HIV2 chronic infection of promonocytic cells

(~) INSTITUT PASTEUR/ELSEVIER

Paris 1990

Res. Virol. 1990, 141, 267-278

HIV2 C H R O N I C I N F E C T I O N OF P R O M O N O C Y T I C CELLS

P. Innocenti and J.-M. Seigneurin Laboratoire de Virologie, Centre Hospitalier Universitaire, 38043 Grenoble (France)

Summary. HIVI infection of mononuclear phagocytes is now well established, whereas H!V2 infection of these cells is less well documented. In this work, we studied the replication of the HIV2RoD strain in the U-937 promonocytic cell line and compared it with that of the HIVIBRq strain, p24 antigen and RT (reverse transcriptase) activity were assessed at regular intervals in cell-free supernatants, as was the infectiousness of the produced virus. In the case of HIV2, after a phase of high cell mortality, a chronically infected cell population releasing infectious virions was obtained (the infection remained stable after 60 days of culture). By contrast, for a given multiplicity of infection (i.e. 10,000 cpm RT/106 cells), HIVI replication in U-937 cells was only transient (i.e. 14 days), leading to the synthesis of slightly infectious and probably defective viral particles. Abortive infection was finally obtained, as confirmed by a polymerase chain reaction which failed to detect any proviral HIV1 DNA in the cell line. These results indicate a marked difference between HIVI and HIV2 in their in vitro interaction with mononuclear phagocytes. KEY-WORDS: HIVI, HIV2, Monocyte, Replication; Virus/cell interactions.

Introduction. The human immune deficiency virus (HIV), the retrovirus causing the acquired immune deficiency syndrome (AIDS), principally infects human T4 lymphocytes (helper/inducer subset) that express the CD4 (T4) molecule. This antigen, or part of it, constitutes an important component of the HIV receptor, as demonstrated by blocking experiments with monoclonal antibodies to the T4 and T4A epitop¢ (Maddon et al., 1987; Dalgleish et ai., 1984; Klatzmann et al., 1984). In the course of infection, a reversal in the ratio of T4+/T8 + cells in peripheral blood is observed, which appears to be the major immunological abnormality of AIDS in vivo. However, these latter cells are not the only ones to be involved in HIV infection. It has been shown that this virus was able to infect cells of the monocytic lineage. The presence of virus particles in macrophage-like cells from brains of AIDS patients has been demonstrated by in situ hybridization and electron microscopy (Vazeux et al., 1987; Koenig et al., 1986; Wiley et al., 1986). Virus has been isolated from peripheral blood monocytes and from macrophage-like cells in lung, lymph nodes and skin of HIV-infected individuals (Gartner et al., 1986 ;.Salahuddin et ai., 1986). The infection of these cells is possible because of expression of the CD4 molecule

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on their surface ~(Asj6 et al., 1987; Wood et al., '1983), even though :its density is low, and because of the likely existence of other non-specific mechanisms of entry (Takeda et ai., 1989; Pauza and Price, 1988). Such cells not only migrate towards different tissues, but they also play a major role in initiating the immune response; they are effector cells in immune reactions and therefore seem to be important actors in the pathogenesis of AIDS. Thus, it has been suggested that mononuclear phagocytes can serve as a reservoir for the virus and as the principal agents for its spread to other target cells (Bender et al., 1988; Fauci, 1988; Roy and Wainberg, 1988). Healthy donor monocytes (Gendelman et ai., 1988; Ho et al., 1986; Nicholson et ai., 1986), as well as myeloid progenitor cells (Folks et al., 1988; Busch et al., 1986), can be infected in vitro. Moreover, several functions of monocytes, such as chemotaxis, phagocytic and bactericidal capacities, antigen presentation and cytokine secretion, could be altered, al~ough some reports seem contradictory (Petit et al., 1987; Salahuddin et al., 1986; Smith et al., 1984). At present, two distinct types of HIV are known: the prototype HIVI (previously referred to as HTLV-III, LAV-I, ARV) and HIV2 (HTLV-IV, LAV-2), isolated in 1983 and 1986 respectively (Guyader et ai., 1987; Clavel et aL, 1986; Gallo et al., 1984; Barr~-Sinoussi et al., 1983). Although these two viruses share ultrasWuctural and biologi~ properties, the alignment of their nucleotide sequences shows a distant homology, from 60 % for the more conserved gag and pol genes to 30-40 % for the other viral genes and the LTR (long terminal repeats). HIV2 was essentially found in West Africa, but has recently begun to appear in the United States and Europe. This latter virus could be less pathogenic than HIV1, but there is as yet no real agreement on this point (Poulsen et aL, 1989; Kanki et al., 1988). Our aim was to study HIV2 replication in vitro and compare it with that of HIVI. In addition, we chose to study the interactions with cells of the monocytic lineage rather than with T lymphocytes which are now well known targets of HIV.. In this present work, :the ~human promonocyte,celi :line U-937, akeady shown~o ,be susceptible to infection with HIV (Folks et al., 1988; Petit et al., 1987), was used as a model. We observed that HIV2 replication in these cells differed from that of HIVI. In the case of HIV2, after a phase of high cell mortality correlated with virus production, chronic infection was obtained in the cell line. In contrast, HIV1 infection of Xhese ~ulv*.,ILIt~ l g . ¢ U llLilLlglJult~f t •

