions from FL/Mol cells in spite of the presence of apparently normal B N A and the positive IF results led us to s t u d y t h e major translation products in the NP cell line as compared to producer cells. Steady state (I-h) labelling of the cell cultures was followed by IP and P A G E analysis. No qualitative difference was remarked
a
b
180-
30FzG. 5. - - PAGE analysis on gradienl ( 8 % - 1 6 % ) gel o/ anti-Pr65gag immunoprecipitales from aH-leueine and 8It-valine sleady slate (1 h) labelled lysates. FL/Mol cells (lane a) and A~89
A
cells (laue b). Molecular weights are expressed in Kd,
B
C
D < 85 41 70
FIG. 6. - - P A G E analysis on gradient ( 8 % - 1 6 % ) gel of anli-gpTO immunoprecipitales from 3H-leucine and 8H-z~aline cellular lysales. FL/Mol cell lysate labelled tar 1 h (lane A) and chased for 16 h (lane C). A Y J M o l cell lysate labelled for 1 h (lane B) and chased for 16 h (lan e D). Molecular weights are expressed in Kd.
NON-PRODUCTIVE FIETROVIRUS INFECTION
13
between AV3/Mol and FL/Mol cell lysates immunoprecipitated with antigag serum. Prl80gag-p ~ Pr65gag and mature p30 were identified by this method in both cell lines, even though the intensity of the FL/Mol gag bands was constantly weaker than that of the AV3/Mol gag bands (fig. 5). Steady-state labelling followed by IP with anti-gp70 serum allowed the detection of an apparently identical Pr85 env in both cell lines, while the detection of mature gp70 env was possible only after pulse- and chaselabelling (fig. 6). The same experiments carried out after 3H-glucosamine labelling indicated t h a t both the Pr85 ~ and the gp70 ~nv are actually glycosylated in both cell lines.
DISCUSSION We report the establishment and characterization of NP chronic infection of a human cell line by a murine xenotropic type-C retrovirus: Mol-MuLV-X. The NP-chronically infected cell line FL/Mol contains the totality of virus genetic information in the form of proviral DNA covalently linked to the cellular genomic DNA. This is shown by liquid-phase hybridization of total cellular DNA and blot hybridization of EcoRI-digested cellular DNA with virus cDNA. The integration pattern of Mol-MuLV-X in NP FL/Mol cells seems to differ markedly from the integration pattern in the producer AV3/Mol cell line. Since no integration-site specificity has up to now been demonstrated for type-C retroviruses, finer analysis of the cellular provirus junction sequences is needed before considering this fact to be related to t h e NP state. Virus DNA is transcribed in FL/Mol cells, and 24 S and 35 S virus RNA transcripts are indistinguishable, by Northern blot hybridization, from those found in normally productive cell lines. Analysis of the major intracellular virus proteins, carried out by specific immunoprecipitation and P A G E shows that the gag gene products synthesized by FL/~Iol cells co-migrate with those synthesized by AV~/Mol cells. A glycosylated Pr85 ..v is produced and processed to yield a gp70 e"v in both cell lines. The gp70 ".v present in FL/Mol cells was found to constantly migrate slightly faster than its counterpart in AV~/Mol. Preliminary quantitative studies carried out by radioimmunoassay (S, Gisselbrecht, personal communication) showed that the amount of virus proteins per cells is about two times higher in FL/Mol than in AV~/Mol. Since our IP experiment,, showed that the virus protein band intensity of FL/Mol lysates was constantly weaker than or equal to the virus protein band intensity of equivalent AV~/Mol lysates, these date indicate that the lack of virion production might lead to the accumulation of virus proteins in FL/Mol cells. Infectious virus presenting the same antigenic and host-range characteristics as the input Mol-MuLV-X is easily rescued from cloned subpopulations o f FL/Mol cells co-cultivated with permissive dog cells in
14
F. CAVALII~RI AND COLL.
tile presence of a fusing agent These results suggest either that the FL/Mol cells which contain non-defective provirus have a cloning advantage over the rest of the population or that a large majority, if not the totality, of the FL/Mol cells contain biologically intact virus information regardless of the NP state. The fact t h a t 80% of FL/Mol cells were positive in murine type-C-virus-specific IF is strongly in favour of the second possibility. Both biological and biochemical evidence strongly suggest t h a t the restriction is due to the cellular environme:,t alone. Viral RNA synthesis is at least partially ruled by the cellular machinery, and the 35 S genomic BNA has been shown to form a pool separated from t h a t of the 35 S RNA responsible for gag protein synthesis [18]. A host-cell-dependent shift in equilibrium between the genomic and the messenger 35 S viral BNA pool could seriously hamper infectious virion production. Interestingly, it should be kept in mind that the level of BNA expression is 10 times lower in FL/Mol than in AY~/Mol. Alternatively, even slight alterations in the viral gp70 could affect its membrane-binding ability and therefore seriously compromise the budding process, or some undefined properties of the cell membrane itself could impair some step(s) of virion assembly. To our knowledge, this is the first report of a chronic non-productive infection of a non-lymphocytic mammalian cell line by a non-defective and non-transforming murine retrovirus. Beeently, Ceecherini-Nelli el al. [4], showed t h a t the expression of integrated primate type-C virus proviral DNA is restricted in non-productively infected human B lymphoblasts. The restriction mechanism appears to be quite different from those already known. Tl,is restriction mechanism seems to be selective, since FL is able to be productively infected by several other xenotropic type-C viruses of non-murine origin [33]. In fact, proviral DNA integration, transcription and translation do occur in FL/Mol cells, while this is not the case either under Fv-1 restriction, which appears to occur before the integration event itself [14], or in the case of embryonal teratoeareinoma cells, where the proviral DNA is integrated [34, 8] but not expressed [26, 6]. Mammalian cells are also known to be non-productively infected by avian sarcoma retroviruses. Virus DNA of Bous sarcoma virus (RSV) can be integrated with low efficiency only after infection with certain RSV stocks [39]. The frequency of transformation is low, and virus is hardly ever produced, even though it can often be rescued by fusion with chick cells [3, 38]. Nevertheless, when comparing restriction of mammalian cells for replication of RSV with the NP FL/Mol cell line, several import a n t differences do appear: RSV is a transforming virus, while Mol-MuLV-X is not; the processing of R S u RNA largely favours 21 S m B N A over 38 S m R N A [29], while in FL/Mol cells, 24 S m R N A and 35 S m R N A are present at about the same level; finally, R S \ r polyproteins are synthesized but not cleaved, while Mol-MuLV-X p30 and pg70 are synthesized in FL/Mol ceils. Murine cells chronically infected by eeotropie MuLV and human cells
NON-PRODUCTIVE RETROVIRUS INFECTION
15
infected by several xenotropic retroviruses are known to switch off virus production when treated with interferon [2, 27 I. Interferon is apparently not implicated in the non-productiveness of FL/Mol cells for two reasons: 1) no interferon activity is detectable in FL/Mol culture supernatants (Rhodes-Feuillette and P~ri~s, unpublished results); 2) the inhibition of retroviral production by interferon affects virion release. Chronically infected treated cells show large clusters of i m m a t u r e virus particles associated with the cell membranes, which is not the case in FL/Mol e v e n after extensive EM observation. The establishment of chronic semi-silent infection of h u m a n cells in vitro raises the question of the possible extension of these findings in vivo. It has been reported t h a t the expression of murine retroviral-like sequences can be detected in various types of h u m a n leukaemia cells [16]. Moreover, the recent isolation of a non-germ-line-transmitted retrovirus from h u m a n leukaemia cells [28, 30], seems to reopen the debate on the possible implication of exogenous retroviruses in some h u m a n lymphoproliferative diseases. Non-defective murine leukaemia viruses do not carry any known transforming gene; nonetheless, they can induce leukaemia, albeit with low efficiency and after long incubation periods, possibly by promoting the transcription of cellular genes via their LT13 {long terminal repeat) [36]. If our findings are extended to an in vivo system, one cannot rule out the possibility t h a t an intact b u t d o r m a n t xenotropic retroviral genome would act in the same way.
