Replication of human immunodeficiency virus in HL-60 cells

© INSTITUTPASTEUR/ELsEVIER Paris 1992

Re.8. ViroL

1992, 143, 249-258

Replication of human immunodeficiency virus in HL-60 cells M. Semmel (1) (*), A. Macho (2), V. Morozov 0), D. Coulaud 0), A. Alileche 0), S. Plalsance 0), J. Aguilar (2) and C. Jasmin 0) ¢1~ Unitd 268 INSERM, 14-16, av. Paul Vaillant-Couturier, 94800 Villejuif (France}, t2j Laboratoire de Chimie des Protdines, IRSC, 8, rue Guy Mocquet, Villejuif (France), and ¢3~IGR rue Camille Desmoulins, Villejuif (France} SUMMARY

HL-60 cells carrying the CD4 marker could be productively infected with human immunodeficiency virus (HIV) but did not form syncytia, though 11-14 days p.i., there was a transient decrease in cell multiplication and viability. After prolonged passage, a subpopulation of HL-60 cells was selected. The virus produced differed from the initial input virus grown on CEM cells: the virions lacked knobs, were either empty or had abnormal cores, had a higher ratio p24/gp41,gp110 and were less infectious. After prolonged passage, virus was produced which was fully infectious for CEM but not for fresh HL-60 cells, and which ressembled the Input virus with respect to morphology and p24/gp41,gp110 ratio.

Key-words: HIV, Glycoprotein, CD4, Replication, HL-60 cell line; Virions, Ultrastructure, RT, Model.

INTRODUCTION HL-60 cells have been described as being poor producers of HIV essentially because the cells tested originally did not carry many CD4 molecules, the principal receptor for the envelope glycoprotein 110 of HIV (Levy et al., 1985; Dalgleish et al., 1984; Clapham et al., 1987). Other authors have shown that cellular tropism may also be determined by the env gene region of the virus (Shioda et al., 1991 ; Cheng-Mayer et al., 1991). HL-60 cells could constitute an interesting model for the study of the relationship between differentiation and HIV infection since these promyeloid cells can be induced to differentiate along the granulocyte pathway

SubmittedMarch 23, 1992,acceptedMay 13, 1992. (') Corresponding author.

(Breitman et aL, 1980) by retinoic acid and along the macrophage pathway by calcitriol (Mangelsdoff et aL, 1984). We have tried to infect an HL-60 cell line which carries the CD4 marker with HIV, and we present here the results of these experiments.

MATERIALS AND METHODS Cells and vlrus

HL-60 cells (Collins et al., 1977) a gift from G. Milon (Institut Pasteur, Paris); CEM clone 11, a Tcell line, a gift from F. Barr6-Sinoussi (Institut Pasteur, Paris); and HT4-LacZ1 cells, a gift from

250

M. S E M M E L E T A L .

J.F. Nicolas (Institut Pasteur, Paris) were used. CEM and HL-60 cells were grown in RPMI-1640 medium (Gibco) containing l0 070 heat-inactivated foetal calf serum, 2 mM glutamine, 100 units penicillin, l0 ttg streptomycin and 0.25 I~g amphothericin/ml; HT4-LacZ1 cells were grown according to Rocancourt et al. (1990). Cells were seeded at 2x105 cells/ml and incubated at 37°C in a humidified atmosphere containing 5 07o CO 2. Medium was renewed and the cell concentration readjusted twice weekly. HIV type l strain LAI (formerly BRU) was a gift from F. Barr~-Sinoussi. Stock virus was grown on CEM clone l l cells and stored at - 8 0 ° C . HT4-LacZ1 cells were grown according to Rocancourt et al, (1990). HL-60 cells were infected by adding cell-free supernatant to the culture (2 x 104 cpm RT HIV per l06 cells). Cell-free supernatant was stored at - 8 0 ° C . Determination of reverse transcriptase (RT) and tissue culture infectious units