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fect, and which aborted rapidly.

Materials and methods.

Cells.

The U-937 cell line derived from a histiocytic lymphoma (Sundstr6m and ~lilsson, 1976) expresses the CD4 molecule on its surface. For the present study, we chose a clone containing 90 % of CD4+ cells, as determined by flow cytometry using monoclonal anti-CD4 antibody (IOT4A tmmunotech).

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antigen. enzyme-linked immunosorbent assay. phosphate-buffered saline. polymerase chain reaction.

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P H A = phytohaemagglutinin. RT = reverse transcriptase. SDS = sodium dodecyl sulphate. SSC = standard saline citrate.

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Virus infection. U-937 cells were infected for 1 h at 37°C either with stock HIVIBRu or HIV2RoD at me same multiplicity of infection, i.e. 10,000 cpm RT (reverse transcriptase) per 106 cells (virus strains were kindly provided by Prof. Montagnier, Institut Pasteur, Paris). The cells were washed 3 times in RPMI-1640, grown in RPMI-1640 supplemented with 10 070 foetal calf serum, 200 mM glutamine, penicillin/streptomycine/kanamycine, and incubated at 37°C in a humidified incubator with 5 °70 CO~ in air. Culture medium was completely changed every 3-4 days and cells were maintained at densities of 1 x 106 cells per m!. Supernatants were harvested every day for the first week post-infection, then every two days for two months and stored at - 8 0 ° C . At different times, 10T cells were centrifuged, washed twice in phosphate-buffered saline (PBS) and stored in pellets at - 8 0 ° C .

Cull viability and morphology. Viability was assessed by the trypan blue dye exclusion technique. The cells were counted with a "Neubauer" chamber and the percentage of cell death was calculated. This was carried out several times so as to estimate the margin of error: (1) < _+3 °70 when cel! death was low, and (2) < +_10 070when cell death was high (i.e. > 50 070). At different times, 106 cells were pelleted, washed twice in PBS pH 7.4, then cytocentrifuged on slides and inactivated 15 min in methanol. The slides were stained with May-Grfinwald-Giemsa and examined with a "Zeiss Axioscope" microscope.

Antigen (Ag) capture assay. Supernatants from HIVI- and HIV2-producing cultures were tested for viral Ag by an Ag capture ELISA assay (ELISA-p24, DuPont). This test detected both HIV 1 and HIV2, with these major core proteins being quite similar in the two viruses, and showing antigenic_ cross-reactivity with ser a from HIVl- or HIV2-infected patients. Data were expressed as OD (optical density) units and considered positive when greater than 3 times the mean value obtained for mock-infected U-937 cells.

Reverse transcriptase (RT) assay. The RT assay was carried out as follows: supernatant viral particles were precip'tated with a solution of polyethylene glycol (PEG-6000 30 070 w/v in 0.4 M NaCl) at a ratio of 2/1 (v/v) for one night at 4°C. They were collected by centrifugation, 45 min at 1,000 g (4°C), resuspended in 501~l of Tris-HCl pH 7.6 and 20 V.I of thi~ suspension was added to the RNA-directedDNA polymerase reaction mixture. Iu order to increase the sensitivity of the technique and to detect earlier virus production, we used ~'-1;2"-3H-TTP. Data were expressed as cpm/ml of culture medium. Under our conditions, supernatant from non-infected cells gave a mean value of 2,000 cpm/ml; therefore, results were considered positive when greater than 5,000 cpm/ml.