INFECTION
CHRONIQUE ET NON PRODUCTIVE DE CELLULES PAR UN RETROVIRUS XI~NOTROPIQUE DE TYPE C
HUMAINES
Nous d6crivons l'~tablissement et la caract6risation d'une lign6e cellulaire humaine (FL/Mol) non productive (NP), infect6e chroniquement par un virus x6notropique murin de t y p e C originaire de la souris M u s molossinus (MoI-Mu-MLV-X). Les cellules chroniquement infect6es ne contiennent aucune particule virale visualisable par microscopie electronique, et elles ne lib~rent aucun virion d6tectable par 6valuation de l'activit6 r6verse ~ranscriptase ou par inoculation des surnageants de culture /1 des cellules permissives. Par immunofluorescence antivirale sp~cifique, no,ts montrons que la majorit6 des cellules sont infeet6es. De plus, la totalit6 du g~nome viral est pr6sente sous forme d'ADN proviral d6tectable par hybridation mol6culaire, et la coculture avec des cellules permissives en presence d'un agent fusionnant (le poly6thyl~ne-glycol) donne lieu ~ la production de virions infectieux. L ' A D N proviral int6gr6 est transcrit et les deux familles d ' A R N viral (24 S e t 35 S) sont synth6tis6es. Les d~terminants antigeniques aussi bien de la prot6ine virale p30*ag que de la glycoprot6ine virale gp70 e''v
16
F. CAVALlt~III AND COLL.
sont I a c n e m e n t nus en Ovidence. L ' a n a l y s e sur gel de polyaerTlamide des lysats des eellules F L / M o l immunoprdeipites n'a permis de m e t t r e en 0videnee aueune anomalie de la svnthbse de la prot6ine virale p30."g ni de ses prdeurseurs. L ' i m m u n s e r m n anti-gp70, par contre, reconnait le l)rceurseur gpr85 el une gp70 ..... qui migre 1O~Orement plus r i t e que la ..7(b,,,,, que ]'on t r o u v e dans d'autres eellules humaines infeet6es et produetriees. 3I(~TS-(:LL.'S : R f t r o v i r u s , lnfeclion non p r o d u c t i v e , X 6 n o t r o p i s m e , B0plication ; T y p e C, Souris, Cellules hunaaines, A D N , A B N , Prot~ines. ACKNOWIA'2DGEMENTS
\\re thank J. Lasneret for electron microscopy, S. Gisselbrecht for performing the radioimmunoassav, Y. Poirot-Lossfelt and M. Garcette for excellent technical assistance and M. C. "Pceuf and M. Pasquet for typing this paper. Partial support for these studies was provided by INSERM (Institut National de la Santa el de la Recherche M6dicale) grant 7841421 and by DGRST (D616gation "9 Gdndrale ~ la Recherche Scientifique et Technique) grants A.650 1926 and 81L0z,8.
REFERENCES 11] Avlv, H. & LEDEL, P., Purification of a biochemicallv active globin mRNA by chromatography on oligothymidylic acid-cellulose. Proe. hal. Acad. ,qci. (Wash.), 1972. 69, 1408-1412. [2] BILLAU, A., I-]EREMANS, H., ALLEN, P. T., DE ~{AEYER-GUIGNARD, J. & DE SOMER, P., Trapping of oncornavirus particles at the surface of interferon-treated cells. Virology, 1976, 73, 537-542. [3] BOETTIGER, D., Virogenic non-transformed cells isolated following infection of normal rat kidney cells with B77 strain Rous sarcoma virus. Cell, 1974, 3, 71-76. [4] CECCHEm~-NELLI,L., DALLA-FAvEnA, R., Mm~KHAM,P. D., RUSCETTI,F. W., \VONc--STAAL, F., GALLO, R. C. & I:{EITZ,M. S., Restricted expression of integrated primate tvpe-C virus (Gibbon ape leukemia virus-Simian sarcoma virus) proviral DNA in non-productively infected human B lvmphoblasts. Virology, 1982, 117, 195-206. [5] CHAT~rOPADHYAY,S. K., LANDER, M. R., GUPTA, S., RANDS,E. & LowY, D. R., Origin of nfink cytopathic focus-forming (MCF) viruses: comparison with ecotropic and xenotropic murine leukemia virus genomes. Virology, 1981, 113, 465-483. [6] D'AuRIOL, L., YANG, \V. K., TOBALY,J., CAVALIERI,F., PEmi~S, J. & EMANOIL-RAVICOVITCH, R., Studies on the restriction of ecotropic murine retrovirus replication in mouse teratocarcinoma cells. J. 9en. Virol., 1981, 55, 117-129_. [7] GAIITLER, S. M., Apparent HeLa cell contamination of human heteroploid cell lines. Nature (Lond.), 1968, 217, 750-771. [8] GAUTSCH, d. \V. & WILSON, M. C., Delayed de nooo methylation in teratocarcino,na suggests additional tissue-specific mechanisms for controlling gene expression. Nature (Lond.), 1983, 301, 32-37. 19] GOLDSBY, R. A. & ZH SEn, E., The isolation and replica plating of mammalian cell clones. Exp. Cell Res., 1969, 54, 271-275.