HIV replication was assayed by measuring RT activity in the supernatant according to OystenJonassen 0986). Briefly, 50 izl cell-free supernatant were incubated for 1 h at 37°C in a mixture containing 0.05 % Triton-X100, 50 mM KCI, 3 mM dithiothreitol, 30 mM TRIS-HCI pH 7.8, 0.3 mM EGTA, 0.13 units poly-rA-oligo-dT as primer (Pharmacia) and 0.0378 mCi ~H-deoxythymidine triphosphate (Amersham). The reaction was stopped with 10 070 trichloroacetic acid (TCA) containing 2 mM sodium pyrophosphate. The precipitate was washed 6 times with 5 070TCA containing 12 mM sodium pyrophosphate, using an "Automash 2000 Harvester" (Dynatech)and "Skatron" filtermats. The filters were counted in scintillation liquid in a "Beckman" counter. Tissue culture infectious units (TI2IL0 were determined by titrating the virus in the supernatant according to Rocancourt et al. (1990). Electron microscopy

acid, dehydrated with ethanol and included in " E p o n " resin by the usual techniques. Cell-free supernatant containing virus was fixed in 4 07o glutaraldehyde and ultracentrifuged for 1 h at 100,000 g. The pellet ws resuspended in S6rensen's buffer, filtered on 100-~n f'llters (MiUipore) and postfixed as described by Barbieri et al. (1970). Sections of filters or cells were coloured with uranyl acetate and lead citrate and observed with a "Zeiss EM 902" microscope. Optical contrast was obtained by selecting the elastic electrons at the slit of the spectrophotometer. Immunofluorescence

For determination of CD4 antigen, cells were labelled with anti-CD4 monoclonal antibody (mAb) linked to fluorescein (clone 13B2, Immunotech). For typing, the cells were labelled with mAb (Coulter) linked to either fluorescein (FITC) or phycoerythrin (PE). Labelled cells were analysed with an "EPICS Profile II Analyzer" (Coulter). Nucleic acid and protein assays

DNA was extracted from 1-2 x 107 cells according to Sambrook et al. (1989). Ten i~g of each DNA were digested with HindIII (Boehringer) and analysed by electrophoresis on a 0.8 °7o agarose gel. HindIII fragments of lambda phage DNA were used as size markers. Transfer of DNA to 0.45-~tm nitrocellulose filter (Sartorius) was done according to Southern (1975). Blots were hybridized for 18 h at 42°C with a a2p-HIV1 full genomic probe (PBT 1, a gift from M.C. Lang, Institut Pasteur~ Paris), prepared according to the instructions from the supplier (Multiprimer Labeling System, Boehringer). After hybridization, filters were washed 5 times for 10 min itl 2 x SSC at room temperature and twice for 15 rain in 0.1 x SSC, 0.1 070 SDS at 50°C. Washed filters were exposed to "Hyperfilm TM M P " (Am~rsham) with an amplifying screen (Dul~Ont, Quanta IIIT) at - 8 0 ° C .

Cells were fixed in 4 °7o glutaraldehyde in phosphate-buffered saline and pelleted at low speed. The pellet was washed in Sorensen's buffer (67 mM phosphate buffer pH 7.4), post-fixed in 2 % osmic

PCR was performed according to the instructions from the supplier of the "Cetus/Perkin Elmer" kit. I4iVI gag-specific primers were used (position 692-712: 5'-CTG ACA CAG GAC ACA OCA GTC AGG-3'; position 1392-1415: 5'-TCC TCC TAC TCC CTG ACA TGC TOT-3'). The following pro-

FITC LTR mAb PCR PE

p.i. RT SDS TCA TCIU

= = = = =

fluoresceinisothiocyanate. longterminal repeat. monoclonalantibody. polymerasechain reaction. phycoerythrin.

= = = = =

post-infection. reversetranscriptase. sodiumdodecyl sulphate. trichloroaceticacid. tissueculture infectiousunit.