Infectivity o f secreted viruses. At different times, the same dose of supernatant culture medium containing new viruses was tested for infectivity by infecting in parallel: (1) T lymphocytes (PHA-

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activated cord blood lymphocytes) and (2) U-937 cells, as described above. H9-IIIB supernatant culture medium was used as positive control. Viral replication was assessed in each case by the Ag ELISA test described above.

Polymerase chain reaction (PCR). PCR was performed with HIV1- and HIV2-infected U-937 cells collected and stored as described. The pellets were resuspended in the DNA extraction buffer (1 ml per 10x 106 cells). The cells were then lysed by adding 0.5 070 sodium dodecyl sulphate (SDS) and incubated with 100 izg/ml RNase and 100 i,tg/ml protease K. After phenol/chloroform extraction, the DNA was precipitated with ethanol and redissolved in sterile distilled water. The oligo primers and the oligo probe corresponded to the 5' portion of the highly conserved gag gene of HIV1 (kindly provided by Prof. B. Mach, Medical University of Geneva, Switzerland). Amplification of a specific segment of DNA by PCR with a thermostable DNA polymerase purified from Thermus aquaticus (Taq DNA polymerase) was carried out as follows: 1 l~g of each DNA sample was mixed with 1.5 mM of each dATP, dTTP, dCTP and dGTP, 1 x enzyme buffer (7 mM Tris-HCI pH 8.8, 16 mM ammonium sulphate, 10 mM magnesium chloride, 10 mM [~-mercaptoethanol, 0.02 070gelatin, 7 l.tM EDTA), 1 ~tM primer I and 1 IzM primer II. After incubation at 98°C for 7 mm (denaturation), the samples were incubated for 1 min at 40°C (annealing of primers). Then, 3 units of Taq polymerase (New England Biolabs) were added and the tubes were incubated for 3 rain at 70°C (DNA synthesis). This cycle was repeated 28 times, with all subsequent denaturation steps limited to 1 min at 92°C and without further addition of enzyme. Ten microlitres of each amplified sample were then adjusted to 0.6 M sodium hydroxide for denaturation, 10 min at room temperature, neutralized with 20 i~l of 2 M ammonium acetate and applied to a nitrocellulose membrane (pore size 0.45 ~tm) with a slot-blotter apparatus (Schleicher and Schiill). The filter was prehybridized for 3 h at 510C in 4 x SSC, 5 mM EDTA, 5 x Denl~rdt (Denhardt = 0.02 070 FicoU, 0.02 070polyvinylpyrolidone, 0.02 070bovine serum albumin), 0.1 070SDS, 100 ~tg/ml h ~ r r l r l g sperm DNA • Then, h- ~v ja h. .r .; .r.i.;.~. ,. a. .¢. ;. n. .n. . . . . . . .a. t. . . .t.h. e . q ' t ~ r r n i n a l was . . .a. r. r. .i , ~ r l .d. p. .r. l.~. .t.n. .r.~. .d. . . . t, out by the addition of an oligonucleotide probe specific for the amplified fragment, labelled with 32p at 51°C overnight (5xSSC, 5 x D e n h a r d t , 2 0 m M sodium phosphate, 5 070 SDS, 100 ~.g/ml denatured herring sperm DNA, 106 cpm/ml of labelled probe). This was washed twice in 3 x SSC, 8 × Denhardt, 5 070 SDS at 50°C for 15 min and autoradiographed for 5-6 h with an intensifying screen at - 8 0 ° C .

Results.

Experiments were carried out twice at 3-month intervals and the same results were found in each case. The replication of HIV1 and HIV2 in U-937 cells was assessed by measuring the RT activity and the gag Ag content of cell-free supernatants. As shown in figure 1, HIV2 expression in these cells was characterized by a lag phase of about 5 days before Ag productian. Then, peak virus production was reached at approximately day 10; this was followed by chronic infection, still present after 60 days of culture. Figure 2 shows the RT activity of the same supernatants which is closely correlated with the Ag released. However, in our hands, the RT assay, as we expected, appeared more sensitive than detection of core protein by ELISA assay and allowed us to reveal virus replication in the cell-free supernatant at day 3 instead of day 5

HIV2 I N F E C T I O N OF P R O M O N O C Y T I C CELLS

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as f o u n d for gag Ag. F r o m this time on, R T activity also increased rapidly and r e m a i n e d stable at values o f a p p r o x i m a t e l y 10 million c p m / m l when using T-1 ; 2 ' - 3 H - T T P .