NON-PRODUCTIVE R E T R O V I R U S INFECTION
17
[10] GROsS-BELLARD,M., 0UDET, P. & CHAMBON, P., Isolation of high molecular weight DNA from mammalian cells. Europ. J. Biochem., 1973, 36, 32-38. [11] HILGERS, J., NOWKINSKI,R. C., GEERING, G. • MORDY, W., Detection of avian and mammalian oncogenic RNA viruses by immunofluorescence. Cancer Res., 1972, 32, 98-106. [12] HINT, B., Selective extraction of polyoma DNA from infected mouse cell cultures. J. real. Biol., 1967, 26, 365-369. [13] HSIUNG, G. D., FANG, C. K. Y. & AUGUST, IV[. J., The use of electron microscopy diagnosis of virus infectious. An overview. Progr. reed. Viral., 1979, 25, 133-159. [14] JOLIC~UR, P. & RASSART, E., Effect of the Fv-1 gene product on synthesis of linear supercoiled viral DNA in cells infected with murine leukemia virus. J. Viral., 1980, 33, 183-195. [15] LAEMMLI,U. K., Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature (Land.), 1970, 277, 680-685. [16] LAnSEN, C. J., IV[ARTY, M., HAMELIN, 1:{., PI~RII~IS, J., BOIRON, M. & TAVITIAN, A., Search for nuclei and sequences complementary to a murine oncornaviral genome in poly(A)-rich RNA of human leukemic cells. Proc. nat. Acad. Sci. (Wash.), 1975, 72, 4900-4904. [17] LEONG, J. A., GARAPIN, A. C., JACKSON, N., FANSHIER, L., LEVINSON, W. & BIsHoP, J. M., Virus-specific ribonucleic acid in cells producing Rous sarcoma virus: detection and characterization. J. Viral., 1972, 9, 891-902. [18] LEVIN, J. G. & I:~OSENAK,i~r J., Synthesis of murine leukemia virus proteins associated with virions assembled in actinomycin-D-treated cells: evidence for persistence of viral messenger RNA. Proc. nat. Acad. Sci. (Wash.), 1976, 73, 1154-1158. [19] LEVY, J. A., Xenotropic viruses: murine leukemia viruses associated with N I H Swiss, NZB and other mouse straipr Science, 1973, 182, 1151-1153. [20] LEVY, J. A., Xenotropic type C viruses, in (( Current topics in microbiology and immunology ,, 79 (p. 111-213). Springer Verlag, Berlin, Heildelberg, 1978. [21] LIEBER, M., SHERR, C. J., POTTER, iV[. & TODARO, G., Isolation of type C viruses from the asian feral mouse M u s musculus molossinus. Int. J. Cancer, 1975, 15, 211-220. [22] MuapnY, E. C., CAMPOS, D. & ARLINGHAUS, R. B., Cell-free synthesis of Rauscher murine leukemia virus gag and <( env )) gene products from separate cellular mRNA species. Virology, 1979, 93, 293-302. [23] NELSON-REss, W. A., OWENS, R. B., ARNSTEIN, B. & KNIAZEFF, J., Source, alterations, characteristics and use of a new dog cell line (Cf2Th). In vitro, 1976, 12, 665-669. [24] 0IE, H. K., RUSSELL, E. K., DOTSON, J. H., RHODES, J. M. & GAZDAR, A. F., Host-range properties of murine xenotropic and ecotropic type-C viruses. J. nat. Cancer Inst., 1976, 56, 423-426. [25] PEEBLES, P. T., An in vitro focus-induction assay for xenotropic murine leukemia virus, feline leukemia virus C, and the feline-primate viruses RD-114/CCC/M-7. Virology, 1975, 67, 288-291. [26] PERILS, J., ALVES-CAaDOSO, E., CANIVET, M., DEBONS-GUILLEMIN, M. C. ~:: LASNERET, J., Lack of multiplication of ecotropic murine C-type viruses in mouse teratocarcinoma primitive cells. J. nat. Cancer Inst., 1977, 59, 463-465. [27] P~nlEs, J., CANIVET, ~'W[.,RHODES-FEUILLETTE,A. c~ TODARO, G. J., Effect of interferon on chronic infection of human cells by xenotropic type C viruses, lnt. J. Cancer, 1979, 23, 798-802. [28] POIESZ, B. J., lqUSaETTI, F. W., GAZDAR, A. F., BUNN, P. A., MINNA, J. D. & GALLO, R. C., Detection and isolation of type C retrovirus particles from fresh and cultured lymphocytes of a pati,nt with cutaneous T-cell lymphoma. Proc. nat. Aead. Sci. (Wash.), 1980, 77, 7415-7419. Ann. Viral. (Inst. Pasteur), 135 E, n~ 1, 19~4
18
F. CAVALIERI AND COLL.
[29] QUINTI~ELL, N., HUGHES, S. H., VARMUS, H. E. & BISHOP, J. M., Structure of viral DNA and RNA in malnmalian cells infected with avian sarcoma virus. J. tool. Biol., 1980, 143, 363-393. [30] REITZ, M. S., POIESZ, B. J., RvsaE'rXl, F. W. & Gm.LO, R. C., Characterization and distribution of nucleic acid sequences of a novel type C retrovirus isolated from neoplastic human T lylnphocytes. Proc. nat. Acad. Sci. (Wash.), 1981, 78, 1887-1891. [31] Ross, J., SCOLXlCK, E. M., AAI~ONSON, A. & TODal~O, G. J., Separation of murine cellular and murine leukemia virus DNA polymerases. Nal~lre New Biol. (Lond.), 1971, 231, 163-169. 132] SAAL, F., CA.NIVET, M., SAMSO,A. & PI~RII~S, J., Replication of mouse sarcoma virus (Moloney) and its helper in a human heteroploid cell line of malignant origin. Path. Biol., 1977, 25, 705-709. [33] SA/~L, F., ROSETO, A., TOBALY, J. & PI-~RII-.S, J., I~tude de la susceptibilite des ccllules humaines cultivees in vitro ~ l'infection par des retrovirus xGnotropiques de type-C. C. B. Acad. Sci. (Paris) (S~r. D), 1981, 292, 12031206. [34] STE~VAI/T, C. L., STUItLMANN, H., JAHNER, D. & JAENISCH, R., De novo methylation expression and infectivity of retroviral genomes introduced into embrvonal carcinoma cells. Proc. nat. Acad. Sci. (Wash.), 1982, 79, 4098-4"102. [35] TA~'LOR, J. M., ILLMENSEE, R. & SUMMERS, J., Efficient transcription of RNA into DNA by avian sarcoma virus polymerase. Biochim. biophys. Acta (Amst.), 1976, 442, 324-330. [36] TEMIX, H. M., Function of the retrovirus long terminal repeat. Cell, 1982, 28, 3-5. [37] THOMAS, P., Hybridization of denatured BNA and small DNA fragments transferred to nitrocellulose. Proc. nat. Acad. Sci. (Wash.), 1980, 77, 5201-5205. [38] TUREK, L. P. & OPPERMANN,H., Spontaneous conversion of non-transformed avian-sarcoma-virus-infected rat cells to the transformed phenotype. J. Virol., 1980, 35, 466-478. [39] VAttMUS, H. E., VOGT, P. K. & BISHOP, J. M., Integration of deoxyribonucleic acid specific for Rous sarcoma virus after infection of permissive and non-permissive hosts. Proc. nat. Acad. Sci. (Wash.), 1973, 70, 30673071.