HIV REPLICATION

tocol for amplification was used : 990C 5 min, 75°C 10 rain, 37°C hold, 94°C l min, 55°C 1 min, 75°C 1 min, 30 cycles, 75°C 10 rain. Thirty ~l of each PCR mixture were analysed in a 1.5 °70 agarose gel in the presence of ethidium bromide (0.5 ~tg/ml). After electrophoresis, the gel was blotted on a "HybondN " filter (Amersham) according to the manufacturer's instructions. Blots were hybridized overnight with the 32p-labelled probe (PBT 0 at 45°C in 5 x SSC, 0.5 °70 SDS and 100 Izg denatured salmon sperm DNA. The filters were washed 3 times in 2 × SSC, 0.1 °70 SDS for 10 rain at room temperature and twice with 0.1 × SSC, 0.1 o7o SDS for 30 rain at 50°C and exposed to "Hyperfilm TM M P " (Amersham) with an amplifying screen (Dupont, Quanta III T) at - 8 0 ° C . Total RNA was extracted from 2-3 x 107 cells according to Chomczinski and Sacchi (1987). Thirty to forty ~tg of each RNA sample were denatured with formaldehyde-formamide, then size-fractionated by electrophoresis through a 1 °70 agarose gel in the presence of 6.7 °70 formaldehyde, 40 mM 3N morpholinopropane sulphonic acid pH 7.5, l mM sodium acetate, 1 mM EDTA. Size standards consisting of a 12-~tg RNA ladder (BRL, Bethesda) were loaded and stained with ethidium bromide. On completion of migration, RNA was transferred by the capillary method overnight in 20 x SSC to a 0.45-~tm pore size "Biodyne" filter (Pall Industry). Filters were baked for 30 min at 80°C, then prehybridized at 42°C for 15 rain to l h in 50 % (v/v) formamide, 5 x SSPE, 0. l °70 SDS, 5 x Denhardt's solution prepared according to Sambrooke et aL (1989) and 100 ~.g/ml salmon sperm DNA (Sigma) denatured by sonication. Hybridization was performed by adding l06 cpm of 32p-cDNA/ml to the prehybridization buffer at 42°C and incubation overnight. The eDNA

251

IN HL-60 CELLS

probe was labelled at a specific activity of about 109 cpm/~tg by nick translation (Amersham kit). After hybridization, filters were washed twice at room temperature in 2 x SSC, 0 . 1 % SDS and subjected to autoradiography at - 80°C. For the subsequent use of filters, the hybridized probe was removed by incubation in boiling 0.1 °7o SDS. To analyse viral proteins, a viral pellet corresponding to 3 x 106 cpm RT HIV was suspended in 50 ~l Laemmli's buffer (Laemmli, 1970) and heated for 3 min at 100°C. SDS polyacrylamide gels (10-15 %) were run using the " P H A S T " system (LKB-Pharmacia) and blotted on nitrocellulose membranes (Schleicher and SchiiU) with the PHAST transfer unit. Membranes were saturated with 5 % bovine serum albumin fraction V (Boehringer) in phosphate-buffered saline, incubated overnight with the primary antibody, washed and incubated with biotinylated anti-species antibody, washed and incubated with streptavidin linked to alkaline phosphatase and revealed with the appropriate substrate according to the manufacturer's instructions (Amersham). Rainbow molecular weight markers were purchased from Amersham. Anti-HIV mAb were a gift from Dr. Traincard (Institut Pasteur, Paris, Hybridolab) (anti-gp41 (A9-11) and anti-gp110 (A105-34)) or were purchased from NEN (anti-p24) or from TEBU (antigag, anti-gp41 and anti-gpll0).

RESULTS

Evolution of HL-60 cells infected with HIV Table I summarizes the markers present on the H L - 6 0 cells used in these experiments and

Table I. FACS analysis of surface markers on HL-60 cells. Antibody CD33(IgG2B) CD 14 (IgG2B) CD13 (IgG1) CD3 (IgG1) CD4 (IgGl) CD8 (IgGl) CD20 (IgG1) CD10 (IgG2A) CD2 (IgGl) CD25 (IgG1) ~'~Fluorescein. ~"~Phycoerythrin.

Linked to: FITC (') FITC PE t'.) PE t..) FITC FITC FITC FITC FITC PE t*')

uninfected 93.9 1.1 96.7 1.5 92.1 0 0 0.3 0 0.8

o70 Positive cells 120 days p.i. 91.5 3.1 97.9 2.2 2.7 1.3 11.0 18.2 0 3.7

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M. SEMMEL E T AL.

Table II. Growth and viability of HL-60 cells after HIV infection.