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Following infection of U-937 cells with either HIV2 or HIV 1 strains, supernatants were harvested at regulm" intervals and tested for the presence of viral RT by RT assay. In order to optimize this technique and to detect the viral particles produced earliest, we used ~.-1,2 J H - T T P for greater sensitivity. According to values ohtmned from non-infected U-937 supernatants, positive data were considered as those that were higher than 5,000 cpm/ml. [] HIV2-infected U-937 cells, [] HIVl-infected U-937 cells, A uninfected U-937 cells.

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In contrast, the same number of H!VI particles induced an acute and immediate infection: p24 Ag was detected as early as 5 h post-infection (fig. 1). This was, however, a transient infection, since virus production decreased rapidly and stopped 14 days post-infection. This replication restriction was still present at day 90 (data not shown). Furthermore, the parallel observed with HIV2 between the two detection techniques was not seen with HIV1 infection of U-937 cells, since no RT activity could be detected in the supernatants, although a peak of p24 Ag production was reached between day 3 and day 7. Whereas no significant degree of cell death ( < 10 %) was observed in the course of HIVI infection (fig. 3), HIV2 induced a high degree of cell lysis (average 60 to 70 % of the cells, with a peak of 90 °7o at day 17), but no syncytium formation could be seen. For 3 weeks, this amount of cell death corresponded closely to Ag production. Then, as cell death decreased to less than 10 °70, cells continued to produce Ag and show strong RT activity, leading therefore to the establishment of a chronically infected population. In HIV2 culture, as shown in figure 4A, giant cells with an increased number of cytoplasmic vacuoles could be seen. In contrast, HIVI infected U-937 cells conserved the same morphology as control cells (fig. 4B and 4C).

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At regular intervals, the number of viable and dead cells was assessed by the trypan blue dye exclusion technique, thus enabling a deduction of the percentage of cell death.

FIG. 4. -- Morphology of cultured celts. A = U-937 + HIV2 (DI4); B = U-937 + HIVI (DI4); C = U-937 control (DI4). May-GriinwaldGiemsa stain, x 260.

HIV2 I N F E C T I O N OF P R O M O N O C Y T I C CELLS

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P. I N N O C E N T I A N D J.M. S E I G N E U R I N

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Because of these results, two questions had to be answered: (1) was an important signal in Ag capture a n d / o r RT activity synonymous with production of infectious viral particles ? and (2) was U-937-secreted virus more infectious for these latter cells ? Culture media from the previous experiments were therefore inoculated into U-937 cells and into PHA-activated T lymphocytes. Within a week, day 14 HIV2 supernatant inoculated into T-cell culture (fig. 5a) gave positive Ag capture results, thus showing that it c o a t , n e d numerous infectious virions. Within the same period, day 3 HIVI supernatant (fig. 5b), although taken at the time of maximal Ag production, remained 8

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days FIG. 5. - - Infectivity of newly synthesized viruses'. Supernatants from the previous experiments were inoculated on U-937 cells and, in parallel, on PHA-activated T lymphocytes. Viral replication was assessed by Ag capture, a) infectivity of new HIV2 viruses; b) infectivity of new HIVI viruses; - - on U-937 cells; . . . . on T lymphocytes; & HIV2 DI4 supernatant (Spn); A HIV2 DI Spn; [] HIVI D3 Spn.

1-11I/2 I N F E C T I O N

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negative, olfly becoming infectious i 9 days after inoculation into T lymphocytes. H IV2 virions grew slightly more slowly in U-937 cells than in T lymphocytes, whereas HIV1 virus showed no sign of replication in these former cells, even after 42 days of culture. On the other hand, it is to be noted that, in spite of negative Ag capture and RT assays, day 1 HIV2 supernatant contained enough infectious virions to enable replication in both cell types within 2 weeks, suggesting that culture assay is the more sensitive method for detecting HIV. The rapid restriction of HIV l replication observed in U-937 cells led us to search for viral genome in these cells, using PCR, in an attempt to determine whether it was an abortive infection or a latent one. As shown in figure 6, hybridization with the specific probe did not reveal any signal, it was therefore concluded that HIV1 had been eradicated from cell culture and that the decrease in, and arrest of, p24 Ag production indicated an abortive infection.

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Discussion.