135 E - -
No 1
JANVIER-MARS
1984
ANNALES VIROLOGIE
DE
NON-PRODUCTIVE OF BY
A
XENOTROPIC
CHRONIC
HUMAN MURINE
INFECTION
CELLS TYPE-C
RETROVIRUS
by F. Cavali&ri, F. Saal, L. D'Auriol, R. Emanoil-Ravicovitch and J. Pgri~s (*) U. 107 I N S E R M , LOI-CNRS, Ddparlrnent d' Oncologie Exp~rimentale, Inslitul de Recherches sur les Maladies du Sang, H6pilal Sainl-Louis, 75475 Paris C~dex 10
SUMMARY We report the establishment and characterization of a non-productive (NP) chronic infection of a h u m a n cell line (FL) by a xenotropic murine type-C retrovirus from M u s molossinus mouse (MoI-MuLV-X). The NP chronically infected cells (FL/Mol) do not show any virus particles detectable by electron microscopy, nor do they release any virus detectable either by reverse transcriptase activity assay or by inoculation of culture supernatants onto permissive cells. Most of the FL cells are shown to be infected, as demonstrated by specific imnlunofluorescence. The totality of provirus DNA genetic information is present, as demonstrated bv specific molecular hybridization, and infectious Mol-MuLV-X virions can be rescued from co-cultures ,~,ita permissive cells after fusion with polycthylene glycol. The integrated provirus DNA is transcribed, and both main species (24 S and 35 S) of viral R N A are synthesized. Antigenic determinants of both virus p30gag and gp70 e''v proteins are easily detected. M a n u s c r i t re~,'u le 24 m a i 1983, accept6 le 16 n o v e m b r e 1983. (-) To w h o m r e p r i n t r e q u e s t s s h o u l d be a d d r e s s e d .
4
F. CAVALII~RI AND COLL.
Polyacrylamide gel electrophoresis analvsis of i m n m n o p r e c i p i t a t e d cell lysates of NP FL/Mol cells (lid not show anv anomaly in the synthesis of the virus p30~"~ protein and its precursors. Anti-gp70 i m m u n e serum precipitated the 85 kd gp70 precursor and a gl)70 which migrated slightly faster t h a n that tound in productively infected h u m a n cell lines. I{EY-WORDS: 1-tetrovirus, Non-productive infection, Xenotropisln, lteplication; Type C, Mouse, H u m a n cells, DNA, I .NA, Proteins.
INTBODUCTION Molossinus xenotropic type-C retrovirus has been isolated from the Asian M u s rnolossinus mouse [21]. It is considered as being a xenotropic virus because of its ability to infect cells from several different non-murine species. Murine xenotropic type-C retrovirus (MuLV-X), although integrated in mouse chromosomal DNA, can productively infect cells only from species foreign to the host of origin [19]. The variety of sensitive hosts includes most m a m m a l s as well as man [24, 33]. This characteristic, along with the ubiquity of xenotropic ~'iruses in mice, was at the origin of major research efforts aimed at understanding t h e biological and pathological significance of such agents. While some data are currently available concerning t h e p u t a t i v e role(s) [20] played by MuLV-X in different life processes of their species of origin, little is known about t h e impact of the viruses on their sensitive hosts. The study of the mechanisms which permit and modulate MuLV-X virus replication in one given target cell should provide better u n d e r s t a n d i n g of these virus-host interactions. In this context, t h e establishment and characterization of the chronic non-productive (NP) infection of a h u m a n cell line by a Mol-MuLV-X described in this report offers an experimental model for the study of long-term M u L V - X - h u m a n cell association.
M A T E R I A L S AND M E T H O D S Cells and virus.
Several cell lines were employed: FL and AV3, both from human amniotic origin but showing HeLa-specific markers [7], and Cf2Th, from canine thymus [23]. cDNA EM FFU IF IP LTR MuLY-X
= = = = = = =
complementary DNA. electron microscopy. focus-forming unit. immunofluorescence. immunopreeipitation. long terminal repeat. murine xenotropic type-C virus.
retro-
i
NP PAGE PEG PTA RT RSV SDS
= = = = = = =
non-predu,:.tive. polyacrylamide gel e l e c t r o p h o r e s i s . polyethylene glycol. phosphotungstic acid. reser~,e t r a n s c r i p t a s e . Bous sarcoma virus. sodium dodecyl sulphate.
NON-PRODUCTIVE RETROVIRUS INFECTION
5
All three cell lines were obtained through the American Type Cellular Collection (Rockville, Maryland, USA). Sarcoma-positive leukaemia-negative (S+L-) mink cells [25] were kindly furnished by C. Todaro (National Cancer Institute, Bethesda, USA). All cell lines were maintained in McCoy's culture medium supplemented with 10% foetal calf serum (Flow Laboratories, Scotland). Xenotropic murine type-C retrovirus from M u s molossinus mouse (MolMuLV-X) was a kind gift from R. Callahan (National Cancer Institute, Bethesda, USA). It was produced by the chronically infected Cf2Th/Mol cell line and titred on S+L- mink cells (see ~cVirus assay ,,). Infection of human cell lines was carried out as follows: 10 ml of clarified, filtered (0.45 ~m Millipore) CI2Th/Mol supernatant fluids (105 FFU/ml) were inoculated onto 10e cells. After 1-h incubation at 370 C, the inocula were removed and fresh culture medium was added. Cultures were conserved in CO., incubator at 37 ~ C and split every 7 days. Cell cloning was performed in microplates as described elsewhere [9]. Virus assay.