Cell number doubling time (h) Uninfected Infected

Days p.i. 0- 4 4- 7 7 - 11 I 1 - 14 14- 18 18-21 120 - 160

36.9 28.2 35.5 38.6 28.4 30.3 30.5 ::t:6

establishes their myelocytic lineage: the cells reacted strongly with antibodies directed against the markers for myeloid cell lines (CD33 and CD13), and either not at all or very weakly with antibodies directed against markers of other cell types (CD14, CD3, CD8, CD20, CD10, CD2 and CD25) (Dorken et ai., 1989). Most of the cells of this particular strain carried the CD4 marker. When cells were chronically infected with HIV, the number of cells carrying the CD4 marker decreased to very low levels, whereas the number of cells carrying the CD20 and CD10 antigens increased. Moreover, cell multiplication and viability decreased 7-14 days p.i. when most progeny virus were produced, but were the same in infected and uninfected cells later on (table II). These results suggest that HIV causes the selection of a subpopulation of HL-60 cells. Table III shows the evolution of the CD4 marker after HIV infection. Eleven days after infection of HL-60 cells with HIV, expression of CD4 antigen decreased, and 60 days p.i., only traces of the antigen could be detected. Thus, in this respect, HL-60 cells behave like T cells: downregulation of CD4 antigen has been described in T4 + lymphocytes (Klatzman et aL, 1984) and in several T-cell lines (Hoxie et al., 1986).

Virus production

in H L - 6 0

cells

In figure 1, virus production in HL-60 and CEM ceils are compared. The production of in-

% Living cells Uninfected Infected

33.6 30.6 91.4 68.5 33.5 28.6 30.8 + 6

95 96 95 92 95 95 94

95 89 85 88 96 96 93

Table III. Evolution of CD4 marker after HIV infection. Cells

CD4(')

Uninfected CEM HL-60 (control) 4 days p.i. 7 days p.i. 11 days p.i. 18 days p.i. 100 days p.i.

1.162 1.676 1.244 1.279 0.320 0.178 0.034

(')Mean of relative fluorescence intensity emitted per cell (5,000 cells tested).

fectious virus by CEM clone 11 cells started on day 11 p.i. The peak was reached 14 days p.i. (fig. 1B); from then on, the cells produced infectious virus continually without cytopathic effect (not shown). In the supernatants of HL-60 and CEM cells, comparable amounts of RT activity could be detected 11 days p.i. Subsequently RT was found irregularly in the supernatant of HL-60 cells with a sharp increase 64 days p.i. (fig. 1A). The supernatant of HL-60 cells did contain infectious virus from day 11 to day 17 p.i. though less than the supernatant of CEM cells. Later on and up to 62 days p.i., no or very little infectious virus was found. Sixty-four days p.i. and later on, HL-60 ceils produced as much infectious virus as CEM cells, without any cytopathic effect (fig. 1B), though the virus did cause syncytia in HT4-LacZ1 cells. In parallel experiments, fully infectious virus was produced

H I V REPLICA TION I N HL-60 CELLS 8

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Fig. 1. Multiplication of HIV. A) RT activity in the supernatant of CEM cells (xxx) and HL-60 cells (---) infected with HIV harvested from CEM cells 11 days p.i. B) TCIU in the supernatant of CEM cells (xxx) and HL-60 cells (---) infected with HIV harvested from CEM cells 11 days p.i. C) RT activity in the supernatant of fresh HL-60 cells infected with virus harvested from CEM cells 11 days p.i. (xxx) or with virus harvested from HL-60 cells 71 days p.i.

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41 and 70 days p.i. (not shown). However, when this virus was compared with virus grown on CEM cells by infecting fresh HL-60 cells, RT activity and the amount of infectious virus were very similar 11-18 days p.i. (fig. IC and D), i.e. less than when CEM cells were infected with these viruses (not shown). Analysis of virus produced in HL-60 cells

Figure 2 shows electron micrographs of the virions produced by CEM and HL-60 cells that