In the present study, a chronic infection of the promonocyte cell line U-937 was obtained with the HIV2RoD strain. Following a 3-day period during which new viral particles were not detected, a peak of virus production was rapidly reached, remaining stable for 60 days. Correlated with the beginning of virus release, there was drastic cell lysis which was, however, transient. A clone of cells was probably selected in vitro, which came about through infection and continued to grow, harbouring and secreting HIV2. The presence of different cytopathic HIV clones has been shown within the same isolate (Sakai et al., 1988), and two low-cytopathic HIV2 strains (HIV2uc] and HIV2sT) were recently isolated (Evans et ai., 1988; Kong et al., 1988). This could therefore explain why a few cells were not affected by viral lysis, and which then gave rise to a chronically infected cell population. Alternatively, it is suggested that the 10 % of cells that did not express the CD4 molecule were infected in a nonspecific manner which, directly or indirectly, would be less destructive for them. Finally, newly synthesized HIV2 viruses seemed to be particularly infectious and virulent, even in low numbers, both for T lymphocytes and U-937 cells.

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In comparison, the HIVIaR U strain led to an acute infection of the promonocyte cell line without any significant cell death. This latter point is in agreement with other data (Fauci et al., 1988; Nicholson et al., 1986). The HIVI life cycle was initially productive, becoming restricted for the following 10 weeks with no viral replication. The same pattern has already been observed in these cells (Pauza et al., 1988; Folks et al., 1987). However, in our case, HIVI replication was followed by an abortive infection, since no viral genome could be detected in U-937-infected ceils using a method as sensitive as the PCR. Although there was an important production of p24 Ag between day 3 and day 7, no RT activity could be detected despite the increased sensitivity of our assay. Moreover, infectivity of the culture medium was very low, i.e. very few infectious particles were released. Therefore, two hypotheses could be raised: either there was a high production of soluble p24, or defective viruses were synthesized. The former would suggest that "too few" viral particles were present to be detectable by RT assay and that an abnormally high production of free p24 protein occurred. The latter hypothesis would suggest that an important release of defective viruses was responsible for the absence of positive RT results (because of no or modified enzyme?) and the weak infectivity seen. Thus, supernatants may contain the major core protein without necessarily being infectious for cells. Recently, it has been asked whether such defective HIV viruses could be responsible for AIDS (Weiss, 1989), as has already been demonstrated for some murine (Aziz et al., 1989) and feline retroviruses. HIV2 replication is not comparable to HIVI replication in monocytic cells, although initially the same amount of virus was inoculated under the same conditions. With HIV2, after an important degree of cell death, a subpopulation of cells chronically producing infectious virions was obtained. In contrast, the HIV-1 infection appeared earlier, but was transient; infected U-937 cells released very few infectious viruses which were then eradicated from the culture, leading to an abortive infection. The interactions between HIV and the monocyte/macrophage lineage are complex. U-937 cells, although not a true physiological system, provide a model of monocyte cell line susceptible to HIVI and HIV2 infection which should contribute tc e.lucidating virus persistence in in vivo monocyte/macrophage cells. R~sum~. INFECTION CHRON1QUE DE CELLULES PROMONOCYTAIRES PAR LE

VIH2

Alors qu'il est bien 6tabli que le VIHI est capable d'infecter les cellules du syst6me des phagocytes mononucI66s, le comportement du VIH2 vis-fi-vis de ces cellules a 6t6 beaucoup moins 6tudi6. Dans cette 6tude, nous avons suivi, dans les cellules de la lign6e promonocytaire U937, la cin6tique de r6plication de la souche de r6f6rence VIH2RoD que nous avons compar6e ~ celle obtenue avec le VIH 1,as;u. A chaque stade~ de la cin6tique, nous avons recherch6 l'antig6ne p24, dos6 l'actlvlt6 de la transcriptase inverse (RT) darts le surnageant des cultures et 6valu6 le caract6re infectieux des virus produits. Avec le VIH2, nous avons obtenu, apr6s une phase de forte mortalit6 cellulaire, l'6mergence d'une population chroniquement infect6e (toujours stable 60 jours apr6s l'infection) et produisant des virions infectieux. En comparaison, pour un m~me inoculum initial (10.000 clam RT/106 cellules), rinfection par le VIH1 n'a 6t6 que transitoire (14 jours), conduisant/t la synth6se de particules virales peu infectieuses et probablement d6fectives. Une infection abortive a finalement 6t6 obtenue, confirm6e par l'absence de g6nome proviral dans les cellules apr6s amplification enzymatique. Ces r6sultats indiquent que l'interaction VIH/cellules monocytaires in vitro diff6re selon qu'il s'agit du VIHI ou du VIH2.

MOTS-CLI~S" VIH1, VIH2, Monocyte, R6plication; Interactions virus/cellule.

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