The S+L - test on mink cells was carried out as described by Peebles [25]. Briefly, log~o virus dilutions were prepared in culture medium and inoculated onto S+L - mink cells, which had been incubated overnight in the presence of 1 ~g/ml polybrene. Focus-forming units (FFU) were estimated by counting the transformation loci under a light microscope 5-6 days post-infection. Titres were expressed in FFU/ml. Reverse transcriptase (RT) activity was tested in culture supernatants following the method described by Ross [31]. The clarified (10,000 g, 10 min) culture supernatants were ultracentrifuged (100,000 g, 90 rain) through a 20% glycerol cushion. Then the pellets were resuspended in Tris-HC1 50 mM pH 7.8 and disrupted by a final concentration of 0 1% NP40. The incorporation of tritiated thymidine monophosphate (from 4 mM of deoxytriphosphate nucleotide, 3H-TTP used as precursor, with specific activity 47 Ci/mM; Amersham) on polyriboadenosine as an exogenous template and oligodeoxythymidine as an exogenous primer (poly-A oligo dT(12-18)) was estimated by trichloroacet~c acid precipitation and nuclear scintillation counting. The presence of virus particles was studied by electron microcoscopy (EM) observation of cell monolayers and pelleted supernatant fluids. Cells were fixed with glutaraldehyde and osmium acid and double-stained with uranyl acetate and lead citrate as described by Hsiung el al. [13]. Pellets from supernatant fluids were stained with phosphotungstic acid (PTA). Infectious virus was also assayed by inoculation of the concentrated supernatant fluids onto canine permissive cells [32], and by direct co-cultivation of infected FL cells with the same highly permissive ceil line. Nucleic acid extraction.
The DNA of the infected cell cultures was extracted after 1 ~/o sodium dodecyl sulphate lysis followed by proteinase K (Merck, Darmstadt, RFA) digestion, phenol chloroform deproteinization and dialysis according to the method of GrossBellard el al. [10]. RNA was removed by alkaline hydrolysis (10 -~ M Na2C03, 37 ~ C, 24 h). DNA was then sonicated to 300-500 nucleotide', in length prior to liquid hybridization. Free provirus forms were extracted from acutely infected Cf2Th cells 16 h post-infection bv the method of Hirt [12]. Total cellular RNA was recovered by pelleting (16,000 g, 20 rain) the cell lysates obtained from the overnight incubation in 6 M urea 3 M lithium chloride of the mixer-blender disrupted cells. The pelleted RNA was phenol-chloroformextracted after proteinase K digestion and precipitated in cold ethanol. The polyadenylated RNA fraction was recovered by elutioa of the total RNA through an oligo-dT cellulose column at uifferent ionic strengths [1].
F. CAVALIERI AND COLL. }~estricLk.. e.~b,.,e ~cr was purcnase0 trom Boehringer Mannheim, France, and DNA digestion was carried out in the presence of k phage internal markers under conditions recommended by the manufacter.
Probe preparalion and molecular hybridizalion. Radiolabelled viral complementary DNA (eDNA) was prepared on a 70 S genomic template obtained from purified MoI-MuLV-X. The reaction was performed in the presence of avian myeloblastosis virus RT (obtained through 3. Beard, Resources and Logistics, National Cancer Institute, Bethesda, USA) as described by Taylor el al. [35]. For Cot and Crt kinetics, the cDNA was synthesized wiLh aI:J-TT]J (50 Ci/mM; Amersham). 3~P-dCTP (25,000 Ci/mM NEN) was used to obtain radiotabelled cDNA for Southern blots. A liquid hybridization reaction were performed in a final mixture volume of 20 tzl containing 10 mM Tris-HC1 (pH 7.4) 750 mM NaCI 2 mM EDTA 0.02% SDS. Hybridization was initiated by heating the reaction mixtures at 98 ~ C for 10 nain, cooling on ice and incubating at 68 ~ C. Hybrids were tested by S: nuclease resistance [17].
Blo[ h qbridizalion. Nucleic acid electrophoresis was carried out on 0.7% agarose bed gels for DNA and 1% agarose bed gels for 1RNA. Nitrocellulose paper transfer and blot hybridization were performed according to a modification of the technique of Southern published by Thomas [37] in the presence of 5xSCC, 50% formamide at 42 ~ C.
31elabolic labelling and immunoprecipilalion (IP). IP was carried out according to Murphy el at. [22]. Briefly, radiolabelled cell cultures were pelleted, washed in a balanced salt solution and lysed in a buffer containing 1% Triton X100, 0.5% sodium deoxychlolate, 0.1% sodium dodecyl sulphate (SDS), 0.2 M sodium chloride, 0.001 M EDTA, 0.05 Tris HC1 pH 7.5. The cell lysates were cleared by overnight incubation at 4 ~ C in the presence of goat normal serum followed by protein A precipitation. The precleared supernatants were adjusted to 1M NaC1 and immunoprecipitated by specific g o a t antisera which are directed against virus gag protein precursor Pr 65 (code n ~ 76S-454) and against the viral envelope glycoprotein gpT0 (code n ~ 6 S-0431) obtained from the National Cancer Institute, Bethesda, USA. Immune complexes were recovered by centrifugation, dissociated in adequate buffer according to Laemmli [15] and resolved by electrophoresis on polyacrylamide gel gradients.
Immunofluorescence. Indirect immunofluorescence (IF) tests for detecting virus p30 protein antigens were performed according to Hilgers et al. [11], using specific anti-murine p30 goat antisera (catalogue n ~ 5 S-123) obtained from the National Cancer Institute, Bethesda, USA). RESULTS
Establishment of ihe N P
chronically infected F L / M o l cell line.
In t h e c o n t e x t of a s y s t e m a t i c s t u d y on t h e susceptibility of h u m a n cell lines to several different M u L V - X viruses, F L cells and AV3 cells were s i m u l t a n e o u s l y infected w i t h Mol-MuLV-X inocula a t a m u l t i p l i c i t y
NON-PRODUCTIVE RETROVIRUS INFECTION
7
of infection of about 1 F F U per cell. From t h e time of infection, the cell cultures were split once a week. Virus production was evaluated by the techniques mentioned in r Materials and Methods ,. Virus antigen expression was monitored by IF with anti-murine p30 virus core protein antiserum. During the first six m o n t h s post-infection, these tests were performed once every two weeks; later t h e y were carried out once every two m o n t h s o v e r one year. The results of these tests were stable and are summarized in table I. No R T activity was detected in FL/Mol culture s u p e r n a t a n t fluids over two years. No infectious viral particles detectable by the S + L - assay of culture s u p e r n a t a n t fluids were released by FL/Mol cells. Furthermore, no virus infectivity was detected in concentrated supern a t a n t fluids inoculated onto permissive cells. EM observation of cellular monolayers and of s u p e r n a t a n t pellets always gave negativc results concerning t h e presence of virus particles. Moreover, the induction of FL/Mol cells by t r e a t m e n t with halogenated pyrimidines (IdUr 40 txg/ml for 24 h) or with halogenated pyrimidines and dexamethasone (IdUr 40 ~g/ml; dexamethasone 10 -" M, 24 h) gave negative results by R T assay and EM observation (data not shown). On the contratry, AV3/Mol culture s u p e r n a t a n t fluids contained both RT activity and 10' to 106 F F U / m l . EM showed numerous particles in cells and s u p e r n a t a n t fluids. Nevertheless, viral p30 antigenic determinants were easily detected by IF in both cell lines, therefore suggesting t h a t the viral genome w a s at least partially present and active in FL/Mol cells despite the absence of virus TABLE I.