253

appeared either empty (fig. 2c) or much larger (fig. 2d) than the input virus (fig. 2a and b) and some particles contained several cores (fig. 2d and e). These virions had either no or fewer knobs than virions grown on CEM cells, and the remaining knobs appeared to be distributed irregularly (fig. 2c, d and e). Sixty-eight days p.i. the virions from HL-60 cells and those from CEM cells appeared to be identical (fig. 20. When viral structural proteins were analysed, virus from HL-60 cells harvested 11 days p.i. contained as much p24 (fig. 3A and B, lanes a and b) but less gp41 than virus from CEM cells (fig 3A, lanes c and d, and fig. 3B, lanes d and e). The other env-coded protein, gp110, also appeared to be less abundant in virus from HL-60 cells than in virus from CEM cells: less antigp110 (A105-34) antibody was bound to gp110 from HL-60 cells than to gp110 from CEM cells (fig. 3A, lanes e and f), but an apparently equal amount of another anti-env antibody (anti-env TEBU) was bound to g p l l 0 from HL-60 and CEM cells (fig. 3B, lanes g and h). Thirty days p.i., the same results were obtained (not shown). When virus was harvested 68 days p.i., virus from HL-60 cells contained as much p24 but less pr45 than virus from CEM cells (fig. 3B, lanes a and c); the amounts of gp41 and gp110 were the same in virus from HL-60 and CEM cells (fig. 3B, lanes d, f, g and i). As expected, the HIV genome was integrated into HL-60 cells as shown by the 6.5-kb fragment obtained after digestion with H i n d I I I (Muesing et al., 1985), though at lesser amounts than in CEM cells (fig. 4). The HIV genome was barely detectable in Southern blots of infected HL-60 cells 4 days p.i. (fig. 4A) but was clearly present when the PCR technique was used (fig. 4B). Four days p.i., the HIV genome could be easily detected in CEM cells by either technique. Dot blots showed that the HIV DNA integrated into HL-60 cells was transcribed (fig. 5A). In the Northern blot (fig. 5B), 3 bands corresponding to the 9.6-, 4.2- and 2-kb mRNA could be distinguished.

254

M. SEMMEL E T AL.

Fig. 2. Electron micrographs of HIV. Thin sections of cell pellets (a, c, f), magnification x 130,000, and of millipore filters (b, d, e), magnification x 150,000. Bar--0.1 gm. a and b -- HIV grown on CEM clone I l cell, I l days p.i. ; c, d and e -- HIV grown on HL-60 cells, I l days p.i. ; f = HIV grown on HL-60 cells, 68 days p.i.

255

H I V R E P L I C A T I O N I N HL-60 CELLS

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Fig. 3. Western blots of structural proteins of HIV produced on CEM and HL-60 cells. A) Comparison of virus produced 11 days p.i. by CEM cells (a, c and e) and by HL-60 cells Co, d and f); a and b are revealed with anti-p24 antibody (NEN); c and d are revealed with anti-gp41 antibody (A9-11); e and f are revealed with anti-gpll0 antibody (A105-34). B) Comparison of structural proteins from virus produced by CEM cells l I days p.i. (a, d and g), HL-60 cells I l days p.i. (b, e and h) and HL-60 cells 68 days p.i. (c, f and i); a, b and c are revealed with anti-gag serum (TEBU); d, e and f, by anti-gp41 serum (TEBU); and g, h and i, by anti-gp110 serum (TEBU).

DISCUSSION The HL-60 cells used in these experiments carried m o r e C D 4 markers than the C E M cells which were used to p r o d u c e virus stock. This w o u l d explain w h y our results differ f r o m those o f earlier investigators. Nevertheless, the HL-60

cells used in these experiments initially p r o d u c e d less infectious virus than C E M cells: though R T activity was c o m p a r a b l e in supernatants o f H L - 6 0 and C E M cells, the f o r m e r contained 5 times less infectious virus than the latter. Electron microscopy showed that virions f r o m C E M occasionally contain lateral bodies and that there

256

M. S E M M E L E T A L . a

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Fig. 4. Analysis of HIV DNA. A) Southern blots in CEM and HL-60 cells 4 days p.i. after digestion with HindIII: a = uninfected CEM cells; b = infected CEM cells; c = uninfected HL-60 cells; d = infected HL-60 ceils. B) Products of PCR reaction (a, b, c and d, as for A).