-
Comparison of the viral state in PL/Mol
-
and A V 3 / M o l cells. AV3/Mol
FL/Mol Time after infection
15 days
45 clays
52 103 46
196 105 204
379 105 297
EM(,)
(+)
(+--)
(++)
I F (5)
30
60
Viral detection
R T a s s a y (1) S+L - test (2) I n o c u l a t i o n of supern a t a n t s on permissive cells (s)
3 months
60
1 year
15 days
45 days
3 months
201 104 ND
2 0 1.4
3 0 2.2
1.8 0 2.1
(+)
(-)
(-)
(--)
ND
50
50
80
1 year
2.6 0 ND
(-) ND
(t) Values r e p r e s e n t the average of results from triplicate c u l t u r e s u p e r n a t a n t s . T h e y are expressed as o p m • 103 of aH-TMP i n c o r p o r a t e d into p o l y - d T p r o d u c t in 60 min. (2) Values represent the average titre from triplicate culture s u p e r n a t a u t s expressed as F F U / m l . (a) Permissive dog-cell m o n o l a y e r s were inoculated w i t h 10 ml of filtered (0.45 ~m Miilipore) F L / M o l or A V 3 / M o l s u p e r n a t a n t s . Cultures were considered as being positive when R T activity of more t h a n 2 • 104 c p m was detected three m o n t h s after inoculation. (4) (;ells anti s u p e r n a t a n t s were studied as described in ,, Materials anti Methods ,,. ( + ) = some t y p e - C particles; ( + + ) = n u m e r o u s type-C particles ; ( - - ) = absence of particles. (s) Percentage of specifically fluorescent cells was established b y c o u n t i n g positive ceils from at least 300 ceils. N D = N o t done.
8
F. CAVALII~RI AND COLL.
production. Moreover, tile high percentage (80~ see table I) of IF-positive FL/Mol cells indicaLes that a large majority of the cells were infected. Characterizalion of lhe viral nucleic acids present in F L / M o l cells. Liquid-phase molecular hybridization experiments were performed between MoI-MuLV-X virus-specific radiolabelled eDNA and cellular DNA from both FL/Mol and AV3/Mol cells. Figure 1 shows the typical aspect of the Cot kinetics obtained in these experiments. These results show t h a t the totality of viral genetic information is present as proviral DNA in FL/Mol cells. The Cot 1/2 ratio indicates t h a t AV3/Mol contains roughly twice as many proviral genome copies as FL/Mol. In order to ascertain that proviral DNA is eovalently linked to cellular DNA in FL/Mol cells, the total cellular high inoleeular weight DNA was digested with EcoBI restriction enzyme, migrated in a 0.7% agarose gel and analysed by Southern blot hybridization with specific Mol-MuLV-X eDNA. EcoRI was
~AV/
/
J
.
MOI_
. .o {'LIMOL
/ tO
,i
O N
/,'"
,I
~5 -T
4 (.2 12.
........ 1 I . ..... .....
.......... 11)~
10 :~
cot (molex secllitcr) Fro.
Liquid phase molecular hgbridizalion between virus specific triliated 3H-eDNA and high molecular weighl celhtlar D N A , lesled by S 1 nuclease resislance.
1. - -
9 = A V J M o l D N A ; 9 = F L / M o l D N A ; 9 = u n i n f e c t e d AV~ D N A ; o = u n i n f e c t e d C o t 1 / 2 v a l u e s a r e 4.5 x 10 2 f o r F I / M o l a n d 1.9 • 10 2 f o r A ' ~ / M o l .
FL
DNA.
NON-PRODUCTIVE RETROVIRUS INFECTION
9 <
9
9
9
,_
ti
.
Ill .
:"';~ "
-6.0
,
I
Fro. 2. - -
I :
-2.9
Nitrocellulose paper blot hybridisation between oirus-specific a~P-cDNA and cellular D N A .
Free proviral DNA intermediate from the Hirt s u p e r n a t a n t fraction of Mol-MuLV-X acutely infected dog cells (lane 1). EcoR[ digests of high molecular weight DNA from: Cf2Th/Mol cells (lane 2), AVJ.Mol cells (lane 3) and FL/Mol cells (lane 4). No hybridization bands were detectable in uninfected negative controls.
chosen because it cuts only once into the provirus of MoI-MuLV-X [5]. Figure 2 shows t h a t the specific virus eDNA probe recognizes fragments of a molecular weight higher than that of free proviral forms, as expected in the case. of integrated provirus. The results of liquid-phase hybridization between FL/Mol cytoplasmic RNA and Mol-MuLV-X cDNA are shown in figure 3. Crt kinetics demonstrate, within the limits of tile technique, that the proviral DNA present in NP FL/Mol cells is transcribed into virus-specific FINA. The Crt 1/2 ratio shows t h a t virus RNA expression is about 10 times lower in FL/Mol than in AV3/Mol cells. The largest families of viral RNA transcripts were analysed by Northern blot hybridization (fig. 4). Both 24 S and 35 S transcripts are present in FL/Mol cells. Moreover, both transcripts are recovered only in poly-A-enriched fractions of cellular RNA, which suggests t h a t they may be able to serve as mRNA. When several clones derived from the original FL/Mol population (see below) were analysed, the results obtained concerning RNA species were
10
F. CAVALII~RI AND COLL.
..o ......... ~ AV IMOL
lOtl
.,.'
f
2 /2 o,.
~FLJ MOL
/ .,:""
/~
-t 40
~ .~176
g.
~176
..-" 2o
FL ~0.e
10 3
i04
Crt (mole x ser,liler) Fla.
3.
~
Liquid phase molecular hybridization belween virus-specific tritiated aH-cDNA and total cellular ICNA, tested by S~-nucleasc resislance.
9 == A V a / M o l F I N A ; o = F L / M o l t : I N A ! V -~ u n i n f e c t e d A V 3 o r F L Y I N A . Crt 1 / 2 v a l u e s are 2 • 10 s Io:' F L / M o l a n d 2 • 10 ~ f o r AV.~/Mol.
strictly similar to those obtained in the total population (results not shown).
Evaluation of the infectious potential of FL/MoI virus information. Our results showed that the totality of proviral D N A information is present in FL/Mol cells, and is apparently fully and normally transcribed into virus-specific 24 S and 35 S poly-A RNA. Nevertheless, no virions are detectable in FL/MoI cultures by any of the very sensitive methods employed in virus assays. However, the hybridization techniques employed do not allow the detection of mutations or small deletions in the virus nucleic acids, while such events could lead to virus defectiveness. The infectious potential of FL/Mol virus information was therefore tested by co-culture of FL/Mol cells with permissive dog cells in the presence
NON-PRODUCTIVE RETBOVIRUS INFECTION
11
of a fusing agent (PEG 1550). The co-culture s u p e r n a t a n t fluids always contained both detectable R T activity and infectious virions, as shown by the S+L - test. Moreover, ten out of ten one-cell-derived FL/Mol sub-, clones which were also totally negative in terms of virus production gave rise to productive infection when co-cultivated and fused with the permissive dog cell line. The rescued infectious v i r u s presented t h e same host range and antigenic determinants as Mol-MuLV-X (data not shown). Since FL/Mol clones were picked at random, this result suggests t h a t at least a large majority of the FL/Mol cells contain functional virus information. :
A::
9 /[
C
"
i
.
D
,
FIG, 4. - - Nitrocellulose paper blot hybridization between virus-speeifie 3~P-eDNA and eellutar R N A .
Poly-A-enriehed lqNA: FL/Mol cells (lane A) and AV#Mol cells (lane B). Poly-A-negative RNA: FL/Mol cells (lane C) and AVz/Mol ceils (lane D). Arrows indieate the positions of 28 S and 18 S ribosomal RNA.
Characterization of the virus-coded polgpeptides present in FL/Mol cells. The total absence of m a t u r e virions from FL/Mol cells in spite of the presence of apparently normal B N A and the positive IF results led us to s t u d y t h e major translation products in the NP cell line as compared to producer cells. Steady state (I-h) labelling of the cell cultures was followed by IP and P A G E analysis. No qualitative difference was remarked
a
b
180-
30FzG. 5. - - PAGE analysis on gradienl ( 8 % - 1 6 % ) gel o/ anti-Pr65gag immunoprecipitales from aH-leueine and 8It-valine sleady slate (1 h) labelled lysates. FL/Mol cells (lane a) and A~89
A
cells (laue b). Molecular weights are expressed in Kd,
B
C
D < 85 41 70
FIG. 6. - - P A G E analysis on gradient ( 8 % - 1 6 % ) gel of anli-gpTO immunoprecipitales from 3H-leucine and 8H-z~aline cellular lysales. FL/Mol cell lysate labelled tar 1 h (lane A) and chased for 16 h (lane C). A Y J M o l cell lysate labelled for 1 h (lane B) and chased for 16 h (lan e D). Molecular weights are expressed in Kd.
NON-PRODUCTIVE FIETROVIRUS INFECTION
13
between AV3/Mol and FL/Mol cell lysates immunoprecipitated with antigag serum. Prl80gag-p ~ Pr65gag and mature p30 were identified by this method in both cell lines, even though the intensity of the FL/Mol gag bands was constantly weaker than that of the AV3/Mol gag bands (fig. 5). Steady-state labelling followed by IP with anti-gp70 serum allowed the detection of an apparently identical Pr85 env in both cell lines, while the detection of mature gp70 env was possible only after pulse- and chaselabelling (fig. 6). The same experiments carried out after 3H-glucosamine labelling indicated t h a t both the Pr85 ~ and the gp70 ~nv are actually glycosylated in both cell lines.
DISCUSSION We report the establishment and characterization of NP chronic infection of a human cell line by a murine xenotropic type-C retrovirus: Mol-MuLV-X. The NP-chronically infected cell line FL/Mol contains the totality of virus genetic information in the form of proviral DNA covalently linked to the cellular genomic DNA. This is shown by liquid-phase hybridization of total cellular DNA and blot hybridization of EcoRI-digested cellular DNA with virus cDNA. The integration pattern of Mol-MuLV-X in NP FL/Mol cells seems to differ markedly from the integration pattern in the producer AV3/Mol cell line. Since no integration-site specificity has up to now been demonstrated for type-C retroviruses, finer analysis of the cellular provirus junction sequences is needed before considering this fact to be related to t h e NP state. Virus DNA is transcribed in FL/Mol cells, and 24 S and 35 S virus RNA transcripts are indistinguishable, by Northern blot hybridization, from those found in normally productive cell lines. Analysis of the major intracellular virus proteins, carried out by specific immunoprecipitation and P A G E shows that the gag gene products synthesized by FL/~Iol cells co-migrate with those synthesized by AV~/Mol cells. A glycosylated Pr85 ..v is produced and processed to yield a gp70 e"v in both cell lines. The gp70 ".v present in FL/Mol cells was found to constantly migrate slightly faster than its counterpart in AV~/Mol. Preliminary quantitative studies carried out by radioimmunoassay (S, Gisselbrecht, personal communication) showed that the amount of virus proteins per cells is about two times higher in FL/Mol than in AV~/Mol. Since our IP experiment,, showed that the virus protein band intensity of FL/Mol lysates was constantly weaker than or equal to the virus protein band intensity of equivalent AV~/Mol lysates, these date indicate that the lack of virion production might lead to the accumulation of virus proteins in FL/Mol cells. Infectious virus presenting the same antigenic and host-range characteristics as the input Mol-MuLV-X is easily rescued from cloned subpopulations o f FL/Mol cells co-cultivated with permissive dog cells in