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.h Fig. 5. Blotting of RNA extracted from HL-60 cells. A) Dot blots: a = uninfectedcells; b = cellsinfectedwith HIV, 7 days p.i. B) Northern blots: a = uninfectedceils; b = cells infected with HIV, 7 days p.i. ; upper panel: hybridizedwith an HIV probe; lower panel: hybridizedwith an actin probe.

are few " e m p t y " forms. Virions from HL-60 cells were " e m p t y " and their membrane ressembled budding immature forms, attached to the cell membrane. However, these structures were either not attached to the cells or else tethered

with a thin stalk to the cell membrane, resembling virions mutated in p6 gag (G6ttlinger, 1991). Most of the virions found in the supernatant contained several cores different from lateral bodies and appeared larger than the input virus. Such forms have been observed before (Gelderblom, 1991) and it has been suggested that the extra cores contain excess p24. Analysis of structural viral proteins favours this hypothesis : initially, virus from HL-60 cells contained as much p24 but less e n v - c o d e d proteins than virus from CEM cells. Loss o f envelope glycoprotein and loss of infectivity have been associated (Gelderblom et al., 1985, 1987; McKeating et al., 1991). ChengMayer et al. (1991) report that altered host range is associated with modifications o f gpl l0 after passaging of HIV on different cells. Shedding of knobs with cell ageing has been reported (Layne et al., 1991). However, delays of assaying were the same for virus grown on CEM and HL-60 cells. Unless virus produced on HL-60 cells sheds knobs more readily than virus produced on CEM cells, age of the preparation could not be the cause o f the apparent deficiency of the virus produced on HL-60 cells. Adaptation of HIV to HL-60 cells appears to be due to selection of a cell population rather than to adaptation o f the virus either through mutation of the virus a n d / o r selection of a variant virus. Indeed, several mechanisms can intervene in the selection of a particular variant present in an originally heterogeneous population: viral entry independent of CD4 may be a factor of selection (Kim et al., 1990), the genome, in particular v p u and the N and C termini o f the e n v sequence, can determine the productivity of infection (Westervelt et al., 1991), whereas the LTR of HIV (Pomerantz et al., 1991) and modifications in the gag-pol region do not appear to be a major determinant of cellular tropism, though post-transcriptional modifications of the env protein do modify the host range (Cheng-Mayer et al., 1991). It is not possible at present to exclude the selection of a variant virus by any o f these mechanisms in addition to the selection o f a cell population.

HIV REPLICA TION IN HL-60 CELLS

However, after prolonged passage, the progeny virus, fully infectious for C E M cells a n d capable of causing syncytia in HT4-LacZI cells, does not differ from the input virus when assayed on fresh HL-60 and CEM cells and has the same morphology and p24/gp41, gp110 ratio as the input virus. It therefore seems more probable that infection of HL-60 cells with HIV causes the death of a subpopulation of cells and the selection of another subpopulation more favorable to HIV multiplication than the original cell line. We conclude that (a) HL-60 cells can be infected by HIV though the virus does not cause syncytia or massive cell death, and can produce virus which, though initially deficient, is fully infectious after prolonged passaging; (b) HL-60 cells contain a factor which interferes with the maturation of HIV originally grown on CEM cells ; and (c) prolonged passaging selects a subpopulation of cells in which the virus can reproduce efficiently presumably because the interfering factor(s) are absent.

Acknowledgements We would like to thank Dr. A. Flamant for critical revision of the manuscript. This work was financed by the "Agence Nationale de Recherches sur le SIDA".

R6plication du virus de rimmunod6ficience humaine dans des cellules HL-60 Des cellules de la lign6e HL-60, porteuses de l'antig6ne CD4, peuvent &re infect6es par le virus de l'immunod6ficience humaine (VIH) et produire du virus, mais elles ne forment pas de syncytia. Entre le 11e et le 17, jour apr6s l'infection, la vitesse de multiplication et la viabilit6 des cellules diminuent transitoirement. Une sous-population de cellules est s61ectionn6e apr6s plusieurs semaines de culture. Le virus produit en un premier temps diff6re du virus inocul~: les virions n'ont pas de spicules A leur surface, ils sont rides ou ils ont des noyaux anormaux, le rapport p24/glM1 ,gp110 e.st sup~rieur et leur pouvoir infectant e.st inf~rieur. Apr~s plusieurs semaines de culture, le VIH produit darts les cellules HL-60

257

ressemble au virus inocul6: la morphologie, le rapport p24/gp41,gp110 et le pouvoir infectant pour les cellules CEM et HL-60 sont les m~mes. Mots-cl~: VIH, glycoprotdine, CD4, Rdplication, L i g n ~ HL-60; Virions produits, Ultra.structure, RT, ModUle.

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