14
F. CAVALII~RI AND COLL.
tile presence of a fusing agent These results suggest either that the FL/Mol cells which contain non-defective provirus have a cloning advantage over the rest of the population or that a large majority, if not the totality, of the FL/Mol cells contain biologically intact virus information regardless of the NP state. The fact t h a t 80% of FL/Mol cells were positive in murine type-C-virus-specific IF is strongly in favour of the second possibility. Both biological and biochemical evidence strongly suggest t h a t the restriction is due to the cellular environme:,t alone. Viral RNA synthesis is at least partially ruled by the cellular machinery, and the 35 S genomic BNA has been shown to form a pool separated from t h a t of the 35 S RNA responsible for gag protein synthesis [18]. A host-cell-dependent shift in equilibrium between the genomic and the messenger 35 S viral BNA pool could seriously hamper infectious virion production. Interestingly, it should be kept in mind that the level of BNA expression is 10 times lower in FL/Mol than in AY~/Mol. Alternatively, even slight alterations in the viral gp70 could affect its membrane-binding ability and therefore seriously compromise the budding process, or some undefined properties of the cell membrane itself could impair some step(s) of virion assembly. To our knowledge, this is the first report of a chronic non-productive infection of a non-lymphocytic mammalian cell line by a non-defective and non-transforming murine retrovirus. Beeently, Ceecherini-Nelli el al. [4], showed t h a t the expression of integrated primate type-C virus proviral DNA is restricted in non-productively infected human B lymphoblasts. The restriction mechanism appears to be quite different from those already known. Tl,is restriction mechanism seems to be selective, since FL is able to be productively infected by several other xenotropic type-C viruses of non-murine origin [33]. In fact, proviral DNA integration, transcription and translation do occur in FL/Mol cells, while this is not the case either under Fv-1 restriction, which appears to occur before the integration event itself [14], or in the case of embryonal teratoeareinoma cells, where the proviral DNA is integrated [34, 8] but not expressed [26, 6]. Mammalian cells are also known to be non-productively infected by avian sarcoma retroviruses. Virus DNA of Bous sarcoma virus (RSV) can be integrated with low efficiency only after infection with certain RSV stocks [39]. The frequency of transformation is low, and virus is hardly ever produced, even though it can often be rescued by fusion with chick cells [3, 38]. Nevertheless, when comparing restriction of mammalian cells for replication of RSV with the NP FL/Mol cell line, several import a n t differences do appear: RSV is a transforming virus, while Mol-MuLV-X is not; the processing of R S u RNA largely favours 21 S m B N A over 38 S m R N A [29], while in FL/Mol cells, 24 S m R N A and 35 S m R N A are present at about the same level; finally, R S \ r polyproteins are synthesized but not cleaved, while Mol-MuLV-X p30 and pg70 are synthesized in FL/Mol ceils. Murine cells chronically infected by eeotropie MuLV and human cells
NON-PRODUCTIVE RETROVIRUS INFECTION
15
infected by several xenotropic retroviruses are known to switch off virus production when treated with interferon [2, 27 I. Interferon is apparently not implicated in the non-productiveness of FL/Mol cells for two reasons: 1) no interferon activity is detectable in FL/Mol culture supernatants (Rhodes-Feuillette and P~ri~s, unpublished results); 2) the inhibition of retroviral production by interferon affects virion release. Chronically infected treated cells show large clusters of i m m a t u r e virus particles associated with the cell membranes, which is not the case in FL/Mol e v e n after extensive EM observation. The establishment of chronic semi-silent infection of h u m a n cells in vitro raises the question of the possible extension of these findings in vivo. It has been reported t h a t the expression of murine retroviral-like sequences can be detected in various types of h u m a n leukaemia cells [16]. Moreover, the recent isolation of a non-germ-line-transmitted retrovirus from h u m a n leukaemia cells [28, 30], seems to reopen the debate on the possible implication of exogenous retroviruses in some h u m a n lymphoproliferative diseases. Non-defective murine leukaemia viruses do not carry any known transforming gene; nonetheless, they can induce leukaemia, albeit with low efficiency and after long incubation periods, possibly by promoting the transcription of cellular genes via their LT13 {long terminal repeat) [36]. If our findings are extended to an in vivo system, one cannot rule out the possibility t h a t an intact b u t d o r m a n t xenotropic retroviral genome would act in the same way.
INFECTION
CHRONIQUE ET NON PRODUCTIVE DE CELLULES PAR UN RETROVIRUS XI~NOTROPIQUE DE TYPE C
HUMAINES
Nous d6crivons l'~tablissement et la caract6risation d'une lign6e cellulaire humaine (FL/Mol) non productive (NP), infect6e chroniquement par un virus x6notropique murin de t y p e C originaire de la souris M u s molossinus (MoI-Mu-MLV-X). Les cellules chroniquement infect6es ne contiennent aucune particule virale visualisable par microscopie electronique, et elles ne lib~rent aucun virion d6tectable par 6valuation de l'activit6 r6verse ~ranscriptase ou par inoculation des surnageants de culture /1 des cellules permissives. Par immunofluorescence antivirale sp~cifique, no,ts montrons que la majorit6 des cellules sont infeet6es. De plus, la totalit6 du g~nome viral est pr6sente sous forme d'ADN proviral d6tectable par hybridation mol6culaire, et la coculture avec des cellules permissives en presence d'un agent fusionnant (le poly6thyl~ne-glycol) donne lieu ~ la production de virions infectieux. L ' A D N proviral int6gr6 est transcrit et les deux familles d ' A R N viral (24 S e t 35 S) sont synth6tis6es. Les d~terminants antigeniques aussi bien de la prot6ine virale p30*ag que de la glycoprot6ine virale gp70 e''v
16
F. CAVALlt~III AND COLL.
sont I a c n e m e n t nus en Ovidence. L ' a n a l y s e sur gel de polyaerTlamide des lysats des eellules F L / M o l immunoprdeipites n'a permis de m e t t r e en 0videnee aueune anomalie de la svnthbse de la prot6ine virale p30."g ni de ses prdeurseurs. L ' i m m u n s e r m n anti-gp70, par contre, reconnait le l)rceurseur gpr85 el une gp70 ..... qui migre 1O~Orement plus r i t e que la ..7(b,,,,, que ]'on t r o u v e dans d'autres eellules humaines infeet6es et produetriees. 3I(~TS-(:LL.'S : R f t r o v i r u s , lnfeclion non p r o d u c t i v e , X 6 n o t r o p i s m e , B0plication ; T y p e C, Souris, Cellules hunaaines, A D N , A B N , Prot~ines. ACKNOWIA'2DGEMENTS
\\re thank J. Lasneret for electron microscopy, S. Gisselbrecht for performing the radioimmunoassav, Y. Poirot-Lossfelt and M. Garcette for excellent technical assistance and M. C. "Pceuf and M. Pasquet for typing this paper. Partial support for these studies was provided by INSERM (Institut National de la Santa el de la Recherche M6dicale) grant 7841421 and by DGRST (D616gation "9 Gdndrale ~ la Recherche Scientifique et Technique) grants A.650 1926 and 81L0z,8.
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NON-PRODUCTIVE R E T R O V I R U S INFECTION
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F. CAVALIERI AND COLL.
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